Brunauer-Emmett-Teller Bjh

Barrett-Joyner-Halenda Bp

ATRP Atom transfer radical polymerization BET

Kλ

Absorption Weight fraction of interphase Materials Institute (IS2M).

Firstly, I want to thank my two supervisors Dr. Abraham CHEMTOB and Dr. Julien POLY. During last 3 years, they taught me a lot with different skills, starting from soft and ending with professional ones. I think, that under this supervision I have made progress in managing my schedule, presenting data, writing manuscripts and treating scientific data. Thanks again for this!

In the next turn, I want to acknowledge all my collaborators:

-Dr. Camélia MATEI-GHIMBEU for our fruitful work on the synthesis of mesoporous carbon materials and for assistance in data interpretation.

-Dr. Laurent PIEUCHOT and Dr. Tatiana PETITHORY for the the studies of cellular interactions with confocal microscopy.

-Dr. Jean-Pierre MALVAL for his help in the fluorescence data acquisition and interpretation.

-Dr. Marc SCHMUTZ for the possibility to shed light onto core-shell morphology with cryo-TEM technique.

-Dr. Florence BALLY-LE GALL for providing me the possibility to teach in a European educational laboratory.

This PhD project had a lot of directions. As consequence, I worked with a lot of collaborators Thanks to Loïc VIDAL for his patience in acquisition of hundreds TEM samples. To Cyril VAULOT for gas adsorption/desorption analysis. To Gautier SHRODJ for modulated differential scanning calorimetry analysis. To Bénédicte RETY for assistance in thermal treatments procedures. To Philippe KUNEMANN for tensiometry analysis. To Samar HAJJAR-GARREAU for XPS analysis. To Dr. Adrian BEDA for his quick feedback concerning Raman test experiment required by reviewers.

Finally, I want to thank to all the administrative staff who helped me a lot in preparing trips for conferences and who facilated my everyday life at IS2M.

Thanks again to everyone!

General Introduction

Within almost two decades from now, polymerization-induced self-assembly (PISA) has confirmed its position as a major method for producing amphiphilic block copolymer nanoparticles (NPs). There are three main drivers of this success: firstly, the possibility to combine PISA with a range of reversibledeactivation radical polymerizations (RDRP); secondly, the single-step access to a range of NP morphologies (from conventional spheres to vesicles or fibers); thirdly, the higher solids content compared to conventional methods such as nanoprecipitation. But these genuine advances should not mask the fact that a substantial majority of PISA-based nano-objects result from a thermally-initiated polymerization. Since 2015, ultra-violet (UV)-visible (VIS) photopolymerization ─ to date implemented primarily as a film curing process ─ has emerged as one of the most promising methods to upgrade PISA process. [START_REF] Jasinski | [END_REF] Photochemically initiated PISA (photoPISA) opens avenues for improved process conditions for the synthesis of diblock copolymer nano-objects in regards to reaction efficiency, energy consumption and safety. Additionally, NPs can be achieved with a higher level of control through adjustments of parameters such as irradiance,

wavelength not accessible with conventional means (i.e. using thermal radical initiators). As core element of this doctoral dissertation is the development of an original photoPISA process relying on a photomediated reversible-addition fragmentation chain transfer (RAFT) mechanism. Three key features characterize our process: an ambient temperature reaction, the use of visible light to start and temporally control the reaction, and the absence of radical initiator. The possibility to avoid the use of any exogenous initiator relies on photosensitive RAFT agents which undergo homolytic β-cleavage under visible light, resulting in formation of primary initiating radicals. To distinguish this mechanism from a photoinitiated RAFT involving a radical photoinitiator, it is referred to as photomediated RAFT.

Since its early developments 5 years ago, photoPISA has been studied by a small number of research groups. Most studies were conducted by the polymer community, who has focused on popular topics such as molecular weight control and broad screening of experimental parameters able to control particle morphology. By contrast, investigating the mechanism of nucleation in this system and how it differs from a thermally-initiated one, or understanding how optical properties change throughout the reaction have been generally overlooked. In addition, characterizing the core-shell particle architecture that plays a critical role in NP properties, or finding relevant application for the block copolymer nano-objects have been much less discussed. This PhD dissertation consisting of 4 chapters aims at shedding light onto several of the aforementioned issues. It begins with a bibliographical chapter providing some general considerations about RAFT, RAFT-mediated PISA and the main contributing factors to the development of photoPISA. It ends with a state-of-the-art review of RAFT-mediated photoPISA, which has been classified according to the three different ─ photoinitiated, photomediated, and photocatalyzed ─ mechanisms. The next three chapters cover the experimental results obtained during this PhD with three main areas of interest:

Chapter I: Emerging trends in RAFT-mediated photoPISA Introduction Self-assembly of amphiphilic diblock copolymers enables the preparation of nano-objects with different morphologies such as spherical micelles (S), worms (W), lamellae (L) and vesicles (V), which have found broad applications in enzyme catalysis, 2 drug delivery, 3,4 nanostructured films, 5 stimuli-responsive smart nanomaterials. 6 Traditionally, the synthesis of these supramolecular structures is conducted from a preformed diblock copolymer. The two main methods are nanoprecipitation and micellization, though many variants exist. 7 Micellization aggregates a portfolio of processes leading to thermodynamically controlled self-assembled particles. These structures are dynamic in the sense that they exhibit exchange of single molecules (unimers) and can be disrupted upon decreasing the copolymer concentration below the critical micellar concentration (CMC). In water, micellization typically requires dissolving the diblock copolymer in a water-miscible organic solvent followed by dialysis against water. Alternatively, micellization can be induced by gradual addition of water, and aggregation is driven subsequently by slow solvent evaporation.

By contrast, nanoprecitation, that was first developed by Fessi et al.,8 is a kinetically controlled process. As described previously, the copolymer is first dissolved in a water-miscible solvent, then the solution is added to a large amount of water. Solvent diffusion leads to a supersaturation of the copolymer, and the formation of micelles. These two methods have the advantage to be compatible with drug loading and can yield uniform, nanosized nano-objects. However, they involve the use of organic solvents that pose potential health risk and solutions. In addition, the challenge is that they are not productive since the typical concentration of polymer is less than 1 wt%, and post-processing steps such as dialysis (that are not easily scalable) are usually necessary. To unlock the potential commercial applications of diblock copolymer NPs, the challenge remains to consistently prepare at high concentrations and in minimum of steps NPs with a narrow, predictable size-distribution and morphology, and using a variety of continuous phases. Since 2003, particular attention has been paid to an original process referred to as PISA. Among its advantages, there is the possibility to conduct the reaction process at higher solids content (from 10 to 50 wt%) [9][10][11] with fewer processes and purification steps 12 since diblock copolymer synthesis and self-assembly takes place concomitantly.

PISA starts from a solvophilic block usually prepared through solution polymerization, and playing the role of steric stabilizer. This soluble polymer is chain extended with a second monomer to form a second solvophobic core-forming block through a judicious choice of solvent. 13 Upon reaching a critical chainlength, this second block drives a spontaneous copolymer self-assembly as shown in Scheme 1.1.

Depending on the initial solubility of the second monomer, two types of polymerization can be distinguished. Emulsion polymerization, that is usually conducted in water, involves an insoluble monomer, 3,14,15 while a soluble monomer is used for dispersion polymerization. 16 With the exception of recent studies resorting to ring-opening metathesis polymerization (ROMP) 17 and non-controlled polymerization mechanism, 18 the development of PISA has been mostly fueled by reversible deactivation radical polymerizations (RDRP). Also referred to as controlled radical polymerizations (CRP), this family of polymerization techniques has one similarity: the existence of an equilibrium between active species (propagating free radical species) and dormant species. In favorable conditions, this equilibrium allows all chains to grow simultaneously nearly at the same rate and therefore have the same chemical composition and length. This results in polymers with predictable degree of polymerization (DP) and narrowly distributed molecular weight dispersity (Đ). RDRP has made possible to synthesize a broad range of diblock copolymer nano-objects through PISA process. Three main techniques have been applied to PISA (1) nitroxide-mediated polymerization, [19][20][21] (2) transition metal-mediated radical polymerization techniques (such as atom transfer radical polymerization (ATRP), 22,23 cobalt mediated polymerization 24 organotellurium-mediated radical polymerization 25 ) and (3) reversible additionfragmentation chain-transfer-mediated polymerization (RAFT), with most emphasis on the latter one.

Owing to its versatility in terms of compatible monomers and experimental procedures, RAFT is by far the most widely used RDRP technique for PISA. 11 A majority of studies relies on conventional thermally initiated RAFT processes. Much less investigated is photochemically-induced PISA despite many distinctive advantages compared to the thermal method as higher solids content and possibility to operate the polymerization at ambient temperature. As entry into this PhD dissertation, this bibliographical chapter Mechanism. The typical mechanism of a RAFT-mediated polymerization is depicted in Scheme 1.2.

Following initiation (step I, radical generation from a non-radical species called initiator) and beginning of propagation (step II, multiple radical addition to monomer), the macroradical Pn  adds to the C=S double bond of the RAFT agent in a pre-equilibrium step (step III). An instable intermediate radical is formed, that can rearrange by releasing the R  radical, and forming a macroRAFT agent or macro-CTA Pn-SC(Z)=S. R  can reinitiate a new chain (step IV, reinitiation). If we look back briefly to step III, a radical displacement (transfer reaction) takes place (from Pn  to R  ) through the CTA, but proceeds in 2 steps in contrast to typical CTA: a first radical addition, followed by as second fragmentation reaction. Of high importance is that the polymeric dithioester Pn-SC(Z)=S resulting from this reaction can also act as transfer agent, making the chain-transfer reaction reversible. Finally, there is no change in the total number of radicals during the addition-fragmentation process. Therefore, the chain transfer reaction can be termed degenerative. This explains why an initial source of radical (initiator, or photoinitiator (PI)) is required in a RAFT process.

Once all the CTA has been consumed, a main equilibrium (step V) can take place consisting of a reversible equilibrium between a dormant macro-CTA species (Pn-SC(Z)=S) and a propagating chain (Pm  ).

General considerations. This equilibrium is responsible for the simultaneous growth of chains during the course of the polymerization. The reversible nature of this chain transfer mechanism allows the production of polymer chains with predictable DP (related to the initial amount of CTA) and with relatively narrow Ð < 1.5. For an effectively controlled polymerization, the rate of chain transfer should be higher than that of propagation, so there should be less than one monomer unit added between activation (release of propagating radical) and deactivation (reaction of propagating radical with macro-CTA). Only, under these conditions, all chains can have a small molecular weight dispersity at a given time. However, irreversible bimolecular terminations involving primary and propagating radicals (step VI) can yield dead chains. This can be minimized however by decreasing the free radical concentration across the course of the polymerization, for example by selecting a high enough ratio [CTA]/ [I]. The products of a RAFT polymerization are chains with and without the TTC end-group at the ω-end (controlled and dead chains, respectively). There are also two types of polymeric chains with regards to the nature of the initiation (αend): chains initiated by the source of radicals (typically an initiator fragment, step II) and chains initiated by the RAFT agent R-group (step IV).

Effect of CTA structure. R-and Z-group play important roles in the function of RAFT agent.

Intermediate radical (S- CH(Z)-S, see step III) formation and stability depend on the Z-group attached to C=S bond. Therefore, the Z group mainly determines addition rate of propagating radicals (pre and main equilibrium). From the other hand, R-group should be a good leaving group and form a sufficiently stable R  radical to drive forward the chain transfer reaction (fragmentation reaction). Additionally, R  must instable enough to promote reversible chain transfer and enable efficient reinitiation.

Control of DP.

The number of chain Xn is determined by the amount of reacted RAFT agent, the amount of initiator decomposed, and the monomer conversion:

𝑋 𝑛 = 𝑝[M] 0 𝑝 ′ [RAFT] 0 +2𝑓𝑝 ′′ [I] 0 (1)
where [M]0, [RAFT]0 and [I]0 are the initial concentrations of monomer, RAFT CTA, and initiator. p, p' and p'' are the fractional conversions of monomer, RAFT agent and initiator, respectively. In the case of initiation by thermal initiator such as diazo or peroxide compounds, the fractions of primary radicals which are unsuccessful in initiating a polymerization is known from initiator efficiency (f). 26 1.2. RAFT as a tool for mediating PISA It is established that amphiphilic AB diblock copolymers can undergo self-assembly in an appropriate solvent for one of the two blocks. 27 In principle, PISA can be conducted using any type of RDRP technique, 28,29 but in practice, the majority of literature's examples are based on RAFT-mediated polymerization. 30,31 The reason behind is that RAFT is tolerant towards many functional monomers, and can be performed in a wide range of solvents, including water, 32,33 polar solvents (in particular alcohols), 34,35 non-polar solvents. 36,37 In this view, RAFT has made possible to synthesize nano-objects with a high level of control and reproducibility that is difficult to achieve using other polymerization processes.

In the early 2000s, Hawkett and co-workers were the first to report RAFT aqueous emulsion polymerization using water-immiscible monomers such as methyl methacrylate, n-butyl acrylate or styrene (2-(butylsulfanyl)-carbonothioyl sulfanyl propanoic acid was used as RAFT agent). 14,38 They showed that the key point to form self-assembled block copolymer micelles was to prevent monomer droplet formation through starved-feed reaction mode. Taking inspiration of this pioneering study, RAFT-mediated PISA technique has rapidly expanded not in emulsion, but rather in aqueous [START_REF] Charleux | Hybrid Latex Particles[END_REF][START_REF] Charleux | [END_REF] and organic dispersion. 41 The symbiotic link between RAFT and PISA is exemplified by the publication of twenty reviews since 2008, covering general considerations 42,43 or more specific topics including biomedical applications. 44,45 A new impulse in the field of RAFT-mediated PISA story took place in 2015, when Cai and co-workers, reported for the first time a photochemically-induced PISA process. 46 1.3. From RAFT PISA to RAFT photoPISA There has been intense interest in using alternate forms of initiation to drive RAFT-type polymerizations such as (photo)redox catalysts, 47 enzymes, [48][49][50] organic acids (radical 51 and cationic 52 mechanisms), and direct activation of the RAFT agent itself. [53][54][55] In particular, the use of electromagnetic radiation to initiate RAFT polymerization has been described employing a broad range of wavelengths including gamma, 56 ultraviolet (UV), 57,58 visible, 47,59 near infrared (NIR) 60 and microwave. 61 However, most RAFT-mediated PISA syntheses have been performed over the last decade using thermal radical initiators.

As briefly addressed in the introduction, photoPISA has several unique advantages, the most important is the mild conditions related to ambient temperature reaction. In 2015, as an example, mild conditions allowed

Tan and co-workers to encapsulate bovine serum albumin (BSA) as a model thermally unstable protein inside photoPISA-derived NPs without losing their biological activity. BSA demonstrated 90% activity as compared to native BSA during photoPISA approach, on the contrary, classical conditions of thermally initiated PISA (70 °C for 2 h) revealed only 35% activity. 62 In 2016, the validity of this approach was also confirmed by Boyer and co-workers which encapsulated a model drug (Nile Red) in PISA-derived NPs. 63 Another advantage of photoPISA relies on the possibility to reach a high level of special and temporal control due to facile turn ON/OFF reaction mode. In 2017, O'Reilly and co-workers, demonstrated that under the same conditions photo-and thermally initiated PISA led to different morphologies. In the case of photoPISA, the authors found that predicting the final morphology was much easier than for a thermally initiated approach. 12 The wavelength of irradiation has also a strong impact on NP morphology. The fine tuning of irradiation wavelength in photoPISA was thus emphasized as a key advantage compared to thermally initiated method. 63 It is a very important aspect because NPs are formed throughout PISA, thus causing an attenuation of the incident photon flux due to backscattering. Turbid media scatters more light and polymerization rates can be decreased due to a lower light penetration. This limitation can be mitigated by using higher irradiation wavelengths. From this view, Boyer and co-workers showed that using specific photocatalyzed systems made possible to conduct photoPISA under yellow and red light. [64][65][66][67] 2. Three photochemical mechanisms of RAFT-mediated photoPISA Three photochemical routes have been mainly employed to initiate RAFT-mediated PISA that differ by the way reactive species are generated,

(1) A photoinitiated approach using a PI,

(2) A photomediated approach by direct activation of the CTA

(3) A photocatalyzed approach relying on a photocatalyst (PC).

These three pathways are described in Scheme 1.3. All lead to the formation of a propagating radical Pn  , which can be involved in a conventional RAFT described at the top of the scheme (main equilibrium, for a complete RAFT mechanism see Scheme 1.2). and TPO-Na (that is water-soluble) and bis-acylphosphine oxide (BAPO). They produce benzoyl and phosphinyl radicals, which are both able to initiate a polymerization. TPO and BAPO derivatives are commercially available, and can be used with LEDs, ranging from 365 to 405 nm. Their red-shift absorption compared to other type I PI (a spectral shift towards higher wavelengths i.e. lower energy and lower frequency) is relevant to limit the absorption of the RAFT agent. The second approach is photomediated RAFT, which relies on the reversible β-cleavage of electronically excited (TTC) compound (Scheme 1.4). This photochemical reaction caused the cleavage of R-S bond, yielding an initiating alkyl radical R  and a thiyl radical. As result, TTC acts not only as a conventional CTA but also as a source of initiating radicals, which explains why it is sometimes referred to as a photoiniferter (for photoinitiator, transfer agent, and termination agent). The reactivity of TTC compounds has been known for a long time. 68 However, their interest in the framework of photo-mediated RAFT mechanism has been renewed only recently, 69 owing to the discovery that this photolysis can occur not only under UV irradiation but also under visible light. Using visible light (typically blue light), a better control of RAFT-mediated polymerizations was reported in solution possibly as a result of minimized side/termination reactions involving photogenerated thiyl radicals. 59,63,[70][71][72][73] In this work, an emphasis was made on photomediated RAFT polymerization, we used CMDTC (Scheme 1.4) as RAFT agent. The RAFT process in a solution using CMDTC as RAFT agent revealed wellcontrolled polymerization with a good match between theoretical and experimental molecular weights.

Photomediated RAFT polymerization

Temporal control and good chain-end fidelity were also demonstrated. These latter features are especially important for conducting PISA because chain extension to form the second solvophobic block requires an efficient reinitiation.

Although this route obviates the need of additional PI or PC, it is at present the least developed in photoPISA. This may seem surprising when we consider that the photochemistry of thiocarbonyl compounds has been well known since the 1980s, 74 and that in the field of polymerization, the use of dithioesters as photoiniferters dates back to the 1980s. 75 As regards UV-Vis absorption, thiocarbonyl double bond chromophore show strong (π, π*) absorption at 280 to 350 nm (S0 → S2), and weak long-wavelength band at 380 to 550 nm assigned to (n, π*) absorption (S0 → S1) (Scheme 1.4). Consequently, UV irradiation can cause excitation into either S1 or S2 bands, but in contrast to (aryl)ketone (see description of type I PI in previous section), homolysis of the (S=)C-S bond adjacent to the electronically excited chromophore (αcleavage) is scarcely favored due to the lower energy of the C=S excited state compared to its C=O analogue. Interestingly, photocleavage is much more common with TTC (S=)C-S-C, in which the carbonsulfur simple bond is sufficiently weak for its homolytic cleavage (β-cleavage) to be energetically feasible from the lowest excited state of the substrate. Indeed, the bond dissociation energy of S-C linkages in common thioether (S-C) compound is about 30 kcal mol -1 weaker than C-C bonds found in alkane, and also significantly smaller than C-O analogues by 10 kcal mol -1 . 76 However, the β-fragmentation of TTC with UV that is reversible has resulted in controlled polymerizations for a few cases only, most of them in solution. 77,78 In the literature, it is often referred to as "reversible photolysis", "direct photolysis" or even "direct photoactivation".

The interest of this photomediated approach is to remove the need of external initiator since initiation radical species, mostly carbon-centered radical species can be generated through β-cleavage of the CTA.

Additionally, macro-CTA bearing a TTC end group can be also subjected to β-cleavage, this enables a photochemical activation of dormant macroradical species without relying on a conventional additionfragmentation mechanism. The main mechanism governing chain growth is still degenerative transfer reaction, allowing for control over the polymerization similarly as a thermal analogue.

Photocatalyzed RAFT polymerization

Like photomediated RAFT, photocatalyzed RAFT is another route free of exogenous PI since they both rely on the RAFT agent to generate initiating radicals. Consequently, there is no risk of "uncontrolled" chains derived from an initiator. Another benefit includes reduced termination under certain conditions when the concentration of initiating radicals can be finely tuned. 79 In 2014, Boyer and co-workers were the first to prove that PCs (Figure 1.4) such as Ir(ppy)3 and [Ru(bpy)3]Cl2 could be used to mediate a RAFT polymerization under visible light in solution. 80 This process was named photoinduced electron transfer -RAFT (PET-RAFT) and proposed to proceed according to the mechanism provided in Scheme 1.3. 81 In a typical PET-RAFT mechanism (oxidative quenching mechanism represented in Scheme 1.3), a PC, for example fac-[Ir(ppy)]3, generates an electronically excited species (PC*) under irradiation, acting as a reductant of TTC compounds (CTA or macro-CTA) via a PET reaction, resulting in the production of a oxidized PC + species and a TTC radical anion. It is hypothesized that this instable anionic species can spontaneously fragment to give initiating carbon radicals (R • with CTA and Pn • for macro-CTA) and a TTC anion -SC(Z)=S. The radical Pn • and R • may propagate, or either participate in a conventional reversible degenerative RAFT process. In addition, they can be subjected to a back electron transfer reaction with the oxidized by PC + species to regenerate the initial PC and form a cation Pn + . This latter can react with the TTC anion -SC(Z)=S to regenerate the dormant species Pn-SC(Z)=S, thereby completing the catalytic cycle.

Like the previous process, PET-RAFT impacts both initiation and activation of dormant species, since the electronically excited reductant (PC*) can react both with CTA (R-SC(Z)=S) and macro-CTA (Pn-SC(Z)=S). This process is compatible with a range of solvents and monomer systems and proved to be possible under homogenous and heterogeneous (PISA) conditions. In addition, the PC can be used at much lower concentrations compared to traditional radical initiators (~0.05 wt% to monomer), and generally provides a greater DP control compared to the other routes described previously. But, the main advantage over other techniques is the possibility to select a broad range of irradiation wavelengths in the visible range, for example in the green while photomediated RAFT is usually limited to blue irradiation. In this domain, there is surely no absorption overlap with RAFT agent. (2,4,5,7-tetrabromo-6-oxido-3-oxo-3H-xanthen-9-yl)benzoate, ZnTPP -5,10,15,20-tetraphenyl-21H,23Hporphine zinc, ZnTMPyP -5,10,15,20-tetraphenyl-21H,23H-porphine zinc. 3. State of the art in RAFT-mediated photoPISA This third section is a state-of-the-art and critical review of photoPISA organized according to the photochemical routes described previously: photoinitiated RAFT, photomediated RAFT relying on direct activation of RAFT agent, and PET-RAFT. All monomers and RAFT agents employed for these three photoPISA routes are summarized in Figure 1.5.

Photoinitiated RAFT PISA

The first examples of photoPISA were based on photoinitiated RAFT due to the fact that it simply involves the replacement of a conventional radical thermal initiator by a radical PI. All PIs used in this section are summarized in Figure 1.2. A summary of the investigations published is shown in Table 1.1. In 2015, Cai and co-workers reported the first example of visible light photoinitiated dispersion PISA (Scheme 1.5) using SPTP as water-soluble PI, CEPA as RAFT agent and a medium-pressure mercury light source (with a 400 nm filter). 46 In this work, PHPMAm based macro-RAFT was chain extended with DAA Recently, this group introduced a new PISA in which particle shape and size uniformity can be controlled by reaction temperature. 83 They studied photoPISA of DAA monomer using PHPMAm macro-CTA (using CEPA as RAFT agent) and SPTP PI in water at 25-70 °C. DLS and cryo-TEM data evidenced that diblock copolymer lamellae formed at 25 °C while the copolymer morphology evolved from S to W to

V during polymerization at 60 °C, which is above the lower critical solution chain length of the coreforming block. The particle shape and size uniformity could be controlled by reaction temperature. V to L and V to W transitions were achieved upon directly cooling the reaction dispersions from 70 °C to 25 °C, leading to transformation of initially free-flowing liquids to physical hydrogels (Scheme 1.7).

In 2017, Tan and co-workers used visible light photoinitiated PISA to produce a series of welldefined all-acrylic PHEA-b-PIBOA diblock copolymer nano-objects. 85 Polymerizations proceeded rapidly with high monomer conversion achieved within 30 min. SEC demonstrated that good control was maintained throughout the photoPISA process, and the final diblock copolymers exhibited relatively low Ð ≤ 1.55. By virtue of the high Tg value of PIBOA, a diverse set of diblock copolymer nano-objects having different morphologies were prepared. The same group also explored a novel type of photoinitiated seeded RAFT dispersion polymerization for the synthesis of epoxy-functionalized triblock copolymer nano-objects at room temperature. 86 Seeded RAFT polymerization exploits diblock copolymer nano-objects as the seeds for further chain extension. This method facilitates the preparation of triblock copolymer nano-objects with O'Reilly and co-workers demonstrated that PISA using the same monomers and CTA and based on thermal and photochemical initiation can lead to structures that are both chemically and morphologically distinct. 12 PEG113-CEPA was used as macro-CTA and chain extended with HPMA to generate PEG113-b-PHPMA300 diblock copolymer. PP-OH was used as PI and VAZO was used as thermal initiator. Their investigations revealed a loss of RAFT end group in photoinitiated PISA responsible for the formation of higher order structures (Scheme 1.12). This is driven by the loss of chain-end fidelity leading to the increase of theoretical molecular weight of the second solvophobic block, as fewer chains can be propagated. It results in the increase of the packing parameter, causing the formation of higher-order morphologies.

Similarly, a loss of end group was also observed upon post-synthetic light irradiation, and caused likewise a shift in morphology towards higher order structures. Additionally, when the morphologies were similar for identical block ratios with near-identical molecular weight distributions, the polymers may not be chemically identical by virtue of the differences in end-group fidelity, which may be important for applications where the ω-end group is required.

Scheme 1.12. Comparison between isothermal photoinitiated PISA (route A) and thermally initiated PISA (route B). 12 Finally, Boyer and co-workers investigated the synthesis of POEGMA-b-PBzMA based NPs of different morphologies via photoinitiated and photomediated RAFT PISA in alcoholic solvents in continuous flow (Scheme 1.13). 90 Polymerizations of BzMA using a POEGMA-based macro-CTA were performed using blue light. In an attempt to speed up the reaction, optimization of the protocol was investigated by the addition of several PIs at various concentrations and irradiances. As no significant change in polymerization rate was observed, a change in co-solvent (DMSO) was made, leading to enhanced polymerization rates with full monomer conversion being reached in just 4 h. By varying the DP of the second block between 50 and 200, a variation in morphology from S to W and V was observed. It was also showed that in the case of methacrylate based macro-CTA there is no significant impact of additional PI on the polymerization rate due to weaker C-S bond. On the contrary, acrylate based macro-CTA can undergo substantial reaction rate acceleration. 63 The steric stabilizer for the PISA process was first synthesized using the thermally initiated RAFT polymerization of OEGMA in the presence of CDPA RAFT agent. Chain extension of macro-CTA was performed subsequently under blue or green light by direct activation of TTC compound. These initial studies were conducted at a total solids content of 10 wt% in ethanol with targeted DP of 200. High monomer conversion (93%) was attained but only after 20 h with a good control over the molecular weight and Đ throughout the polymerization (Đ < 1.3). This approach was harnessed to encapsulate a model hydrophobic drug (Nile Red). As previously stated, encapsulation can be an issue in conventional thermal and photoinitiated PISA processes. Photomediated RAFT PISA stands out by the absence of external initiator. Therefore, there is no need to remove residual PI or catalyst species which could be a potential source of toxicity. O'Reilly's group exploited this advantageous feature to prepare polymersomes encapsulating enzymes. Enzyme loading in such kind of nano-objects requires permeability to enable interaction with the external environment.

O'Reilly and co-workers described a synthetic route towards intrinsically permeable PEG-b-PHPMA vesicles formed by a one-step aqueous visible light photomediated PISA route using only commercial reagents (Scheme 1.16). 92 The PEG-CEPA shell and PHPMA internal membrane were highly hydrated to allow size-selective transport of small molecules while retaining the protein inside the vesicle interior.

Compartmentalization and retention of protein functionality was demonstrated using green fluorescent protein as a macromolecular chromophore. The authors claimed that such encapsulation approach could be also applied to a range of macromolecules. In the continuity of this work, O'Reilly and co-workers assessed the impact of different types of small molecule surfactants on aqueous photomediated PISA processes for the formation of polymer/surfactant complex nano-objects. 54 PEGA-CEPA macro-CTA was chain-extended by dispersion polymerization conditions using a water miscible monomer HPMA as the core-forming block. PEG113-b-PHPMA400 nano-objects were synthesized in the presence of various surfactants. It was demonstrated high tolerance towards non-ionic surfactants. In contrast, ionic surfactants had high impact on photoPISA process since morphological transitions occurred towards lower-ordered structures upon increasing ionic surfactant concentration. The zwitterionic surfactants caused a V-to-W-to-S morphology transition. Furthermore, following the same route, this group demonstrated an efficient approach for the fabrication of polypeptoidbased diblock copolymer nano-objects with predictable morphologies at high concentrations (10-20 wt%) by combining living N-carboxyanhydride (NCA) ROP and aqueous photomediated RAFT via PISA process. 93 As can be seen in Scheme 1.17, PSar served to form an hydrophilic stabilizer block for a subsequent RAFTmediated chain extension with methacrylate monomer in aqueous dispersion polymerization conditions.

Small molecule CTA (CEPA-CTA) suitable for methacrylate monomers was introduced to the (PSar59) chain ends to afford a PSar-based macro-CTA. A diverse set of nano-object morphologies including higher-order structures was obtained as evidenced by the construction of a phase diagram. A single V phase was selected and colloidal stability and ability to encapsulate hydrophilic enzymes were thoroughly assessed.

The results show great promise for use of the developed materials in various biomedical applications.

Scheme 1.17. Synthesis of PSar59-b-PHPMAn (n = 100, 200, 300, and 400) diblock copolymer nano-objects via aqueous RAFTphotomediated PISA using PSar59-based macro-CTA. 93 In the latest work of this group, fluorine-containing diblock copolymer nano-objects were prepared by photomediated RAFT PISA (Scheme 1.18). 94 Polymerizations of PFMA was carried out in anhydrous DMSO in the presence of PEG113-CEPA as macro-CTA. This resulted in the formation of diblock copolymer NPs that possessed a stabilizing PEG shell and a PPFMA block-forming core. The presence of the penta-fluorophenyl ester groups was exploited to attach amino compounds to yield cross-linked polymeric nano-objects. Morphologies were retained after the post-polymerization modification process.

Disassembly of the disulfide cross-linked NPs occurred in the presence of intracellular concentrations of glutathione. The cytotoxicity of the cross-linked materials was evaluated in vitro.

Scheme 1.18. Preparation of PEG113-b-PPFMA200 nano-objects via photomediated PISA in DMSO (M1), followed by crosslinking reactions using either ethylene diamine (P1) or cystamine (P2). 94 3.3. Photocatalyzed RAFT PISA PET-RAFT PISA). In 2015, Boyer's group reported the first photoredox-catalyzed PISA approach for the preparation of various NP morphologies. 80 The structures of all PCs used in this section are gathered in In a next study combining also PET-RAFT and PISA, ZnTPP PC was employed and low irradiances (3.1 mW cm -2 at 560 nm of and 0.7 mW cm -2 at 635 nm). 95 By varying DP of the hydrophobic block, NPs of different morphologies were formed at high monomer conversion (>98%). Interestingly, the encapsulation of the ZnTPP catalyst into the NP core was achieved by dialysis against water with no significant change in NP morphology. It was also shown that ZnTPP-loaded NPs showed potential in the field of photodynamic therapy owing to their ability to generate singlet oxygen under visible light irradiation. Additionally, a singlet oxygen quencher (ascorbic acid) was used to irreversibly consume photosensitized oxygen. The corollary is the possibility to conduct the polymerization without traditional deoxygenation procedures.

In continuation to the abovementioned work, Boyer and co-workers, also proved that organic dyes such as EY and (riboflavin 5′-monophosphate) FMN in conjunction with ascorbic acid as a reducing agent were efficient catalysts to activate a PET-RAFT mechanism without prior deoxygenation and in small reaction volumes (20-4400 μL). 64 It was found that in such kind of photopolymerization process EY undergoes discoloration under green light (Scheme 1.20). This phenomenon is due to photoreduction of dyes in the presence of appropriate reducing agent, in this particular caseascorbic acid. The reduced specie is semiquinonic structure of the dye. The original dye is regenerating through interaction with oxygen which presented in the reaction mixture resulting in the formation of hydrogen peroxide species. Another redox reaction converted hydrogen peroxide to reactive hydroxyl radicals thanks to the excess of ascorbic acid that allows to initiate RAFT polymerization. This photocatalyzed RAFT PISA proved to control a range of monomer families (acrylamides, acrylates, methacrylates) for the synthesis of NPs. The ability to perform multiple parallel polymerizations in low volumes without deoxygenation has the potential to greatly increase the rate at which different reaction parameters can be synthetically screened. ZnTPP can also generate singlet oxygen (from molecular oxygen) through an oxidative quenching responsible from the conventional PET-RAFT mechanism. Boyer and co-workers proved that DMSO was not necessary to quench singlet oxygen, in situ deoxygenation can be also effective by irreversibly reacting the singlet oxygen with external chemical quenchers such as 9,10-dimethylanthracene (DMA). 65 Using this approach, oxygen tolerant PET-RAFT can be achieved in a range of non-quenching solvents (Scheme 1.21).

Using for example EtOH, NPs of different morphologies (S and V) were synthesized using a PISA approach.

Scheme 1.21. Synthetic approach towards performing oxygen tolerant PET-RAFT polymerization under red light. Oxygen tolerance is achieved by ZnTPP mediated photosensitization of molecular oxygen into singlet oxygen which can then be irreversibly reacted with a singlet oxygen quencher. This process is tolerant to different organic solvents and can be used to synthesize polymeric NPs according to a PISA approach. 65 Recently, a similar PET-RAFT PISA involving a self-quenching monomer MTEMA was proposed by Boyer and co-workers. 66 This is a variant method to mediate oxygen-tolerant PET-RAFT polymerization by trapping singlet oxygen with appropriate thioester function of monomer. In fact, the polymerization kinetics were not affected by the presence of air as near identical polymerization kinetics were observed for non-deoxygenated and deoxygenated systems, which is attributed to the singlet oxygen quenching ability of MTEMA (Scheme 1.22). In both cases, well-defined polymers were obtained with good control over the molecular weight and Đ. It was demonstrated that MTEMA could be involved in a PISA process starting from a POEGMA based macro-CTA to yield well-defined polymeric NPs of various morphologies. These NPs were rapidly disassembled after exposure to visible light due to the formation of singlet oxygen by the encapsulated ZnTPP and subsequent oxidation of the thioether groups.

Scheme 1.22. The PET-RAFT polymerization of MTEMA in the presence of ZnTPP and CDTPA as PC and RAFT agent, respectively. 66

In 2018, Boyer and co-workers reported a benchtop process for the synthesis of cross-linked polymeric NPs by exploiting wavelength-selective photochemistry to perform orthogonal photoPISA and photoinduced cross-linking. 67 Water-soluble ZnTMPyP catalyzed the aqueous PET-RAFT dispersion polymerization of HPMA. As previously described, photoPISA process could be conducted under low irradiance of red LED and without deoxygenation due to the action of a singlet oxygen quencher (biotin or vitamin B7), which allowed for the synthesis of a range of NP morphologies (S, W and V). To perform NP cross-linking, a photoresponsive comonomer was used: (7-[4-(trifluoromethyl)coumarin] methacrylamide (TCMAm)) without affecting the particle morphology (Scheme 1.23). Under red light (first step), the authors checked that only photoPISA occurred, with no evidence of TCMAm dimerization. Subsequent exposure to UV radiation (second step) resulted in a rapid cross-linking, leading to the retention of NP morphology (kinetically frozen particle). Boyer and co-workers had developed a continuous photoPISA in a flow reactor setup based on a photoinitiated RAFT mechanism. In 2019, they developed a similar process by employing this time Eosin Y/triethanol amine catalytic system. 49 PET-RAFT was used for the chain extension of a macro-CTA in dispersion polymerization using DMAA/DAA (BTPA was used as RAFT agent). Polymer NPs were produced with a variety of solid contents and lengths for the solvophobic and solvophilic blocks. Reactions were compared in both batch and flow conditions. Compared to batch conditions, higher order morphologies such as W and V could be accessed in flow.

Conclusions

PISA has recently emerged as one of the most promising approaches for producing block copolymer NPs. Much less investigated is photo-induced PISA despite many distinctive advantages compared to the thermal method. In this first chapter, we described how RAFT polymerization mechanism has been leveraged to produced new block copolymer NPs via a PISA process. Limitations of conventional thermally initiated RAFT-mediated PISA approach were described, that promoted the emergence of photoPISA. After commenting on the three photochemically-driven (photoinitiated, photomediated and photocatalyzed) RAFT mechanisms, a state-of-the-art of RAFT-mediated photoPISA according to these different mechanisms was proposed.

The next chapter focuses on the synthesis of PHEA-b-PS diblock copolymer based NPs via RAFT photomediated PISA process in dispersion, which is the least exploited photoRAFT mechanism despite its greater potential. Our approach proceeds indeed at moderate temperature, under visible light, and without the need of any external photoinitiator. Such original photochemical mechanism was exploited to synthetize consecutively the two copolymer blocks of PHEA and PS. Unlike other studies related to NP morphology control, emphasis has been made for the first time on the investigation of particle formation/growth mechanism and on the characterization of optical properties throughout the polymerization process.

Introduction

Over the last decade, polymerization-induced self-assembly (PISA) has emerged as a versatile method for the preparation of well-defined diblock copolymer NPs with various morphologies. 98 In a PISA mechanism, self-assembly occurs during the synthesis itself of the amphiphilic diblock copolymer, when the first solvophilic block is chain-extended with the second solvophobic one. 13 Reaching a critical chain-length for this latter block results in the spontaneous copolymer self-assembly. The solvophilic block acting as a steric stabilizer is generally prepared via solution polymerization, while the insoluble block can be formed via two different methods: aqueous emulsion polymerization involving insoluble monomers, 3,14,15 or dispersion polymerization, using on the contrary, soluble monomers. 16 There are two main advantages of PISA over more conventional post-polymerization self-assembly techniques: [9][10][11] (i) higher solids content (10-50 wt% instead of <1 wt%); 99 and (ii) reduced processing and purification steps. 12 Scheme 2.1. Overview of the photomediated controlled radical polymerization mechanism combining RAFT with the reversible photolysis of the chain-transfer agent.

Therefore, nano-objects derived from PISA have attracted much attention for a diverse range of applications, including enzyme catalysis, 2 drug delivery, 3,4 nanostructured films, 5 stimuli-responsive smart nanomaterials 6 etc. With the exception of recent works resorting to ring-opening metathesis 17 or noncontrolled 18 mechanisms, the fast development of PISA has been mostly driven by reversible-deactivation radical polymerizations. Also referred to as controlled radical polymerizations (CRPs), these mechanisms have made possible to synthesize a broad range of diblock copolymer structures. Among the different reported CRPs, reversible addition-fragmentation chain transfer (RAFT, Scheme 2.1) utilizing thiocarbonylthio compounds as chain-transfer agents (CTA) is by far the most widely used in PISA, owing to its versatility in terms of compatible monomers and experimental procedures. 11 RAFT-mediated PISA has been developed mostly using thermal initiators, but the alternative use of radiation-mediated processes grouped under the term "photoPISA", can be an even more advantageous route. 81 A major asset is the possibility to perform PISA over a wide temperature range. For example, ambient temperature contributes to reducing energy consumption or making the process safer. In addition, the use of low temperatures has proved beneficial for the synthesis of polymer nanoparticles encapsulating thermally sensitive proteins, 62 possessing CO2-or thermo-responsive properties, 100-102 and bearing reactive functional groups. 103 Exploiting the different photo-induced RAFT mechanisms, 105 three photoPISA approaches have been already reported in the literature. The most common strategy consists simply in using a PI 47,54,57,58,85,87,93,[106][107][108][109][110] rather than a thermal initiator. In order to avoid non desired absorption by the RAFT agent, near UV 57,58 or visible irradiation 47 is usually preferred. In 2014, a second approach was proposed by Boyer and co-workers, who demonstrated that visible PCs such as Ir(ppy)3 and [Ru(bpy)3]Cl2 can be used to activate the RAFT agent via a photo-induced electron transfer. 65,67,80,95 At the center of the current study is a third approach based on the reversible 𝛽-cleavage of the excited state, formed under irradiation, of the TTC compound (Scheme 2.1), which yields an initiating alkyl radical and a thiyl radical.

As result, the CTA acts also as a PI. Another distinctive feature of this mechanism over its photoinitiated homologue is the temporal control of polymerization. Because the RAFT agent photoactivation becomes reversible, it is possible to (re)initiate or to terminate reversibly the propagating chains by switching on or off the light source. The photochemical reactivity of TTC compounds has been known for a long time. 56,78 However, its interest for photomediated polymerization has been recently revived thanks to the discovery that photolysis can occur not only under UV irradiation but also visible light. In this latter case, the weak absorption corresponding to the (n,*) transition is exploited instead of the main and dramatically more intense (,*) band. 59,73 Using visible light, a better control of RAFT-mediated polymerizations was reported in solution, possibly as a result of minimized side/termination reactions involving the thiyl radicals. 57,70,63,78,111,112 Although this route obviates the need of additional PI or PC, it is surprisingly the least developed in photoPISA. There has been only two reports on RAFT-mediated photoPISA relying on the reversible photolysis of a CTA, namely 4-cyano-4-((dodecylsulfanylthiocarbonyl)sulfanyl)pentanoic acid, under visible light at 460 nm. In these studies, the dispersion polymerization of benzyl methacrylate 63 and hydroxypropyl methacrylate 91 was performed in presence of a solvophilic macro-CTA based on poly(oligo(ethylene oxide)). In order to synthesize nano-objects based on amphiphilic diblock copolymers, a series of hydrophilic macro-CTAs was first synthesized by photomediated RAFT polymerization of hydroxyethyl acrylate (HEA) in 1,4-dioxane at ambient temperature (25 °C). The initiator-free synthesis was carried out under blue light irradiation (provided by a 472 nm LED) and using the CMDTC as RAFT CTA (Figure . 2.1). CMDTC was selected because of its β-cleavage ability at this wavelength 70 and the fact that relatively unstable primary alkyl radicals can be produced. A short radical lifetime drives fast radical recombination allowing therefore to minimize degradation reactions. 112 Figure 2.2a shows a typical kinetic profile for the synthesis of PHEA58-TTC. The determination of the conversion of HEA by 1 H NMR evidenced a pseudo first order kinetics, in agreement with a constant concentration of propagating species during the polymerization. A conversion of 58% was achieved within 5 h of irradiation. The SEC traces revealed narrow dispersities (Figure 2.2b) over the reaction, suggesting a controlled mechanism. In addition, the linear relationship between the measured molecular weights and conversions (Figure 2c) fully supported a well-controlled RAFT synthesis. Following the same procedure, other PHEA macro-CTAs exhibiting different chain lengths (DPn = 25, 52 and 82) were successfully synthesized.

Synthesis of PHEA-PS by photomediated RAFT dispersion polymerization of styrene

Kinetic study

The PHEA macro-CTAs discussed above were then chain-extended with St at 35 °C in a methanol/water mixture (95/5 wt%) using a similar photomediated RAFT approach (λ = 472 nm), and in absence of any external photoinitiator. Consistently with a PISA process in dispersion, the reaction medium progressively turned turbid as polymerization proceeded (see pictures of the reaction medium taken at different reaction times in Figure 2.3). As depicted in Figure 2.4a, the polymerization rate was slow and a monomer conversion of approximately 60% was achieved after 70 h. Note that even conventional RAFT alcoholic dispersion polymerizations of St carried out at higher temperatures (T ≥ 70 °C) is known to suffer from significantly incomplete conversion; 41,113 a problem somehow mitigated by the addition of water. 114 In our case, the slow polymerization can be mostly ascribed to the dramatic effect of temperature on the propagation rate constant of St (kp = 132 L mol -1 s -1 at T = 35 °C vs kp = 482 L mol -1 s -1 at 70 °C). 115 This substantially differs from the first step of solution polymerization where a single linear regime was obtained (Figure 2.2a). Irrespective of the initiation method employed, this dual rate behavior was reported by many authors in RAFT dispersion polymerization, 85,109 and was interpreted broadly as the transformation from a "homogeneous" to "heterogeneous" polymerization. In Interval I, lasting typically 30-40 h, the reaction rate was extremely slow (15% after 30 h). As can be seen in Figure 2.3 (t = 0 -30 h), the medium was macroscopically homogeneous at the beginning, then slightly scattering at the end (t = 30 h). This suggests that this first regime includes not only the solution polymerization of the PHEA25-b-PSn-TTC diblock copolymer chains but also the formation of micellar particle by self-assembly (particle nucleation).

Starting after 35 h, interval II was marked by a significant increase of the polymerization rate (2 times greater). Meanwhile, the medium became increasingly scattering ( The acceleration of St conversion translates the change of polymerization site from continuous phase to copolymer particles. The particles become favored as the main reaction site since, firstly, most copolymer chains have precipitated, and secondly, the monomer concentration in particles [M]p is higher to the one in solution [M]s. The high local monomer concentration within the particle core, similar to bulk monomer concentration, drives a higher propagation rate in this second interval. To support this assumption, a control experiment of PS extension performed in solution showed only a single and slow regime reaching only 30% conv. after 70 h under irradiation. As polymerization proceeds, the monomer diffuses from the continuous phase into the polymer particles to replace that which has been reacted. An equilibrium (saturation) monomer concentration is thus maintained in the particles, accounting for the steady polymerization rate observed. Because the reaction is stopped before completion, there is no hypothetical interval III where monomer depletion within the particles could cause a decrease of reaction rate. Narrow dispersities were measured (Đ ~ 1.1), accordingly with a controlled mechanism. The controlled character was definitely confirmed by the linear relationship between the measured molecular weights and conversions (Figure 2.4c). We thus demonstrate the versatility and broad applicability of the photomediated PISA synthesis by showing that the photolysis of the RAFT chain-end can be exploited to synthesize successively the solvophilic (PHEA) and solvophobic (PS) blocks. After 47 h of irradiation, spherical and narrowly distributed nanoparticles with increasing mean diameters were observed. Particle nucleation is assumed to take place earlier, but at shorter times, the PS block is likely too short to drive a high enough glass transition temperature necessary to preserve the integrity of the particles under the TEM beam. In agreement with this assumption, the samples taken before 30 h showed a coalesced structure. After 47 h of irradiation, narrow particle size distributions (Đ = 1.05) were obtained, irrespective of the samples, with a mean diameter increasing from 22.5 nm for PHEA25-PS68 (47 h) to 31.6 nm for PHEA25-PS118 (70 h) as detailed in Table 2.1. As a result, varying irradiation time proved to be a suitable means of controlling particle size in the size range 20-40 nm. DLS studies (Figure 2.4a) confirmed the particle growth as a function of time at a rate similar to that of the TEM data. As expected, substantially larger mean particle diameters (+ 20 nm) were measured by DLS (intensity-average diameters) that takes into account the solvation of the PHEA layer on the nanoparticle surface and the swelling of the polymer particles by residual monomer. It is also worth mentioning that these diblock copolymer nanoparticles invariably possess aspherical morphology whatever the length of the PS chains. The causes do not lie in the specificities of the mechanism (RAFT agent photolysis, low temperature) but rather in the choices made as regards DP of the stabilizer and core-forming blocks. Other trials, which go beyond the framework of this study, showed that utilizing a relatively short PHEA block and targeting sufficiently high DP for the PS block enabled to access higher-order morphologies, as extensively reported in literature. 11

Colloidal study

Progress of RAFT-mediated dispersion photo-PISA

Although the synthesis of diblock copolymer nanoparticles during RAFT dispersion polymerization is well documented, 85 only a few studies addressed the issues of nucleation process and the way particles grow. 116 The fact that our system displays slow kinetics of polymerization and a single uniform spherical morphology provides a simplified and appropriate framework to explore these mechanistic points.

Micellization

A conventional dispersion polymerization 16 starts from a homogeneous solution. 63 Given that PHEA25-CMDTC contains both a lyophilic group (PHEA block) and a lyophobic moiety (-SC12H25 as -Z group of the RAFT agent), one might legitimately wonder about its capacity to form micelles initially in the alcoholic reaction medium. Figure 2.6a displays the evolution of surface tension σ as a function of PHEA25-CMDTC concentration for measurements performed in MeOH/H2O (95/5, %w/w), containing also St to reproduce reaction medium. No significant surface tension reduction was observed in the concentration range 0 -10 -2 mol L -1 , proving the absence of surface activity under our reaction conditions. By contrast, surface tension reduction was demonstrated in water in which PHEA25-CMDTC has probably a lower affinity (72 mN m -1 in pure water vs 52.5 mN m -1 at 10 -3 mol L -1 ). As a conclusion, micelles were not present at the beginning, and polymerization of St undoubtedly took place in solution initially. This accounts from the slow polymerization in Interval I since the monomer concentration was low. To detect when nucleation (micellization) starts, we used a DLS-based method originally developed to determine the critical micellar concentration of amphiphilic diblock copolymers formed in solution. 117 Our hypothesis is that the onset of micellization (beginning of particle nucleation) should be marked by a sudden increase in the intensity of scattered light (Is).

Particles growth

To elaborate the mechanism of photoPISA, several colloidal data were determined quantitatively: particles number (Np, concentration of polymer particles in units of number of particles per liter), mean aggregation number (Nagg, i.e. the number of block copolymer chains in a single nanoparticle), surface area stabilized by a copolymer chain (Sagg). Table 2.1 summarizes their evolution for different mean DP (m) of the PS core-forming block (details on calculation are provided in Annex). These data were obtained from a combination of TEM and 1 H NMR spectroscopy analysis for the dispersion polymerization of the PHEA25 macro-CTA. Because TEM particle size values were accessible from Interval II, only the mechanism of particle growth could be examined in detail.

Between 47 and 70 h, the particle size increase (from 22.5 nm to 31.6 nm) was accompanied by a 2fold decrease in Np (from 1.3⋅10 19 L -1 to 6.7⋅10 18 L -1 ) and larger Nagg (from 368 to 686). In this case, the steric stabilization of PHEA25 block is likely to be insufficient to withstand the growth of the PS core and the resultant increase of particle surface area. In accordance with this hypothesis, Sagg was found to be relatively constant (~4.3-4.6 nm 2 ), regardless of PS chain length or particle size. Such mechanism where particle growth requires the incorporation of new copolymer chains to stabilize the newly formed surface area was already reported by the groups of Zhang and Armes. 116,118 This trend was exacerbated when the reaction was carried at 55 °C instead of 35 °C (Table 2.1), which resulted in larger particle diameters (42.1 nm). In this case, the drop of Np was even more pronounced because the larger DP of PS (160 instead of 118) can drive the incorporation more polymer chains. Another plausible explanation is that a higher temperature causes more particles coalescence. Table 2.2 provides similar colloidal characteristics for PISA syntheses performed with PHEA52-CMDTC and PHEA85-CMDTC. In both instances, Np and Nagg remain unchanged during the polymerization.

Consistently, the area occupied by PHEA blocks on the particle surface is larger for longer blocks and tends to increase with conversion. This result can be understood on the basis of the stronger steric stabilization of longer solvophilic blocks, which makes possible particle growth without the need for additional copolymer chains in the particle. This indicates that the observed increase in particle size in this case is simply the result of the growth of the PS core.

General mechanism of particle nucleation/growth

Two intervals (I, II) can be discerned in the RAFT-mediated dispersion photoPISA presented in

Optical properties

Despite its impact on polymerization progress, very few studies on photoPISA address the issue of optical properties. 55 In regular transmission, the apparent absorbance 𝐴 λ of a diluted diblock copolymer latex may be expressed as:

𝐴 λ = 𝐸 λ 𝑙 = (𝐾 λ + 𝑆 λ )𝑙 (1)
where The challenge is the quantitative differentiation of both absorption 𝐾 λ TTC (cm -1 ) and scattering 𝑆 λ (cm - 1 ) coefficients. Because there is no absorption between 500 nm and 800 nm (𝐾 λ TTC = 0), in this range,𝐸 λ is thus simply equal to 𝑆 λ (eq. 2). Given the small droplet diameters (less than 0.1 times the wavelength),

𝐸 λ : extinction coefficient [cm -1 ] 𝐾 λ : absorption coefficient [cm -1 ] 𝑆 λ : scattering coefficient [cm -1 ] 𝑙:optical path length [cm]
Rayleigh theory applies and the experimentally determined 𝑆 λ may be proportional to the inverse of the wavelength to the power four. In agreement with this hypothesis, a range of simple fitting plots 𝑆 λ Fit = 𝑘𝜆 -4 showing an excellent agreement with the experimental 𝑆 λ plots could be built (Figure S1 in Annex).

The validity of our fitting was also supported by the linear correlation between 𝑆 λ Fit and the intensity-average diameters of particles (Dp) to the power six predicted by the Rayleigh theory (Figure 2.7b). The final step consisted in using the calculated 𝑆 λ Fit data in the 280-500 nm range to estimate separately the absorption coefficient 𝐾 λ TTC = 𝐸 λ -𝑆 λ Fit . Figure 2.7c shows the 𝐾 λ TTC values during the dispersion polymerization.

Clearly, a decrease of 𝐾 λ TTC within the time of reaction can be observed. An analogous study performed in solution revealed a similar trend, suggesting that the change in the microenvironment of the RAFT agent, from solution to particles, was not accountable. As emphasized by Qiao and co-workers, a more likely reason is the partial photolytic degradation of RAFT agent. 112,120 Up to 12% of photodegradation was determined after 15 hours of irradiation by these authors. In our case, longer irradiation (70 h) together with higher irradiance (547 mW cm -2 ) explain the higher extent of degradation.

Conclusions

Well-defined spherical nanoparticles were prepared by an original approach combining PISA in dispersion with a photomediated RAFT mechanism under visible-light irradiation. PHEAs were first synthesized in solution before being used as macro-CTAs for a subsequent chain-extension with St in dispersion. Besides the confirmation of the control of the polymerization, the kinetic studies evidenced two distinct regimes related to particle nucleation and particle growth. In the second interval, the high number of particles swollen by monomer resulted in much faster polymerization rate. UV-visible measurements revealed that absorption due to the TTC functions decreased during a first phase of the polymerization, before reaching a constant value, suggesting that a partial degradation of the terminal functions could first occur in the early stage of reaction. Complementary investigations are carried out for a better understanding of this observation. As expected, it was possible to modify particle size by playing on the respective lengths of the solvophilic and solvophobic blocks. Interestingly, photo-PISA could be conducted at ambient or lower temperatures than the ones commonly used in conventional PISA. This wider range of possible experimental conditions is expected to broaden the scope of PISA to new applications, involving for instance thermally sensitive compounds. The temporal control of the polymerization is undoubtedly a supplementary advantage, which could lead to more finely controlled or even adaptable morphologies.

Part I: An electron microscopy approach

Introduction

One major interest of amphiphilic block copolymers lies in their ability to form aggregates of high morphological complexity. In the literature, there is an abundance of studies using amphiphilic block copolymers of different compositions and block lengths that are able to self-assemble into a variety of supramolecular aggregates, such as spheres, rods, lamellae, vesicles, etc. 121 The manipulation of interfacial curvature plays a central role in the control of aggregate morphology. It should be noted that not all these block copolymer supramolecular structures can be deemed nano-objects (or nanoparticles), only those whose three dimensions span from approximately 1 nm to 100 nm. 122 Spherical nanoparticles are the most common and investigated copolymer nano-objects. 123 Among the plethora of methods developed for the formation of spherical amphiphilic block copolymer nanoparticles, nanoprecipitation and polymerizationinduced self-assembly (PISA) are the most popular. Nanoprecipitation involves the dissolution of a preformed copolymer in an organic water-miscible solvent. Upon addition to the aqueous phase, the organic solvent immediately diffuses out leading to the formation of nanoparticles. In PISA, a lyophilic block is chain-extended using a monomer whose corresponding homopolymer is insoluble in the chosen solvent. 13 After reaching some critical degree of polymerization (DP), the second block eventually becomes insoluble resulting in a spontaneous copolymer self-assembly and the formation of block copolymer nanoparticles.

Regardless of the synthetic pathway, block copolymer nanoparticles have attracted much attention for a diverse range of applications 124 including therapeutics delivery, 48,125 imaging contrast agents, 126 nanostructured films, 5 stimuli-responsive nanomaterials 127,128 and templating of inorganic materials. 55,129 Crucial for many applications is the core-shell architecture, in which the inner region mainly composed of the lyophobic blocks of the amphiphile is surrounded by a brush-like corona rich in lyophilic blocks. 130 Various examples show a strong link between core-shell morphology and nanoparticles' properties. In the field of nano-delivery systems for instance, the inner core is thus able to act as cargo space for the solubilisation of lipophilic drugs, proteins or nucleic acids. 131 In bioimaging, the hydrophobic core can serve as nano-depot for accommodation of fluorescent agent. 132 Engineering of the shell can be also utilized to attach bioactive ligands (antibodies, peptides, etc.) in order to facilitate cell entry for targeted drug delivery, but also to prepare supported catalyst 133 or diagnostic systems. 134 Finally, latexes containing a soft shell and a hard core can be used as building block for the creation of nanostructured coatings with improved mechanical properties and film-forming ability. 135 Although a core-shell architecture has been claimed in many studies reporting spherical block copolymer nanoparticles, detailed morphological characterization is usually missing. 136 In most cases, justification for core-shell structure is based only on thermodynamic incompatibility between blocks or their difference of solubility in continuous phase. There are only few examples where core and shell components have been visually identified and differentiated, mostly through transmission electron microscopy (TEM). [137][138][139] Other imaging techniques such as scanning electron microscopy (SEM) 140 or atomic force microscopy (AFM) 141 have been generally overlooked. The barriers to imaging particle core-shell architecture include small diameters (sometimes well below 100 nm), low differential contrast, limited thickness of the lyophilic shell, etc. Considering the growing number of studies on particles prepared by PISA, 142 and more generally, the recent developments of polymeric micelles and nanoparticles in experimental medicine and pharmaceutical sciences, 143 there is a need to carry out a thorough review of the imaging techniques to substantiate claims of core-shell architecture.

In Comparative study enables to assess how the DP of the PHEA stabilizing block affects nanoparticle morphology.

Conventional TEM

Unstained samples

We began with a TEM analysis of air-dried unstained specimens obtained from PHEA-b-PS diblock copolymer nanoparticles. Conventional TEM is indeed the most straightforward and widely applied method for studying the structure of PISA-derived particles, although it can also suffer from non-negligible problems. The main obstacle is the poor contrast between the different morphological features. Contrast is hindered by the fact that most polymer blocks are amorphous, similar in electron density and have a limited scattering cross-sections. 144 In addition, the need for specimen drying or high-energy radiation imposed by conventional TEM may create some artefacts by altering the sample's structure, topography or chemical composition. This may be particularly the case when block copolymer nanoparticles contain a polymer segment with a sub-ambient glass transition such as PHEA. dried nanoparticles obtained without staining preparation. The two objects have in common to exhibit spherical shape and narrow particle size dispersity (𝑃𝐷𝐼 ≤ 1.05). However, the particles also display a number of differences, particularly as regards size, morphology, state of aggregation and surface structure, that merit more thorough discussion. Size. In both instances, the number average diameters (𝐷 n ) are well below 100 nm. PHEA85-b-PS130

features significantly smaller sizes than PHEA23-b-PS130: 19.7 ± 3.0 nm (the value after sign "±" is standard deviation) and 36.4 ± 4.8 nm respectively. The difference of particle size can be attributed to the length of the lyophilic block that determines particle stability against coalescence. Measurement uncertainty was evaluated for each sample: u PHEA23-PS130 = 0.5 nm and u PHEA85-PS130 = 1.4 nm (uncertainty was evaluated by measuring 50 times of the same particle). The values show that a reliable particle sizing is possible. The fact that PHEA85-b-PS130 particles appear with less details and sharpness account for the higher uncertainty value.

Morphology. Close inspection of TEM images reveals other differences than particle diameters.

While image 2a shows a single particle morphology, image 2b exhibits a vast majority of homogenous spherical particles (accounting for more than 95% of objects) and a few isolated vesicles readily recognisable through their inside space (lumen) appearing brighter (indicated by arrows in image 2b shows signs of coalescence. Our interpretation is that there are drying-induced artefacts due to insufficient steric stabilization. Firstly, because much less aggregated particles are visible when decreasing particle concentration (Figure S2 in Annex). Secondly, DLS data do not show any evidence of aggregates.

Surface structure. Of particular interest is that no domain formation assigned to shell and core regions are visible in TEM images. However, clear differences in the aspect of particle surface can be seen.

Only PHEA85-b-PS130 particles boasting the long PHEA stabilizing block show unsharp particle boundaries, while the PHEA23-b-PS130 particles display relatively sharp margins. This result raises two questions. Firstly, are dried samples representative for the native samples present in dispersion? Secondly, do visible particles boundaries reflect the entire core-shell architecture? Because the lyophilic PHEA phase has a sub-ambient 𝑇 g PHEA (-7.3 °C), the shell is susceptible to significant structural changes upon drying. Our assumption is that PHEA segments could collapse upon drying and form a thin film surrounding the PS core. Under these conditions, the poor mass-thickness contrast of the PHEA phase could prevent its visualization, and only the PS core (or PS rich domains) may be imaged. Because PS chains possess a much higher 𝑇 g PS (99.0 °C), distortions induced by desolvation and exposure to electrons are minimized. Therefore, in conventional TEM, image contrast from the background originates from the locally increased thickness of the bumpy PS core. While the lyophilic shell cannot be imaged unambiguously, the lack of surface sharpness for PHEA85b-PS130 particles compared to PHEA23-b-PS130 particles may translate a higher excluded volume for the longer PHEA block.

In summary, TEM analysis of dried particle samples largely evaluates the dimension of the core due to collapse of the lyophilic shell upon drying. Hence, misinterpretation of TEM data can result in errors in the determination of the particle diameter.

Positive staining

To overcome the aforementioned limitations of conventional TEM, heavy-element positive staining can be used in order to increase specimen contrast, and possibly resolve the PHEA shell unseen with unstained samples. Only in a few isolated studies, staining methods have been used to preferentially decorate one phase of core-shell copolymer nanoparticles. [145][146][147][148][149] In the case of positive stain EM, the higher contrast is generated primarily by the differential electron scattering due to more electron dense stained regions inside the particles and less electron dense unstained surrounding environment. However, the second sample with the short PHEA does not display the similar darker annular contrast and control experiment with pure PS nanoparticles of similar size subjected to the same staining procedure shows again the same artefact (Figure S3 in Annex). The inner ring of darker contrast has been already described in the literature, 155 and can be related to two issues: a higher stain concentration at particle surface that makes the particles boundaries more scattering, 145 or the result of drying with the preferential adhesion of organic residue on particle surface against the TEM support. 156 In our case, purification of particles by dialysis has not removed the dark ring, suggesting the minor role played by stain surface accumulation.

Morphology. Since RuO4 reacts with both polymer segments, it is not possible to achieve a nonambiguous differentiation of particle core and shell. In order to preferentially decorate the shell, OsO4 was used because of its inefficiency to oxidize PS phase. Unfortunately, the images are similar to those of unstained samples (Figure 3.1.2), indicating that OsO4 staining has been ineffective and cannot be selectively incorporated in one individual component.

In summary, RuO4 stain cannot reveal the multiphase structure of composite PHEA-b-PS particles.

However, this positive staining is able to shape boundaries of polymer particles, which is of high interest to get a complete picture of the particle. However, there are justified doubts about positive staining's ability to preserve the native structure of the specimen, and a trend to overestimate particle diameter.

Negative staining

Another complementary way to analyze soft nanomaterials is negative stain TEM, which has been very popular for biological structures but much less employed for polymer colloids. 145,147 In negative staining, the stain does not penetrate the particles, but rather adsorbs to the surface and is dissolved in the continuous phase. After solvent evaporation, the particles are enveloped by an amorphous matrix of electron dense stain compound, with the advantages of increasing the contrast, and the image is therefore a kind of finger-print. 157,158 When it is properly performed, negative staining with UAc can also preserve the sample's integrity, morphology, and size. Contrast. As indication that negative stain has been successful, a contrast is produced between the background and the particles. The particles appear as light areas because of their low electron scattering power relative to the dense surrounding stain, which scatters the electrons more and appears darker.

However, a negative stain is unable to penetrate the object, therefore, internal structural details cannot be deduced. Clearly, PHEA23-b-PS130 is much amenable to visualization by this method than PHEA85-b-PS130.

In this case, the copolymer assemblies are more fragile because of a poorer steric stabilization resulting from a shorter PHEA stabilizing block. As a result, the particles are more likely to collapse or disassemble upon adsorption, staining or drying on the grid. translates to a maximum end-to-end distance of 15 nm for a fully stretched polymer chain. This value is thus much larger than that of the new region revealed by negative staining. Two reasons are suggested to explain this gap: firstly, PHEA chains are not likely to be fully stretched as this conformation is entropically unfavorable, and secondly, this soft shell is susceptible to contraction by drying despite negative staining.

In summary, negative staining may be more efficient in preserving the pristine state of copolymer nanoparticles, and shape the particular boundaries in a more faithful manner than positive staining. A comparison with unstained image can give an indirect access to shell thickness when spatial resolution is sufficient. However, no all specimens are amenable to visualization by negative staining and core-shell phase differentiation remain inaccessible. Size. We notice that the average diameter of PHEA23-b-PS130 particles (37.0 ± 5.5 nm) is relatively similar regardless of the preparation method: 36.4 nm with drying, 41.8 and 41.3 nm with positive and negative staining respectively. In this case, the shell is thin (< 2 nm) resulting in minimal size deviations between techniques which can resolve PHEA region (negative / positive stain TEM) and the others that cannot. Conversely, PHEA85-b-PS130 presents a more extended shell, and its apparent diameter is consistently more dependent on the EM preparation method. Interestingly, particle size calculated from the cryo-TEM image (21.6 ± 3.2 nm) is relatively similar to that of unstained dried sample (19.7 nm), and therefore, substantially lower than diameters derived from positively stained TEM (31.1 nm) and negatively stained TEM (29.7 nm). This suggests that analysis of undried specimen by cryo-TEM also largely evaluates the dimension of the core. This time, the explanation behind is not the collapse of the hydrophilic shell, but the difference of conformation between lyophilic and lyophobic chains. The PHEA chains are likely to be partially stretched whereas the PS core is more closely packed due to its hydrophobic nature. The difference of chain density between core and shell chains accounts for a mass-thickness contrast of PHEA significantly smaller than that of PS, leading to a poorer capacity to scatter electrons. The fog surface together with the irregular shape can be assigned to a brush structure with density fluctuation as opposed to a dense core. This feature is reminiscent of a core-shell architecture, although no indisputable proof is provided because no morphology domains can be imaged within the particle.

Morphology

In summary, cryo-TEM cannot resolve the nanoparticle shell because the lyophilic PHEA in stretch conformation form regions with low chains density, and therefore poor electron capacity. When shellforming block is long enough, its presence is manifested by unsharp envelope and dark spots inside the PS core suggestive of chains perpendicular to the plane.

SEM

SEM has distinctive advantages compared to TEM and cryo-TEM, in particular, finer surface structure images can be obtained upon operating at lower accelerating voltages. Under these conditions, the penetration and diffusion area of incident electrons is shallow. Surface structures is thus gained because the number of secondary electrons emitted from the surface is maximized compared to backscattered electrons generated from within the specimen. 159 SEM can be employed to analyze nanoparticle size, shape and surface structure. a PS latex was prepared using semi-continuous emulsion polymerization. In this method, surfactant (SDS, 0.2024 g, 0.701 mmol) and initiator (ammonium persulfate, 0.0264 g, 0.116 mmol) were dissolved in water (40 g) and added to a glass reactor (Schlenk tube) equipped with a stainless steel stirrer, a reflux condenser, a sampling device, a nitrogen gas inlet tube, and a temperature probe. When the reaction temperature was reached (80 °C), the monomer (8.8 g, 84.5 mmol) was continuously fed (49 mg min-1) over a period of 3 h. At the end of monomer feeding, the system was maintained at the reaction temperature for 60 min in order to minimize the amount of residual monomer.

Particle size. The number average particle diameters of PHEA85-b-PS130 (22.9 ± 2.9 nm) and PHEA23-b-PS130 (34.8 ± 5.3 nm), as well as size dispersities (PDI ≤ 1.04), are comparable to those of TEM (without staining) and cryo-TEM. This result suggests that SEM also fails to resolve the PHEA shell of the PHEA85-b-PS130 specimen. However, particle core size and shape can be determined with precision. Like previous TEM images, the PHEA23-b-PS130 latex exhibiting the shortest PHEA block shows signs of coalescence attributed to drying.

Surface structure. Using PS latex as benchmark, a thin bright corona attributed to edge effects can be visualized in image 6c. 160 It is well established that particle edges (see schematic explanation in Figure S4 in

Scanning transmission electron microscopy (STEM).

The same tandem of nanoparticles (PHEA85-b-PS130 and PHEA23-b-PS130) was also analyzed using a STEM detection mode. This transmission working mode is known to provide better spatial resolution than SEM because of a stronger mass-thickness contrast.

Accordingly, measurement uncertainties are of the same order of magnitude or even weaker than for TEM data: 𝑢 PHEA85-𝑏-PS130 = 0.70 nm and 𝑢 PHEA23-𝑏-PS130 = 0.85 nm. In addition, the accelerating voltage is much lower than in conventional TEM (30 kV versus 200 kV), implying fewer risks of damaging the sample, especially the PHEA layer. The results gathered in Table 3.1.1 show a strong resemblance between STEM and TEM data as regards average particle diameter and measurement uncertainty. Again, staining reveal the entire particle volume without contrasting shell and core, while non stained samples show only the PS core. All STEM images are available in Figure S5 in Annex. In summary, edge effect in SEM might be harnessed to evidence the presence of protruding PHEA chains at particle surface (although more evidence is needed). Nevertheless, PHEA shell cannot be resolved in a way to allow a precise determination of particle size and shape. Therefore, the apparent diameter is largely the particle core. Interestingly, STEM mode can be considered a viable alternative to TEM for the imaging of block copolymer nanoparticles.

Conclusions

Amphiphilic block polymer nanoparticles display core-shell architecture, but precise imaging of their multiphase structure has long been elusive and problematic. Herein we reported morphological characterization of two PHEAx-b-PS130 copolymer nanoparticles bearing a short and long PHEA stabilizing block (x = 23 and 85) using TEM, cryo-TEM and SEM. The shell extent of PHEA23-b-PS130 is too short to be resolved, regardless of the method. This explains why particle size given by the three EM techniques are in the same average order. By contrast, PHEA85-b-PS130 particles boasting the longest PHEA stabilizing block shows significant differences depending on the technique. TEM analysis of air-dried unstained sample can only image the particle core, and a blurred interface provides the hint of the shell existence. Staining is thus essential for shaping the particle boundaries, and negative staining using uranyl acetate seems more amenable than positive staining (RuO4) to fix the particles in their pristine form and obtain a precise measurement of particle size. However, no staining method was able to reveal the multiphase structure.

Cryo-TEM ensures that the specimens can be studied in their native form, but the solvophilic PHEA in stretch conformation exhibits a too poor electron capacity to allow visualization, and only protruding chains appear as dark spots from particle interior. Like TEM and cryo-TEM, SEM largely evaluates the PS core.

However, fine surface structures reminiscent of PHEA shell can be imaged through "edge effect", leading to highly bright particles with unsharp interface. In conclusion, no technique allows a satisfactory imaging of core and shell regions, however their combination gives access to particle size, shell thickness and specific morphological features suggestive of shell existence. We believe that these findings will open new avenue in the rationale use of microscopy techniques for the analysis of soft self-assembled polymer nanostructures.

Part II: Physicochemical characterization

Introduction

Core-shell latexes are part of structured composite particles. They feature at least two separate bulk polymer phases of different chemical composition preferentially located on particle surface and at the center. 130 Such latex architecture with two spatially separated components has attracted interest in a variety of applications, some of them characterized by high tonnage productions, such as impact modifiers, waterborne coatings and adhesives. 161 Core-shell latex particles are typically synthesized by interfacial polymerization in dispersed media, in which a shell-forming monomer is polymerized by emulsion polymerization on the surface of seeded "core" particles. 162 The typical core-shell latex particle that results from this method contains two polymeric phases composed of two distinct homopolymers or random copolymers. 147 In addition to the two-step method, other variant methods have been described in the literature 130 , as well as more complex particle architectures including raspberry-, confetti-or sandwich-type 163 . Generation of two separate polymer domains within the confined space of a core-shell particle is driven by the interplay of thermodynamic factors (interfacial tension of polymer phases, reaction temperature, cross-linking, etc.) and kinetic factors (initiator type and concentration, feeding rate of the shell-forming monomer, etc.). To evidence the multiphase structure of composite latex particle, mostly imaging techniques such as TEM, 140 or in less extent, AFM 164 are typically used. 136 Other non-microscopic techniques such as DSC 165 or solid-state NMR 166 have been employed to obtain indirectly some data on the quantitative fraction for the various phases.

Recently, a new class of core-shell nanoparticles based on amphiphilic graft or block copolymers has emerged. 167 They consist of a solvophobic polymer core coated with a solvophilic stabilizing polymer shell.

In contrast to conventional core-shell particles, the two polymer components are covalently bonded and the particles can be accessed via single-step polymerization methods without the need of external surfactant.

The preferred synthesis method is based on self-assembly of amphiphilic block copolymers such as PISA, 124 but other techniques also exist such as free radical copolymerization of hydrophilic macromonomers with hydrophobic monomers 168 or graft copolymerizations from water-soluble macroinitiators. 169 The applications for these particles are also different, including diagnostic testing, bio-separations, controlled release of drugs and other biological agents. 125 Unlike the first type of core-shell particles, characterizing the multiphase structure of these self-assembled core-shell particles is generally very challenging. In a few isolated studies only, [137][138][139]170 TEM has provided indisputable evidences of the core-shell morphology through the contrasted observation of multiple polymeric domains within the particle. However, in a majority of studies, usage of electron microscopy is made difficult by particle sizes smaller than 100 nm. In addition, the low glass transition temperature of the shell component leads to particles highly susceptible to structural changes upon drying or exposure to radiation of electrons. 171

In the quest for alternative solutions, we report herein the advantages and limitations of two analytical techniques for the morphology study of core-shell amphiphilic diblock copolymer nanoparticles: DSC and X-ray photoelectron spectroscopy (XPS). The spherical particles were prepared by visible-light photoPISA In DSC, the change in heat capacity for each block segment (PHEA or PS) across the Tg can be used to determine the amount of bulk polymer phase. Therefore, DSC is able to determine quantitatively the weight fractions of PS in the core and PHEA in the shell, the remainder corresponding to the interface. This latter can be defined as a region of finite thickness within which the composition varies continuously from one bulk phase to the other. Note that an interfacial region is formed within the particle even when the two polymer segments forming the block copolymer are incompatible, because of the interdiffusion of a fraction of the two copolymer phases. [172][173][174] DSC has already proved to be a valuable technique to study the coreshell morphology of 2-component polymer particles produced by seeded emulsion polymerization, 136,175,176 but never with amphiphilic copolymer nanoparticles. Originally reported by Tembu-Nzudie et al. in 1994 for poly(butadiene)/poly(methyl methacrylate) latex, 177 the method was further elaborated. 175 In particular, the groups of Hourston, 178,179 Karlsson,180,181 and Sunberg 182,183 have emphasized the interest of DSC or modulated temperature DSC for determining the interfacial content of composite latex.

XPS has a typical analysis depth of less than 7 nm. Therefore, only the latex "surface" is likely to be probed, thus shedding light into the shell composition. Given its sensitivity to atomic composition, XPS could thus distinguish the solvophilic PHEA from the solvophobic PS block because it is the only block containing oxygen atoms. XPS has previously been used to quantitatively assess the surface composition of polymeric particles, 184 and recently the morphology nanoparticles of poly(styrene)/poly(methyl methacrylate) core-shell latex. 185 The presence of a surface stabilizer 186 and the efficiency of particle surface modification were also studied by XPS. 187 Results and Discussion 1. Cryo-TEM

nm

). The particles have seemingly a homogeneous structure devoid of apparent polymer domains. This result is in contrast with the common representation of a core-shell particle where both copolymer blocks are assumed to phase separate at nanoscale to form a PS core surrounded by a stabilizing PHEA palisade. A more in-depth study of the nanoparticles obtained at high magnification (Figure 3.2.2b) show that particles display a granular structure that was assigned to protruding PHEA chains. The stabilizing chains oriented in the z direction give rise to a higher contrast, which is manifested by black dots in the internal structure of particles. Conversely, the hydrophilic blocks arranged around the PS core (i.e. with the solvent in background) lack of contrast because they cannot adopt this specific orthogonal orientation. Unlike the PS chains aggregated in the core, the highly solvated PHEA chains cannot form dense domains and their thickness is too small to generate a contrast with the background. Consequently, it is not possible to visualize the archetype of core-shell architecture: a corona composed of PHEA brushes surrounding a spherical PS core. In conclusion, the formation of composite particles can be evidenced by cryo-TEM, but only by expert eyes. In addition, the quantitative assessment of the size and mass distribution of the individual elements in the polymer composite has remained inaccessible by electron microscopy. 2. Modulated differential scanning calorimetry (MDSC)

Study of lyophilized sample by SEM

Prior to studying PHEA85-b-PS130 block copolymer nanoparticles by DSC, the first step was to lyophilize the latex sample to maintain the integrity of the core-shell morphology (see details in the experimental conditions). Lyophilization has the dogma to preserve the core-shell architecture. However, it is not straightforward that freeze-dried structure mirrors that of the starting colloidal dispersion, in particular when particles have a soft polymer shell. In our case, dried sample are required for DSC analysis given that PS block has a glass transition of 100 °C. As can be seen in

MDSC study of lyophilized PHEA-b-PS latex

To account for the formation of distinct PS and PHEA phases within the copolymer particle, we refer to the difference of glass transition temperatures of these two blocks. Some values of respectively of -7. It is seen that 17% of the total copolymer is in the PHEA phase (shell, 𝛿 PHEA s ), 14% in the PS phase (core, 𝛿 PS c ) and 69% is estimated to be the interfacial region (𝑊 i = 𝛿 PS i +𝛿 PHEA i

). The fraction of mixed phase is significant, but the result is not surprising given the partial miscibility of both blocks. For the PS block itself, only 24% forms a bulk core phase while 76% is entrapped in the interface. In addition, the fact that the glass transitions are shifted compared to pure polymers are indicative that the two separated phases are not pure. [178][179][180][181][182][183] The glass transitions of PHEA shell is found at 28.1 °C versus -7.

Effect of annealing time on phase separation of lyophilized PHEA-b-PS latex

Because of the partial immiscibility of the core and shell phases, we speculate that phase separation in core-shell lyophilized particles could be improved upon annealing. As can be seen graphically in Figure 3.2.5a, increasing time of the thermal annealing at 150 °C (0 -120 min) causes significant changes of the dCp/dT vs. temperature signal. The magnitude of the dCp/dT signals for the PHEA and PS phases increases, which suggests that the ΔCp value also grows, and that the fractions of the pure PHEA and PS components build up. This is a clear confirmation that further and gradual phase separation takes place upon annealing.

Upon thermal annealing, we can clearly see that the Tg of the PS phase rises gradually to reach after 120 min a value similar to that of the PS homopolymer. This shift translates the progressive departure of the PHEA plasticizing chains from the PS core. ) were summarized in Table 3.2.1. 𝑊 i decreases from 69% for the as-lyophilized sample to 50% after 120 min annealing. As shown in Table 3.2.1, the reduction of the interfacial zone mainly results from PS chains diffusing out of the interface with time. The weight fraction of PS in the interface 𝛿 PS i thus decreases from 44% to 30% after 2 h.

Effect of sample preparation

Lyophilization is the most conventional way to preserve the core-shell morphology when particles change from dispersion to dry state. To more precisely assess the impact of sample preparation on phase separation, we have changed the way the PHEA85-b-PS130 copolymer was dried. Firstly, a film was prepared from a dispersion aiming to produce a nanostructured sample by coalescence of core-shell particles. Conversely, an amorphous film where phase aggregation is minimized can be obtained from a solution where the copolymer was firstly dissolved. The preparation and the MDSC results for these two latex and solution films are described below and compared with those of the lyophilized analogue.

Solution film. The copolymer was dissolved in a good solvent (THF) to disrupt the particles and a film was prepared from the solution by casting the dispersion and evaporating the continuous phase under ambient conditions. Interestingly, the dCp/dT vs. T plot (not represented) shows no clearly defined feature which could be identified as the pure component of the copolymer. Consequently, it is not possible to reveal 2 distinct regions of PHEA and PS phases. This result suggests that in this sample the two PHEA and PS domains are intimately mixed, and that no polymer domain is formed. This is an example where the two blocks are trapped in non-equilibrium states due to kinetic restrictions. The slow diffusion of the polymer chains or/and the insufficient difference in solubility parameters of the two blocks has prevented the occurrence of a phase separation (although the precise value for both polymers is not accessible), hence no individual peaks can be identified in the DSC trace. Noteworthy is that even 2 h annealing at 150 °C had no effect on this sample, and no phase segregation was observed.

Latex film. To prepare this sample, the latex was cast and the continuous phase evaporated under ambient conditions as previously described. Figure 3.2.6a shows the differential of heat capacity signal for different annealing times. Before treatment (t = 0), copolymer film derived from latex after solvent evaporation shows again two individual peaks related to PHEA and PS phases. However the magnitude of the dCp/dT signals for the PS is much higher than that of PHEA. The DSC trace bears some ressemblance with that of the lyophilized sample (Figure 5a). This result supports that a composite structure can be also preserved during latex film formation due to the limited diffusion ability of PS chains at ambient temperature. The weight fraction of the interface of 60% is even smaller than that of lyophilized particles (69%). Similarly, the effect of annealing on 𝑊 i and 𝑊 lost i is shown in Figure 3.2.6b. As in the previous case, annealing caused a gradual phase separation of the core and shell components in the latex film, leading to an interface reduced to 25% after 2 h at 150 °C. As can be seen in Table 3.2.2 summarizing the weight fractions, the increase of PHEA and PS phases both account for the reduction of the interface. Lyophilized block copolymer nanoparticles have a higher initial fraction of interfacial layer (69%) comparing to air-dried film (60%) that may require a longer annealing time to achieve a phase separation comparable to that of the air-dried film.

XPS characterization

XPS offers qualitative and quantitative data about the surface structure of particles. However, it should be noted that XPS analysis probes only the elemental and chemical composition of the particles up to approx. 7 nm in depth, which makes this method valuable only to determine the surface composition of core-shell particles. 188 Three types of PHEA85-b-PS130 block copolymer samples were analyzed: lyophilized particles (a), latex film resulting from dispersion evaporation (b) and solution film obtained by evaporation of a THF solution (c). Figure 3.2.7 shows their XPS spectra with the surface composition of oxygen and carbon expressed as an atomic percentage (%) and determined from the integrated peak area of all elements present in a sample. In our two-component system, PS has only carbons while PHEA contains carbons and Annealing time min oxygen. Therefore, oxygen can act as a unique elemental marker for the PHEA phase. O1s peak but also C1s band are easily identified in the three spectra, even in the lyophilized (a) and nanostructured films (b) which are assumed to have a core-shell structure. This means that a significant amount of PS chains can be found at the surface of these two samples. Elemental analysis of lyophilized particle and nanostructured film reveal an enrichment in oxygen up to 15.01% and 15.59% compared to the theoretical value of 13.28% for PHEA85-b-PS130 diblock copolymer (corresponding to an isotropic distribution of polymer segments). This

𝛥𝐶 p PHEA J g -1 °C-2 𝛥𝐶 p PS J g -1 °C
shows that these two specific samples have a higher fraction of the PHEA solvophilic phase at their surface.

By contrast, the amorphous film (c) has the lower content in oxygen at 9.84%, indicative of a mixed phase. higher than the amorphous sample c. However, the presence of PS remains important, and contradict the picture of a surface only occupied by PHEA chains. The first explanation relies on the diffusion of PS chains in the PHEA shell phase, which is consistent with the partial miscibility of PS in PHEA phase as proved by DSC data. Another more plaisible reason is that in the lyophilized and dried latex, the shell is strongly contracted by dehydration and the dried shell layer could have a thickness lower than 7 nm given the relatively small DP of the PHEA chain. The consequence is that not only the shell but also a part of the interface could be probed by XPS. Therefore, XPS seems less adapted than DSC to the analysis of amphiphilic copolymers nanoparticles, in particular when the spatial extent of the solvophilic shell is small.

In agreement, DLS analysis revealed an intensity-average diameter of only 40 nm.

Conclusions

The morphology of conventional core-shell particles prepared by interfacial polymerization has been extensively characterized by MDSC. In this study, we show that a similar characterization can be used for composite latex particles composed of amphiphilic block copolymers. Starting with a model PHEA85-b-PS130 amphiphilic copolymer nanoparticles, the lyophilized sample exhibits three phase behaviors assigned to shell (PHEA), core (PS) and interface where the two blocks are homogeneously mixed. The composition of core and shell phases, the weight fraction of interface, and its evolution over annealing time can be obtained. The method can thus characterize indirectly but quantitatively the state of mixing of the two blocks within the nanoparticle while direct morphology characterization by electron microscopy can be equivocal. The method can be also exploited to investigate the extent of phase separation in the copolymer latex film after solvent evaporation, paving the way to the study of nanostructured films composed of separate polymer nanodomains.

Part I: Core-shell PISA-derived nanoparticles as template for mesoporous carbons

Introduction

Latex templating has proven very successful for designing macroporous materials with pore sizes ranging from 50 nanometers to a few micrometers. [189][190][191] Conversely, this route has been used only to a limited extent for the preparation of mesoporous materials with smaller pore sizes (between 2 nm and 50 nm), 151 in particular if compared to the countless reports involving a polymerization within surfactant liquid crystalline (LC) phases. 192,193 Despite the enormous diversity of polymers, latex templating essentially relies on standard monodisperse PS, [194][195][196][197] poly(methyl methacrylate) 198 or poly(ionic liquid) 199 nanoparticles stabilized by adsorbed molecular ionic surfactant. Their use has made possible the synthesis of carbon 194,195,197,198 or silica 151,196,199 mesoporous materials with ultralarge-pores (30 -50 nm), difficult to achieve with existing conventional methods. Unlike inorganic nanoparticles, organic colloids can also be easily removed by calcination at moderate temperature (300 -400 °C). Despite these advantages, latex templating suffers from two major drawbacks. Firstly, their use for mesoporous materials' synthesis necessitates particles with a diameter smaller than 50 nm. 200 However, only a handful of polymerization processes in dispersed media can achieve this size range, including microemulsion polymerization. 201 Despite a significant utilization of this technique in the literature, 194,198 low monomer content (5-10 wt% of the total mass) and much higher surfactant concentration (~ 10 wt%) are required in a microemulsion polymerization compared to its analogue in emulsion (~ 1-2 wt%) due to the necessity of stabilizing a larger interfacial area. [START_REF] Jasinski | [END_REF] This restricts the potential use of microemulsion-polymerized particles in a template synthesis since high particles contents and low surfactant levels are desirable for this application. A second limitation of colloidal templating is that the latex spheres are regarded only as spatial fillers: 202 the polymerizing medium acts as matrix, whose growth at the interface simply "coats" the template. It follows that the role played by latex surface properties is often overlooked, although they are the key to matrix/template compatibility, pore architecture control and pores' interconnectivity. This aspect has been insufficiently researched because it remains very challenging to adjust precisely the composition and the dimension of organic particles' surface. 203 In order to expand the usage of latex templating for mesoporous materials, new synthetic methods are therefore needed to create organic colloids with tunable sizes, small diameters (< 50 nm) and welldefined surface properties. These developments are of a major importance for mesoporous carbons since the design of pores larger than 10 nm is difficult to obtain by using classical silica hard template and/or surfactant soft template approaches. 204,205 For many applications, large mesopores are known to improve the accessibility and the transport efficiency of large molecules such as biomacromolecules, organic molecules, bulky dyes and viruses. [206][207][208] Moreover, large mesopores can be employed as nanoreactors to confine metal-based nanoparticles, drug and gene molecules, acting as catalyst, energy storage material, adsorption site or biomedical carrier, leading thus to hybrid materials with improved performances. [209][210][211] Currently, most ultra-large mesoporous carbons were synthesized using LC mesophases based on specific amphiphilic poly(ethylene oxide)-based block copolymers. [212][213][214] Thus, polymer nanoparticles could open new avenues for mesoporous carbons with ultra-large and tunable pore sizes and featuring an open porosity. Extensively investigated over the last 10 years, PISA represents an efficient and versatile route to block copolymer nano-objects. 215,216 In particular, PISA has proven successful to create sub-50 nm nanoparticles with a core-shell morphology. 130,136 In addition to having a suitable diameter range and size distribution, our diblock copolymer nanoparticles do not require additional surfactant since stabilization is ensured by PHEA chains. Unlike electrostatic stabilization, such steric stabilization leads to a more robust template able to disperse in various organic monomer solutions. 217 Recently, a PISA templating approach was described involving the immobilization of reactive precursors within the shell of the nano-objects to yield inorganic hollow nanoparticles. 129 Our process resulting in mesoporous carbon materials is significantly different.

Typically, our core-shell nanoparticles were dispersed in a mixture of phenolic resin precursors (phloroglucinol and glyoxylic acid). 218,219 The solvophilic PHEA block acts as steric stabilizer forming an outer shell in contact with the polymerizing medium. The hydroxyl groups of PHEA chains favor hydrogen bonds and even covalent coupling with the phenolic resin. The particle core is made up of the phase segregated solvophobic PS block. After polymerization and solidification of the phenolic polymer network, template decomposition and carbonization are performed in a single step to generate a mesoporous carbon structure. PHEA and PS form two separated shell and core domains, respectively, whose sizes can be finely and independently adjusted by controlling the DP of the two polymer blocks. As a result, such core-shell copolymer particles have a well-defined structure, particularly as regards surface composition. This makes PISA-derived particles a much more versatile and complex template than a conventional latex. We demonstrate here that pore size can be varied within the mesopore range by changing the PS core diameter, while the wall thickness can be controlled by the extent of the stabilizing PHEA shell, which delimitates two neighboring particles. Additionally, mesoporous carbons with original pore topology can be achieved starting from non-spherical PISA particles such as vesicles.

Results and discussion

Synthesis and characterization of core-shell nanoparticle template

We used RAFT polymerization, a well-established controlled radical polymerization method to mediate the PISA process and produce the PHEA-b-PS core-shell nanoparticles. Thoroughly described in a previous publication, 55 the synthesis procedure consists in two separates steps: a PHEA block was first synthesized in solution (1,4-dioxane), then subjected to a chain-extension with styrene in a mixture of methanol/water (95/5 %w/w) in which PS is insoluble. This results in a spontaneous self-assembly of PHEA-b-PS chains after PS block reaches a critical DP. In the DSC thermograms (Figure S7 in Annex), the occurrence of two Tgs in the diblock copolymer system demonstrates that the hydrophilic and hydrophobic blocks selected are thermodynamically incompatible and form different domains. This is supposed to drive nanophase separation in dispersion, and consequently, the formation of particles endowed with core-shell morphology. Another advantage of a RAFT mechanism is the precise and independent control of DP for the two blocks as size exclusion chromatrography (SEC) traces reveal narrow dispersities. The PISA syntheses were all conducted at 20 wt% solids content allowing to comply with request of high latex concentration for the subsequent carbon templated synthesis. the PHEA shell is likely to be melted down by the electron beam. Our assumption is that it may form a transparent thin film surrounding the PS core that is not visible because of a too weak contrast. Therefore, only the PS core possessing a much higher Tg (90 °C, Figure S8 in Annex) can be detected by TEM due to its higher stability under the electron beam. To support this assumption, no domain formation can be seen in the inner core area, and a sharp interface is clearly visible. The collapse of the PHEA shell impedes the use of TEM for particle sizing, but provides a useful insight into the PS core's dimension, which eventually determines the size of the future mesopore in the carbon materials. Therefore, only indirect methods such as DLS can give information on full particle size and size distribution. As can be seen in the inset of ). Furthermore, the latex shows a high colloidal stability over time, and no significant variation of particle diameter is observed by TEM over a period of 12 months (𝛥𝐷 TEM 𝑙 .= +1.9 nm). The efficiency of steric stabilization is also reflected by the ability to re-disperse the nanoparticles in water through dialysis without significant aggregation. Similarly, no sign of destabilization is found when PHEA25-b-PS118 particles are dispersed in the mixture of phenolic precursors for the preparation of the mesoporous material. As expected from a steric stabilization, the acidic solution containing glyoxylic acid and phloroglucinol precursors did not affect stability.

Mesoporous carbon through core-shell nanoparticle templating

Mesoporous materials were prepared following a conventional templating procedure schematically illustrated in pyrolysis. Therefore, our pore size data lend confidence in a faithful replica of the original template. This mechanism is consistent with the fact that PISA particles are kinetically and mechanically stable templates with less risks to be disrupted in the course of the phenolic resin cross-linking than dynamic structures based on LC mesophases or microemulsions. 220 The wall thickness (𝛿 TEM p ) of the mesoporous material is estimated to be ~ 4-5 nm from TEM pictures, which is relatively significant compared to other materials prepared by particle/emulsion templating. 194,201 Indeed, when two particles approach each other, steric repulsion between PHEA layers solvated by phenolic precursors limit the overlap, resulting in thick and robust framework walls. However, we were unable to establish from TEM imaging whether the foam structure was made up of interconnected pores due to poor contrast between cells, windows and the carbon framework. In order to reveal in greater details the 3D structure of this sample, electron microscopy tomography was performed. experiments without template revealed a material devoid of mesopores, but also microporous. In this case, Vmicro ranges between 0.15 and 0.20 cm 3 g -1 , suggesting that microporosisty in our latex-templated sample results primarily from carbon walls. At higher relative pressures, the isotherm shows a steep hysteresis of H1-type with a characteristic symetrical shape. This feature is indicative of a narrow range of uniform and open mesopores where networking effects are limited. As supported by TEM data, the porous structure is composed of large spherical cells, interconnected by windows or channels that are sufficiently large to prevent pore blocking effects. Consequently, the desorption branch is not delayed, as is the case in networked structures where nitrogen evaporation can be hindered by narrow pore constrictions. 222 The surface area was evaluated by the Brunaeur-Emmet-Teller (BET) method, and SBET was estimated to be 691 m 2 g -1 which is similar to carbon templated by surfactant. 223 Pore size distribution (see inset of The inset provides the meso-and micropore size distributions obtained from BJH and NLDFT methods, respectively. Thermal properties. The hybrid nanoparticle/phenolic resin solid was also subjected to thermogravimetric analysis under nitrogen in order to shed light into the template decomposition and the conversion of phenolic resin into carbon material. As shown in Figure 4.1.6, the total weight loss of pyrolyzed carbon is 80%. The TGA thermogram shows two distinct and separate weight-loss steps: a gradual and limited weight loss (~10%) between 50 and 300 °C due to water desorption and degradation of short polymer chains, and a much more significant weight-loss (~70%) in the temperature range 350 -420 °C. This second weight change transition is highlighted by plotting the corresponding derivative on the right side y-axis. This results in a well-defined and intense peak centered at 405 °C, assigned to the simultaneous decomposition of PHEA25-b-PS118 copolymer and phenolic resin. A control experiment where PHEA25-b-PS118 was analyzed individually corroborates this conclusion, and also leads to a complete weight loss. This latter result illustrates that none of the polymer blocks serves as carbon precursor, and both are sacrificed upon pyrolysis. 224 We believe that the high thermal stability for copolymer particles is key to the success of our latex templating approach. By delaying the elimination of the template, further polycondensation reactions between phloroglucinol and glyoxylic acid can take place in a way that template's shape and morphology can be efficiently imprinted. As a result, the material is able to withstand drying and carbonization without collapse of the mesostructure.

Effect of various experimental parameters.

Effect of PHEA and PS blocks length. An advantage resulting from the controlled radical polymerization process is the precise and independent tuning of solvophobic and solvophilic block's DP.

Using PHEA25-b-PS118 block copolymer as reference (run a, Table 4. clarify the effect of the solvophilic block. In all cases, the dispersity of the copolymer chains was estimated to be 1.2-1.3, and spherical particles with a narrow size distribution were achieved. As summarized in Table 4.1.1, the general trend of DLS data is that increasing the PS block for PHEA25-b-PS160 causes the formation of larger particles: 𝐷 DLS 𝑙 = 45.0 nm versus 38.9 nm for PHEA25-b-PS118. PHEA chain length being unchanged, PS growth causes an increase of particle surface area, which can be only accommodated by the incorporation of new copolymer chains to stabilize the particles, resulting in a particle size increase. nm). This supports PS core dimension as a key parameter controlling mesopore size. Like carbon a, the sample b templated by PHEA25-b-PS160 copolymer exhibits also a type IV isotherm characteristic of mesoporous materials. As expected, changing the PS length does not change the BET surface area (SBET = 702 m 2 g -1 ) and micropore volume (Vmicro = 0.34 cm 3 g -1 ). Additionally, the total pore volume is higher for the PHEA25-b-PS160 (1.58 cm 3 g -1 ) compared to PHEA25-b-PS118 material (1.3 cm 3 g -1 ) due to a larger diameter of pores induced by the larger template size and higher mesopore volume. In contrast to the reference system a exhibiting a H1-type hysteresis, a typical H2-type hysteresis is found with a broader and almost flat saturation plateau at high P/P 0 . This reflects a delayed desorption step of the gas molecules due to some pore confinement. A similar quantity of copolymer was added in the two samples, but larger size of PHEA25-b-PS160 latex leads to a lower density of particles in b compared to a (approximately 2 times lower), thus creating a greater interparticle distance. Compared to PHEA25-b-PS118, the structure is thus moving from an array of interconnected pores in a to an assembly of cavities connected by much narrower constrictions in b. Consequently, the spherical shape of the pores is less distorted and appear more resolved in the TEM picture. In such pore blocking model found in carbon b, pore emptying is delayed and proceeds presumably through a percolation process which accounts for the asymmetrical shape of hysteresis loop. In H2-type hysteresis, a precise pore size distribution is generally difficult to obtain, [225][226][227] and accordingly, the average mesopore size (𝐷 BJH p = 22 nm, Table 4.1.1) is significantly underestimated compared to TEM data

(𝐷 TEM p = 29 nm).
For the two other carbons prepared with the latex exhibiting a longer PHEA stabilizing block and similar PS block (run c and d), smaller pores (𝐷 TEM p = 16 -19 nm) are found compared to carbon a in accordance with the decreased size of the PS core (𝐷 TEM 𝑙 = 20 -24 nm). In addition, these two samples seem characterized in the TEM images by a higher extent of closed porosity with more isolated and not connected mesopores. As a result, the pores possess a well-defined spherical shape, and a much thicker wall separates neighboring pores: 𝛿 TEM p ~ 7-8 nm in carbon c (PHEA52-b-PS110) and 9-10 nm in carbon d (PHEA85-b-PS114). This structure is in contrast to the carbon a derived from PHEA25-b-PS118 that gives rise to an open porosity manifested by a highly interconnected mesoporous structure. Consequently, a much lower density of pores is thus generated, leading to a 5-fold decrease of the mesopore volume (Vmeso = 0.1 -0.2 cm 3 g -1 ) as determined from the adsorption isotherms. A type H5 loop can be also recognized in We therefore conclude that wall thickness can be adjusted by the spatial extent of the stabilizing layer (particle shell). Steric stabilization results from solvated PHEA chains anchored on the particle surface, which hinders the approach of other particles and thus acts as a mechanical barrier to flocculation or coalescence. The steric barrier surrounding the particle will be extended upon increasing the DP of PHEA.

Because the stabilizing shell is highly solvated by the phenolic resin, it results in a broadening of carbon wall surrounding the pores after carbonization. In agreement with this reasoning, a system of densely packed copolymer particles under TEM show that the average distance separating two PS cores (pore precursor) tends to increase with PHEA length (Figure S9 in Annex). As a last comment, a significant drop of microporous volume is also noted: Vmicro= 0.1-0.19 cm 3 g -1 versus 0.34 cm 3 g -1 for the reference sample a templated by PHEA25-b-PS118 and 0.19 cm 3 g -1 for a template-free carbon. This result could be explained by a change of the environment of phenolic resin upon increasing the PHEA shell. In samples c and d, a higher fraction of phenolic network is in close contact with PHEA which affects its chains conformation and seems to hinder the process of micropore formation. To understand this result, PHEA homopolymers with differents lenghts were employed as template. However, no decrease of microporosity was observed compared to a template-free sample (see Table S1 and Figure S10 in Annex), which therefore means that a more complex mechanism is likely to proceed with a combined effect of the two copolymer blocks on the microporous domain.

Effect of template concentration. In a new series of experiments depicted in Figure 4.1.9, the molar ratio r of copolymer (PHEA25-b-PS118) in the experiment to the copolymer used in experiment a was varied:

1 (a), 0.8 (e), 0.6 (f), 0.4 (g) and 0.2 (h). Similar pore average diameters (Table 4.1.1) and pore size distributions (Figure S10 in Annex) were obtained in all instances, consistently with the fact that the same latex was systematically employed. The samples exhibit larger wall thickness 𝛿 TEM p upon decreasing template concentration, for example 5-6 nm in carbon g and 6-7 nm in carbon h. For r ≤ 0.4 (g and h), the pores are more widely spaced due to the combined effect of short stabilizing chain (PHEA25) and lower particle concentration. Consequently, both wall and pore shapes become clearly distinguishable in the TEM photos. One can draw an analogy with the mesoporous carbons c and d prepared with longer solvophilic block (PHEA52 and PHEA85), and where steric stabilization acts also as spacer between PS cores further converted into mesopores (Figure 4.1.7). For r ≥ 0.6 (a, e and f), the close packing of spheres results in a dense arrangement where the particles come into close contact. Larger contact area between touching spheres leads to an open porosity with interconnected pores distorting the cell spherical shape. This shift of pore topology depending on template concentration is clearly reflected in the adsorption isotherms shown in As r decreases, a gradual broadening of the hysteresis loop is observed with a clear transition from a H1 loop (for r ≥ 0.6) to a H2 loop for r ≤ 0.4. This change translates the shift from a narrow distribution of interconnected mesopores to a more complex pore network with narrow pore openings. The plateau region of hysteresis loop gradually lowers due to the decrease of mesopore volume: 1.01 cm 3 g -1 (r = 1) to 0.47 cm 3 g -1 (r = 0.2). This decrease directly results for the lower density of particles in the material.

Nevertheless, the BET surface areas is almost unchanged (640 -714 m 2 g -1 ) because the micropore volume remains similar regardless of template content. This series of experiment demonstrates that wall thickness can be independently varied to pore size by varying the latex content.

Comparison with PS and silica colloidal template

To emphasize the particularity of core-shell nanoparticle templating, mesoporous carbons were produced from two more conventional particles templates: PS and silica nanoparticles.

PS Latex

The PS nanolatex (run i, Table 4.1.2) stabilized by SDS was prepared following a specific semibatch process 200 where continuous monomer feeding to a PS seed enables to maitain a small particle size. As expected, TEM imaging ( The main porous properties obtained after carbonization were summarized in Table 4.1.2. The most distinctive feature in TEM image of mesoporous carbon (Figure 4.1.10, image i) is pore wall thinning, with the consequence of pore windows occupying a larger area of the surface of the carbon surface. We think that this structure is related to a much thinner SDS stabilizing layer compared to PHEA layer, which favors the building of a sphere-touching network. In addition, its well known that electrostatic stabilization favors a closer packing of particles because repulsion forces are operative at shorter distances compared to steric stabilization. 229 In contrast to reference sample a templated by copolymer particles, TEM tomography analysis reveal in this case that the structure is only partially interconnected by large windows. As shown in Compared with carbon a, a significantly much larger mesopore volume (Vmeso = 2.60 cm 3 g -1 ) was obtained.

This result can be understood on the basis of a higher size and density of pores due to thinner walls. In addition, a perfect agreement was observed between the diameter value determined by TEM (𝐷

TEM p = 38
nm) and the one estimated with BJH model (𝐷 BJH p = 40 nm). Colloidal silica. Commercially available silica nanoparticles (𝐷 TEM 𝑙 = 23 nm) was selected as another conventional template to produce mesoporous carbon material. It is worth noting that silica nanoparticles is a hard template which cannot be eliminated during the calcination stage in contrast to polymer particles. Silica template was thus selectively removed after carbonization through washing with a concentrated soda solution (5 M). The TEM images of mesoporous carbon before after template removal are provided in Figure 4.1.10. The hybrid mixture phenolic resin/SiO2 (image j0) shows a very small interparticle space caused by high SiO2 cohesion properties, which make resin domains impossible to distinguish even by high resolution TEM. In the mesoporous carbons (image j), very thin walls separating an array of highly interconnected mesopores are clearly visible consistently with the previous observation.

Compared to copolymer template, there is an increase of both micropore (Vmicro = 0.38 cm 3 g -1 ) and mesopore (Vmeso = 1.56 cm 3 g -1 ) volume. If the first value is related to different interactions between surface groups of silica nanoparticles, the second is the direct consequence of higher dense packing driven by smaller walls. Surface area is SBET = 952 m 2 g -1 (40% higher than in reference sample a), and a good agreement about pore size is found between TEM data (𝐷 TEM p = 20 nm) and gas sorption data (𝐷 BJH p = 23 nm).

Vesicle templating

By a judicious variation of the packing parameter, which is determined by the relative volume fractions of the stabilizer (PHEA) and core-forming (PS), it is possible through RAFT aqueous dispersion polymerization to synthesize copolymer particles with a higher order morphology. Compared to PHEA25-b-PS118 latex used as reference (run a, Table 4.1.2), PHEA17-b-PS136 (run k) shows a higher mean DP of the PHEA stabilizer block, while the mean DP of the hydrophobic PS block increases. This leads to an increase of the packing parameter, which accounts for the progressive evolution in copolymer morphology from spheres to vesicles as observed in the TEM image k0 of

Conclusions

This study shows the new potentialities offered by well-defined core-shell PHEA-b-PS nanoparticles as template for the synthesis of mesoporous carbons. Diblock copolymer nanoparticles with diameters ranging from 20 to 40 nm were prepared by an approach combining PISA in dispersion with a controlled RAFT mechanism. Fine tuning of textural properties is achieved by varying template concentration and block DP. Compared with a conventional latex templating approach, a core-shell particle morphology stands out by its ability to provide independent control of mesopore diameter and wall thickness. As replicate of the hydrophobic core, the mesopore size can be adjusted by changing the PS block DP. Wall thickness and thus interconnectivity can be modulated by the spatial extent of the PHEA steric stabilizing layer that will be more extended with a higher DP. The same effect can be reached by decreasing template concentration, thus leading to completely isolated mesopores. The interconnectivity of carbon material is very important for application as this feature responsible for molecules, cations, anions, proteins, etc. migration efficiency.

Therefore, core-shell PHEA-b-PS nanoparticles prove to be a complex and dual template because it ensures both a conventional transcriptive synthesis thanks to PS core acting as "impenetrable" filler, but also a morphosynthesis through a spatially-controlled polymerization in PHEA shell. Finally, we have also reported the synthesis of mesoporous carbon material based on a vesicular template prepared also by PISA that is not achievable using conventional soft templates.

Part II: Stable sub-50 nm PISA-derived nanoparticles for bioimaging

Introduction

Biomedical and pharmaceutical applications including drug delivery and diagnostic imaging are increasingly reliant on nanoscale vehicles. 230 A key reason is their ability to solubilise (encapsulate) poorly water-soluble drugs or contrast agents, a central requirement for improving efficacy and reducing side effects (e.g. from cytotoxic molecule). The portfolio of synthetic and biological nanoscale materials includes liposomes, 231 albumin, 232 inorganic nanoparticles (NPs), 233 and amphiphilic diblock copolymer NPs. 234 This latter represents one of the fastest growing classes of nanoscopic carriers, 235 and examples based on poloxamer are already approved excipients for pharmaceutical use. 236 Driven by self-assembly, the formation of AB diblock copolymer NPs proceeds via nanoscale phase separation of a shell-forming solvophilic block A acting as steric stabilizer and a core-forming solvophobic block B entrapping generally the drug cargo or imaging agent. Flory-Huggins interaction parameter (AB), solvent quality and block volume fraction are key parameters governing the assembly mechanism. 237 The interest of diblock copolymer supramolecular aggregates has focused in the targeted delivery of anticancer drugs owing to the ease with which ligands can be attached, and particle diameter tuned. 238 Indeed, size range of several tens of nanometers and surface functionalization have proven to be the most critical factors for maximizing therapeutic efficacy. 239 By contrast, the contribution of diblock copolymer NPs to bioimaging is still modest.

To date, their use is limited to fluorescence optical imaging, 132,[240][241][242][243][244][245][246][247][248] and to a lesser extent, magnetic resonance imaging. 249 In most studies, the interactions with biological matter have been scarcely addressed. 250 Fluorescent NPs are currently playing a pivotal role in optical imaging of cells and tissues, but the field is dominated by inorganic NPs including quantum dots or dye-doped silica NPs that remain the workhorses. 251 However, fluorescent diblock copolymer micelles may offer more potential because of their versatility in terms of size, surface properties, composition and range of fluorophores available. 252 For this reason, they can be better customized for the needs of fluorescent imaging where characteristics such brightness, emission properties, particle diameter, protein adsorption, biocompatibility and biodegradability must be balanced depending on the application. Typically, the fluorophore molecule can be incorporated into or onto the NP via ionic interaction, physical trapping, or covalent conjugation. Unlike other dye-doped polymer NPs, fluorescent diblock copolymer assemblies are advantageously self-stabilized by the hydrophilic block of the constituting diblock copolymer. Therefore, their surface is well-defined, and no additional surfactant is required, which can cause cytotoxicity problems and disturb cellular internalization. 253 Despite these advantages, the use of fluorescent diblock copolymer NPs has remained limited. The two fundamental impediments to a more widespread use are their kinetic stability and the current methods to synthesize them. Traditionally, the particles are not generated directly upon polymerization, but from a preformed diblock copolymer, using either nanoprecipitation or micellization.

Micellization in water requires dissolving the diblock copolymer in a water-miscible organic solvent followed by dialysis against water. Alternatively, micellization can be induced by gradual addition of water, and aggregation is driven subsequently by slow solvent evaporation. In nanoprecitation, the copolymer is again dissolved in a water-miscible organic solvent, but the solution is added this time to a large amount of water. Solvent diffusion leads to a supersaturation of the copolymer, and the formation of micelles. These methods have the advantage to be compatible with fluorophore loading and can yield uniform and nanosized micelles. However, they involve the use of organic solvents that pose potential health risks. In addition, the challenge is that they are not productive since the typical concentration of copolymer is less than 1 wt%, and post-processing steps are necessary.

Since 2008, particular attention has been paid to an original and direct pathway to diblock copolymer nano-objects referred to as PISA. 254,255 Among its advantages, there is the possibility to conduct the reaction at high solids content up to 50 wt%, [9][10][11] and in a single step 12 since copolymer synthesis takes place concomitantly with particle formation. PISA process starts from a solvophilic block A usually prepared through reversible-deactivation radical polymerization in solution, mainly RAFT polymerization. This soluble polymer acting as steric stabilizer is chain extended with a second monomer to form a second solvophobic block B. When this core-forming block reaches a critical DP, a spontaneous copolymer selfassembly takes place through a judicious choice of solvent. Very few RAFT-mediated PISA studies deal with the synthesis of fluorescent NPs. Physical trapping of Nile red, 256 pyrene, 257 merocyanine, 258 or Rhodamine B 259 in PISA-prepared NPs was reported. But in neither case were their spectroscopic properties studied, and bioimaging application proposed.

The present work explores the synthesis of fluorescent poly((oligo(ethylene glycol) acrylate)-blockpoly(benzyl acrylate) NPs (POEGA-b-PBzA) via a photomediated RAFT polymerization at ambient temperature. OEGA was chosen as monomer instead of PHEA due to the well-known biocompatibility of polyethylene oxide chains. The distinctive feature of this approach lies in the absence of external initiator that may have undesired toxic side effects. 55 Initiating radicals are generated by β-cleavage of electronically excited TTC CTA. 81 Like conventional RAFT polymerization, a reversible degenerative transfer reaction involving the CTA is still the main mechanism dictating chain growth. The resulting spherical diblock copolymer NPs are narrowly distributed and have an average size of 10-50 nm controllable through the DP of the solvophilic POEGA block. Sub-50 nm NPs are known to be well suited to cellular imaging owing to their higher penetration ability. After synthesis, the NP were encapsulated through a swelling procedure with various concentrations of Nile red, a lipophilic visible light fluorophore non covalently bound to the copolymer. Nile red has an emission range in 550-650 nm adapted to optical confocal microscopy imaging, and its highly hydrophobic nature (predicted logP of 3.6) can favor its retention inside the particle. To ensure minimal toxicity and high kinetic stability, the POEGA-b-PBzA particles initially synthesized in an alcoholic medium were dialyzed against water to form kinetically stable spherical NPs. This change of solvent leads to an increase of the interfacial tension between PBzA core-forming block and continuous phase, and trapping of NPs in a non-equilibrium state. In the so called "kinetically frozen" micelles, dissociation and exchange of diblock copolymer chains (unimers) are hindered by a large energetic barrier, allowing better and longer retention of loaded fluorophore in NPs. 238 Additionally, the particles are much less perturbed by change in concentration, ionic strength, pH and temperature. Those features are crucial for imaging purposes, since micelles must remain intact in biological medium to prevent fluorophore cargo release and minimize side effects. We show that this innovative approach towards Nile red-doped POEGAb-PBzA NPs stripped of initiator residue with high structural stability and adjustable size has high potential for cellular imaging. To preserve the sample's integrity, negative staining using uranyl acetate was performed. 145,147 shows typical TEM images of four negatively stained samples taken at different moments of the synthesis.

The minimal time for observation of NPs is 14 h, which is in accordance with conversion-time curves indicating that interval II (marking the end of nucleation) does not start before 12 h (image a). As evidence that the negative staining is successful, the particles appear as light areas because of their low electron scattering power relative to the dense surrounding stain, which scatters the electrons more and appears darker. The average diameters are consistent with particle growth (whose mechanism is discussed in the next it is imperative to have well-defined particles with narrow size distributions for bioimaging application.

For understanding how particle grow after their nucleation, Table 4.2.1 gathers the values of three colloidal parameters obtained at different times of interval II during the synthesis of POEGA27-b-PBzA172:

particles number (Np), mean aggregation number (Nagg), and surface area stabilized by a copolymer chain (Sagg). Between 14 and 17 h, particle size increase (from 23.4 nm to 31.7 nm) was accompanied by a 40% decrease in Np (from 2.02 × 10 19 L -1 to 1.22 × 10 19 L -1 ) and larger Nagg (from 155 to 257). This suggests that steric stabilization of POEGA27 chains is likely to be insufficient to withstand the growth of the PBzA core driven by monomer consumption. Therefore, the increase of particle surface area must be sustained by additional copolymer chains, brought by diffusion through aqueous phase and particle coalescence. 116,118 In accordance with this hypothesis, Sagg was found to be relatively constant (∼11.1-12.3 nm 2 ), regardless of concentration of NR in swelling solution (from 0.25 to 1.5 mM). Indeed, no size variation (DLS and TEM data) was reported over more than 6 months of storage. A high fluorophore loading (> 85%) was determined for this trio, though the amount of encapsulated dye ω(Nile red) remains limited (0.15 wt% of copolymer). This suggests that a saturation occurs for low value of Nile Red, and advocates the use of low concentrated swelling solutions. Our findings also underline the importance of not using high volume fraction of swelling solution. The explanation is possibility related to the fact that THF can swell both POEGEA and PBzA blocks. As a result, higher volumes of swelling solution could dissolve the NPs and cause a destabilization after THF removal by dialysis.

Kinetic stability of POEGA-b-PBzA NP

The issue of dye-loaded amphiphilic copolymer NP stability has been often overlooked on the basis that critical micelle concentration (CMC) of non-ionic surfactants is much lower than for ionic surfactants. 261 However, this view is based only thermodynamic considerations, and disregards kinetic aspects. For self-assembled diblock copolymer NPs, kinetic stability is defined by the exchange rate of chains between micelles, or between micelles and unimers. 262 Dilution, change of pH or ionic strength can impact the exchange rates, and cause micelle disassembly and loss of cargo molecules in the case of encapsulated nanovehicles. Destabilization can take place non only with thermodynamically controlled micelles, but also with kinetically trapped micelles (out of equilibrium). Like nanoprecipitation, PISA is a kinetically controlled process. 215 In literature, the kinetically trapped morphology of PISA-based NPs was usually easily overcome upon change of temperature or solvent composition, leading to a change of morphology. 263 Different strategies have been designed to ensure NP kinetic stability and that the encapsulated fluorophore is not released prematurely. Trapping micelles in a kinetically stable state (or frozen state) can be achieved by cross-linking 264 or chemical modification of the core-forming block. 265 In this study, we relied on a selective solvent able to increase the interfacial tension of the core-forming block.

Interfacial tension (core/solvent) is known to be a key parameter playing on kinetic stability. 123 In our case, when the continuous phase was changed to water, the copolymer chains were driven to transcend the kinetically trapping phenomenon and further reorganize to a kinetically frozen self-assembly. Upon To qualitatively estimated the local polarity, spectra of Nile red dissolved in THF and methanol were also acquired.

Steady-state and time-resolved fluorescence

Following the absorption experiments, steady-state fluorescence spectra of Nile red were acquired in solution using different solvents, and in dispersion using POEGA27-b-PBzA172 NPs. A total of 5 organic solvents were tested, and some representative emission spectra are shown in Figure 4.2.9. The spectra were normalized with respect to their peak maxima. As expected, the peak maximum shifts to the right (red shift)

as the solvent polarity increases (from hexane to acetonitrile). 269 Meanwhile, the spectra obtained in apolar solvent such as hexane exhibited several peaks which merged into a single broad peak with increasing solvent polarity. It is worth noting that the emission spectrum in acetonitrile exhibits a red-shifted peak at 618 nm. This peak is close to the one observed at 617 nm in dye-loaded NPs in water. To better understand the solvent polarity effect, the Lippert-Mataga relation was applied. This relation has been widely used to correlate the energy difference between emission (he) and absorption (ha), also known as Stokes' shift, To study the quenching effect, quantum yield (Φfluo) of POEGA27-b-PBzA172 NPs including two different loadings in Nile red (0.15 wt% and 0.29 wt%) were determined (Table 4.2 This supports again a preferential location of the fluorophore within the polar shell.

Brightness and photostability

Theoretical brightness per NP (Bp) was also determined. Bp is defined as product of extinction of fluorophore at maximum absorption (), quantum yield yield (Φfluo) and number of dye par particle (Ndye). 126

General conclusion

This PhD dissertation is a contribution to the rapidly growing field of photochemically driven PISA.

As entry into the subject, chapter I described generalities about RAFT, RAFT-mediated PISA, genesis of photoRAFT, and a state-of-the-art account of research and development of photoRAFT-mediated PISA from 2015 to today. Chapter II was devoted to the development of a RAFT photomediated PISA process.

Emphasis was made on the mechanism governing nanoparticles formation and how optical properties As achievement, this thesis manuscript has extended the scope of photoPISA through the development of an initiator-free RAFT photomediated variant activated under blue light. As regards NP growth, the length of the solvophilic block was proven to be a critical parameter not only to control morphology but also to define the final number of particles (particle size), which is a very important characteristic for applications. TEM and SEM can be used as routine techniques to determine the diameter of particle core, but negative stain TEM has turned to be essential to have a "complete picture" of the coreshell NP dimension. In this sense, the document praises the use of a multi-technique approach to have a reliable characterization of particle size and extent of bulk domain. When used as nanotemplate, PISAderived NPs can afford nanoporous carbons with interconnectivity and controlled size of mesopore and wall thickness, a combination of properties scarcely achievable with regular latex templates. As last achievement, we showed that biocompatible fluorescent nanovehicles could be produced, whose kinetically frozen state can be exploited to offer long term kinetic stability and a latex stripped of unimers. Important for applications in bioimaging and drug delivery, these characteristics are not easily attainable with block copolymer NPs formed by micellization or nanoprecipitation.

To conclude, this study opens the door to interesting perspectives and ways for improving or upgrading photoPISA. Firstly, most TTC RAFT chain-end shows an absorption range related to the (n,*) transition ranging from 400 nm to 520 nm. This upper limit suggests that TTC β-cleavage and reversibledeactivation reaction could be extended to higher wavelength, typically green irradiation. Light penetration could be enhanced upon increasing wavelength irradiation due to smaller light attenuation by scattering.

Therefore, limited final conversions and poor polymerization rates, common in photomediated RAFT, could be pushed up upon switching from blue light initiated to green light initiated polymerization. The second advantage of using green light is to provide better control over methacrylate derivatives' polymerization, while in the case of blue light, tertiary carbon shifts the equilibrium towards higher concentration of active species that leads to limited control over polymerization. Despite an exhaustive study on template synthesis of mesoporous carbon with PISA-derived NPs, we were not able to achieve highly ordered mesoporous carbons, that are of particular interest for application in batteries. A PISA process driving more narrowly

distributed NPs could open new horizons in this domain. Another field of interest in terms of application might be the production of nanostructured films through the coalescence of core-shell PISA-derived NPs.

Soft shell-forming block could ensure film-forming properties to the latex, while a core-forming block endowed with higher glass transition would drive higher thermo-mechanical performances. Annex (OEGA; Sigma-Aldrich; > 96%) was purified by passing through a basic alumina column (Sigma-Aldrich) prior to use. Benzyl acrylate (Alfa Aesar; ≥ 98%) was purified by passing through a basic alumina column (Sigma-Aldrich) prior to use. Ethanol (Fischer Chemical; ≥ 99%), methanol (Fischer Chemical; ≥ 99%).

tetrahydrofuran (THF; Sigma-Aldrich; ≥ 99.9%), 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid (DDMAT; Sigma-Aldrich; 98%), diethyl ether (Sigma-Aldrich; ≥ 99%), Nile red (TCI, ≥ 98%) were used without purification.

Syntheses

Photomediated RAFT polymerization of HEA in solution (ex. PHEA25-TTC). In a typical synthesis, HEA (4.04 g, 34 mmol), TTC (0.396 g, 1.2 mmol) and dioxane (12.16 g) were charged into a Schlenk tube equipped with a stirring bar. The mixture was degassed by three freeze-pump-thaw cycles before being placed under nitrogen. The tube was then placed in a pre-heated oil bath at 25 °C and irradiated during 5 h in an immersion type photoreactor containing a blue LED spot light (λ = 472 nm, 547 mW cm -2 ) (Figure S13 in Annex). Aliquots were regularly withdrawn to determine the conversion by 1 H NMR analysis (0.1 mL dissolved in 0.5 mL of D2O). After 5 h (HEA conversion of 58%), the polymer was recovered by precipitation into 200 mL of diethyl ether, before being dried under vacuum overnight to give ∼1.5 g of purified PHEA. The macro-CTA was then characterized by SEC in THF (𝑀 n ̅̅̅̅ = 6.36 10 3 g mol -1 , Đ = 1.26).

Synthesis of PHEA-b-PS diblock copolymer particles via photomediated dispersion PISA of St (ex.

PHEA25-b-PS118). In a typical synthesis, PHEA25-TTC (0.266 g, 0.082 mmol), St (1.721 g, 16.5 mmol) and a methanol/water mixture (8.18 g/0.43 g, 95/5 %w/w) were charged into a Schlenk tube equipped with a stirring bar. The mixture was degassed by three freeze-pump-thaw cycles before being placed under nitrogen. The tube was then placed in a pre-heated oil bath at 35 °C and irradiated during 70 h in an immersion type photoreactor (λ = 472 nm, 547 mW cm -2 ). In the kinetic study, the mean degree of polymerization of the PS block was systematically varied by adjusting the irradiation time. The syntheses involving PHEA macro-CTA of different lengths were performed at the same monomer/solvent weight ratio of 1/3. Three aliquots of 0.1 mL were regularly withdrawn during reaction. The first one was used for 1 H NMR analysis (after dissolution in 0.5 mL of CDCl3) to determine St conversion. The second one was employed for SEC measurements to determine polymer molecular weights. Finally, DLS and TEM measurements were carried out using the third aliquot (see next section for details).

Synthesis of mesoporous carbon materials templated by PHEA25-b-PS118 copolymer nanoparticles

(run a). In a typical procedure, phloroglucinol (0.205 g, 1.63 mmol) and glyoxylic acid monohydrate (0.152 g, 2.05 mmol) were dissolved under stirring at room temperature in methanol (1.96 g). This solution was then added to a non-purified PHEA25-b-PS118 copolymer dispersion (4.96 g). This leads to a molar ratio phloroglucinol:glyoxylic acid:template = 1:1:0.020. Methanol (0.723 g) was added to the reaction mixture to obtain a total weight of 8 g, which was also kept constant in all runs. The turbid sample was transferred into smooth glass dishes and left at room temperature for 6 h in order to evaporate methanol solution and the non-reacted styrene. Subsequently, a thermal consolidation of the phenolic resin was performed at 60 °C overnight to increase the cross-linking density of the phenolic resin framework. The resultant material was pyrolysed at 600 °C under argon for 2 h. In the series of experiments involving a variation of template concentrations (run e-h), the molar ratio 𝑟 = n(copolymer) n(copolymer) run a ⁄ was changed from 1 to 0.2.

In these cases, the molar ratio phloroglucinol:glyoxylic acid:template = 1:1:0.020r.

Synthesis of PS nanolatex. PS latex was prepared using semi-continuous emulsion polymerization. In this method, surfactant (SDS, 0.2024 g, 0.701 mmol) and initiator (ammonium persulfate, 0.0264 g, 0.116 mmol) were dissolved in water (40 g) and added to a glass reactor (Schlenk tube) equipped with a stainless steel stirrer, a reflux condenser, a sampling device, a nitrogen gas inlet tube, and a temperature probe. When the reaction temperature was reached (80 °C), the monomer (8.8 g, 84.5 mmol) was continuously fed (49 mg min -1 ) over a period of 3 h. At the end of monomer feeding, the system was maintained at the reaction temperature for 60 min in order to minimize the amount of residual monomer.

Photomediated RAFT polymerization of OEGA in solution (ex. POEGA22-DDMAT). OEGA (5.000 g, Cryo-TEM: 5 µL of the sample (0.1 wt%) were applied onto a 400 mesh Cu grid covered with a lacey carbon film that was freshly glow discharged to render it hydrophilic (Elmo, Cordouan Technologies).

The grid was rapidly plunged into liquid ethane slush by using a homemade freezing machine with a controlled temperature chamber. The grids were then mounted onto a Gatan 626 cryoholder and observed under low dose conditions on a Tecnai G2 microscope (FEI) operating at 200 kV. The images were recorded with a slow scan CCD camera (Eagle 2k2k FEI). Uncertainty of particle diameters are u PHEA85-b-PS130 = 1.30 nm and u PHEA23-b-PS130 = 1.70 nm.

Scanning electron microscopy (SEM): High resolution SEM images were obtained with a JEOL JSM-7900F scanning electron microscope in GBSH (gentle beam super high resolution) mode. In this mode, an accelerating voltage of 5.5 kV and a specimen bias voltage of -5 kV specimen were applied. The STEM images were acquired at 30 kV on the same microscope using DEBEN GEN5 annular STEM detector. The previously prepared TEM grids were reused for the SEM and STEM observations.

UV-Spectroscopy. UV-Vis spectra were obtained using a JascoV730 UV/Vis/NIR spectrophotometer. The selected range of wavelengths was 280-800 nm.

LED lamps. The emission spectrum and irradiance of the LEDs were determined using a Field Max II (COHERENT) optical spectrum analyzer and a Power Max PM10 powermeter at a distance of 1 cm.

Nitrogen adsorption measurements. N2 adsorption isotherm were obtained by using Micromeritics ASAP 2420. The samples were first outgassed at 573K during 12h and weighted to determine the real mass.

After, the samples were outgassed a second time on the analysis port at 573 K during 2 h before analysis.

The tubes were not backfilled with N2 before analysis. The free volume measurements were realized after analysis in order to avoid the pollution and/or the not-reversible capture of He in the materials. The isotherms were deconvoluted with different models in order to extract the textural properties. In particular, we have used Brunauer-Emmet-Teller (BET) model 1 for the surface determination. The Rouquerol procedure 2 was used to avoid any subjectivity in evaluating the BET monolayer capacity. On the other hand, the porosity distributions were calculated by applying Density Functional Theory model (global calculation)

and by Brunauer-Joyner 3 (mesoporous distribution).

Differential Scanning Calorimetry (DSC).

A Mettler Toledo DSC1 was used for thermal analysis and glass transition temperature determination. The macro-CTA and copolymer samples were heated from -50 to 150 °C, cooled to -50 °C and re-heated to 150 °C. The heating and cooling rates were +/-10 °C/min. Glass transition temperatures was determined during the 3 rd heating cycle.

Modulated differential scanning calorimetry (MDSC). MDSC of lyophilized copolymer nanoparticles was performed with a Mettler-Toledo DSC1. The sample was heated from -50 °C to 150 °C with an oscillation amplitude of 1.5 °C, an oscillation period of 60 s, and a heating rate of 3 °C min -1 . Only the data of the first scan were analyzed so that the state of mixing in the original sample was not altered by measurement. Total heat flow was separated into two components attributed to reversible and non-reversible heat flows. Only the reversible (or in-phase) component was used for the calculation of Cp and dCp/dT.

Details on the mathematical treatment of the reversible component were provided in ESM. Similar analysis was also carried after various thermal annealing times (1, 2, 5, 10, 30, and 120 min) at 150 °C.

In each instance, it was possible to determine the amount of interface and bulk phases in core-shell particles from the increment of heat capacity of PHEA and PS phases across the Tg in PHEA85-b-PS130 copolymer (𝛥𝐶 p ) and in equivalent PHEA and PS homopolymers (𝛥𝐶 p0 ). filled with the copolymer dispersions and placed into a freeze dryer (Cryonext, Cryotec France). The samples were frozen in the freeze dryer; the shelves being cooled at -0.5°C min-1. The samples were then kept for 3 h at -40°C in order to ensure a complete freezing. Drying was performed at a pressure of 10 Pa.

The temperature was first maintained at -30°C during 8 h, then -15°C for 5 h and finally 0°C for 3 h. A desorption step allowing complete drying of the sample was then applied. The samples were maintained in the freeze dryer, the temperature was set at 25°C and the pressure was increased first at 1 Pa for 12 h then at 10 Pa for 8 h.

Latex film preparation. The continuous phase of the dispersion was evaporated to create a nanostructured film. For this, the films were prepared by spreading a thin layer (0.1 to 0.3 mm) of a copolymer dispersion on a clean glass substrate or in a DSC sample pan. The latex films were left 12 h at ambient conditions to dry, and then analyzed without annealing. The film was clear and free of cracks.

Solution film preparation. The nanoparticles were dissolved in a good solvent to disrupt the selfassembly, then solvent was evaporated to create a film displaying in principle homogeneously mixed polymer blocks. For this, 5 mL of THF was added to 1 mL of copolymer dispersion to dissolve the nanoparticles. The 50 μL of homogeneous solution was cast in a DSC pan or a XPS mold. Then, the solution was dried at room temperature for 12 h.

Calculations

Colloial data calculations

Number of particles (𝑵 𝒑 ):

𝑁 p = 6𝜏 𝜋𝜌 𝑃𝑆 𝐷 p 3 (1)
Where 𝜌 PS : density of polystyrene = 1.047 [g cm -3 ] 𝑁 agg : mean aggregation number []

Mathematical treatment of MDSC data

A Mettler-Toledo DSC1 was used for thermal analysis, glass transition temperature, heat capacity and derivative of heat capacity determination. The lyophilized block copolymer nanoparticles were heated from -50 to 150 °C after prior thermal annealing step (1 min, 2 min, 5 min, 10 min, 30 min and 120 min).

An oscillation amplitude of 1.5 °C and an oscillation period of 60 s with the heating rate of 3 °C min -1 were used for the measurements of the heat flow and the heat capacity: 𝑉 micro cm 3 g -1

Additional tables

𝑉 meso cm 3 g -

Scheme 1 . 1 .

 11 Scheme 1.1. Schematic representation of PISA mechanism.

Scheme 1 . 2 .

 12 photomediated, and photocatalyzed ─ mechanisms.

Figure 1 . 1 . 1 . 2 .

 1112 Figure 1.1. Photolysis mechanism of Type I PI with two conventional examples (a) diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO) and (b) 2-methyl-4′-(methylthio)-2-morpholinopropiophenone (MMMP, also referred to as Irgacure 907) .

Figure 1 . 2 .

 12 Figure 1.2. List of type I radical PIs used in photoinitiated RAFT-mediated PISA. SPTPsodium phenyl-2,4,6trimethylbenzoylphosphinate; SBAPO sodium bis(mesitoyl) phosphinic acid; TPO -2,4,6-trimethylbenzoyldiphenylphosphinate; HMPP -2-hydroxy-2-methylpropiophenone; MMMP -2-methyl-4′-(methylthio)-2-morpholinopropiophenone; PP-OH -2-hydroxy-4′-2-(hydroxyethoxy)-2-methylpropio-phenone.

Scheme 1 . 4 .

 14 Scheme 1.4. General mechanism of radiation-induced β-cleavage of S-cyanomethyl-S-dodecyltrithiocarbonate (CMDTC) RAFT agent. UV-visible absorption spectra of this RAFT CTA in dioxane is shown below.

  photomediated RAFT polymerizations are represented in Figure 1.3.

Figure 1 . 3 .

 13 Figure 1.3. RAFT agents used for photomediated RAFT polymerization

Figure 1 . 4 .

 14 Figure 1.4. Structure of main PCs used in photocatalyzed RAFT processes. Ru(bpy)3Cl3tris(bipyridine)-ruthenium(II) chloride, Eosin Ysodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxo-3H-xanthen-9-yl)benzoate, ZnTPP -5,10,15,20-tetraphenyl-21H,23Hporphine zinc, ZnTMPyP -5,10,15,20-tetraphenyl-21H,23H-porphine zinc.

Figure 1 . 5 .Scheme 1 . 5 .Scheme 1 . 6 .

 151516 Figure 1.5. CTAs used in photoinitiated, photomediated and photo-catalyzed PISA processes: CEPA -4-cyano-4-ethylsulfanylthiocarbonylsulfanyl-pentanoic acid; CPADB -4-cyano-4-((phenylcarbonothioyl)thio)pentanoic acid; DDMAT -S-1-dodecyl-S′-(α,α′-dimethyl-α″-acetic acid) trithiocarbonate; BTPA -2-(nbutyltrithiocarbonate)-propionic acid; CDPA -4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl] pentanoic acid. The list of solvophilic monomers used in photoinitiated, photomediated and photo-catalyzed syntheses of macro-CTA precursors: PEGpoly(ethylene glycol); GlyMAglycidyl methacrylate; OEGMAoligo(ethylene glycol) methyl ether methacrylate (n = 9); HEAhydroxyethyl acrylate; HPMA -2-hydroxypropyl methacrylate; DMAAdimethyl acrylamide; Sarsarcosine; HPMAm -2-hydroxypropyl methacrylamide; MEAmethylethyl acrylate; GMAglycerol monomethacrylate. The list of solvophobic monomers used in photoinitiated, photomediated and photo-catalyzed PISA processes during chain-extension. DAAdiacetone acrylamide; HPMA -2hydroxypropyl methacrylate; DMAAdimethyl acrylamide; MTEMA -2-(methylthio)ethyl methacrylate; IBOAisobornyl acrylate; BzMAbenzyl methacrylate; t-BAtert-butyl acrylate; PFMApenta-fluorophenyl methacrylate; MMAmethyl methacrylate; DMAEMA -2-(dimethylamino)ethyl methacrylate.

Scheme 1 . 9 .Scheme 1 . 10 .

 19110 Scheme 1.9. Synthesis of diblock copolymer and triblock copolymer nano-objects by photoinitiated RAFT dispersion polymerization and photoinitiated seeded RAFT dispersion polymerization.86

Scheme 1 . 13 .

 113 Scheme 1.13. Synthesis of POEGMA-b-PBzMA NPs via methacrylate-based photoPISA. 90

Scheme 1 . 14 .Scheme 1 . 15 .

 114115 Scheme 1.14. Synthetic approach towards generating self-assembled POEGMA-b-PBzMA NPs via visible light photomediated RAFT PISA.63

Scheme 1 . 16 .

 116 Scheme 1.16. (A) Preparation of inherently permeable protein-loaded nanoreactors by aqueous PISA. (B) Cryo-TEM image and fluorescence micrograph of empty vesicles (I, II) and confocal optical microscopy image of green fluorescent protein-loaded vesicles (III, IV). 92

Figure 1 . 3 .Scheme 1 . 19 .

 13119 Figure 1.3. The ruthenium based PC, Ru(bpy)3Cl2 was able to regulate the dispersion polymerization of BzMA in ethanol using a POEGMA macro-CTA under blue light irradiation (Scheme 1.19). Self-assembled NPs with different morphologies, such as S, W, and V were produced. In addition, external temporal regulation by light was demonstrated by "ON/OFF" experiments.

Scheme 1 . 20 .

 120 Scheme 1.20. Proposed mechanism of photoinitiation by EY and AscA in the presence of oxygen.64

Scheme 1 . 23 .

 123 Scheme 1.23. ZnTMPyP mediated PET-RAFT dispersion polymerization of HPMA under red light and in the presence of biotin as a singlet oxygen quencher.67

Following a similar

  methodology, new spherical and narrowly distributed diblock copolymer nanoparticles(20 -40 nm) were synthesized by RAFT dispersion polymerization of styrene (St) at 35 °C and 20 wt% solids using a PHEA based macro-CTA as steric stabilizer block (Figure2.1). Both solvophilic PHEA block and solvophobic PS block were obtained via photomediated RAFT polymerization by leveraging the reversible 𝛽-fragmentation of the commercial CMDTC RAFT CTA under blue light (λmax = 472 nm). Emphasis was made on two domains poorly investigated so far in photo-PISA: the mechanism of particle formation/growth, as well as the characterization of optical properties throughout the dispersion polymerization.

Figure 2 . 1 .

 21 Figure 2.1. Synthesis of poly(hydroxyethyl acrylate) macro-CTA via photomediated RAFT polymerization in 1,4dioxane.Subsequent chain extension via dispersion photo-PISA of styrene in a methanol/water mixture.

Figure 2 . 2 .

 22 Figure 2.2. Visible light photomediated polymerization of HEA in solution during the synthesis of PHEA58-TTC. (a) Polymerization kinetics, conversion measured by 1 H NMR; (b) SEC chromatograms (RI detector) in THF; (c) Number-average molecular weight (𝑀 n ̅̅̅̅ ) and dispersity (Đ = 𝑀 w ̅̅̅̅̅ /𝑀 n ̅̅̅̅ ) with conversion; dashed line: theoretical molecular weight calculated with conversion. Experimental conditions: HEA : CMDTC =100 : 1; [HEA]0 = 2.97 M in dioxane, blue LED (547 mW cm -2 , 25 °C).

Figure 2 . 3 .

 23 Figure 2.3. Series of photographs showing the evolution of the reaction medium during the photomediated RAFT dispersion polymerization of St (synthesis of PHEA25-b-PSm diblock copolymer particles).

  Two distinct intervals (I, II) corresponding to different rate behaviors were observed in the ln([M]0/[M]) versus time plot of Figure 2.4a.

Figure 2 . 3 ,

 23 t = 47-70 h), most likely because of the gradual increase of particle (micelle) diameter (see TEM and DLS size data in Figure 2.4a).

Figure 2 . 4 .

 24 Figure 2.4. (a) Polymerization kinetics for the synthesis of a PHEA25-b-PS118 copolymer. Conversion measured by 1 H NMR Evolution of particle size determined by DLS and TEM. (b) SEC chromatograms in DMF at 50 °C (RI detector) of PHEA25-CMDTC and PHEA25-b-PSm diblock copolymers synthesized via photomediated RAFT polymerization in dispersion. (c) Evolution of 𝑀 n ̅̅̅̅ and dispersity index with monomer conversion; dashed line: theoretical molecular weight calculated with conversion. Experimental conditions: PHEA25-CMDTC:St = 1:200; [PHEA25-CMDTC]0 = 7.68 mM in methanol/water mixture (95/5 wt%), blue LED (547mW cm -2 , 35 °C).

Figure 2 . 5

 25 Figure 2.5 shows a series of representative TEM images obtained from samples withdrawn at different intervals during the dispersion photopolymerization of St starting from PHEA25-TTC macro-CTA.

Figure 2 . 5 .

 25 Figure 2.5. TEM images of PHEA25-PSm samples taken at different times during dispersion PISA of St.

Figure 2 . 6 .

 26 Figure 2.6. Surface tension of a MeOH:H2O (95/5, %w/w) mixture for different concentrations of PHEA25-CMDTC as determined by the Wilhelmy plate method. The typical concentration of macro-CTA used in photomediated RAFT polymerization in dispersion is 7.68 mM. (b) Scattered intensity (mcps-mega counts per second, at low concentration is proportional to particles concentration) determined by DLS technique from aliquots withdrawn during photo-PISA via RAFT polymerization in dispersion.

Figure 2 .

 2 6b shows the evolution of scattered intensity as function of time within the first 20 h of irradiation. DLS measurements were performed without any dilution and at constant incident light intensity enabling comparisons between samples. In the first 8 h following light exposure, the scattering intensities had constant and low values consistently with a homogeneous medium devoid of colloidal clusters. As early as 10 h (well before interval II), scattered intensity displayed a linear and sharp increase, which marked the formation of the first micelles. St conversion was only 3-4% at this time, leading to a critical degree of polymerization of ~6-8 units for the PS block (PHEA25-b-PS6-8-CMDTC).

Table 2 . 1 .

 21 Colloidal data of PHEA25-b-PSm particles formed by dispersion photo-PISA at two different temperatures (35 and 55 °C). Dp -Diameter of particles determined by TEM. Np -Number of particles per liter of the reaction mixture. PDI -Dispersity index. 119 Nagg -Number of RAFT chain-ends per particle. Sagg -Area occupied by 1 RAFT chain-end function on the surface of the particles.

Scheme 2 . 2 .

 22 Scheme 2.2. Particle nucleation occurs in Interval I. The main mechanism for the formation of polymer particles is homogeneous nucleation involving the self-assembly of solution-polymerized copolymer chains.As revealed by DLS analysis, nucleation begins as early as 8 h (only 6-8 St units), making monomer partitioning inside the PS core-forming block of the micelles possible. Nevertheless, the polymerization remains slow due to a low number of particles. A long nucleation period occurs with particle number increasing slowly with time, and the medium becoming more turbid. Its duration varies only in the conversion range 4-15% (DP of St increasing from 6 to 30), but it runs between 8 and 35 h because of the slow propagation of St in solution. At the end of Interval I, nucleation stops and no new particles are generated (but Np is not necessarily constant). In interval II, polymerization proceeds in the polymer particles, and the system undergoes change of reaction kinetics. The high number of polymer particles swollen by monomer drives both a constant and higher polymerization rate. A steady-state number of particles can be preserved when steric stabilization is adequate (PHEA52 and PHEA85). With shorter PHEA stabilizers (PHEA25), the particle diameter increase requires the addition of new copolymer chains through possibly because of the particle coalescence.

Scheme 2 . 2 .

 22 Scheme 2.2. Schematic representation of photo-PISA mechanism. (Np is the number of particles per liter of the reaction mixture).

Figure 2 .

 2 Figure 2.7a shows a series of absorption spectra of PHEA25-CMDTC macro-CTA in the range 300 -400 nm taken throughout St dispersion polymerization. They clearly show that the extinction coefficient 𝐸 λis the combined result of both absorption and light scattering as soon as particles are formed (t > 47 h). The λmax (310 nm) of the absorption band of the π,π * -transition of TTC-based RAFT agent is clearly visible in all spectra, while scattering contribution increases during irradiation. In the selected spectral range > 280 nm, only the RAFT agent absorbs (St, PS and PHEA have a negligible absorption above 300 nm). Consequently, the eq. 1 can be simplified as:

Figure 2 . 7 .

 27 Figure 2.7. (a) UV-Vis absorption spectra of PHEA25-CMDTC macro-CTA in the range 300 -400 nm throughout St dispersion polymerization. (b) Fitted scattering coefficient(𝑆 λ Fit ) at 500 nm versus particles average diameter (Dp). (c) Estimated absorption coefficient 𝐾 λ TTC of PHEA25-b-PSm during dispersion photo-PISA.

  this study, we started from soft PHEA-b-PS particles prepared via dispersion PISA described in the previous chapter. Relying on PHEAx-b-PSy nanoparticles 55 (where x and y refer to the DP of the respective blocks), we chose a copolymer composition located in the spherical aggregate regime for reasons of simplification. Subsequently, model nano-objects having different lengths of PHEA shell-forming block were studied by three electron microscopy (EM) techniques: conventional TEM with and without staining, cryo-TEM and SEM. The objectives of this present study are twofold: to learn which techniques are appropriate for imaging the core-shell architecture of PHEA-b-PS particles, and more generally, to define best practices for EM analysis of PISA-prepared particles. This is very important because the polymer community is currently making intensive use of EM to study soft block copolymer nanoparticles without being always aware of common errors in the interpretation of EM data: artefacts, sample reorganization induced by drying, image distortion caused by radiation damage, etc. Results and discussion 1. Synthesis of amphiphilic diblock copolymer nanoparticles PHEA-b-PS Two types of model amphiphilic diblock copolymer nanoparticles, each containing a constant DP PS core-forming block (130) and varying DP of PHEA stabilizing block (23 and 85), were synthesized with an alcohol-based PISA method described elsewhere. 55 The compounds had relatively narrow molecular weight dispersity: Đ = 1.11 (PHEA23-b-PS130) and 1.43 (PHEA85-b-PS130). In the light of the dynamic light scattering (DLS) data, the samples were found monodisperse, with z-average (intensity weighted mean hydrodynamic diameter) of 79 nm (PHEA23-b-PS130) and 40 nm (PHEA85-b-PS130). Using Mie theory and optical properties of nanoparticles, number-weighted average diameters were also estimated -39 nm (PHEA23-b-PS130) and 21 nm (PHEA85-b-PS130)essentially for comparative purpose with EM data. Due to the highly hydrophobic nature of PS, the growth of a polar PHEA block in a methanol/water mixture leads to selective solvation of the chains, inducing phase separation and particle formation. As sketched schematically in Figure 3.1.1, the expected core shell architecture is thus made up of a hard

Figure 3 . 1 . 1 .

 311 Figure 3.1.1. PHEA-b-PS core-shell nanoparticles are thought to contain 3 different phases: two bulk phases of pure PS (core) and PHEA (shell) as well as an interfacial phase composed of a mixture of both copolymers.

Figure 3 . 1 . 2

 312 Figure 3.1.2 shows high magnification TEM images of PHEA85-b-PS130 (a) and PHEA23-b-PS130 (b)

Figure 3 . 1 . 2

 312 Figure 3.1.2 Conventional TEM images of (a) PHEA85-b-PS130 and (b) PHEA23-b-PS130 block copolymer nanoparticles. The inset shows the size distribution obtained from TEM measurements. The red arrows in image b highlight vesicular structures.

Figure 3 . 1 . 3 .

 313 Figure 3.1.3. Positive stain EM images of (a) PHEA85-b-PS130 and (b) PHEA23-b-PS130.

Figure 3 . 1 . 3

 313 Figure 3.1.3 shows the same samples PHEA85-b-PS130 (a) and PHEA23-b-PS130 (b) after rutheniumtetraoxide (RuO4) positive staining. RuO4 is a strong scattering agent that reacts with polymer regions inside the particle improving the contrast.150 Because of its high oxidative properties, RuO4 is presumed to stain both PHEA and PS domains, by reacting respectively with hydroxyl groups and aromatic unsaturations.151- 153 Four different structural features can be highlighted:Contrast. Regardless of the sample, the action of the stain has improved considerably the image contrast against the support film. Image contrast against grid background can be further enhanced after latex dialysis to remove copolymer chains soluble in the continuous phase. As noted previously, PHEA23-b-PS130 exhibits a pronounced tendency to form pools of clustered particles while PHEA85-b-PS130 particles are well separated.Size. Interestingly, an increase of the average diameter 𝐷 n-RuO 4 is also observed compared to unstained analogues: 50% for PHEA85-b-PS130 (31.1 ± 3.8 nm) and 15% for PHEA23-b-PS130(41.8 ± 7.2 nm). This increase is consistent with our hypothesis that the superficial PHEA-rich region (shell) might be undetected with unstained specimen, and requires staining to be revealed. As further evidence, we note that the size increase for PHEA85-b-PS130 particles is much larger than for PHEA23-b-PS130, which is consistent with the longer PHEA block and a more extended lyophilic shell. It is worth noting that the diameter of positive stained specimen significantly departs from the number-weighted mean diameters estimated by DLS measurements: 21 nm (PHEA85-b-PS130) and 39 nm (PHEA23-b-PS130.). It is well reported that direct comparison between TEM of dried particles and DLS data involving solvated particles is not always relevant.154 Halo. In Figure3.1.3a, positively stained PHEA85-b-PS130 shows a darker halo contouring the spherical particles. It is therefore tempting to interpret this feature as a sign of a core-shell architecture.

Figure 3 . 1 . 4 .Figure 3 . 1 . 4

 314314 Figure 3.1.4. Negative stain electron microscopy of (a) PHEA85-b-PS130 and (b) PHEA23-b-PS130

  Size and shell thickness. The number average diameters 𝐷 n-UAc = 29.7 ± 4.2 nm (PHEA85-b-PS130) and 41.3 ± 5.1 nm (PHEA23-b-PS130) -are substantially larger than those of the unstained samples. This suggests that negative coating at particle surface may be efficient in revealing the particles boundaries, which is a requirement for precise measurement of particle size. Slightly smaller values are found compared to positively stained samples for PHEA85-b-PS130 (-5.1 nm) but not for PHEA23-b-PS130. This could indicate that positive staining may over-estimate the dimensions of the nano-objects, but only when the shell is extended. By comparing the negatively stained images (𝐷 n-UAc ) where particles are presented in a most faithful way possible particle and unstained image (𝐷 n ) representative only of particle core, the thickness δ of the shell can be tentatively estimated (δ = (𝐷 n-UAc -𝐷 n )/2). For PHEA85-b-PS130, δ ~ 5.0 ± 1.8 nm while δ of PHEA23-b-PS130 is in the error range and cannot be accessed with precision due to the limited extent of PHEA domain. PHEA85-b-PS130 is composed of 85 repeat units for the lyophilic backbone segment, which

3 .

 3 Cryo-TEM Previous analyses have shown that PHEA-b-PS particles are problematic specimens for conventional TEM, in particular due their proneness to dehydration in the vacuum of the electron microscope. In what follows, the two same samples were analyzed by cryo-TEM, a useful technique to visualize particle in their dispersion state by rapid vitrification in a thin layer of solvent. Though cryo-TEM requires more complex specimen preparation, it is the only technique able to image nanoparticles in their native form.Figure 3.1.5 represents two typical cryo-TEM images of PHEA85-b-PS130 (a) and PHEA23-b-PS130 (b) nanoparticles at low magnification.

Figure 3 . 1 . 5 .

 315 Figure 3.1.5. (a) Cryo-TEM image of PHEA85-b-PS130 particles. Particle surface displays some dark spots, better seen in the inset. Indicated by arrows, these spots correspond to PHEA chains pointing parallel to the electron beam trough the continuous phase. The chains non-parallel to the beam are no clearly seen as the signal-to-noise ratio is too low. (b) Cryo-TEM image of PHEA23-b-PS130 particles. Two particle morphologies can be distinguished: spherical particles and vesicles. The inset shows vesicle. The darker rim corresponds to the PHEA chains (short arrow), then the light grey the PS, and finally again a darker thin rim for the PHEA chains in contact with the inner space (long arrow). (c) Pure PS nanoparticles. Used as benchmark latex, the particles appear homogeneous with a smooth surface.

  . A more in-depth examination of the PHEA85-b-PS130 specimen obtained at high magnification (see the inset of Figure3.1.5a) reveals dark spots inside the particles. These features are not artefact and can be rationalized on the basis of extended PHEA chains pointing in a direction parallel to the electron beam. Conversely, chains non-parallel to the beam have a much lower scattering efficiency and cannot be imaged. Due to fast vitrification, the PHEA molecules usually adopt random orientations in the amorphous ice layer, consequently, the PHEA chains at the edge of the particle are unlikely to adopt this particular orthogonal orientation, making not possible the visualization of lyophilic shell. In contrast, PHEA23-b-PS130 does not display a similar patched structure since the short PHEA chains do not have a sufficient scattering efficiency. In the same sample, it is noteworthy that some isolated vesicles can be detected in the inset of Figure3.1.5b, supporting that their presence in conventional TEM is not an artefact caused by drying or concentration effect. In addition, no sign of coalescence is visible (as noted previously in TEM images), thus confirming the previous assumption of drying-induced aggregation (vide supra). Surface structure. The micrograph 5a shows that PHEA85-b-PS130 particles present a fuzzy surface that resembles an envelope as opposed to the sharper surface of PHEA23-b-PS130 (Figure 3.1.5b) and pure polystyrene (PS) particles (Figure 3.1.5c) serving as benchmark. a

Figure 3 . 1 . 6

 316 Figure 3.1.6 displays SEM images of PHEA85-b-PS130 (image a) and PHEA23-b-PS130 (Figure 3.1.6b) nanoparticles.To ease interpretation, a control analysis with PS nanoparticles was also carried out (image c). In all instances, an accelerating voltage of 5.5 kV was used during image acquisition in order to emphasize surface structures. Thanks to a -5 kV specimen bias voltage, the electrons eventually strike the sample with a landing voltage of only 500 V, thus preventing it from damages.

Figure 3 . 1 . 6 .

 316 Figure 3.1.6. SEM images of (a) PHEA85-b-PS130, (b) PHEA23-b-PS130 diblock copolymer nanoparticles, (c) PS latex used as model latex. All images were obtained at a landing voltage of 500 V.

  Annex) allows greater electron beam penetration into surface region. This results in the generation/escape of a larger number of secondary electrons, giving rise to a typical bright particle surface. By comparison, PHEA23-b-PS130 (Figure 3.1.6b) displays a much thicker bright corona due to the emission of more secondary electrons. PHEA85-b-PS130 particles (Figure 3.1.6c) appear even brighter because the surface may have a higher secondary electron emitting capacity. The more pronounced brightening of copolymer particles compared to PS particles could be reconciled with the presence of protrusions onto particle surface. The longer the PHEA chains, the greater the edge effect. Another non-direct indication of core-shell morphology relies on extremely blurred interphase particularly for PHEA85-b-PS130 based nanoparticles.

Fig. 3 . 2 . 1 .

 321 photomediated reversible-addition-fragmentation-transfer (RAFT) polymerization. The resulting PHEA85-b-PS130 particles have an intensity average-diameter of 40 nm (dynamic light scattering data) and their typical core-shell structure is sketched in Figure 3.2.1.

Figure 3 . 2 . 2

 322 Figure 3.2.2 shows two cryo-TEM pictures at low (a) and high magnification (b) obtained from PHEA85-b-PS130 latex. Cryo-TEM was chosen over other imaging techniques because it is recognized as the most amenable to the examination of soft nanomaterials. Indeed, the particles can be studied in their most native state without the need of specimen drying. As can be seen on the TEM image a, the spherical particles exhibit a narrow polydispersity index (𝑃𝐷𝐼 TEM = 1.03) and a small apparent diameter (𝐷 n = 21.6 ±

Figure 3 . 2 . 2 .

 322 Figure 3.2.2. (a) Cryo-TEM image of PHEA85-b-PS130 latex at low magnification. (b) High magnification image corresponding to the framed area shown in image a. Concentration of copolymer dispersion = 0.1 wt%.

Figure 3 . 2 . 3 ,

 323 a SEM image of lyophilized particles shows that the core-shell morphology has been mostly maintained although the soft PHEA shell led to a partial coalescence of the particles boundaries.

Figure 3 . 2 . 3 .

 323 Figure 3.2.3. SEM image of lyophilized PHEA85-b-PS130 block copolymer nanoparticles.

3

 3 °Cand 99.0 °C were found when analyzing by DSC PHEA85 and PS130 homopolymers of similar degree of polymerization as the PHEA85-b-PS130 block copolymer (FigureS6ain Annex). However, the DSC thermogram of as-lyophilized PHEA85-b-PS130 latex shows a broad endotherm (FigureS6bin Annex), which makes challenging the precise assignment of the glass transition regions of PHEA and PS blocks. It is well established that conventional DSC faces problems of overlapping between thermodynamic and kinetic phenomena. To overcome this issue, modulated DSC (MDSC) has made possible to separate the total heat flow of DSC into two components: a reversible signal induced by changes in heat capacity (e.g. glass transitions) and a non-reversible signal involving only kinetic processes (enthalpic recovery and residual solvent evaporation). From the reversible signal, one can thus determine the change in heat capacity without the interference of kinetic events. Figure 3.2.4a shows the variation of heat capacity (Cp) as a function of temperature data for lyophilized PHEA85-b-PS130 nanoparticles obtained from the reversible heat flow. We took only the data of the first scan, which is the true reflection of the morphology in the original latex particle state. On the same plot are also given the results obtained from PHEA and PS homopolymers representing respectively the idealized pure shell and core phases. Although transitions can be clearly distinguished in the three heat capacity traces, it is difficult to draw conclusions due to their low intensity. To gain further information, Figure 3.2.4b shows dCp/dT, vs. temperature. Hourston et al. have highlighted the utility of treating DSC data in the form of temperature differential of the heat capacity as a function of temperature.178,179 Accordingly, the lyophilized copolymer sample (trace 1) displays now two single and well-defined peaks at 28.1 °C and 85.5 °C, which can be assigned respectively to a shell phase rich in PHEA and a core phase rich in PS. The pure polymer phases of PHEA (trace 2) and PS (trace 3) show two intense peaks at -7.3 °C and 99.0 °C, in agreement with their Tg values. However, the trace of the lyophilized copolymer sample 1 is not well separated and the broad baseline suggests a significant fraction of interfacial region. By measuring the increment of heat capacity in the two resolved glass transition regions of PHEA and PS (𝛥𝐶 p ) and knowing the increments of pure phases (𝛥𝐶 p0 ) from traces 2 and 3, it has been possible to determine the amount of different phases. The calculations are detailed in the experimental section. Clearly, the heat capacities 𝛥𝐶 p PHEA and 𝛥𝐶 p PS in the different copolymers are systematically lower than 𝛥𝐶 p0 . This suggests the existence of an interfacial region and the partial miscibility of the two PHEA and PS segments.

  3 °C for the pure PHEA reference. The PS core has a transition at 85.5 °C in the copolymer whereas the pure PS sample shows a sharp glass transition centered at 99.0 °C. The plasticizing effect of PHEA chains trapped into PS matrix, and the rigidification induced by PS inclusion into PHEA domains account for this difference. It is another evidence of the partial miscibility of both polymers.

Figure 3 . 2 . 4 .

 324 Figure 3.2.4. (a) Variation of heat capacity (ΔCp) as a function of temperature obtained from the reversible heat flow for lyophilized PHEA85-b-PS130. (b) Temperature differential of the heat capacity (dCp/dT) as a function of temperature

Figure 3 . 2 .

 32 5b shows the evolution of the weight fraction of the interface 𝑊 i (left y axis) and the total change of lost weight fraction of polymer in the interface (𝑊 lost i ) as a function of annealing time. In addition, all 𝛥𝐶 p values and weight fractions of PHEA in shell (𝛿 PHEA s ), PS core (𝛿 PS c ) and interface (𝑊 i = 𝛿 PS i + 𝛿 PHEA i

Figure 3 . 2 . 5 . 3 . 2 . 1 .

 325321 Figure 3.2.5. (a) Temperature differential of the heat capacity (dCp/dT) of core-shell lyophilized PHEA85-b-PS130. particles as a function of temperature for different annealing times at 150 °C. (b) Evolution of weight fraction of the interface (solid line) and weight fraction of lost interface (dashed line) for different annealing times

Figure 3 . 2 . 6 .

 326 Figure 3.2.6. (a) Temperature differential of the heat capacity (dCp/dT) of latex film obtained after solvent evaporation as a function of temperature for different annealing times at 150 °C for latex film obtained after solvent evaporation. (b) Evolution of weight fraction of the interface 𝑊 i (solid line) and weight fraction of lost interface 𝑊 lost i (dashed line) for different annealing times.

Figure 3 . 2 . 7 .

 327 Figure 3.2.7. XPS survey spectra of PHEA85-b-PS130 copolymer in different forms: (a) lyophilized particles, (b) latex film and (c) solution films

Figure 3 . 2 . 8

 328 Figure 3.2.8 shows a high resolution analysis of the C1s peak region for the same three samples. In all instances, a fitting with five peaks was performed: the two main features were assigned to C-C and C-H (285.00 eV) and C-O (286.70 eV), and the three weak signals were attributed to O=C-O (289.5 eV), C=O (291.7 eV) and C-COO (285.4 eV). Because they are better resolved, we have paid attention to the two main resonances of C-O and C-C/CH. Both structured samples obtained by lyophilization (a) and latex drying (b) show comparable content of C-C/C-H, respectively 76.7% and 75.9%. This value is lower than theoretical value of 81.8% for a PHEA85-b-PS130 diblock copolymer, confirming an enrichement of the surface in PHEA. The amorphous sample (c) with a C-C/CH signal of 81.53% agrees with an isotropic distribution of PHEA and PS phases. The integration area of the C-O band is again similar in samples a and b, and 35%

Figure 3 . 2 . 8 .

 328 Figure 3.2.8. C1s core-level XPS spectrum and their deconvolution for PHEA85-b-PS130 copolymer in different forms: (a) lyophilized particles, (b) latex film and (c) solution films.

Figure 4 . 1 . 1 .

 411 Figure 4.1.1. PHEA-b-PS core-shell nanoparticles with a diameter ranging from 20 to 40 nm synthesized via PISA. The chain ends result from the RAFT CTA (CMDTC) used to mediate the controlled radical polymerization.

Figure 4 . 1 . 2 .

 412 Figure 4.1.2. TEM image of PHEA25-b-PS118 copolymer nanoparticles prepared in methanol:H2O mixture (95:5 %w/w) solution. The inset shows the number size distribution obtained from DLS data.

Figure 4 . 1 . 2

 412 Figure 4.1.2 shows a representative TEM picture obtained from PHEA25-b-PS118 latex (𝑀 n (PHEA) = 2900 g mol -1 and 𝑀 n (PS) = 12300 g mol -1). The spherical particles exhibit a narrow particle size dispersity

Figure 4 . 1 . 2 ,

 412 the DLS distribution reveals a small polydispersity (𝑃𝐷𝐼 DLS 𝑙 = 0.174) and a higher average diameter of 𝐷 DLS 𝑙 = 38.9 nm. Taking into account the soft poly(acrylate) shell, the DLS-derived value is consistently higher, and provides a rough estimation of the solvated shell (δ = 5-10 nm) by taking the difference between DLS and TEM particle diameters (δ = 𝐷 DLS 𝑙 -𝐷 TEM 𝑙

Figure 4 . 1 . 3 .

 413 As described above, a colloidally stable dispersion based on PHEA25-b-PS118 / phenolic resin mixture containing a template content of 60 wt% with respect to the monomers was aged at room temperature for 6 h. The objective was to ensure slow condensation and removal of most solvent and residual monomer by evaporation. Condensation was then completed at 60 °C for 12 h. At this stage (i.e. prior to pyrolysis), TEM analysis shows a homogeneous template/phenolic resin composite, where the intimate mixing of the two polymers does not allow for the discrimination of nanoparticles. The brown composite residue was further pyrolysed at 600 °C in argon to decompose the phenolic polymer leading to the carbon network creation and to eliminate the organic polymer nanoparticles in a single step, resulting in the mesopore formation.Morphology and structure.

Figure 4 . 1 .

 41 4a-b shows some typical TEM images at different scales of the mesoporous carbon derived from PHEA25-b-PS118 copolymer nanoparticles. The characteristic structure of a mesostructured cellular foam can be recognized, which consists of a disorder array of spherical mesopores framed by the carbon struts.

Figure 4 . 1 . 3 .

 413 Figure 4.1.3. Process for the synthesis of mesoporous carbon using core-shell diblock copolymer nanoparticles.

Figure 4 . 1 .

 41 Figure 4.1.4c shows a tilt series of five characteristic images where windows connecting the cells are clearly visualized. Pore connectivity in the whole sample is proven from the full tilt series of 1000 images.

Figure 4 . 1 . 4 .

 414 Figure 4.1.4. (a) TEM images of mesoporous carbon templated by PHEA25-b-PS118 copolymer nanoparticles, (b) higher magnification image of the same sample, (c) electron tomography tilt series where interconnected pores are indicated by solid white lines.

Figure 4 . 1 . 5 )

 415 based on Barrett-Joyner-Halenda (BJH) model reveals a mesopore distribution ranging from 10 to 30 nm.The average mesopore size of 22 nm (𝐷 BJH p ) is fully consistent with TEM results (𝐷 TEM p = 22 nm). The size distribution of micropores determined by NLDFT method shows a small population at 0.5 nm together with a larger one at 1.4 nm.

Figure 4 . 1 . 5 .

 415 Figure 4.1.5. Nitrogen adsorption/desorption isotherms of carbon material template by PHEA25-b-PS118 copolymer nanoparticles.The inset provides the meso-and micropore size distributions obtained from BJH and NLDFT methods, respectively.

Figure 4 . 1 . 6 .

 416 Figure 4.1.6. Thermogravimetric plots showing the variation of weight loss (solid line) and its derivative (dashed line) of PHEA25-b-PS118 copolymer nanoparticles and the composite PHEA25-b-PS118 nanoparticle/phenolic resin.

  1.1), three other diblock copolymers templates were employed for the nanoporous carbon synthesis: PHEA25-b-PS160 (run b) to assess the influence of the length of the solvophobic block; PHEA52-b-PS110 (run c) and PHEA85-b-PS114 (run d) to

  Conversely, the stronger steric stabilization of PHEA52-b-PS110 and PHEA85-b-PS114 yields smaller particles, 𝐷 DLS 𝑙 = 30.1 and 33.2 nm respectively. As expected, the TEM size data (see 𝐷 TEM 𝑙 in Table4.1.1)show a similar range of variations. However, smaller values are obtained because only the PS core can be imaged by this technique as previously underscored.

Figure 4 . 1 . 7

 417 shows a series of nitrogen adsorption/desorption isotherms for all these carbon materials (left) together with the corresponding TEM images (right). Like reference sample a, TEM imaging shows that all carbon materials possess a disordered mesoporous structure with uniform pores. However, clear differences are noticeable as regards cell size, shape and pore interconnectivity.

Figure 4 . 1 . 7 .

 417 Figure 4.1.7. Left: Nitrogen adsorption/desorption isotherms of carbon materials templated by PHEA25-b-PS118 (a), PHEA25-b-PS160 (b), PHEA52-b-PS110 (c) and PHEA85-b-PS114 (d). Right: TEM images of the corresponding materials (𝐷 TEM pis the average pore size). In this series, a steady molar ratio phloroglucinol:glyoxylic acid:template = 1:1:0.020 was chosen.

Figure 4 . 1 . 7 ,

 417 which is reminiscent of complex and heterogeneous porous structures containing both open and partially blocked mesopores.228 To provide a definitive answer on the effect of the solvophilic shell on pore architecture and interconnectivity, scanning electron microscopy (SEM) was used because of its topographic contrast imaging capacity. The SEM images in Figure 4.1.8 highlight most clearly the contrast between the highly interconnected structure of carbon a (PHEA25-b-PS118) and the closed porosity of carbon d (PHEA85b-PS114), which agrees closely with both TEM and adsorption data.

Figure 4 . 1 . 8 .

 418 Figure 4.1.8. SEM images of carbons a and d templated respectively by PHEA25-b-PS118 and PHEA85-b-PS114 diblock copolymers

Figure 4

 4 Figure 4.1.9. As r decreases, a gradual broadening of the hysteresis loop is observed with a clear transition

Figure 4 . 1 . 9 .

 419 Figure 4.1.9. Effect of PHEA28-b-PS134 template concentration on textural properties of mesoporous carbons. r is the molar ratio of copolymer compared to reference experiment: r = 1 (a), 0.8 (e), 0.6 (f), 0.4 (g) and 0.2 (h). Left: nitrogen adsorption/desorption isotherms. Right: TEM pictures of corresponding mesoporous carbon materials.

Figure 4 . 1 . 10 ,

 4110 image i0) shows particles with an average diameter of 𝐷 TEM 𝑙 = 47 nm in agreement with DLS measurements (𝐷 DLS 𝑙 = 55 nm). To allow a relevant comparison with PHEA25-b-PS118 latex, a similar weight fraction in polymer template was used for the fabrication of carbon.

Figure 4 . 1 . 10 ,

 4110 Figure 4.1.10, the type IV isotherm exhibits a well-defined and a symmetrical type-H1 hysteresis loop.

Figure 4 . 1 . 10 .

 4110 Figure 4.1.10. Left: Nitrogen adsorption/desorption isotherms of carbon material templated by PHEA25-b-PS118 (a), PS nanolatex (i) and silica nanoparticle (j). Right: corresponding TEM images of PS latex (i0), mesoporous carbon derived from PS latex (i), phenolic resin/SiO2 hybrid before NaOH treatment (j0), and mesoporous carbon derived from SiO2 nanoparticles (j).

Figure 4 . 1 . 11 .

 4111 There is an obvious interest to use such kind of non-spherical templates in mesoporous carbon synthesis since this type of morphology is not achievable by LC mesophases. TEM picture k of mesoporous carbon obtained with this vesicular copolymer nano-object shows a complex structure, with lighter and darker regions reflecting the morphology of the orginal template. Darker regions display a similar size as voids of vesicular templates suggesting the ability of phenolic precursors to cross the PS membrane, fill the vesicle interior and undergo a spatially controlled polymerization. As can be seen in Figure4.1.10, the nitrogen adsorption/desorption isotherm of carbon material k features a coonventional type IV isotherm. The surface area is relatively unchanged (SBET = 683 m 2 g -1 ) compared with the reference sample a. A sharp H1-type hysteresis is observed, despite difficulties to distinguish mesopores from the TEM picture. A decrease of mesoporous volume (Vmeso = 0.74 cm 3 g -1 ) compared with the reference sample a (Vmeso = 1.01 cm 3 g -1 ) could reflect a decreased stability of vesicular nano-objects. Weaker stabilization or morphology change of the copolymer vesicules is also suggested during carbon preparation given that BJH model reveals an average 𝐷 BJH p = 28 nm, while the starting vesicular nano-objects are characterized by larger sizes and dispersity (𝐷 TEM 𝑙 = 30 -170 nm).

Figure 4 . 1 . 11 .

 4111 Figure 4.1.11. Left: nitrogen adsorption/desorption isotherms of mesoporous carbon prepared from PHEA17-b-PS136 copolymer vesicles. Right: TEM images of the copolymer vesicles (k0) and the corresponding mesoporous carbon (k).

Results and discussion 1 .Figure 4 . 2 . 1 .

 1421 Figure 4.2.1. Synthesis of POEGA macro-CTA via photomediated RAFT polymerization in ethanol. Subsequent chain extension via dispersion photo-PISA of benzyl acrylate in a methanol/water mixture.

Figure 4 . 2 . 2 .

 422 Figure 4.2.2. Visible light photomediated polymerization of OEGA in ethanol for the synthesis of POEGA22-DDMAT macro-CTA. (a) Monomer conversion measured by 1 H NMR; (b) SEC chromatograms (RI detector) in DMF; (c) number-average molecular weight (𝑀 n ̅̅̅̅ ) and dispersity (Đ = 𝑀 w ̅̅̅̅̅ /𝑀 n ̅̅̅̅ ) as a function of conversion; dashed line: theoretical molecular weight calculated with conversion. Experimental conditions: OEGA : DDMAT = 35 : 1 mol; [OEGA]0 = 0.82 M in ethanol, blue LED (0.8 mW cm -2 , 30 °C).

Figure 4 . 2 . 3 .

 423 Figure 4.2.3. Synthesis of POEGA27-b-PBzA172 via visible light photomediated polymerization of benzyl acrylate in methanol/water starting from POEGA22-DDMAT macro-CTA. (a) Benzyl acrylate conversion as a function of irradiation ( 1 H NMR data). (b) SEC chromatograms in DMF at 50 °C (RI detector) for different irradiation times. (c) Evolution of (𝑀 𝑛 ̅̅̅̅ ) and Đ with monomer conversion; dashed line: theoretical molecular weight. Experimental conditions: POEGA27-DDMAT : BzA = 1 : 200 mol; [POEGA27-DDMAT]0 = 5.21 mM in methanol/water mixture (70/30 %w/w), blue LED (0.8 mW cm -2 , 30 °C).

Figure 4

 4

  section): 23.4 ± 3.3 nm (14 h, image b), 28.7 ± 4.6 nm (16 h, image c) and 31.7 ± 4.7 nm (17 h, image d) at the end of the reaction. The objects exhibit a spherical shape and narrow particle size dispersity (PDI ≤ 1.04). Since the size strongly influences the biodistribution of NPs and the way of internalization into cells,

Figure 4 . 2 . 4 .

 424 Figure 4.2.4. Negative stain TEM images of POEGA27-b-PBzAm samples during RAFT photomediated PISA of benzyl acrylate in methanol/water mixture.

Figure 4 . 2 . 6

 426 illustrates the effect of these two parameters on the relative fraction of incorporated fluorophore (dye loading, %) for POEGA27-b-PBzA172. To determine dye loading, the as-prepared NR-doped NP were dissolved in THF, and their absorbance was recorded and compared to a calibration plot. Among the 15 samples prepared, the three ones possessing the highest concentration and volume fraction of swelling solutions (violet bars) led to instant NR precipitation, translating the limited loading capacity of the NP. The rest of the samples did not exhibit any sign of precipitation or colloidal destabilization after the freezing/encapsulation procedure.Nevertheless, gradual fluorophore leakage took place for a majority of samples after 1 week of storage (green bars), as attested by Nile red precipitation due to the extremely low water-solubility of NR.Ultimately, only 3 systems (red bars, marked 1-3) prepared with the lowest volume fractions of swelling solution(5-10 vol%) and NR concentration (0.25 -0.50 mol L -1 ) displayed superior and long stability.

Figure 4 . 2 . 6 .

 426 Figure 4.2.6. Loading of Nile Red inside POEGA27-b-PBzA172 diblock copolymer based NPs after encapsulation/freezing procedure. 3 systems marked as 1, 2 and 3 turn out to be highly stable.

  replacing methanol/water (70/30 %w/w) with water, (PBzA/solvent) is assumed to increase significantly (measurement in progress), indicating the formation of kinetically frozen micelles in water. There are other examples in the literature showing that such values of interfacial tension are sufficient for creating kinetically stable spheres. PB-PEO, PS-PMA, and PS-PEO were thus described in the literature as kinetically frozen structures.266 In PISA, most examples of PISA of kinetically frozen structures were obtained in water using an aqueous emulsion polymerization process.267 To assess the kinetic stability and dye retention of our fluorescent NPs in water, variation of particle average diameter and absorption properties of POEGA27-b-PBzA172 NPs were investigated using DLS and UV-Vis spectroscopy respectively. Using particles with a polymer concentration of 1.6 mg mL -1 and a dye concentration of 2.4 μg L -1 (ω(Nile red) = 0.15 wt% to copolymer), no significant change in size and absorbance occurred during more than 6 months of storage. As commented below, Nile red is a wellestablished polarity probe, and change of absorption properties would have suggested change of environment of micelle disruption. In addition, retention of size and dye was observed after 8 times-fold dilution in water. Similarly, particles diluted in NaCl (0.1 and 1 M), or subjected to a temperature stress (80 °C, 30 min) exhibited high stability (Figure4.2.7).

Figure 4 . 2 . 7 .

 427 Figure 4.2.7. Retention of NR (%) for different treatments undergone by the latex. During thermal stability treatment the system was heated up to 80 °C during 30 min and then was cooled down.

Figure 4 . 2 . 8

 428 shows that dye-labelled NPs exhibit an absorption maximum at 550 nm, the value is 40 nm red-shifted in comparison to the spectrum of Nile red in THF, but match the Nile Red spectrum in more polar methanol. For NPs, the higher extinction observed for smaller wavelength is not related absorption but to Rayleigh scattering causing an attenuation of the incident photon flux. These data suggest a more polar environment of encapsulated Nile red compared to THF. The strong resemblance with the spectrum in methanol are consistent with a preferential localization of Nile red in the outer POEGA layer despite its high hydrophobicity and the presence of less polar PbzA inner core. As apparent in Figure 4.2.8, a two-fold increase of Nile red concentration (0.29 wt% to copolymer) did not change the maximum absorption wavelength, and thus the microenvironment of the fluorophore.

Figure 4 . 2 . 8 .

 428 Figure 4.2.8. Normalized absorption spectra of Nile red in THF (thick solid line) and methanol solutions (thin solid line). Corresponding spectra of Nile Red labelled POEGA27-b-PBzA174 NP with a dye content of 0.15 wt% (dotted line) and 0.29 wt% (dashed line).

  with solvent polarity represented by Δf (orientation polarizability).270 This qualitative view indicates that NR is sensitive to solvent polarity and therefore we can mimic the polatity environment using Δf . Embedded NR in NPs reveals high polarity of the local environment. This is again in line with a preferential distribution of Nile red in the outer POEGEA shell. Despite its hydrophobic nature, hydrogen bonding interactions are likely to play an important role in NP bonding of Nile red dye. This result could explain the low saturation threshold of Nile in NP found by dye loading measurements. When localized in hydrated POEGA shell, poorly water-soluble dye molecules are more likely to diffuse out and precipitate than molecules entrapped in the apolar core. In addition, this dielectric model does not take into account the specific H-bond interaction with H2O which can also explain such an effect. The Figure4.2.10 illustrates this specific interaction even if the medium polarity is also increasing upon addition of water.

Figure 4 . 2 . 9 .

 429 Figure 4.2.9. (a) Normalized fluorescence spectra of Nile Red in four different solvents (n-hexane, THF, DCM, ACN) and encapsulated in POEGA27-b-PBzA174 NP with a dye content of 0.15 wt% (dotted line) and 0.29 wt% (dashed line). (b) Lippert-Mataga plot of Nile red in 4 pure solvents (blue dot) and POEGA27-b-PBzA172 (0.15 wt% of Nile red to copolymer). The data are fitted to a straight line (slope) 9700 (R2 > 0.92).

Figure 4 . 2 .

 42 10 shows the polarity-induced effect in the emission of Nile red for different volumes fraction of water added in THF-water mixture. The spectra position of the maximum absorption shifts from 597 nm for THF to 631 nm for a water content of 22 vol%. In comparison POEGA27b-PBzA172 NPs has an emission of 621 nm. Therefore, we have the confirmation that Nile red is preferentially localized in the POEGA shell. This interpretation is in accordance with other reports on coreshell particles were Nile red was found to be preferentially solubilized in the outer polar shell.271,272

Figure 4 . 2 . 10 .

 4210 Figure 4.2.10. (a) fluorescence emission spectra of Nile Red in THF-water mixture. (b) Nile red in THF-water mixture (blue dots) and POEGA27-b-PBzA172 (0.15 wt% to polymer, green dot).

. 2 )

 2 . The respective values of 0.50 and 0.55 were comparable to those reported free Nile red fluorophore in THF. The Φfluo values slightly decrease with dye concentration. However, it is difficult to have a general trend due to the challenge of producing a range of latexes containing different contents of dye. However, the similarity of absorption and emission measurements for the two NP samples suggest that the different of quantum yield does not originate from polarity-related effects, indicating that dye microenvironment is similar regardless of Nile red content. The main reason of decreased Φfluo is probably fluorescence self-quenching, when the density of fluorophores molecules increases within the confined space of particles. Fluorescence lifetimes were also recorded and results were compared between Nile red in solution and in NP. Similarly, to steadystate fluorescence, fluorescence life time are sensitive to solvent polarity.273 For NP specimens, the fluorescence decay of Nile red can be described by a single exponential, with a time constant (τfluo) of approx. 5 ns, indicative of single locus for the fluorophore with no partitioning between core and shell. No significant change of emission properties (intensity, wavelength) was noted over time, demonstrating the absence of spatial redistribution. τfluo values are consistent with those obtained in relatively polar solvents.

For

  POEGA27-b-PBzA172 NPs containing the lowest content in fluorophore (0.15 wt% to polymer), the theoretical brightness was   Φfluo  Ndye = 3.8 10 4  0.55  52 = 1.09  10 6 M cm -1 . The value is one order concentration of dye used for these experiments(15 nM), their high brightness has ensured a good signal to noise ratio. However, because Nile Red is quenched in water, its release from nanoparticles into an aqueous solution could not be detected.274 For bioimaging applications, assessing the non-cytotoxicity and biocompatibility of dye-labelled BC NPs is an important requirement. Starting with POEGA27-b-PBzA172 NPs including the highest content in Nile red (0.30 %w/w of copolymer), in vitro cytotoxicity experiments were performed following a MTT protocol. Two cell lines were used: C2C12 et RAW 264.7. As sown in Fig. 4.2.12d, after 6 h of incubation at two different polymer concentrations (80 and 16 µg mL -1 ), the cellular metabolic activity of the NPtreated samples was found at the same level as control samples devoid of NPs, irrespective of the cellular type used. The highest polymer concentration corresponds to a fluorophore content of 15 nM in water, that

Figure 4 . 2 . 12 .

 4212 Figure 4.2.12. Cellular uptake and cytotoxicity of POEGA-b-PBzA nanoparticles (NPs). C2C12 (a) or RAW 264.7 (b and c) cells were labelled with CellTracker (green) in order to visualize the cytosol and incubated with NPs (magenta) for 30 minutes. The cells were then observed by confocal microscopy. Z-series of optical sections were collected for each cell line. A single optical plane (xy) and the corresponding orthogonal views (xz and yz) are shown. NPs accumulate in the perinuclear regions but are not detected within in the nucleus (c). Cell viability assays (MTT) suggest that the NPs are not toxic for either C2C12 or RAW 264.7 cells (d).

(

  absorption and scattering) change over the polymerization course. These fundamental issues have not been sufficiently elaborated. Chapter III proposed a multi-techniques approach based on electron microscopy imaging and indirect physicochemical methods (mostly temperature modulated DSC) for better characterizing the presumed core-shell nanoparticle architecture of PISA-derived NPs. Chapter IV harnesses the potential of two types of PISA-derived nano-objects in emerging, but still largely unexplored fields: PHEA-b-PS in templated synthesis of mesoporous carbon materials; and POEGA-b-PBzA in bioimaging of cellular uptake.

  10.417 mmol), DDMAT (0.109 g, 0.298 mmol) and ethanol (10.000 g) were charged into a Schlenk tube equipped with a stirring bar. The mixture was degassed by three freeze-pump-thaw cycles before being placed under nitrogen. The polymerization was conducted inside a LED circular photochemical reactor at 30 °C. The photoreactor was constructed by winding a 455 nm LED strip (SMD3528, 60 LED/Meter, Lightingwill, length: 1000 mm) around a quartz cylinder (internal diameter: 60 mm, length: 200 mm). The schlenk tube was introduced in the axis of the quartz cylinder where it received an irradiance of 0.8 mW•cm - 2 . Aliquots were regularly withdrawn to determine the conversion by 1 H NMR analysis (0.1 mL dissolved in 0.5 mL of D2O). After 21 h (OEGA conversion of 61%), the polymer was recovered by precipitation into 200 mL of diethyl ether, before being dried under vacuum overnight to give ∼3.0 g of purified POEGA. The macro-CTA was then characterized by SEC in DMF (𝑀 n ̅̅̅̅ = 9.88  10 3 g mol -1 , Ð = 1.15).Synthesis of POEGA-b-PBzA diblock copolymer particles via photomediated dispersion PISA of BzA (ex. POEGA27-b-PBzA174). POEGA27-DDMAT (0.872 g, 0.067 mmol), BzA (1.721 g, 13.317 mmol) and a methanol/water mixture (7.56 g/3.24 g, 70/30 %w/w) were charged into a Schlenk tube equipped with a stirring bar and thoroughly homogenizes during 10 min. The mixture was degassed by three freeze-pumpthaw cycles, then placed under nitrogen. The tube was then exposed during 17h to the light of a LED circular photoreactor (λ = 455 nm, 0.8 mW cm -1 ), the temperature being kept at 30 °C. In the kinetic study, the mean degree of polymerization of the PBzA block was varied by adjusting irradiation time. The syntheses involving POEGA macro-CTA of different lengths were performed at the same monomer/solvent weight ratio of 3/10. Three aliquots of 0.1 mL were regularly withdrawn during reaction. The first one was used for 1 H NMR analysis (after dissolution in 0.5 mL of CDCl3) to determine BzA conversion. The second one was employed for SEC to determine polymer molecular weights. Finally, TEM characterization was carried out using the third aliquot.fully dried with a piece of filter paper, Tecnai G2 microscope (FEI) operating at 200 kV was used for the imaging of this PHEA23-b-PS130 sample.

  The weight fractions of PHEA in the shell (𝛿 PHEA 𝑠 ) and PS in the core (𝛿 PS 𝑐 ) can be expressed as follows: ωPHEA and ωPS are respectively the weight fractions of PHEA and PS blocks in PHEA85-b-PS130 copolymer. The values ωPHEA = 0.42 and ωPS = 0.59 were derived from the degrees of polymerization obtained by 1 H NMR data. 𝛥𝐶 p PHEA and 𝛥𝐶 p PS are increments of heat capacities for PHEA and PS in copolymer sample at glass transition temperatures, 𝛥𝐶 p0 PHEA and 𝛥𝐶 p0 PS are increments of heat capacities for pure PHEA and PS homopolymers. DSC traces of homopolymer shown in Figure S15 in Annex give𝛥𝐶 p0 PHEA = 0.047 J g -1 °C-2 and 𝛥𝐶 p0 PS = 0.054 J g -1 °C-2 .The weight fractions of PHEA and PS in interfacial phase,

  𝐷 p : diameter of particles determined by TEM images using ImageJ software[nm] 𝜏: mass concentration of copolymer (2) [g L-1 ]Mass concentration of copolymer calculations (𝝉) :𝑛 ∶ number of particles used in manual treating of TEM images with ImageJ software[-] Mean aggregation number calculations (𝐍 𝐚𝐠𝐠 ) -𝐶𝑇𝐴): molar concentration of macro-CTA in reaction mixture [mol L-1 ] 𝑁 a : Avogadro constant = 6.022•10 23 [mol-1 ] 𝑁 p : Number of particles per 1 liter of reaction mixture [L-1 ] Surface area stabilized by a copolymer chain calculation (𝑺 𝐚𝐠𝐠 ) : diameter of particles determined by TEM[nm]

Figure S5 .

 S5 Figure S5. STEM images of (a) PHEA85-b-PS114 and (b) PHEA25-b-PS118 diblock copolymer based nanoparticles. STEM images with RuO4 positive staining of (a) PHEA85-b-PS114 and (b) PHEA25-b-PS118 diblock copolymer based nanoparticles. STEM images with UAc negative staining of (a) PHEA85-b-PS114 and (b) PHEA25-b-PS118 diblock copolymer based nanoparticles.

Figure S6 .

 S6 Figure S6. (a) DSC traces of lyophilized PHEA85-b-PS130 diblock copolymer based nanoparticles and individual blocks. Second run (after polymer memory cleaning). (b) DSC traces of PHEA85-b-PS130 copolymer in different forms: (a) lyophilized particles, (b) latex film and (c) solution films.

Figure S7 .

 S7 Figure S7. DSC curves of different PHEA-b-PS based copolymers with different block lengths. Comparison with PHEA macrophotoinitiators.

Figure S8 .

 S8 Figure S8. (a) PHEA25-b-PS118 particles show a physical contact between the PS cores because the PHEA layer thickness provided by 25 HEA units is minimal only 3 nm. By contrast, drying of a TEM grids where PHEA85-b-PS114 (b) latex was cast yields array of disordered spherical PS cores because 85 HEA units create a more effective stabilizing layer, increasing the distances between PS cores.

Figure S9 .

 S9 Figure S9. (a) Nitrogen adsorption/desorption isotherms and (b) TEM images of carbon materials based on (b) homopolymer PHEA17-CTA and (c) PHEA85-CTA.

Figure S10 .

 S10 Figure S10. Pore size distributions by the BJH method of carbon materials synthesized based on (a) modulation of template size, (b) different template concentrations, (c) conventional soft-templates and (d) using higher order morphologies.

Figure S11 .

 S11 Figure S11. TEM images of (a) POEGA18-b-PBzA186, (b) POEGA27-b-PBzA172 and (c) POEGA35-b-PBzA180 samples. Evolution of particle size highlighted with negative staining approach.

Figure S12 .

 S12 Figure S12. SEC traces in DMF at 50 °C (refractive index detector) of PHEA85-b-PS130 and PHEA23-b-PS130 diblock copolymers.

Figure S13 .

 S13 Figure S13. Immersion-type photoreactor used in this study. (1) LED light source (472 nm). (2) Waveguide. (3) Double-walled Schlenk tube. (4) Rubber septum. (5) Stopcock.

Figure S14 .

 S14 Figure S14. Schematic representation of positive staining technique and set-up.

Figure S15 .

 S15 Figure S15. DSC traces of lyophilized PHEA85-b-PS130 diblock copolymer based nanoparticles and individual blocks. Second run (after polymer memory cleaning).

  photocatalyseurs redox absorbant dans la lumière visible tels que Ir(ppy)3 et [Ru(bpy)3]Cl2 peuvent être utilisés pour activer l'agent RAFT par un transfert d'électrons photoinduit. Dans la présente étude, une troisième approche basée sur le clivage -β réversible des composés thiocarbonyle-thiol sous irradiation a été développée (schéma ci-dessous), qui génère un radical alkyle, amorçant la polymérisation, et un radical thiyle. Une autre caractéristique distinctive de ce mécanisme est le contrôle temporel de la polymérisation.Comme la photoactivation de l'agent RAFT devient réversible, il est possible de (ré)initier ou de terminer de manière réversible les chaînes de propagation en allumant ou en éteignant la source lumineuse. Bien que cette voie rende inutile l'utilisation d'un photoamorrceur ou un photocatalyseur supplémentaire, elle est étonnamment la moins développée dans la photoPISA.

  2.1. Photoinitiated RAFT polymerizationIn a photoinitiated RAFT polymerizations, the radical PI simply replaces the radical initiator used in a thermally initiated RAFT polymerization. Radical PIs are light absorbing compounds which under UV or visible light generate one or more radicals capable of initiating polymerization. Type I PIs generate radicals via a unimolecular homolytic cleavage reaction. This type of initiation (Figure1.1) is predominantly used in photoPISA in order to create primary radicals. For this reason, this PI class deserves to be further described.

Table 1 . 1 .

 11 Summary of studies based on photoinitiated RAFT PISA processes.

	Research	PI	CTA	1
	group

st monomer 2 nd monomer T, °C Irradiation nm, mW cm -2 Morphology Year Ref.

	Cai and	SPTP	CEPA	HPMAm	DAA	25	< 400, 0.2	M	2015	46
	co-workers
		SPTP	CEPA	HPMAm	DAA	25	< 400, 0.2	CV	2016	82
		SPTP	CEPA	HPMAm	DAA	25-60	< 400, 0.2	S, W, V	2019	83

Table 1 .2. Summary

 1 of studies based on photomediated RAFT PISA.

Research group CTA 1 st monomer 2 nd monomer T, °C Irradiation: nm, mW cm -2 Morphology Year Ref.

	Boyer and	CDPA	OEGMA	BzMA	25	460-530, 0.7	S, W, V	2016	63
	co-workers
		CDPA	PEG	HPMA	37	460, 0.7	M, W, V	2018	91
	O'Reilly and	CEPA	PEG	HPMA	37	400-410, -	V	2017	54,92
	co-workers
		CEPA	Sar	HPMA	37	405, -	S, W, V	2018	93
		CEPA	PEG	PFMA	37	405, -	S	2018	94
	See

Table 1 .1 for key to abbreviations Table 1.2 summarizes

 1

	all investigations reporting photomediated RAFT PISA. In 2016, Boyer and
	co-workers reported the first RAFT-photomediated PISA to yield NPs under visible light in the absence of
	any external catalyst or initiator (Scheme 1.14) under blue or green light.

Table 1 .3. Summary on investigations relying on photocatalyzed RAFT PISA Research group PC CTA 1 st mon. 2 nd mon. T, °C Irradiation: nm, mW cm -2 Morphology Year Ref.

 1

	Boyer and	Ru(bpy)3Cl2	CPADB	OEGMA	BzMA	25	460, 0.7	S, W	2015	80
	co-workers
		ZnTPP	CDPA	OEGMA	BzMA	25	560, 3.1	S, W, V	2016	95
							635, 0.7
		Eosin Y	BTPA,	Acrylamides,	25	530, 2.65	S, W	2017	64
			CPADB	Acrylates,
				Methacrylathes
		ZnTPP	CDPA	OEGMA	BzMA	25	635, 3.0	S, W, V	2017	65
		ZnTPP	CDPA	OEGMA	MTEMA	25	635, 1.7-3.0	S, W, V	2017	96
		ZnTMPyP	CDPA	PEG	HPMA	25	365, 10.2	S, W, V	2018	97
							595, 10.2
		Eosin Y/	BTPA	DMAA	DMAA,	25	530, <1	S, W, V	2019	49
		Triethanol amine			DAA
	For abbreviations, refer

to Table 1.1 Table 1.3 summarizes

  all investigations resorting to photocatalyzed RAFT PISA (also referred to as

Table 2 . 2 .

 22 Colloidal data of PHEA52-b-PSm and PHEA85-b-PSm particles formed by dispersion photo-PISA.

	Time	m (PS degree of	Dp	PDI	Np	Nagg	Sagg
	h	polymerization)	nm		L -1		nm 2
			PHEA52-b-PSm (35 °C)
	47	48	20.3 ± 0.4	1.03	1.90⋅10 19	244	5.3
	54	52	21.0 ± 0.4	1.03	1.78⋅10 19	260	5.3
	70	110	24.0 ± 0.4	1.03	1.80⋅10 19	257	7.0
			PHEA85-b-PSm (35 °C)
	47	78	21.0 ± 0.4	1.06	2.76⋅10 19	168	8.3
	54	92	20.0 ± 0.4	1.06	3.45⋅10 19	134	9.4
	70	114	20.5 ± 0.5	1.08	3.57⋅10 19	130	10.2

Dp -Diameter of particles determined by TEM. Np-Number of particles per liter of the reaction mixture. PDI -Dispersity index. 119 Nagg -Number of RAFT chain-ends per particle. Sagg -Area occupied by 1 RAFT chain-end function on the surface of the particles.

  ). The presence of this second minor population suggests a beginning of order-order morphological transition induced by a decrease of PHEA stabilizer DP from 85 to 23. Like previous reports, 55 a sphere-to-vesicle transitions can result from the decrease of the effective volume fraction of the hydrophilic block, that hence increases the packing parameter, P, for the copolymer chains. We checked that a new decrease of stabilizing block DP from 23 to 17 resulted in full transition to vesicle morphology This conclusively supports our hypothesis that a PHEA DP of 23 is probably close to sphere/vesicle phase boundary.

	Aggregation state. It is noteworthy that only the latex exhibiting the shortest PHEA block (image 2b)

Table 3 .

 3

	1.1. Comparison between particle size data determined by TEM and SEM
	Microscopic method	PHEA85-b-PS130	Dp, nm	PHEA23-b-PS130
	TEM	19.7 ± 3.0		36.4 ± 4.8
	TEM with RuO4 positive staining	31.1 ± 3.8		41.8 ± 7.2
	STEM	20.2 ± 1.7		34.8 ± 2.6
	STEM with RuO4 positive staining	28.1 ± 2.2		42.6 ± 4.0

Table 3 .2.2.

 3 Summary data extracted from temperature differential of the heat capacity (dCp/dT) as a function of temperature curves for PHEA85-b-PS130 latex film

Table 4 .

 4

	Template	Run	r	𝑫 𝐓𝐄𝐌 𝒍 nm	𝑺 𝐁𝐄𝐓 m 2 g -1	𝑽 𝐓 cm 3 g -1	𝑽 𝐦𝐢𝐜𝐫𝐨 cm 3 g -1	𝑽 𝐦𝐞𝐬𝐨 cm 3 g -1	𝑫 𝐁𝐉𝐇 𝐩 nm	𝒑 𝑫 𝐓𝐄𝐌 nm
	Effect of PHEA and PS block length
	PHEA25-b-PS118*	a	1	31.6	691	1.30	0.29	1.01	22	22
	PHEA25-b-PS160	b	1	42.1	702	1.58	0.34	1.24	22	29
	PHEA52-b-PS110	c	1	24.0	498	0.41	0.19	0.22	25	19
	PHEA85-b-PS114	d	1	20.5	369	0.28	0.14	0.14	22	16
	Variation of template concentration
	PHEA28-b-PS134	e	0.8	32.1	714	1.25	0.31	0.94	22	20
	PHEA28-b-PS134	f	0.6	32.1	717	1.14	0.31	0.83	22	22
	PHEA28-b-PS134	g	0.4	32.1	687	1.02	0.29	0.73	22	23
	PHEA28-b-PS134	h	0.2	32.1	640	0.73	0.26	0.47	22	23
	𝑟 = Vmicro and Vmeso -Total pore volume, micropore volume and mesopore volume, respectively. 𝐷 BJH n(𝑐𝑜𝑝𝑜𝑙𝑦𝑚𝑒𝑟) . 𝐷 TEM 𝑙 -Average diameter of latex nanoparticles (TEM data). SBET -BET surface area determined by the BET method. VT, n(copolymer) 𝑟𝑢𝑛 𝑎 p -Mesopore diameter calculated by the BJH method from the isotherm adsorption branch. 𝐷 TEM 𝑝 -Mesopore diameter (TEM data).

1.1. Textural and structural properties of carbon materials synthesized using different copolymer template structure and concentration.

Table 4 .

 4

	Template	Run	𝑫 𝐓𝐄𝐌 𝒍 nm	𝑺 𝐁𝐄𝐓 m 2 g

1.2. Textural and structural properties of carbon materials synthesized using different templates.

-1 𝑽 𝐓 cm 3 g -1 𝑽 𝐦𝐢𝐜𝐫𝐨 cm 3 g -1 𝑽 𝐦𝐞𝐬𝐨 cm 3 g -1

								𝑫 𝐁𝐉𝐇 𝐩 nm	𝒑 𝑫 𝐓𝐄𝐌 nm
	PHEA25-b-PS118 (sphere)	a	31.6	691	1.30	0.29	1.01	22	22
	PS latex	i	47	594	2.90	0.28	2.60	40	38
	Silica particles	j	23	952	1.94	0.38	1.56	23	20
	PHEA17-b-PS136 (vesicle)	k	30-170	683	1.02	0.28	0.74	28	-

Table 4 .2.1.

 4 Colloidal data of POEGAn-b-PBzAm particles formed by dispersion photo-PISA with variation of solvophilic/solvophobic ratio. Dp -Diameter of NP determined by negative stained TEM. Np -Number of polymer NP per liter of dispersion. PDI -Dispersity index. 119 Nagg -Average number of diblock copolymer chains per NP. Sagg -Area occupied by a single diblock copolymer chain on the surface of the NP.

	Diblock copolymer NP	Dp	PDI	Np	Nagg	Sagg
		nm		L -1		nm 2
		Effect of PBzA block length
	POEGA27-b-PBzA88	23.4 ± 3.3	1.03	2.02 × 10 19	155	11.1
	POEGA27-b-PBzA160	28.7 ± 4.6	1.06	1.57 × 10 19	199	13.0
	POEGA27-b-PBzA172	31.7 ± 4.7	1.04	1.22 × 10 19	257	12.3
		Effect of POEGA block length
	POEGA18-b-PBzA186	50.6 ± 4.6	1.01	2.86 × 10 18	1096	7.3
	POEGA27-b-PBzA172	31.7 ± 4.7	1.04	1.22 × 10 19	257	12.3
	POEGA35-b-PBzA180	13.3 ± 2.0	1.04	1.89 × 10 20	16	33.5

Table 4 . 2

 42 absorption maximum of Nile Red encapsulated in POEGA-b-PBzA copolymer based nanoparticles, λfluoemission maximum of Nile Red encapsulated in POEGA-b-PBzA copolymer based NP, Φfluoquantum yield, τfluolife-time, Nnumber of Nile Red molecules per nanoparticle, Bptheoretical brightness

	System	λabs	λfluo	Φfluo	τfluo	kr × 10 8	Ndye/particle	Bp × 106
		nm	nm		ns	s-1		M -1 cm -1
	Solution
	n-hexane	490	525	0.47	-	-	-	-
	THF	529	597	0.62	4.3	1.14	-	-
	Dichloromethane	539	604	0.92	4.4	2.07	-	-
	Acetonitrile	527	617	0.75	4.6	1.63	-	-
	Dispersion
	C(Nile red) = 0.15	550	617	0.55	4.81	1.14	52	1.09
	wt%
	C(Nile red) = 0.29	551	622	0.50	4.44	1.12	100	1.91
	wt%
	λabs -

.2. Comparison between fluorescence data of Nile red in solution and in dispersion (POEGA27-b-PBzA172)

Table S1

 S1 Textural and structural properties of carbon materials synthesized using PHEA homopolymer with differents lenghts

	Template	Run	𝐷 TEM 𝑙 m	n	𝑆 BET m 2 g -1	𝑉 T cm 3 g -1

Acknowledgments

This PhD work was carried in the "Functional Materials Department" of the Mulhouse Science

Scheme 1.7. Schematic illustration of responsive diblock copolymer NPs synthesized via photoPISA. 83 Using the same PI (SPTP), Tan and co-workers expanded upon this approach by employing violet LED light (λ = 405 nm) to polymerize HPMA from PEG based macro-CTA agent (CEPA as RAFT agent). 62 Under dispersion conditions, this approach led to a diverse set of morphologies with S, W and V by varying the target DP of HPMA and the total solids content (Scheme 1.8). Scheme 1.8. Preparation of various nano-objects via aqueous dispersion photoPISA. 62 In another study of this group, two POEGMA macro-CTAs (RAFT agent = CDPA) were chain extended via photoinitiated RAFT dispersion polymerization of HPMA in the presence of SPTP PI under violet LED light (λ = 405 nm) in water. 84 Quantitative monomer conversions (> 99%) were achieved within 30 min irradiation, while SEC analysis indicated well-controlled polymerizations. Two phase diagrams were constructed by using POEGMA14-CDPA and POEGMA25-CDPA as macro-CTAs, which can be used as a roadmap for the reproducible synthesis of POEGMA-b-PHPMA nano-objects. The morphology of the final product was very sensitive to DP, monomer concentration, and molecular weight of the macro-RAFT agent.

POEGMA14-b-PHPMA200 prepared at 15 wt% existed as a physical gel at 25 °C, and upon decreasing temperature to 4 °C a degelation occurred.

Chapter III:

Characterizing the coreshell morphology of PISAderived particles Part I adapted from: V. Tkachenko, L. Vidal, L. Josien, M. Schmutz, J. Poly and A. Chemtob, Polymers, 2020, 12, 1656, https://doi.org/10.3390/polym12081656 Part II adapted from: V. Tkachenko, L. Josien, G. Schrodj, S. Hajjar-Garreau, J. Poly and A. Chemtob, Colloid Polym Sci, 2020, 298, 1095, https://doi.org/10.1007/s00396-020-04676-7

Chapter IV: Leveraging PISA-derived particles to develop functional materials Part I adapted from: V. Tkachenko, C. Matei Ghimbeu, C. Vaulot, L. Josien, L. Vidal, J. Poly and A. Chemtob, Langmuir, 2019, 35, 16324-16334. of magnitude smaller than that found in another study describing Nile-red encapsulated PS nanoparticles. 260 In this example, the number of dyes per particle was higher through an encapsulation in the apolar core. As expected, increasing the dye content (0.29 wt% to polymer) enables to produce brighter particles (Bp = 1.91  10 6 M cm -1 ) through a higher number of dye molecules per particle. To study photostability, Nile red solution in THF and Nile-red encapsulated NRs were exposed to the high photon flux of a fluorescent confocal microscope (Irradiance = 76  10 3 W cm -2 , λ = 485 nm). Compared with the sample in solution, confinement inside the NPs conferred much better photostability to the fluorophore due less exposure to atmospheric oxygen. Regardless of dye content, NPs exhibited a similar photobleaching half-time of approx. 125 s, a value 10 times higher than for the control experiment in solution.

Cellular interactions

Internalization of NPs into cells was investigated by CFM. C2C12 cell line was stained with CellTracker Green CMFDA to visualize the entire cells, before subsequent incubation with POEGA27-b-PBzA172 NPs (polymer concentration: 80 µg mL -1 , fluorophore content: 0.30 %w/w of copolymer). Within 30 min, approximately all cells incubated with NPs were fluorescent, consistently with a rapid uptake of encapsulated Nile Red (Fig. 4.2.12a). Intracellular fluorescence was observed throughout the entire cytosol, with a higher density in perinuclear regions. To exclude the possibility that rapid uptake could be cell linespecific, macrophages (RAW 264.7 cells) were also incubated with nanoparticles and analysed by FCM.

The uptake of Nile red in C2C12 cells after 30 min of incubation was very similar to that in RAW 264.7 cells (Fig. 4.2.12b). In both instances, there was no Nile red signal observed inside the nuclei, suggesting that the particles integrity was maintained after internalization (Fig.

4.2.12c). Despite the relatively low

Characterizations Size exclusion chromatography (SEC-THF).

Accurate molecular weights of macro-CTAs were determined by SEC in THF at 35 °C. Samples with precise concentrations around 1-3 mg mL -1 were prepared and filtered (PTFE membrane; 0.20 μm) before injection. The flow rate was 1.0 mL min -1 . The following Agilent 1260 Infinity series setup was used: a G1310B isocratic pump; a G1322A degasser; a G1329B auto-sampler; a G1316A thermostated column compartment equipped with a set of Polymer Laboratories ResiPore columns (nominal particle size: 3 μm; porosity: 2 μm) composed of a guard column (50 × 7.5 mm) and two columns (300 × 7.5 mm); a G1314B variable wavelength detector; a G7800A multidetector suite equipped with a MDS refractive index detector and a MDS viscometer detector.

Universal calibration was performed using a set of EasiVial polystyrene (PS-M) standards. Agilent GPC/SEC software and multi-detector upgrade were used to determine molar mass values and distributions.

Size exclusion chromatography (SEC-DMF).

Molecular weights of PHEA-PS copolymers were determined by SEC in DMF with additive of 0.01 M LiBr at 50 °C. For sample preparation, an aliquot was precipitated in 2 mL of diethyl ether under intense stirring, then the copolymer collected after filtration was dried and dissolved in DMF. Solutions of samples with precise concentrations around 1-3 mg mL -1 were then prepared and filtered (PTFE membrane; 0.20 μm) before injection. The flow rate was 0.9 mL min -1 .

The following Agilent 1260 Infinity series setup was used: a G1311A isocratic pump; a G1322A degasser; a G1313A auto-sampler; a G1316A thermostated column compartment equipped with a set of Polymer Laboratories ResiPore columns (nominal particle size: 3 μm; porosity: 2 μm) composed of a guard column (50 × 7.5 mm) and two columns (300 × 7.5 mm); a G1314B variable wavelength detector; a G7800A multidetector suite equipped with a MDS refractive index detector. Relative calibration was performed using a set of EasiVial poly(methyl methacrylate) (PM) standards. Agilent GPC/SEC software and multi-detector upgrade were used to determine molar mass values and distributions. where n is the number of particles) was determined by using 200 particles. "Polydispersity index" (PDI) (or "dispersity" as recommended by IUPAC) was used to describe the breadth of particle size. PDI is defined as 𝐷 w /𝐷 n , where 𝐷 w = ∑ 𝐷 n 4 / ∑ 𝐷 n 3 . The TEM grid underwent three types of specimen preparations before analysis.

Nuclear magnetic resonance (NMR).

For dried samples, latex samples were diluted 200 times with a methanol/water mixture (to get conc.

= 0.1 wt%), cast onto the TEM grid, and dried overnight at room temperature.

For positive staining TEM, RuO4 vapours was produced in situ, by reacting 0.5 mL of a 13 wt% aqueous solution of sodium hypochlorite with 150 mg of RuCl3•3H2O in a 10-cm diameter petri dish. TEM grid with disposed sample (preparing of the grid according to the same procedure as for dried samples/conventional TEM) was placed close to the reaction mixture and a second petri dish of similar size was positioned above to form a closed chamber. The grid was left for 10 min. A scheme of the experimental set-up is available in Figure S14 in Annex. 153 For negative staining TEM, a rapid flushing method was implemented. The protocol was originally developed by Imai et al., 278 and more recently adapted by Scarff et al. 279 The idea of the method is to minimize the time the sample has to interact with the support of the grid surface before fixation. The goal is to hinder structural changes in the specimen that could occur upon prolonged absorption time on the carbon film or through capillary action. Before sample application, the TEM grid was faced upon a microscope slide, then irradiated in a glow discharge unit (UV/Ozone ProCleaner Plus) for a minimum of 300 s to render it hydrophilic. In a typical procedure, 70 µL of uranyl acetate (1 wt% solution in water) was drawn up into the tip of a 200 µL pipette. 10 µl of the air gap were subsequently drawn up, then a 20 µL of deionized water (acting as wash/mixing agent) followed by another air gap of 10 µL, and finally 10 µL of sample solution (1 wt%). The edge of the grid was gripped with a pair of negative pressure tweezers, holding the tweezers so that the grid is angled at approximately 45° facing away from the researcher. The entire content of the pipette tip was ejected across the face of the TEM grid. The excess of stain was removed by touching the torn edge of a piece of filter paper to the edge of the grid. The grid was left dried over air. Due to difficulties to efficiently adsorb PHEA23-b-PS130 based nanoparticles with the latter protocol, preparation conditions were changed: a 5µl drop of ethanol was applied onto 400 mesh Cu grids covered with a plain carbon film. After 1 min interaction, the excess was removed and a 5 µL drop of latex was applied. After 1 min, the excess was removed by touching the torn edge of a piece of filter paper to the edge of the grid, and immediately a 5 µL drop of 2 wt% uranyl acetate aqueous solution was added. After 1 min, the grid was

The weight fraction of the interface can be expressed as follows:

Thermo-gravimetric analysis (TGA). was used to study the mass loss during phenolic resin/template thermal treatment by heating with 2 °C min -1 up to 600 °C under nitrogen (Mettler-Toledo TGA 851e).

X-ray photoelectron spectroscopy (XPS). XPS experiments were performed in situ (P < 10 -9 mbar) with a VG Scienta SES 200-2 spectrometer using a monochromatic Al-Ka X-ray source (hv = 1468.6 eV).

The Spectra were measured at normal incidence. The depth analysis is about 7 nm. The high resolution spectra and wide scan were collected with pass energy of 100 eV and 500 eV respectively. The deconvolution of the spectra into different components was performed with Gaussian (70%)-Lorentzian (30%) shaped peak using casaXPS software (version 2.3.18) after having subtracted a Shirley-type background. The atomic percent is determined using integrated peak areas of each component and taking into account the transmission factor of the spectrometer, the mean free path, and the sensibility factor of each atom. Determination of fluorophore contents in the particles. The fluorophore loading (%w/w) of the particles was determined by UV-Vis spectrophotometry. After the encapsulation procedure (see previous sub-section), the NP sample was diluted 10 times with THF. This results in disruption of NP and dissolution of copolymer and NR so that Beer-Lamber Law can be applied to determine Nile red concentration in solution using a calibration curve. Dye loading is expressed as the ratio of Nile red molecules encapsulated to the initial amount of NR used for encapsulation.

Lyophilized latex preparation. Lyophilization is meant to preserve, as much as possible, the original core-shell morphology of the wet nanoparticles.

Where 𝐷 w : weight-average diameter [nm]

𝐷 n : number-average diameter [nm] Weight-average diameter calculations (𝐷 w ) :

Where Results are presented below.

Brightness calculations.

The brightness of each particle can be expressed as Bp = Φf × N × ε, where Φf is fluorescence quantum yield, Nnumber of Nile Red molecules per particle, εmolar absorption coefficient (for Nile Red ε = 38000 mol L -1 cm -1 ). N was calculated from the expression as follows: 𝑁 = 𝐶(𝑁𝑅) ×𝑁 agg 𝐶(𝑐𝑜𝑝𝑜𝑙𝑦𝑚𝑒𝑟)

, where 𝐶(𝑁𝑅) is concentration of Nile Red encapsulated in NPs, 𝑁 agg is the number of copolymer chains per particle and 𝐶(𝑐𝑜𝑝𝑜𝑙𝑦𝑚𝑒𝑟) is the concentration of copolymer as determined by gravimetry. 𝑀 n ̅̅̅̅

Additional figures