Martin Fauquignon
email: martin.fauquignon@enscbp.fr

Elise Courtecuisse
email: elise.courtecuisse@enscbp.fr

Romane Josselin
email: romane.josselin@enscbp.fr

Angela Mutschler
email: angela.mutschler@enscbp.fr

Annie Brûlet
email: annie.brulet@cea.fr

Marc Schmutz
email: marc.schmutz@ics-cnrs.unistra.fr

Jean-François Le Meins
email: lemeins@enscbp.fr

Large Hybrid Polymer/Lipid Unilamellar Vesicle (LHUV) at the nanoscale: An insight into the lipid distribution in the membrane and permeability control

Keywords:

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Synthetic vesicles are the subject of an increasing number of studies because of their interest in numerous applications fields. Among them, drug targeting and delivery, biosensor and nanoreactor development (artificial organelle) are particularly considered. Liposomes obtained from the self-assembly of phospholipids have been thoroughly investigated since their discovery in the sixties, because of their biocompatible and biofunctional character. As a drug delivery system, it has been evidenced that liposomes could provide improved therapeutic efficacy and reduced systemic toxicity. But, even though some drug delivery systems based on liposomal suspensions have been approved by the health authorities (Myocet®, Daunoxome®, Doxil®, Onivyde TM …), their use is yet relatively limited despite years of research due to their inherent membrane fragility and high membrane permeability. In the last few decades, polymersomes obtained by self-assembly of block copolymers appeared as kind of analogue of liposomes but with higher functional viability and robustness. [1][2][3][START_REF] Cotton | X-Ray and Light Scattering[END_REF] More recently, hybrid polymer/lipid vesicles appeared to be a promising alternative of their forerunners as they could combine synergistically the advantages of each component. [5][6][7] So far, many studies have sought to highlight the interest of these hybrid polymer/lipid vesicles for different pharmaceutical, biological or biochemical applications like nano bioreactor, [8][9][10][11][12][13][14][15] drug delivery and targeting system, [16][17][18][19][20][21][22] or molecular recognition. 23 A relatively few number of studies have focused on the self-assembly, membrane structure and properties relationship of these hybrid systems. 24,[25][26][27] Evidences of the hybrid nature of these vesicles at nanoscale are rare, and the membrane structure most often remains unknown. Dynamic Light Scattering (DLS), Differential Scanning Calorimetry and Cryogenic Transmission Electron Microscopy (Cryo-TEM) were used to evaluate the hybrid character of LHUV. 19,20,24 Dual-color Fluorescence Cross-Correlation Spectroscopy was also used to prove efficiently hybrid character of Large Hybrid Unilamellar vesicles 28 but information about the membrane morphology are almost inexistent. Recent studies of our group have shown that LHUV presenting lipid domains could be obtained. 29,30 Generally, there is a lack of information about the molecular and process parameters leading to LHUV presenting a homogeneous mixture of components or, in contrast, a lateral phase separation in the membrane. This is mainly due to a lack of systematic approach in literature where polymers used could present different chemical composition, molar mass or architecture from a study to another. It is however important, in order to perfectly exploit the potential of such systems, to increases our knowledge about the relationship between molecular parameters of the constituting blocks of LHUV and their membrane structuration and properties. As these systems are highly regarded for applications such as controlled drug delivery or development of nanoreactors, it is essential to control and rationalize their membrane permeability.

For now, different systems have been analyzed and it has been shown that LHUV composed of poly(butadiene)-b-poly(ethylene oxide) (PBD22-b-PEO14) and 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC) at a molar fraction of 50% show permeability to calcein acetomethoxylester between those of pure liposomes and polymersomes. 31 Similar observations have already been made on the same system at different lipid molar fraction (25, 50 and 75%) during carboxyfluorescein release experiments. 19 In hybrid vesicle obtained from poly(lactic acid)-b-poly(ethylene oxide) (PLA54-b-PEO45), and POPC, the presence of copolymer tends to decrease the release kinetic of carboxyfluorescein compared to pure POPC vesicles. This effect increases with the molar content of the copolymer. 16 All these results correspond to what is « expected » for such mixture. However, it is interesting to note that in other studies it has been reported that the permeability of hybrid membrane towards H + /OH seems to be different, as LHUVs composed of PBD37-b-PEO22 and DOPC, tend to be even more permeable than pure liposomes. 13 Experiments have been performed also on hybrid films based on dipalmitoylphosphatidylcholine (DPPC) and PBD-b-PEO: the release of hydrophobic drug, Paclitaxel, has been shown to be faster with hybrid films than with pure components films. 32 Globally, it is difficult to extract general tendencies from these results, as physicochemical properties of the molecules considered, are very different from one study to another. Moreover, the membrane structure of the hybrid system considered is most often unknown.

In this work, we pursue a systematic investigation that consists in extracting molecular parameters that modulate the membrane structure of hybrid polymer/lipid vesicles. In previous studies, we have demonstrated that the association of poly(ethylene oxide)-bpoly(dimethylsiloxane)-b-poly(ethylene oxide) (PEO-b-PDMS-b-PEO) triblock copolymer associated with DPPC in gel or fluid state led to the formation of LHUV that could present lipid nanodomains. This was evidenced by SANS, cryo-TEM and time-resolved FRET. 29 Here, we present a complete study of the association of PDMS-b-PEO diblock copolymer and a phospholipid (POPC) in a fluid state into LHUV. Their hybrid character and the membrane structure have been studied by DLS, SANS and cryo-TEM. Membrane thicknesses of the polymersomes obtained from those diblock copolymers were similar to those obtained with triblock. Therefore, with such an approach, we hope to decipher the effect of the copolymer architecture on the membrane structure of the corresponding LHUV. In addition, we have evaluated through fluorescein release experiments the influence of lipid content and copolymer molar mass on the membrane permeability.

Materials

POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), partially deuterated POPC-d31 (1palmitoyl-d31-2-oleoyl-sn-glycero-3-phosphocholine) and DOPE-Rhod (1,2-dioleoyl-snglycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)) were obtained from Avanti Polar Lipids Inc (Alabaster, AL, Canada). Fluorescein was purchased from Aldrich.

The different diblock copolymers PDMS-b-PEO and the NBD-labelled PDMS were synthesized and characterized according to protocol described in a previous study 33 . The molecular characteristics are summarized in Table 1.

Compound

Abbrev. Table 1. Molecular characteristics of copolymers and phospholipids used (copolymers names used in previous studies 26,34 and abbreviations used in this article). a Molar mass determined by 1 H NMR. b Membrane thickness determined by SANS in ref 29,33 .

Molar mass

Methods

Preparation of LUV and LHUV

The LUVs and LHUVs were prepared by film rehydration process followed by extrusion through a polycarbonate membrane. Briefly, copolymer/phospholipid mixture was prepared in chloroform at a desired ratio. (15% w/w or 25% w/w of lipid). These ratio were chosen in analogy with previous systematic study 29 with the idea that a majority in polymer would be more interesting in term of stability for potential application in different fields such as drug delivery or nano reactors. This solution was then vacuum-dried until complete solvent evaporation and re-suspended in an adequate aqueous solution at room temperature (at 2 mg.mL -1 for Cryo-TEM characterization, 1 mg.mL -1 for light scattering studies, 10 mg.mL -1 for SANS studies and permeability measurements). For SANS, mixtures of D2O/H2O were used to control the contrast variation while for all other measurements, phosphate buffer saline (PBS) solution with an osmolarity of 300 mOsm.L -1 and pH = 7.4 was used. Afterwards, the LHUVs were obtained by extrusion (21 times) of this suspension through polycarbonate filters with pore sizes of 100 nm at room temperature. Details about DLS, CryoTEM, SANS measurements and fitting methodology are available in Electronic Supporting Information.

Dynamic light scattering

Characterisation of self-assembled nanostructures by dynamic light scattering (DLS) were performed at 20°C using a Malvern Zetasizer Nano ZS90, emitting vertically polarized light at λ= 632.8 nm and signal was detected at 90°. The vesicles present a quite narrow size distribution and the data could be treated with the 2 nd order cumulant analysis with a relatively r low PDI Index (<0.2). The hydrodynamic radius (RH) was determined from the apparent diffusion coefficient (D) using the Stokes-Einstein relation 𝐷𝐷 = 𝑘𝑘 B 𝑇𝑇 (6 π𝜂𝜂 S 𝑅𝑅 H ) ⁄ , with kB the Boltzmann constant and 𝜂𝜂 S the viscosity of the solvent.

Cryo-TEM

Cryo-TEM preparations and observations: The vitrification of the samples was carried out in a homemade vitrification system. The chamber was held at 22°C and the relative humidity at 80%. A 5 µL drop of the sample was deposited onto a lacey carbon film covered grid (Ted Pella) rendered hydrophilic using an ELMO glow discharge unit (Cordouan Technologies). The grid is automatically blotted to form a thin film which is plunged in liquid ethane hold at -190°C by liquid nitrogen. In that way, a vitrified film is obtained in which the native structure of the vesicles is preserved. The grid was mounted onto a cryo holder (Gatan 626) and observed under low dose conditions in a Tecnai G2 microscope (FEI) at 200 kV. Images were acquired using an Eagle slow scan CCD camera (FEI).

Small Angle Neutron Scattering measurements:

SANS experiments were carried out on PACE and PAXY spectrometers at Laboratoire Léon Brillouin (CEA Saclay, France). Different configurations were used to reach a scattering vector range q from 0.002 to 0.15 Å -1 .

Pure lipid or copolymer vesicles were prepared in D20 at a concentration of 10mg.ml -1 . For the hybrid vesicles, the contrast variation technique already used in a previous study 29,30 was performed. Details about the methodology are available in supporting information.

Permeability measurements

For permeability measurements, the LHUVs were rehydrated in 100 mM fluorescein solution in PBS. For drug loading and release experiments, separation of fluorescein-loaded LHUVs and non-loaded fluorescein molecules was performed by gel filtration column (Sephadex gel, G100) using PBS as eluant in iso-osmolar condition to prevent swelling or shrinking of the vesicles. 35 Aliquots were collected and presence of LHUVs was followed by DLS at 90°. Aliquots with the higher scattering intensity in DLS (higher concentration in vesicles) were combined to obtain pure fluorescein-loaded LHUV.

The encapsulation efficiency, loading content and permeability were quantified by UV-vis spectroscopy. The results obtained for each sample are the average of three experiments.

In order to measure the fluorescein release over time, an USP 4 apparatus (Sotax CE7 smart with CP 7 piston pump) was used. This device is connected to an UV-vis spectrophotometer (Specord 200 plus, analytikjena). Flow through cells (22,6 mm diameter) were used in a closed system. The system was temperature controlled at 25 °C. Briefly, 1 mL of LHUV suspension was placed in a dialysis bag, which define the volume of the sample (Vsample) (Float-A-Lyzer, 100 kDa cut-off) and deposited inside a flow through cell. 80 mL of PBS were used as release medium at a flow rate of 16 mL/min. Absorbance of the release medium at 488 nm (corresponding to the maximum absorbance of fluorescein) was measured continuously during 24 h. The concentration of the released fluorescein was quantified by using the Beer-Lambert law and the calibration curve realized with this spectrophotometer.

The apparent permeability of the membranes was calculated as following, assuming that the diffusion of Fluorescein through vesicle membrane is reversible and rate limiting. 36 :

𝑃𝑃 𝑎𝑎𝑎𝑎𝑎𝑎 = 𝑑𝑑𝑑𝑑 𝑑𝑑𝑑𝑑 � × 𝑉𝑉 𝑠𝑠𝑎𝑎𝑠𝑠𝑎𝑎𝑠𝑠𝑠𝑠 × [𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝑑𝑑𝐹𝐹𝐹𝐹𝐹𝐹] 𝑎𝑎𝑎𝑎𝑎𝑎𝑠𝑠𝑎𝑎 × 𝑅𝑅 3 × ∆𝐶𝐶 × [𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝑑𝑑𝐹𝐹𝐹𝐹𝐹𝐹] 𝑠𝑠𝑒𝑒𝑒𝑒𝑎𝑎𝑎𝑎𝑠𝑠𝑒𝑒𝑠𝑠𝑎𝑎𝑒𝑒𝑠𝑠𝑎𝑎 × 𝑉𝑉 𝑟𝑟𝑠𝑠𝑠𝑠𝑠𝑠𝑎𝑎𝑠𝑠𝑠𝑠 𝑠𝑠𝑠𝑠𝑎𝑎𝑚𝑚𝑒𝑒𝑠𝑠
With 𝑑𝑑𝑑𝑑 𝑑𝑑𝑑𝑑 � the variation with time of the fluorescein concentration outside the vesicles, 𝑉𝑉 𝐹𝐹𝑠𝑠𝑠𝑠𝑠𝑠𝐹𝐹𝐹𝐹 the sample volume before dialysis (1 mL), [𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝑑𝑑𝐹𝐹𝐹𝐹𝐹𝐹] 𝑠𝑠𝑑𝑑𝑑𝑑𝐹𝐹𝑑𝑑 the fluorescein concentration of the solution used to hydrate films (100mM) , ∆C the fluorescein concentration gradient inside and outside the membrane, [𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝑑𝑑𝐹𝐹𝐹𝐹𝐹𝐹] 𝑠𝑠𝑒𝑒𝑒𝑒𝑎𝑎𝑎𝑎𝑠𝑠𝑒𝑒𝑠𝑠𝑎𝑎𝑒𝑒𝑠𝑠𝑎𝑎 the fluorescein concentration determined at the end of the release study. The complete release of fluorescein was checked by disrupting the vesicle with addition of Triton X-100. 𝑉𝑉 𝑟𝑟𝑠𝑠𝑠𝑠𝑠𝑠𝑎𝑎𝑠𝑠𝑠𝑠 𝑠𝑠𝑠𝑠𝑎𝑎𝑚𝑚𝑒𝑒𝑠𝑠 is the total volume of the release medium (80 mL). dc dt � was estimated in the linear part of the release curves.

The encapsulation efficiency (EE) represents the percentage of fluorescein that is successfully entrapped into the LHUV:

𝐸𝐸𝐸𝐸 % = 𝑠𝑠 𝑟𝑟𝑠𝑠𝑠𝑠𝑠𝑠𝑎𝑎𝑠𝑠𝑠𝑠 𝑠𝑠 𝑒𝑒𝑡𝑡𝑒𝑒 𝑓𝑓𝑠𝑠𝑒𝑒𝑡𝑡 × 100
With 𝑠𝑠 𝑟𝑟𝑠𝑠𝑠𝑠𝑠𝑠𝑎𝑎𝑠𝑠𝑠𝑠 the mass of fluorescein encapsulated in vesicles, determined via the concentration of fluorescein after release experiment in the release medium, and the volume of the release medium, and 𝑠𝑠 𝑒𝑒𝑡𝑡𝑒𝑒 𝑓𝑓𝑠𝑠𝑒𝑒𝑡𝑡 the mass of fluorescein contained in the hydration solution.

The loading content (LC) corresponds to the ratio of the mass of encapsulated fluorescein to the copolymer (or copolymer and lipid) mass:

𝐿𝐿𝐶𝐶 % = 𝑠𝑠 𝑟𝑟𝑠𝑠𝑠𝑠𝑠𝑠𝑎𝑎𝑠𝑠𝑠𝑠 100µ𝑠𝑠 𝑠𝑠 𝑒𝑒𝑡𝑡𝑎𝑎𝑡𝑡𝑠𝑠𝑐𝑐𝑠𝑠𝑠𝑠𝑟𝑟 × 100
𝑠𝑠 𝑒𝑒𝑡𝑡𝑎𝑎𝑡𝑡𝑠𝑠𝑐𝑐𝑠𝑠𝑠𝑠𝑟𝑟 represents the mass of copolymer. It is determined by gravimetry, drying 100 µL of the purified vesicle suspension. Therefore, 𝑠𝑠 𝑒𝑒𝑡𝑡𝑎𝑎𝑡𝑡𝑠𝑠𝑐𝑐𝑠𝑠𝑠𝑠𝑟𝑟 was obtained as:

𝑠𝑠 𝑒𝑒𝑡𝑡𝑎𝑎𝑡𝑡𝑠𝑠𝑐𝑐𝑠𝑠𝑠𝑠𝑟𝑟 = 𝑠𝑠 𝑒𝑒𝑡𝑡𝑒𝑒 -𝑠𝑠 𝑓𝑓𝑠𝑠𝑒𝑒𝑡𝑡𝑟𝑟𝑠𝑠𝑠𝑠𝑒𝑒𝑠𝑠𝑚𝑚𝑒𝑒 -𝑠𝑠 𝑃𝑃𝑃𝑃𝑃𝑃
With 𝑠𝑠 𝑒𝑒𝑡𝑡𝑒𝑒 the total mass obtained by gravimetry, 𝑠𝑠 𝑃𝑃𝑃𝑃𝑃𝑃 the mass of buffer solution alone, 𝑠𝑠 𝑓𝑓𝑠𝑠𝑒𝑒𝑡𝑡𝑟𝑟𝑠𝑠𝑠𝑠𝑒𝑒𝑠𝑠𝑚𝑚𝑒𝑒 the estimated mass of fluorescein encapsulated in the volume considered for gravimetry.

Results and discussion

Stability of vesicles

The vesicles obtained by film rehydration and extrusion process were analysed by DLS, after the preparation and after 500 days storage at 4°C, in order to estimate the stability of the obtained structures. Globally, the hydrodynamic diameters measured by DLS are in agreement with the size of the polycarbonate filters used for extrusion (100 nm) and present narrow size distribution as illustrated by the low polydispersity index (PDI) values (Supporting Information, Table S1). After 500 days, as illustrated in the Figure 1, the size and size distribution are almost unchanged, excepted for pure POPC vesicles where the size doubles and PDI increases from 0.08 to 0.3. This result illustrates the excellent stability of the polymer/lipid vesicles obtained compared to pure POPC vesicles, with an increase of their size and size distribution that could likely result from the fusion or aggregation of these vesicles over time.

Morphology characterization

To get more information about the morphology of the obtained nanostructures, Cryo-TEM experiments were performed with all block copolymers referenced in Table 1, in association with POPC at 15% w/w content. Cryo-TEM has been performed on the pure polymersomes in a previous study. 33 For the system with the lower molar mass copolymer Si23EO13 /POPC, two main morphologies are observed as shown in Figure 2. The main population is constituted of vesicles with a membrane thickness of 8.3 ± 1.1 nm (Figure 2-B). This thickness value is slightly below the one of pure polymersomes (8.6 ± 1.1 nm). Therefore, this population may correspond to pure polymersomes or hybrid vesicles in which lipid is homogeneously dispersed in the polymer membrane. The second but minor population consists in vesicles with a membrane thickness of 5.5 nm, for which a bilayer structure is visible, typical of phospholipid vesicles (Figure 2-C). Interestingly, for the system with a higher molar mass copolymer Si27EO17/POPC, only vesicles with a thick uniform membrane are observed (Figure 3). The average membrane thickness is 10.2 ± 1.0 nm, which is close to the one reported for pure polymersomes (10.0 ± 1.0 nm). The absence of vesicle with a bilayer membrane excludes the presence of liposomes and therefore suggests an effective mixture between the copolymer and the lipid. The absence of lipid domains, characterized by a thinner membrane, suggests that the lipid is homogeneously distributed in the membrane. Finally, the system with the highest molar mass copolymer Si36EO23/POPC, presents mostly vesicular structures (Figure 4-A andB). Among the present vesicles, mainly homogeneous membranes are observed (Figure 4-A, B and C). A small fraction of vesicles has a bilayer membrane (Figure 4-A andD). Tubular elongated structures are also observed (Figure 4-B).

The thickness of both types of membrane was measured on a few objects. For solid membranes, a thickness of 11.9 ± 1.5 nm is measured, slightly less than that obtained for Si36EO13 polymersomes (13.1 ± 1.5 nm). The bilayer membranes have a thickness of 6.0 nm, close to that of liposomes. To conclude on this part, the formation of vesicles from a mixture of diblock PDMS-b-PEO copolymer and POPC was confirmed by Cryo-TEM. Vesicular structures were observed for all three systems using 15% w/w POPC. A small population of elongated tubular structures were also observed, with the highest molar mass copolymer (Si36EO13). It seems that these morphologies are frequently observed when phospholipids and copolymers of high molar mass are associated to generate LHUV, 24,29 The mechanism of their formation is still unknown so far. The vesicles prepared from Si23EO13 and Si36EO23 copolymers present two types of membranes: mostly homogeneous membranes, but also a small fraction of membranes with a bilayer organization, which could be attributed to the formation of liposomes. Vesicles with thick membranes can be hybrid vesicles with a homogeneous membrane or pure polymersomes. However, given the initial lipid/polymer composition and the small number of lipid vesicles observed, it is likely that a large number of these vesicles are hybrid vesicles. In addition, for vesicles prepared from Si36EO23/POPC, the average membrane thickness is thinner than the one of pure polymersomes, which may be an indication of their hybrid character. It has to be noted that a decrease in membrane thickness has already been observed for PDMS-b-PMOXA vesicles upon addition of a small amount of cholesterol, resulting in a modification of packing density of the molecules in the membranes. 28

Membrane homogeneity in LHUV observed by SANS

To get more information about the hybrid character of the vesicles obtained and their membrane structure, we used SANS with a contrast variation technique to selectively detect the signal of either the lipid, the polymer phase or all together. We studied the hybrid vesicles at two different compositions, 15 and 25% w/w of POPC.

All the curves obtained in full contrast condition could be fitted with a vesicle form factor, as expected from the Cryo-TEM results (SI, Figure S1). The elongated structures observed occasionally in Cryo-TEM for the Si36EO13/POPC were not detected by SANS, attesting their weak proportion. Sizes deduced from the fit to the vesicle form factor are in good agreement with DLS and Cryo-TEM results (Table S2), attesting of the vesicular morphologies.

To prove the hybrid character, we have studied the vesicle suspension in lipid contrast and polymer contrast conditions following a method described in a previous study 29 and summarized in the SI. In both conditions, the SANS curves obtained could be again fitted with a simple vesicle form factor (Figure 5). In our previous study on triblock copolymers, in polymer contrast, data could only be fitted with a perforated vesicle form factor in the case of low mass triblock copolymers, while in lipid contrast the data were fitted reasonably well with a disk form factor. 29 This was due to the presence of lipid nanodomains in the vesicle. Here, we were able to fit the data by a simple vesicle form factor, which allows to exclude a lateral phase separation in the membrane. One can suppose that we could have pure polymersomes with some pure liposomes, but this hypothesis is unlikely: first by observing the membranes of all the vesicles by Cryo-TEM and then looking at the fitted parameters which will be detailed now. Regarding the fitting methodology, scattered intensity in lipid contrast condition was relatively weak. Several parameters set (volume fraction/ radius/ membrane thickness and dispersity) could be used to fit the data as reported in Table S3 (SI) and led to the same fit quality. Therefore, the Kratky-Porod plot (Ln(Iq²) vs. q²) was used to have an average value of the membrane thickness. For a lamella of thickness δ, the intermediate q Guinier approximation of the scattering intensity is defined as:

𝐼𝐼(𝑞𝑞) = 𝐼𝐼(0) 𝑄𝑄 2 𝐹𝐹𝑒𝑒𝑠𝑠 �- 𝑞𝑞 2 𝑅𝑅 𝑔𝑔 2 1 �
where

𝑅𝑅 𝑔𝑔 2 = δ 2 12
Plots are available in SI, Figure S2. Values of membrane thickness thus obtained are indicated in Table 2. They are in between 5-7 nm close to the one obtained for pure lipid membrane (4.7 nm), whatever the lipid fraction used. These values of membrane thickness were therefore set as a fixed parameter in the vesicle form factor fit. Volume fraction, radii and distribution were then determined by the fit. The values obtained are indicated in SI, Table S4-S6. The thickness determined in polymer contrast condition and in lipid contrast condition corresponds respectively to the membrane thickness of the polymer phase and of the lipid phase. The evolution of the membrane thickness with lipid fraction is illustrated in Figure 6. When observing the polymer phase, the membrane thickness decreases with POPC fraction, as it was observed in Cryo-TEM: this suggests interactions between polymer chains and phospholipids and consequently homogeneous distribution of lipid in the polymer membrane. The higher the molar mass of the copolymer, the greater is the contraction of the chains in the membrane. When observing the POPC phase, in the case of Si23EO23 and Si27EO17, larger thicknesses between 6 and 6.8 nm are obtained. Again, this result is a proof of strong interactions and mixing between lipid and polymer. However, for Si36EO23, the membrane thickness value 4.8 nm is relatively close to the one obtained for pure liposomes (4.7 nm) and could suggest fission in the sample (polymersomes and liposomes), but in this case, the thickness of the polymer phase should also remain unchanged, which is not observed. Globally the SANS data in the different contrast conditions are in agreement with the Cryo-TEM observations. Vesicular morphology is obtained independently of the molar mass of the copolymer used. The evolution of the membrane thickness in polymer and lipid contrast conditions suggests interactions between polymer and lipid phase, and therefore a hybrid character. In addition, the fact that data could be fitted with simple vesicle form factor indicates the formation of vesicle with homogeneous distribution of components in the membrane.

It is worth noting that a simple change in architecture of the copolymer, diblock or triblock able to form vesicles with comparable membrane thicknesses, leads to different membrane structures. Whereas lipid nanodomains have been evidenced in LHUV formulated with PEOb-PDMS-b-PEO triblock copolymer and phospholipid in a fluid state 29 , the use of diblock copolymers of same chemical nature leads to LHUV homogeneous membrane structure.

Permeability measurements

In order to evaluate the membrane permeability of these vesicles, fluorescein was encapsulated inside the different LHUVs, and their permeability have been estimated through the measurement of the amount of fluorescein released from vesicle over time. Encapsulation efficiency and loading content have been also estimated.

The evolution of the amount of fluorescein released has been first studied for all pure liposomes and polymersomes (Figure 7-A). The raw data representing the absorbance over time are available in SI, Figure S4. The values of permeability as well as the loading content and encapsulation efficiency are illustrated in Table 3. The low encapsulation efficiency values obtained are inherent to the film hydration, which is a passive encapsulation process. The inner vesicular volume is very small compared to the entire volume of the sample. Concerning the loading content, the obtained values, around 3 to 5%, are relatively classical for such encapsulation procedure. 37,38 As expected, the permeability of pure lipid vesicles is higher than pure copolymer vesicles, except for the lowest molar mass copolymer (Si23EO13). This latter presents a permeability value considerably higher, despite a higher membrane thickness (6.9 nm instead of 4.7 nm for pure POPC liposomes). It has been recognized in literature that the permeability of the polymersomes is modulated by their membrane thickness and that it is much lower than the one of liposomes. 2,36,[39][40][START_REF] Cevc | Phospholipids handbbok[END_REF] However, the variety of block copolymer used to study their membrane permeability is far more restricted than the variety of phospholipids used. It has to be noted that quantitative studies of vesicles permeability are scarce because of technical difficulties that are nicely summarized in a recent study of Castiglione and col. [START_REF] Poschenrieder | [END_REF] Quantitative value of permeability for simple solute (water, protons) can be found so far only for membrane constituted of hydrophobic blocks such as polybutadiene, 39,43 polybutylene oxide 36 or polycaprolactone. 40 Permeability to various molecules has also been estimated in polymersomes made of triblock PMOXA-b-PDMS-b-PMOXA copolymers. [START_REF] Poschenrieder | [END_REF] Therefore this result for Si23EO13 shows that care has to be taken before considering polymersomes far more impermeable than liposomes as « universal », chemical nature of the copolymer as well as its architecture is of importance.

Concerning the hybrid systems, the release profiles are represented in Figure 7-B, C and D. The release profiles of pure liposome and polymersome are represented as reference. The values of the permeability as well as the loading content and encapsulation efficiency are illustrated in Table 3.

In the case of hybrid vesicles constituted with the lowest molar mass copolymer (Si23EO13), the permeability seems to be intermediate between those of pure liposomes and those of pure polymersomes. In that case, the permeability of polymersomes Si23EO13 which is higher than POPC liposomes, decreases when lipid is added. However, for polymersomes with higher membrane thicknesses (Si27EO17 and Si36EO23) that present permeabilities a bit lower than pure liposomes, the permeability does not seem to be affected in a significant way.

This very weak influence of lipid content on the permeability of the polymersomes may be in relation with the absolute differences in terms of permeabilities between pure POPC vesicles and SixEOy polymersomes which were surprisingly low for Si23 and Si27. Therefore even is the membrane Is modified as illustrated by the membrane thicknesses evolution revealed by SANS, when lipids are incorporated, this may not facilitate a precise measurement of intermediate permeability between pure polymersomes and liposomes. However our measurements clearly show that a permeability greater than the pure liposomes, which has already been obtained in literature 13,32 is definitely to discard. Such a greater permeability may be related to the presence of heterogeneities (raftlike domain) in the membrane, but this has not been quantified yet.

Conclusion

In this work, Large Hybrid Unilamellar Vesicles (LHUV) were obtained from the association of PDMS-b-PEO diblock copolymers with different molar masses and a phospholipid (POPC) in a fluid state. In all cases, LHUVs were obtained in the polymer/ lipid composition (up to 25% w/w in lipid) investigated with homogeneous distribution of lipid in the polymer membrane as revealed by Cryo-TEM and SANS experiments. This work reveals the importance of polymer chains conformation in the membrane of hybrid vesicles. Indeed, for triblock copolymer PEOb-PDMS-b-PEO, studied in a previous work, that presents according to a work of Itel et al. 44 , a mixture of hairpin and extended chain conformation in the membrane, the lipid distribution is not homogenous and presence of lipid nanodomains in the vesicle has been revealed. 29 The diblock copolymers used in this study form membrane with a conformation closer from the lipid bilayer conformation, which help the lipid to disperse homogeneously in the membrane. This leads to LHUVs with a permeability intermediate between those of liposomes and polymersomes. Future work could consist in a fine analysis of membrane structure of LHUV for which higher permeability than pure liposomes has been observed 13 ,and establish a correlation with the eventual presence of domain, as it was suggested in hybrid polymer/lipid planar films 32 .

1 Large hybrid polymer/lipid unilamellar vesicle (LHUV) at the nanoscale: An insight into the lipid distribution in the membrane and permeability control.

Time after formation

S2.2. Small angle neutron scattering

SANS experiments were carried out on PACE and PAXY spectrometers at Laboratoire Léon Brillouin (CEA Saclay, France). Different configurations were used to reach a scattering vector range q from 0.002 to 0.15 Å -1 .

Pure lipid or copolymer vesicles were prepared in D20 at a concentration of 10mg.ml -1 . For the hybrid vesicles, the contrast variation technique already used in a previous study 2,3 was performed. A specific solvent mixture can match the scattering length density (SLD) of each component and thus hide its scattering contribution. To have access to the polymer signal, vesicles were prepared in D20/H20, 49/51, vol:vol (SLD = 2.7 10 10 cm -2 ) (named polymer contrast condition) at a concentration in polymer of 10 mg.ml -1 . To measure the lipid signal, vesicles were prepared in D20/H20, 9/91, vol:vol (SLD = 0.07 10 10 cm -2 ) (lipid contrast) at a concentration in lipid of 10 mg.ml -1 . Finally, to detect globally the polymer and POPC-d31, samples were prepared in pure D2O (SLD = 6.38 10 10 cm -2 ) (full contrast) at 10 mg.ml -1 .

The samples were put in quartz Hellma © cells with 1 or 2mm path lengths and measured at room temperature.

The raw spectra were corrected from the empty cell and other sources by conventional procedures 4,i using LAMP program ii in order to obtain the SANS curves in absolute units (cm -1 ). In lipid contrast condition, the vesicle form factor could also be used but different set of parameters fit similarly the data, as indicated in Table S3:

Table S3: Illustration of the difficulty to fit data in lipid contrast condition with the vesicle form factor. *: SLD fixed. Different sets of parameters could equally fit the data of Si27EO17 25% POPC.

The membrane thickness obtained with vesicle form factor fit being relatively variable, we therefore used the approximation of large vesicle measured in the intermediate q range for the vesicle radius and the Guinier range for the membrane thickness in order to obtain an average value of membrane thickness using Krakty-Porod representations shown in Figure S2. The slope of the linear fit in the intermediate q range equals δ 2

12 , where δ is the average membrane thickness.

Fit n°1 Fit n°2 Fit n°3

Background (cm -1 ) 1.015 S1: Parameters used to fit the data for LHUV of Si23EO13 with the vesicle form factor for different conditions. a : Volume fraction calculated from Eq. S3. *: SLD fixed. **: value deduced from the linear fit in the Kratky-Porod representation of SANS (Fig. S7) set fixed for the fit.

S.2.4. Fluorescein encapsulation and release

The quantification of the permeability has been made through measurement of UV absorbance of fluorescein released through the vesicle membrane. A calibration curve has been first performed with fluorescein in PBS 1X (Figure S3).

Figure 1 :

 1 Figure 1: Size distribution (bottom-X; left-Y axis) and autocorrelation function (top-X; right-Y axis) obtained by DLS at 90°, after vesicle formation (solid line) and after 500 days at 4°C (dotted lines).

Figure 2 :

 2 Figure 2: Cryo-TEM pictures of the 85/15 w/w Si23EO13/POPC sample. A-Representative picture of the population observed. Vesicle indicated with a white arrow corresponds to vesicle presenting a bilayer. B-Enlargement of vesicle presenting membrane thickness of 8.3 nm. C-Vesicle with bilayer signature. White scale bars represent 100 nm.

Figure 3 :

 3 Figure 3: CryoTEM pictures of the 85/15 w/w Si27EO17/POPC sample. A, B-Representative pictures of the population observed. C-Enlargement showing the characteristic thick membrane of the vesicles. White scale bars represent 100 nm.

Figure 4 :

 4 Figure 4: Cryo-TEM pictures of the 85/15 w/w Si36EO23/POPC sample. A-Characteristic image obtained for this sample. The presence of vesicles with a bilayer membrane is indicated by white arrows. B-Picture showing the coexistence of the vesicle with tubular structures (white arrows). C-Characteristic image of the majority of vesicles in the sample, characterized by a thick membrane. D-Characteristic image of a vesicle in the sample characterized by a bilayer membrane. White scale bars correspond to 100 nm.

Figure 5 :

 5 Figure 5: SANS curves in polymer contrast and lipid contrast conditions, for LHUVs composed of Si23EO13, Si27EO17 and Si36EO23 and POPC. The data are adjusted by the vesicle form factor (colored lines). The intensity is shifted by the factor indicated in the figure caption for visibility.

Figure 6 :

 6 Figure 6: Representation of the membrane thicknesses of copolymers and POPC as a function of POPC fractions. The data are adjusted by linear regression (colored lines).

Figure 7 :

 7 Figure 7: Fluorescein release experiments. A-Release profiles from vesicles composed of pure POPC (grey line), Si23EO13 (red line), Si27EO17 (blue line) and Si36EO23 (green line). B-Release profiles from vesicles composed of pure POPC (grey line), Si23EO13 (red line), Si23EO13/POPC 85/15 w/w (orange line) and Si23EO13/POPC 75/25 w/w (yellow line). C-Release profiles from vesicles composed of pure POPC (grey line), Si27EO17 (darker blue line), Si27EO17/POPC 85/15 w/w (blue line) and Si27EO17/POPC 85/15 w/w (lighter blue line). D-Release profiles from vesicles composed of pure POPC (grey line), Si36EO23 (darker green line), Si36EO23/POPC 85/15 w/w (green line) and Si36EO23/POPC 85/15 w/w (lighter green line).

Figure S3 :

 S3 Figure S3: Calibration curve of UV absorbance of fluorescein in phosphate buffer saline (PBS) 1X at 25°C.

Figure S4 :

 S4 Figure S4: Evolution of UV absorbance due to fluorescein release from pure liposomes and polymersomes.

Figure S5 :

 S5 Figure S5: Evolution of the concentration of fluorescein during release experiments for pure liposomes, pure polymersomes and hybrid vesicles.

Table 2 :

 2 Membrane thickness obtained by the Krakty-Porod fit in lipid contrast condition.

	Sample	Lipid fraction (%w/w-%mol/mol)	Lipid phase thickness (nm) (Kratky-Porod)
		15-36.7	6.8
	Si23EO13
		25-52.3	6.0
		15-40.2	6.4
	Si27EO17
		25-56	6.6
	Si36EO23	25-63.7	4.8

Table 3 :

 3 Permeability, encapsulation efficiency and loading content obtained for pure liposomes, pure polymersomes and hybrid vesicles.

	Sample	Permeability (10 -9 nm.s -1 )	Encapsulation efficiency (%)	Loading content (%)
	POPC	1.88 ± 0.17	0.32	4.86
	Si23EO13	2.74 ± 0.20	0.15	4.44
	Si27EO17	1.20 ± 0.11	0.17	4.97
	Si36EO23	1.22 ± 0.14	0.12	2.96
	Si23EO13 + 15% POPC	2.18 ± 0.07	0.11	4.24
	Si23EO13 + 25% POPC	2.37 ± 0.10	0.09	4.85
	Si27EO17 + 15% POPC	1.04 ± 0.03	0.11	2.14
	Si27EO17 + 25% POPC	0.94 ± 0.05	0.08	3.17
	Si36EO23 + 15% POPC	1.18 ± 0.03	0.07	1.18
	Si36EO23 + 25% POPC	1.37 ± 0.12	0.12	3.73

Table S1 :

 S1 Hydrodynamic diameter (Dh) and polydispersity index (PDI) of hybrid vesicles after formation t0 and 500 days after formation.

			Dh (nm)	PDI	Scattered intensity (a.u.)
		t0	122	0,08	27189
	POPC
		+ 500 days	337	0,30	1107
	Si23EO13	t0	97	0,09	20707
	+ 15% POPC	+ 500 days	129	0,07	21416
	Si23EO13	t0	120	0,05	11759
	+ 25% POPC	+ 500 days	130	0,11	23775
	Si27EO17	t0	100	0,10	13250
	+ 15% POPC	+ 500 days	113	0,06	12003
	Si27EO17	t0	130	0,06	12982
	+ 25% POPC	+ 500days	136	0,08	27047
	Si36EO23	t0	120	0,16	13704
	+ 25% POPC	+ 500 days	160	0,12	22120

Table S2 :

 S2 Fit parameters to the vesicle form factor obtained for LHUVs in full contrast condition.

	Copolymer	Si23EO13	Si23EO13	Si27EO17	Si27EO17	Si36EO23
	POPC mass fraction	15%	25%	15%	25%	25%	POPC
	Background (cm -1 )	0.068	0.057	0.068	0.068	0.065	0.065
	SLD (x10 -6 Å -2 )	0.471	0.745	0.471	0.745	0.745	2.800
	SLD solvent (x10 -6 Å -2 )	6.360	6.360	6.360	6.360	6.360	6.360
	Volume Fraction	0.0060	0.0071	0.0062	0.0066	0.0053	0.0039
	(Fit)
	Volume Fraction (gravimetry)	0.0055	0.0060	0.0053	0.0055	0.0046	0.0019
	Radius (nm)	39.8	38.7	40.2	41.5	51.2	38.5
	Radius dispersity
	(log-normal	0.250	0.250	0.250	0.250	0.250	0.250
	distribution)
	Thickness (nm)	6.7	6.3	7.4	7.3	8.2	4.7
	Thickness
	dispersity (log-normal	0.106	0.146	0.145	0.132	0.145	0.150
	distribution)

Table S4 :

 S4 Parameters used to fit the data for LHUV of Si36EO23 with the vesicle form factor for different contrast conditions. a : Volume fraction calculated from Eq. S3 after gravimetry. *: SLD fixed. **: value deduced from the linear fit in the Kratky-Porod representation of SANS curve (Fig.S7) set fixed for the fit.

						Si36EO23 Si27EO17
		Full	15% POPC Polymer	Lipid	25% POPC Full	25% POPC Polymer	Lipid
	SLD (x10 -6 Å -2 ) * contrast SLD solvent (x10 -6 Å -2 ) * Volume fraction Radius (nm) Radius dispersity (log-normal distribution) Background Background 0.068 (cm -1 ) (cm -1 ) SLD SLD 0.471 (x10 -6 Å -2 )* (x10 -6 Å -2 )* SLD solvent (x10 -6 Å -2 )* SLD solvent (x10 -6 Å -2 )* 6.360	Full contrast 0.0008 39.3 0.250 contrast contrast 0.065 0.665 1.025 0.745 0.060 2.800 6.360 2.800 0.060	Polymer contrast 2.800 0.060 0.0009 38.3 0.250 contrast 0.705 0.068 0.060 0.745 2.800 6.360	Lipid contrast 0.0011 39.8 0.250 contrast contrast 0.667 1.015 1.017 0.060 2.800 2.800 0.060 2.800 0.060
	Thickness (nm) Volume fraction 0.0062		0.0049		9.6 0.0012	0.0066	7.7	0.0052	6.3 0.0011
	Volume fraction Thickness dispersity (log-normal distribution) Theoretical volumefraction a Theoretical volume fraction a 0.0053	0.0058	0.0053 0.0046 0.0012 0.022	0.0036 0.063 0.0052 0.0055	0.0062	0.0019 0.029 0.0010 0.0010
	Radius (nm)	40.2		39.3		39.9	41.5		36.9	40.5
	Radius (nm) Radius dispersity			51.2		44.3		41.6
	(lognormal distribution) distribution) Radius dispersity (log-normal 0.250		0.250	0.250	0.250	0.250 0.250		0.250	0.250	0.250
	Thickness (nm)	7.4		7.5		6.4 **	7.3		7.1	6.6 **
	Thickness (nm) Thickness			8.2		7.9		4.8 **
	Thickness dispersity distribution) (lognormal distribution) dispersity (log-normal 0.145		0.113	0.145	0.15	0.027 0.132		0.137	0.150	0.119

Figure S2: Kratky-Porod representation of the SANS data for different LHUV samples in lipid contrast condition; from top to bottom Si23EO13 + 15% POPC (red), Si23EO13 + 25% POPC (orange), Si27EO17 + 15% POPC (blue), Si27EO17 + 25% POPC (light blue) et Si36EO23 + 25% POPC (green). Data are fitted by linear regression (dotted line).The slope of the linear equation equals δ 2

12 , where δ is the average membrane thickness of LHUV.

Table S5 :

 S5 Parameters used to fit the data for LHUV of Si27EO17 with the vesicle form factor for different contrast conditions. a : Volume fraction calculated from Eq. S3 *: SLD fixed. **: value deduced from the linear fit in the Kratky-Porod representation of SANS curve (Fig.S7) set fixed for the fit.

				Si23EO13
			15% POPC			25% POPC
		Full	Polymer	Lipid	Full	Polymer	Lipid
		contrast	contrast	contrast	contrast	contrast	contrast
	Background (cm -1 )	0.068	0.675	1.019	0.057	0.675	1.021
	SLD (x10 -6 Å -2 )*	0.471	0.060	2.800	0.745	0.060	2.800
	SLD solvent (x10 -6 Å -2 )*	6.360	2.800	0.060	6.360	2.800	0.060
	Volume fraction	0.0060	0.0051	0.0011	0.0071	0.0052	0.0013
	Theoretical volume fraction a	0.0055	0.0068	0.0013	0.0060	0.0069	0.0013
	Radius (nm)	39.8	38.2	35.9	38.7	37.6	39.4
	Radius dispersity
	(log-normal	0.250	0.250	0.250	0.250	0.250	0.231
	distribution)
	Thickness (nm)	6.7	6.4	6.8 **	6.3	6.2	6.0 **
	Thickness
	dispersity (log-normal	0.106	0.084	0.091	0.146	0.124	0.150
	distribution)
	Table

Author manuscript of article published in Journal of Colloid and Interface Science (2021) https://doi.org/10.1016/j.jcis.2021.06.172

Martin Fauquignon1 , Elise Courtecuisse 1 , Romane Josselin 1 , Angela Mutschler 1 Annie Brûlet 2 , Marc Schmutz 3 and Jean-François Le Meins 1, *

Acknowledgements

M. Fauquignon gratefully acknowledges the Scientific Department of University of Bordeaux for a PhD fellowship.

Author manuscript of article published in Journal of Colloid and Interface Science (2021) https://doi.org/10.1016/j.jcis.2021.06.172

Author manuscript of article published in Journal of Colloid and Interface Science (2021) https://doi.org/10.1016/j.jcis.2021.06.172

Declaration of competing interest

There is no competing interest to declare

Authors contribution

Martin Fauquignon: All experiments and data analysis, manuscript review.

Elise Courtecuisse: Drug release and permeability measurements.

Romane Josselin: Drug release and permeability measurements, manuscript review.

Angela Mutschler: Drug release and permeability analysis, manuscript review.

S1. Materials

S2. Methods

S2.1. Dynamic light scattering

Hybrid vesicles were characterized by Dynamic Light Scattering (DLS) at 90° using a Malvern Zetasizer Nano ZS90 at 20°C. Results are listed in Table S1. A complete characterization of the pure polymersomes by static and dynamic light scattering is available in a previous study. 1 Different models have been used to analyse the scattering curves of the samples. Fits to the form factor of vesicle, disk or various core-shell cylinder were achieved using the SasView program ( http://www.sasview.org/). Another model of perforated vesicle developed in a previous study 2 in order to describe the scattering of phase separated polymer/lipid vesicles was also used.

Volume fraction parameter:

Being aware that extrusion process can lead to a significant mass loss, all samples were quantified again, after the SANS experiments to verify the lipid and polymer volume fraction, which is an important parameter in the fitting procedure. The total polymer/lipid quantities were determined by thermogravimetry measurements. 5 The theoretical volume fractions were determined as follows:

Cdry being the concentration determined after extrusion process via gravimetry, after drying a known volume of vesicle suspension. flipid and fpolymer are the mass fraction of lipid and polymer respectively, and fhydr-lipid, fhydr-poly are the hydrophilic fraction in lipid and polymer respectively. The volume fractions obtained by fitting the SANS curves are in fair agreement with the theoretical volume fractions (Table S2, S4, S5).


Dr Bohdan Andreiuk

Dr Ilya O Aparin

Dr Andreas Reisch

Dr Andrey S Klymchenko
email: andrey.klymchenko@unistra.fr

Bulky Barbiturates as Non-toxic Ionic Dye Insulators for Enhanced Emission in Polymeric Nanoparticles

Keywords: nanoparticles, fluorescence, dyes/pigments, bulky hydrophobic anions, aggregation

Bulky hydrophobic counterions (weakly coordinating anions) can insulate ionic dyes against aggregation-caused quenching (ACQ) and enable preparation of highly fluorescent dye-loaded nanoparticles (NPs) for bioimaging, biosensing and light harvesting. Here, we introduce a family of hydrophobic anions based on fluorinated C-acyl barbiturates with delocalized negative charge and bulky non-polar groups. Similarly to fluorinated tetraphenylborates, these barbiturates prevent ACQ of cationic dye alkyl rhodamine B inside polymer NPs made of biodegradable poly(lacticco-glycolic acid) (PLGA). Their efficiency to prevent ACQ increases for analogues with higher acidity and bulkiness. Their structure controls dye-dye communication, yielding bright NPs with on/off switching or stable emission. They enhance dye encapsulation inside NPs, allowing intracellular imaging without dye leakage. Compared to fluorinated tetraphenylborates known as cytotoxic transmembrane ion transporters, the barbiturates display a significantly lower cytotoxicity. These chemically available and versatile barbiturate derivatives are promising counterion scaffolds for preparation of bright non-toxic fluorescent nanomaterials.

Introduction

Fluorescent nanoparticles (NPs) gained importance in recent years due to their unique optical properties and numerous potential applications, including biological imaging and biosensing [1] as well as light harvesting. [1b, 2] Organic fluorescent nanoparticles are of particular interest as the highly versatile nature of organic materials and fluorescent dyes enable preparation of materials of programmed optical properties, easy functionalization with targeting ligands, antibodies, DNA, stealth groups, etc. On the other hand, bio-applications of organic nanoparticles impose strict requirements such as biocompatibility, biodegradability, low toxicity and eco-friendliness, which remains a challenge even for organic nanomaterials. Important examples of organic NPs include AIE NPs, [3] conjugated polymer NPs, [4] dye-loaded polymeric NPs [3b, 5] and lipid NPs. [6] Dyeloaded polymeric NPs are particles of choice in view of bio-applications, because they present several unique features. On the one hand, by selecting appropriate polymer matrix, NPs with desired biocompatibility, biodegradability and stability can be obtained. For example, poly(lacticco-glycolic acid) (PLGA), approved for human use by the United States Food and Drug Administration and by the European Medicine Agency, can be used to prepare biodegradable NPs, which are generally known in the field of drug delivery. [7] On the other hand, selection of the dye and its loading level allow fine tuning of optical properties of NPs, such as absorption and emission color, [8] brightness, [9] photostability, [10] etc. [5a] However, preparation of bright dye-loaded NPs requires addressing two key challenges.

First, fluorescent dyes should be efficiently encapsulated into the polymer matrix without dye leakage (leaching) in biological media. [11] Second, at high dye loading aggregation-caused quenching (ACQ) is commonly observed, which complicates preparation of bright NPs. [5a] Dye self-quenching is generally caused by the flat aromatic structure of fluorophores, which favors pistacking of dyes into so-called H-aggregates with face-to-face assembly. [12] To address the problem of ACQ, a number of approaches have been proposed, [5a] which include aggregationinduced emission, [3a, 13] the use of bulky side groups, [10,14] and bulky hydrophobic counterions. [15] Our previous works showed that bulky hydrophobic counterions are of particular interest for dyeloaded polymeric NPs, as they can address both the problems of ACQ and dye leakage from NPs for different cationic dyes. [8, 11b, 15a] These counterions are generally weakly coordinating anions, most commonly tetraphenylborate (TPB) derivatives. [16] They are characterized by large size, high hydrophobicity and strong delocalization of the negative charge over the large volume of the anion.

Weakly coordinating anions are often constituents of ionic liquids and they are commonly used for stabilization of reactive cations [17] and for preparation of ion-selective electrodes and electric batteries. [16,18] Earlier works showed that they can minimize self-quenching in pure dye salts, [19] and the most efficient were those characterized by large size and high level of fluorination. [20] As we showed later on, these counterions can form highly hydrophobic ion pairs with a cationic dye, which ensures its nearly quantitative encapsulation inside the hydrophobic polymeric matrix even at high loading during nanoparticle synthesis by nanoprecipitation. [11b, 21] Moreover, these bulky anions serve as spacers (or insulators) between encapsulated cationic dyes, thus preventing their self-quenching through pi-stacking into H-aggregates. [11b, 15a, 22] Therefore, despite very high loading of cationic dyes with these bulky hydrophobic anions (up to 50 wt% vs polymer), the obtained NPs exhibited unprecedentedly high fluorescence quantum yields (~50%). [23] In this way it was possible to obtain NPs 6-100 fold brighter than quantum dots of similar size and emission color. [9, 15a, 24] Owing to the short dye-dye distances inside these highly loaded NPs, the loaded dyes exhibited unique collective behavior due to coupling by ultrafast excitation energy transfer, [15a] which enabled preparation of a giant light-harvesting nanoantenna for single-molecule detection at ambient light. [25] Moreover, the concept of bulky counterions works with both rhodamine and cyanine dyes, which enabled preparation of differently colored ultrabright NPs and development of a cell barcoding system for in vitro and in vivo applications. [8] Such NPs have also been used for the assembly of biosensors for detection of nucleic acids with single-molecule sensitivity [23][24] and live tracking inside mouse brain. [26] However, so far, the counterion approach was mainly limited to fluorinated tetraphenylborate derivatives [8, 11b, 15a] and an aluminate of perfluorinated tert-butanol. [21] These ions can transport cations, including protons, through biological membranes, such as cell plasma membranes and mitochondria, which leads to cytotoxicity at micromolar concentrations. [27] Moreover, bulkier and more fluorinated anions were found to be more cytotoxic because of their stronger capacity to transport cations. [27] Even though dye-loaded polymeric NPs encapsulating these counterions were not cytotoxic, [8,21] questions can be raised about potential toxicity of these anions after biodegradation of the polymer matrix.

Moreover, due to the symmetric nature of these anions, it is synthetically challenging to monofunctionalize them. [28] Therefore, there is a need to look for alternative scaffolds for a hydrophobic ion. One approach, called small-molecule ionic isolation lattices (SMILES) proposed to generate bulky hydrophobic anions by complexing small inorganic anions with a large macrocycle (cyanostar), which allows to significantly decrease ACQ of cationic dyes in bulk polymeric materials [15b] and nanoparticles. [29] Another attractive approach is the design of an organic ion with high charge delocalization ("soft" ion), which would be also highly hydrophobic, similarly to weakly coordinating anions based on borates and aluminates, but without their limitations.

Classical examples of hydrophobic anions are oxonol dyes (Figure 1A), in which a single negative charge is delocalized over a long conjugation system. [30] However, oxonol dyes are colored, which would alter or quench the emission of a cationic dye in the counterion-based approach. Therefore, an oxonol analogue with a much shorter conjugation should be developed.

In the present work, we synthesized a family of organic anions based on a barbiturate scaffold and studied their capacity to prevent ACQ in dye-loaded polymeric NPs. Their weakly acidic methylene group was C-acylated in order to enable formation of the anion with delocalized negative charge after deprotonation (Figure 1,A,B), similarly to oxonols, but without absorption of light in the visible region. Our results showed that acylated barbiturates with perfluorobenzoyl group can form stable ion pairs with a cationic rhodamine dye and ensure its efficient encapsulation and excellent optical properties inside polymer NPs. This work constitutes the first demonstration of an anion with π-delocalized negative charge capable to replace conventional weakly coordinating anions in preparation of dye-loaded polymer NPs. Importantly, the obtained anions are much less toxic than fluorinated tetraphenylborate, which enables preparation of much safer nanomaterials for bioimaging applications and increases their potential for clinical translation.

Finally, a relatively simple chemistry of these anions opens the way to their further functionalization and scale up.

Results and discussion

Primarily, we synthesized barbiturates with (R) groups being cyclohexyl and R' being undecyl (1) or phenyl (2) (Figure 1B, Scheme 1). These molecules were readily obtained from the barbituric acid derivative and the corresponding carboxylic acids using EDC as coupling agent in the presence of DMAP. The obtained C-acyl barbiturates were mixed with octadecylrhodamine B (R18) perchlorate in DCM with excess of triethylamine for deprotonation of barbiturates and ion exchange. However, no stable enough ion pairs of compounds 1 and 2 with cationic dye R18 were formed to be purified by silica chromatography (Figure 1, Supporting Information). This result could be explained by insufficient electron-withdrawing effect of the used acyl groups, which made possible re-protonation of the formed enolate anion on silica, in line with relatively high calculated pKa of 6.51 and 6.04 for compounds 1 and 2, respectively. Therefore, to increase the acidity of the barbiturates, the R' group was changed to perfluorophenyl in molecule BarC6 (Figure 1B), which was obtained following the same methodology (Scheme 1). The anion of the obtained molecule could form ion pairs with the R18 cation that could be detected on silica thin layer chromatograph as a faster moving spot (Figure 1, Supporting Information), similarly to the ion pair R18/F5-TPB. We suppose that lower pKa values (estimated value 3.68) help to generate more stable ion pairs with the R18 dye. Further, we replaced the cyclohexyl (R) groups with diphenylmethyl to obtain the even bulkier perfluorophenyl analogue BarPh2 (Figure 1B, Scheme 1). The anionic form of BarPh2 also appeared to form stable ion pairs with R18 (Figure 1, Supporting Information), in line with similarly low calculated pKa values (3.98). Moreover, the BarPh2 salt migrated faster than that of BarC6, indicating a higher hydrophobicity of the former.

Moreover, in contrast to the salt of BarC6, the BarPh2 salt did not show any trace of the R18 at the start of the TLC, indicating that it is more stable on silica. Both ion pairs of novel barbiturate anions with the R18 dye, namely R18/BarC6 and R18/BarPh2, alongside with the already known R18/F5-TPB and R18/ClO4 as reference compounds, were encapsulated inside biodegradable PLGA (poly(lactide-co-glycolide)) nanoparticles (Figure 1) using a simple method based on nanoprecipitation from the watermiscible solvent acetonitrile. [15a, 31] The dyes were loaded at 5, 20 and 50 mM concentration with respect to polymer.

The size of the obtained nanoparticles was measured by TEM (Figure 2) and DLS (Figure 3A). For both new barbiturates (BarC6 and BarPh2) at 50 mM dye loading, TEM images revealed spherical shape of the obtained NPs, similarly to F5-TPB (Figure 2). The measured size values were similar for all three types of NPs in the range of 33-36 nm. By contrast, PLGA NPs loaded at 50 mM with R18 and small hydrophilic perchlorate counterion (R18/ClO4) yielded large aggregates according to our earlier TEM data. [21] DLS confirmed small size (40-50 nm) of the formulated PLGA NPs, which was independent of dye loading (Figure 3A). By contrast, ion pair R18/ClO4 with the hydrophilic perchlorate counterion induced formation of much larger (>500 nm) and colloidally unstable nanoparticles at 50 mM dye loading. The latter observation, in line with our previous reports, [11b, 15a] can be explained by cationic dye adsorption at the anionic nanoparticle surface, which leads to decrease of surface charge and further NP aggregation. Zeta potential measurements showed strong negative values for PLGA NPs with all three studied bulky counterions at 50 mM dye loading (Figure 2, Supporting Information). In contrast, the absolute value of the zeta potential was significantly lower in case of R18/ClO4, confirming neutralization of the negative surface charge of NPs by cationic R18.

To evaluate quantitatively the dye encapsulation efficiency in NPs, we used our previously developed protocoldialysis for 24h of NPs solutions versus 1 mM beta-cyclodextrin. [11b, 21] During this procedure all the non-encapsulated dyes and those adsorbed on the particle surface are washed off. Then, the comparison of dye concentration before and after dialysis gives us the encapsulation efficiency. The obtained results (Figure 3D) showed poor encapsulation efficiency of R18/ClO4 (51±2%) and almost quantitative encapsulation of R18/F5-TPB (92±4%) at 50 mM (corresponding to ~7.5% w/w) loading. For the barbiturate BarC6 anion, the dye encapsulation efficiency was 74±1%, whereas for the bulkier and more hydrophobic BarPh2 the highest dye encapsulation efficiency was achieved (96±1%). This efficient encapsulation of R18/BarPh2 can be explained by higher hydrophobicity of BarPh2 compared to its cyclohexyl-substituted rival BarC6. At 5 mM dye loading, independently of the counterion, the quantum yields (QY) of all NPs were high (> 70%, Figure 3B). However, upon increase of loading to 50 mM, perchlorate demonstrated its inability to prevent dye self-quenching (QY = 4%), in contrast to R18/F5-TPB loaded NPs, displaying a QY of 55% at this loading. Remarkably, for the bulky barbiturate-based ion pair R18/BarPh2, the QY value was almost as high (52%), suggesting that BarPh2 is as effective in preventing ACQ as the reference counterion F5-TPB. The ion pair R18/BarC6 displayed a QY of about 20% at 50 mM loading, which was 5-fold higher compared to perchlorate, but still not as high as for F5-TPB. These results show that quantum yield and encapsulation efficiency of dye-loaded NPs correlate directly with bulkiness and hydrophobicity of the barbiturates, respectively, in line with our previous data. [32] Fluorescence anisotropy measurement is one of the ways to study dye-dye communication inside NPs, asenergy transfer from dye to dye leads to loss of fluorescence anisotropy. [33] According to our results, already at 5 mM dye loading the fluorescence anisotropy values were very low (>0.01) for the F5-TPB counterion, in line with earlier data. [15a] In case of the BarPh2 counterion, the anisotropy was also relatively low at this loading (Figure 3C). It further decreased with dye loading reaching nearly zero at 50 mM for both counterions, suggesting fast excitation energy transfer in these NPs. In sharp contrast, for the BarC6 counterion, the anisotropy values were relatively high at all loadings, similarly to those for the perchlorate counterion, implying that dye-dye communication inside NPs was weak. Thus, among the two barbiturates only the bulkier and more hydrophobic one (BarPh2) favors dye-dye communication, similarly to F5-TPB.

The structure of the counterion also affected spectroscopic properties of the encapsulated R18 dye. Although at 5 mM loading the effects in the absorption spectra were minor, at 50 mM dye loading they became much more pronounced (Figure 3E). In line with previous studies, NPs loaded with R18/F5-TPB showed a blue-shifted absorption maximum compared to R18/ClO4loaded NPs. The absorption maxima of R18 with barbiturate counterions were intermediate between R18/ClO4 and R18/F5-TPB, though the BarPh2 was slightly red shifted compared to BarC6. Similar tendencies were observed in the fluorescence spectra. The red shifted absorption and emission of the BarPh2 is unexpected, given its similar performance with F5-TPB. One could speculate that, unlike the case of F5-TPB, where negative charge is mostly localized at weakly polarizable fluorine atoms, BarPh2, due to its higher electronic polarizability decreases the energy of the excited state of R18. Single-particle brightness was studied by wide-field microscopy. NPs loaded at 50 mM with R18/BarPh2, R18/BarC6 and R18/F5-TPB as well as commercial quantum dots of similar emission range, QD585, were immobilized on glass and excited by a LED source at 550 nm (Figure 4A-D). All of the tested dye-loaded NPs appeared much brighter than quantum dots QD585: R18/F5-TPB, R18/BarC6 and R18/BarPh2-loaded NPs were, respectively, 33, 29 and 28fold brighter. Surprisingly, the brightness of R18/BarC6 NPs (Figure 4C) was close to those of R18/F5-TPB and R18/BarPh2 NPs (Figure 4B, D), despite having more than two-fold lower quantum yield and lower encapsulation efficiency. This peculiar behavior of these NPs can be explained based on single-particle fluorescence intensity traces (Figure 4E-G). R18/BarPh2 NPs (Figure 4G) underwent ON/OFF fluorescence switching (blinking), similarly to R18/F5-TPB NPs (Figure 4E). Statistical analysis based on the mean of three measurements (>400 particles of each type) suggested that blinking took place for 95+/-0.5% and 78+/-2% of all recorded R18/F5-TPB and R18/BarPh2 NPs, respectively. According to earlier studies of R18/F5-TPB NPs, such behavior is only possible when the dyes are in close proximity inside the nanoparticle, making ultrafast energy transfer possible and in consequence effective quenching of the whole particle by transient dark species (such as triplet/radical states of rhodamine). [9, 15a] Thus, similarly to F5-TPB, the BarPh2 anion may favor clustering of R18 dyes inside the polymer matrix, which is in agreement with the observed low fluorescence anisotropy values (Figure 3C). By contrast, the majority (766%) of R18/BarC6 NPs particles showed constant emission without blinking (Figure 4F). This very different behavior is probably related to inefficiency of dye-dye energy transfer, as suggested by relatively high values of the fluorescence anisotropy. Thus, higher than expected relative brightness of R18/BarC6 NPs could be explained by poor dye-dye communication, which prevents quenching the encapsulated dyes by a single dark states or by singlet-singlet annihilation.

We can speculate that poor dye-dye communication for R18/BarC6 even at relatively high loading originates from rather homogeneous distribution of fluorophores within the polymer matrix of NPs, whereas bulkier and more hydrophobic R18/BarPh2 tends to cluster. In our previous work, we observed similar effects in the fluorescence anisotropy and particle blinking when bulky fluorinated TPBs were compared to the much smaller anion B(CF3)4. [11b] Together with the present data, we have now a solid background to conclude that size and hydrophobicity of the counterion is a tool to control dye clustering in the polymer matrix of NPs. We expect that larger and more hydrophobic counterions favor dye clustering, because their salts with R18 are significantly more hydrophobic than the PLGA polymer, so that during nanoprecipitation they precipitate before the polymer, forming the core with clustered dye. In contrast, R18 salts with counterions of intermediate hydrophobicity (like BarC6 and B(CF3)4) would co-precipitate together with the polymer, thus favoring more homogeneous dye distribution in the polymer with longer dye-dye distances, leading to higher fluorescence anisotropy and stable emission without blinking.

To test the obtained NPs in biological media, they were incubated with KB cells for 3 hours and then the cells were washed and imaged by confocal microscopy. All four counterions at the highest (50 mM) and lowest (5 mM) dye loading were tested (Figure 5). R18/F5-TPB NPs at 5 mM loading (Figure 5B) appeared as bright dots, being localized in the perinuclear regions, and without clear background signal from other parts of the cells. This result is in line with previous data, confirming that after endocytosis of R18/F5-TPB NPs, no significant dye leakage is observed. R18/BarPh2 and R18/BarC6 NPs at this loading showed similar behavior (Figure 5D and 4C, respectively). A significant degree of leakage was observed for R18/ClO4 NPs at 5 mM loading (Figure 5A) as a diffuse fluorescence inside the cells, so that the characteristic perinuclear dots (endosomes/lysosomes) were practically not visible. The same tendency was observed at 50 mM loading, where R18/ClO4 NPs showed strong fluorescence all over the cells, while for NPs with other ion pairs, a clear dotted fluorescence was still observed (Figure 5E-H). Nevertheless, for both R18/F5-TPB and R18/BarC6 NPs at 50 mM loading some diffuse intracellular fluorescence started to be visible (Figures 5F and 5G, respectively), indicating imperfect encapsulation of these ion pairs at the highest loading. Remarkably, for 50 mM R18/BarPh2 NPs, diffuse fluorescence was negligible (Figure 5H), suggesting absence of dye leakage in biological media. The observed cellular data correlated well with the encapsulation studies, showing the highest encapsulation efficiency for R18/BarPh2 and the lowest for R18/ClO4 (Figure 3D). Thus, the novel counterion BarPh2 outperforms our "golden standard" F5-TPB for bioimaging purposes.

In this respect, it is close to the recently reported F9-Al, a strongly fluorinated aluminium-based counterion, [21] showing superior encapsulation of the R18 dye. Finally, we compared the cytotoxicity of the counterions vs our standard F5-TPB. To this end, we prepared salts of BarC6 and BarPh2 with 1.2 eq. of triethylamine and studied them together with the F5-TPB lithium salt in HeLa cells using standard MTT assay. After 24 h incubation, the F5-TPB counterion was found to be the most cytotoxic, showing <50% of cell viability at 3 µM concentration (Figure 6). In sharp contrast, both barbiturates demonstrated virtually no cytotoxicity at this concentration. Moreover, BarPh2 showed low cytotoxicity with cell viability >50% for the studied concentration range up to 30 µM. BarC6, having lower hydrophobicity and slightly lower pKa than BarPh2, displayed intermediate cytotoxicity between F5-TPB and BarPh2, with cell viability <50% at 10 µM concentration. Thus, both barbiturates, especially BarPh2, are less cytotoxic than our standard F5-TPB, which is probably connected to the lower capacity of barbiturates to transport protons and other cations through biological membranes. We can speculate that barbiturates due to their high hydrophobicity and relatively weak acidity (3.68 and 3.98) are bound to lipid membranes in molecular form which prevents them from deprotonation. The fact that the more hydrophobic BarPh2 is much less toxic than BarC6 supports this hypothesis, because the former should have even stronger partitioning into lipids, further decreasing the probability of its deprotonation. By contrast, F5-TPB can preserve the anionic form when bound to the lipid membrane and therefore can operate as an efficient shuttle of protons and other cations.

Conclusions

In conclusion, we describe a new family of hydrophobic anions based on acylated barbiturates with strongly delocalized negative charge, which, similarly to weakly coordinating anions based on boron or aluminium, can enhance encapsulation and emission of cationic dyes inside polymer NPs. Among the studied acyl barbiturates, the bulkiest and the most hydrophobic anion prevents self-quenching of a cationic rhodamine B derivative as efficiently as the "golden standard" tetrakis(pentafluorophenyl)borate and at the same time enables more efficient dye encapsulation with minimal leakage in living cells. Moreover, the chemical structure (hydrophobicity) of the barbiturate counterion can control the dye-dye communication, as evidenced by fluorescence anisotropy and, thus, tune the emission mode of nanoparticles from complete on/off switching (blinking) to steady emission. Finally, the acyl barbiturates displayed significantly lower cytotoxicity compared to tetrakis(pentafluorophenyl)borate. Moreover, the least toxic acyl barbiturate BarPh2 was both the more hydrophobic and slightly less acidic one, which is probably related to its inability to get deprotonated and thus to transfer the proton and other cations through biological membranes. These results provide insights on molecular design of bulky hydrophobic counterions with minimized cytotoxicity. Overall, barbiturate anions, owing to these unique properties, open the route to preparation of non-toxic and eco-friendly dye-loaded polymer NPs, which can find application in biosensing, bioimaging and materials science.

Experimental Section

Detailed protocols of the synthesis and characterization of barbiturates, preparation and characterization of NPs, their studies in cells as well as some data on TLC of the ion pairs and zeta potential of NPs are provided in the Supporting Information.

Figure 1 .

 1 Figure 1. (A) Chemical structures of the oxonol dye scaffold and C-acyl barbiturates scaffolds with their resonance structures. (B-D) Schematic presentation of a fluorescent nanoparticle (D) encapsulated with ion pairs of a cationic dye R18 (C) and bulky hydrophobic counterions (B).

Scheme 1 .

 1 Scheme 1. Synthesis of target barbiturates (in blue) bearing N-cyclohexyl (A) and Ndiphenylmethyl (B) groups: (a) malonic acid, THF; (b) dodecanoic acid, EDC×HCl, DMAP, DCM; (c) benzoic acid, EDC×HCl, DMAP, DCM; (d) perfluorobenzoic acid, EDC×HCl, DMAP, DCM; (e) CDI, THF (f) malonyl chloride, DCM; (g) perfluorobenzoic acid, EDC×HCl, DMAP, DCM.

Figure 2 .

 2 Figure 2. TEM images of NPs loaded with R18 dye with bulky counterions: F5-TPB (A), BarC6 (B) and BarPh2 (C). Graphs on the right of the TEM images correspond to size distribution histograms. The provided diameters are mean values of NPs based on analysis of >200 particles per condition. The error is the half-width at the half-maximum.

Figure 3 .

 3 Figure 3. Properties of the developed NPs, loaded with ion pairs of R18 dye with different counterions. (A) Hydrodynamic diameter according to DLS, (B) fluorescence quantum yield, (C) fluorescence anisotropy and (D) encapsulation efficiency at 50 mM dye loading. (E) Absorption

Figure 4 .

 4 Figure 4. Single-particle fluorescence properties of the tested dye-loaded PLGA NPs. (A-D) Wide-field fluorescence images of QD585 (A), R18/F5-TPB (B), R18/BarC6 (C) and R18/BarPh2 (D) NPs at 50 mM dye loading. Identical imaging conditions were applied for dye-loaded NPs (excitation power density of 4.4 W/cm 2 at 550 nm), ten times higher illumination power was used for QDs. Image size was 15x15 µm. (E-G) Typical single-particle fluorescence traces of NPs loaded with R18/F5-TPB (E), R18/BarC6 (F) and R18/BarPh2 (G), recorded at the same conditions.

Figure 5 .

 5 Figure 5. Confocal images of KB cells incubated for 3h with dye-loaded PLGA NPs. NPs were loaded with R18/ClO4, R18/F5-TPB, R18/BarC6 and R18/BarPh2 at 5 and 50 mM concentrations. Excitation wavelength was 561 nm, emission was collected from 570 to 650 nm. Image size was 58.4x58.4 µm.

Figure 6 .

 6 Figure6. Viability of HeLa cells exposed for 24 h to different concentrations of the three counterion salts: F5-TPB lithium salt, BarC6 and BarPh2 salts with triethylamine (1.2 eq.). Cell viability was tested in quadruplicate and repeated at least two times using MTT. The error bars correspond to the standard deviation.

Acknowledgements

This work was supported by the European Research Council ERC Consolidator grant BrightSens 648528. B.A. was supported by LabEx Chimie des Systèmes Complexes. I.O.A. acknowledges support from Marie Curie post-doctoral research grant (H2020-MSCA-IF-2018, DNANanoProbes 846571) by the European Union.

Entry for the Table of Contents

Bulky hydrophobic anions based on fluorinated C-acyl barbiturates with delocalized negative charge are found to provide insulation of ionic dyes, thus preventing their aggregation-caused quenching in polymeric nanoparticles. Their chemical structure defines dye encapsulation, particle brightness and on/off switching. Compared to currently used anions, C-acyl barbiturates are nontoxic, while offering chemical availability and versatility.

Conflict of Interest

The authors declare no conflict of interest.