Huaiyou Chen

Ali Emre Celik

Angela Mutschler

Antoine Combes

Anne Runser

Andrey Klymchenko

Sébastien Lecommandoux
email: lecommandoux@enscbp.fr.

Christophe Serra
email: ca.serra@unistra.fr.

Andreas Reisch
email: reisch@unistra.fr.

Assembly of Fluorescent Polymer Nanoparticles using different Microfluidic Mixers

Keywords: Microfluidics, Nanoparticle synthesis, Nanoprecipitation, Size control, Fluorescent nanoparticles

Nanoprecipitation is a facile and efficient approach to the assembly of loaded polymer nanoparticles (NPs) for applications in bioimaging and targeted drug-delivery. Their successful use in clinics requires reproducible and scalable synthesis, for which microfluidics appears as an attractive technique. However, in the case of nanoprecipitation, particle formation depends strongly on mixing. Here, we compare 5 different types of microfluidic mixers with respect to the formation and properties of Poly(D-L-lactide-co-glycolide) (PLGA) and Poly(methyl methacrylate) NPs loaded with a fluorescent dye salt: a crossshaped mixer, a multilamination mixer, a split & recombine mixer, two herringbone mixers, and two impact-jet mixers. Size and fluorescence properties of the NPs obtained with these mixers are evaluated. All mixers, except the cross-shaped one, yield NPs at least as small and fluorescent as those obtained manually. Notably in the case of impact-jet mixers operated at

Introduction

Polymer nanoparticles (NPs) have attracted high interest in the biomedical field, notably as carriers for contrast agents and as systems for drug targeting and release. [START_REF] Wang | Conjugated Polymer Nanoparticles for Imaging, Cell Activity Regulation, and Therapy[END_REF][START_REF] Shi | Cancer Nanomedicine: Progress, Challenges and Opportunities[END_REF][START_REF] Pearce | Insights into Active Targeting of Nanoparticles in Drug Delivery: Advances in Clinical Studies and Design Considerations for Cancer Nanomedicine[END_REF] They offer the possibility to use biocompatible or even biodegradable materials, present a high versatility in terms of material and load properties. Their surface properties can now be readily controlled, and through this their fate in biological environments. Over the last two decades, the development of polymer NPs has achieved a level, at which scale-up and reproducibility of particle assembly starts to become a focus, in order to translate this type of materials towards the clinic and commercialization. [START_REF] Wang | Self-Assembly of Colloids Based on Microfluidics[END_REF][START_REF] Roces | Translating the Fabrication of Protein-Loaded Poly(Lactic-Co-Glycolic Acid) Nanoparticles from Bench to Scale-Independent Production Using Microfluidics[END_REF][START_REF] Ma | Microfluidic-Mediated Nano-Drug Delivery Systems: From Fundamentals to Fabrication for Advanced Therapeutic Applications[END_REF][START_REF] Hamdallah | Microfluidics for Pharmaceutical Nanoparticle Fabrication: The Truth and the Myth[END_REF] As an example, so-called fluorescent dye-loaded polymer NPs have gained increasing interest as very bright fluorescent probes. [START_REF] Reisch | Fluorescent Polymer Nanoparticles Based on Dyes: Seeking Brighter Tools for Bioimaging[END_REF][START_REF] Visaveliya | Softness Meets with Brightness: Dye-Doped Multifunctional Fluorescent Polymer Particles via Microfluidics for Labeling[END_REF][START_REF] Wan | Aggregation-Induced Emission Active Luminescent Polymeric Nanoparticles: Non-Covalent Fabrication Methodologies and Biomedical Applications[END_REF][START_REF] Robin | Strategies for Preparing Fluorescently Labelled Polymer Nanoparticles[END_REF] Thanks to different approaches for overcoming aggregation-caused quenching of the dyes inside the particles, they gave rise to fluorescent emitters several orders of magnitude brighter than fluorescent proteins, molecular dyes, or even luminescent quantum dots. These particle systems have already led to various applications, i.e. as ultrabright probes for single-particle tracking in vitro and in vivo, [START_REF] Egloff | Size-Dependent Electroporation of Dye-Loaded Polymer Nanoparticles for Efficient and Safe Intracellular Delivery[END_REF][START_REF] Khalin | Ultrabright Fluorescent Polymeric Nanoparticles with a Stealth Pluronic Shell for Live Tracking in the Mouse Brain[END_REF] cell labelling agents, [START_REF] Andreiuk | Fluorescent Polymer Nanoparticles for Cell Barcoding In Vitro and In Vivo[END_REF] as well as light-harvesting nanomaterials for signal amplification, [START_REF] Trofymchuk | Giant Light-Harvesting Nanoantenna for Single-Molecule Detection in Ambient Light[END_REF] and very sensitive probes for biomolecules. [START_REF] Egloff | Enzyme-Free Amplified Detection of Cellular MicroRNA by Light-Harvesting Fluorescent Nanoparticle Probes[END_REF][START_REF] Lone | Energy Transduction through FRET in Self-Assembled Soft Nanostructures Based on Surfactants/Polymers: Current Scenario and Prospects[END_REF] Polymer NPs can be synthesized either from monomers in various types of emulsion polymerizations or from already formed polymers. [START_REF] Rao | Polymer Nanoparticles: Preparation Techniques and Size-Control Parameters[END_REF][START_REF] Banik | Polymeric Nanoparticles: The Future of Nanomedicine[END_REF] Among the latter, nanoprecipitation is a very attractive approach for the assembly of loaded polymer NPs. [START_REF] D'addio | Controlling Drug Nanoparticle Formation by Rapid Precipitation[END_REF][START_REF] Rivas | Nanoprecipitation Process: From Encapsulation to Drug Delivery[END_REF][START_REF] Sanson | A Simple Method to Achieve High Doxorubicin Loading in Biodegradable Polymersomes[END_REF] In nanoprecipitation, both polymer and load are dissolved in a water miscible solvent and the resulting organic solution is added to an aq ue ous phase, which can or cannot contain surfactant molecules ( Scheme 1 ) . Mixing, and typically interdiffusion, of the organic and aq ue ous phase, leads to a mixture in which the polymer and the load are not soluble anymore, creating supersaturation, which is the driving force for particle formation. The fact that particle formation in this case is a k i netically controlled process allows trapping the load. This means that encapsulation ( and release) of compounds inside polymer NPs is not only controlled by their chemistry and hence their eq uilibrium partitioning between particle and dispersant phase. [START_REF] Rivas | Nanoprecipitation Process: From Encapsulation to Drug Delivery[END_REF][START_REF] Hayward | Tailored Assemblies of Block Copolymers in Solution: It Is All about the Process[END_REF] Indeed, encapsulation efficiency can also be optimiz ed by adj usting the conditions of particle formation, independently of the nature of the loaded cargo. O riginally, nanoprecipitation was mainly performed through manual addition of one of the phases to the other, typically with the aq u eous phase in large excess. [START_REF] Fessi | Nanocapsule Formation by Interfacial Polymer Deposition Following Solvent Displacement[END_REF] Tak i ng into account the fact that often relatively small amounts of valuable compounds have to be encapsulated, one possibility to better control the addition of the two phases is the use of microfluidics. [START_REF] Visaveliya | Softness Meets with Brightness: Dye-Doped Multifunctional Fluorescent Polymer Particles via Microfluidics for Labeling[END_REF][START_REF] Ding | Microfluidic Nanoprecipitation Systems for Preparing Pure Drug or Polymeric Drug Loaded Nanoparticles: An Overview[END_REF][START_REF] Liu | Formulation of Nanoparticles Using Mixing-Induced Nanoprecipitation for Drug Delivery[END_REF][START_REF] Khan | Production of Nanoparticle Drug Delivery Systems with Microfluidics Tools[END_REF][START_REF] Liu | Current Developments and Applications of Microfluidic Technology toward Clinical Translation of Nanomedicines[END_REF][START_REF] Sun | Diverse Particle Carriers Prepared by Co-Precipitation and Phase Separation: Formation and Applications[END_REF] Microfluidics should, in principle, allow automatiz ation of NP assembly with high reproducibility and further scale-up through highly parallel synthesis. However, particle formation in nanoprecipitation depends strongly on the speed of mixing of the two phases, which defines the supersaturation and the homogeneity of conditions during particle formation. In classical microfluidic systems the flow is strongly laminar due to the small dimensions of the channels (corresponding to low Reynolds number). In consequence, mixing is mainly relying on diffusion, which is a slow process resulting in a very low speed of mixing. To circumvent this problem, different types of microfluidic mixers have been developed, making use of external energy input (active mixers) or relying solely on pressure driven flow (passive mixers). [START_REF] Mansur | A State-of-the-Art Review of Mixing in Microfluidic Mixers[END_REF][START_REF] Ward | Mixing in Microfluidic Devices and Enhancement Methods[END_REF] Among the latter, three major strategies exist to increase the speed of mixing: (i) Decrease of the width of the laminar flow phases, resulting in a decrease in the diffusion distances. This principle is notably realized in hydrodynamic flow focusing, [START_REF] Karnik | Microfluidic Platform for Controlled Synthesis of Polymeric Nanoparticles[END_REF] multilaminar mixers, [START_REF] Bally | Improved Size-Tunable Preparation of Polymeric Nanoparticles by Microfluidic Nanoprecipitation[END_REF][START_REF] Yus | Microflow Nanoprecipitation of Positively Charged Gastroresistant Polymer Nanoparticles of Eudragit® RS100: A Study of Fluid Dynamics and Chemical Parameters[END_REF] but also in so-called split & (re)combine mixers. [START_REF] Bresseleers | Tuning Size and Morphology of MPEG-b-p(HPMA-Bz) Copolymer Self-Assemblies Using Microfluidics[END_REF] (ii) Introduction of transversal flow elements, leading to partially chaotic mixing, as in the case of staggered herringbone mixers. [START_REF] Stroock | Chaotic Mixer for Microchannels[END_REF][START_REF] Sanchez-Gaytan | Real-Time Monitoring of Nanoparticle Formation by FRET Imaging[END_REF] (iii) Collision of the flows, leading to a further breakdown into small fluid segments and, in some cases, to actual turbulent mixing, as in impact-jet and vortex mixers. [START_REF] Markwalter | Design of a Small-Scale Multi-Inlet Vortex Mixer for Scalable Nanoparticle Production and Application to the Encapsulation of Biologics by Inverse Flash NanoPrecipitation[END_REF][START_REF] Johnson | Chemical Processing and Micromixing in Confined Impinging Jets[END_REF][START_REF] Nagasawa | Design of a New Micromixer for Instant Mixing Based on the Collision of Micro Segments[END_REF][START_REF] Han | High-Speed Continuous Production of Polymeric Nanoparticles with Improved Stability Using a Self-Aligned Coaxial Turbulent Jet Mixer[END_REF][START_REF] Anton | A New Microfluidic Setup for Precise Control of the Polymer Nanoprecipitation Process and Lipophilic Drug Encapsulation[END_REF] Application of such mixers to the assembly of polymer NPs through nanoprecipitation has, effectively, proven to be a useful approach to reduce the size and, in several cases, the polydispersity of the resulting particles, with a great reproducibility. [START_REF] Lebleu | Self-Assembly of PEG-b-PTMC Copolymers: Micelles and Polymersomes Size Control[END_REF] However, up to now direct comparisons of the performance of these different mixers are scarce, [START_REF] Liu | Formulation of Nanoparticles Using Mixing-Induced Nanoprecipitation for Drug Delivery[END_REF][START_REF] Johnson | Chemical Processing and Micromixing in Confined Impinging Jets[END_REF][START_REF] Abdelkarim | Microchannel Geometry vs Flow Parameters for Controlling Nanoprecipitation of Polymeric Nanoparticles[END_REF][START_REF] Li | Insight into Drug Encapsulation in Polymeric Nanoparticles Using Microfluidic Nanoprecipitation[END_REF] and, in particular, to the best of our knowledge, no such comparison addresses the assembly of fluorescent dye-loaded polymer NPs through nanoprecipitation. In the present work, we therefore evaluated the fabrication of dye-loaded polymer NPs using different microfluidic mixers (Figure 1) for two polymers frequently used for the preparation of NPs: Poly(D-Llactide-co-glycolide) (PLGA) and Poly(methyl methacrylate) (PMMA) (Scheme 1). Both polymers contained small amounts of carboxylic acid groups, on the chain end for PLGA and as co-monomer (1.3 mol%) for PMMA, to better control particle size. [START_REF] Reisch | Charge-Controlled Nanoprecipitation as a Modular Approach to Ultrasmall Polymer Nanocarriers: Making Bright and Stable Nanoparticles[END_REF] These polymers were loaded with the salt of a rhodamine B derivative (R18) with the bulky hydrophobic counterion tetrakis(pentafluorophenyl)borate (F5-TPB). Besides its practical significance for reducing aggregation caused quenching (ACQ), this dye salt has the advantage that it can serve as a model compound, whose fluorescence directly reflects the organization of the load inside the NP. [START_REF] Reisch | Tailoring Fluorescence Brightness and Switching of Nanoparticles through Dye Organization in the Polymer Matrix[END_REF] We first compared the sizes of NPs obtained from these polymers either through manual nanoprecipitation or using one of seven different microfluidic mixers while varying the flow conditions over a wide range of flow speeds. For selected systems, we then evaluated in more detail the fluorescence properties.

Experimental

Materials

Poly(D-L-lactide-co-glycolide) (PLGA, acid terminated, lactide:glycolide=50:50, Mw

Methods

Preparation of NPs: Stock solutions of polymers were prepared at a concentration of 10 g.L -1 in acetonitrile. These solutions were diluted to 2 g.L -1 in acetonitrile containing 5 wt% of R18/F5-TPB (relative to the polymer), where needed. In some cases these solutions were further diluted to 1 or 0.5 g.L -1 . For manual preparation, this solution was quickly added to a 9 -fold volume excess of MilliQ water under shak i ng ( Thermomixer comfort, E ppendorf, 1 0 0 0 rpm at 2 1 °C ) . F or preparation using microfluidics, this solution was mixed in the microfluidic mixer in a ratio 1 : 9 with MilliQ water at various flow rates. The latter were chosen over the range, where a reproducible and stable flow was achieved with the corresponding mixer.

Samples were tak en after about 3 0 s of stabiliz ation at the given flow rates. The particle solutions were analyz ed directly after suitable dilution, without purification in order to better visualiz e the effects of preparation conditions. The following mixers were used for particle preparation (Figure 1

Transmission electron microscopy (TEM):

Solutions of bare dye-loaded NPs (5μL) were deposited onto carbon-coated copper-rhodium electron microscopy grids following amylamine glow-discharge. They were then treated for 20 s with a 2% uranyl acetate solution for staining. The obtained grids were observed using a Tecnai F20 Twin transmission electron microscope (FEI Eindhoven Holland) operating at a voltage of 200 kV. Images (2,048 pixels × 2,048 pixels) were recorded using a US1000 camera (Gatan). After drying, the uranyl acetate remaining on the sample provides the contrast, often negative, but in some cases also in the form of a ring or shadow around the particles. Images were analyzed using the Fiji software. At least 200 particles per condition were analyzed.

Absorption and emission spectra were recorded on a Cary 5000 Scan ultraviolet-visible spectrophotometer (Varian) and on a FS5 Spectrofluorometer (Edinburgh Instruments)

equipped with a thermostated cell compartment, respectively. For nanoparticles formed with the split and recombine mixer, absorption and emission spectra were recorded on a UV double beam spectrophotometer (Specord 200 plus, analytikjena) equipped with a D 2 E and a Halogen Lamp and a Jasco FP-Spectrofluorometer with a Xe arc lamp with a shielded lamp housing (150 W). The excitation wavelength was set to 530 nm and emission was recorded from 540 to 750 nm. QYs were determined from the absorbance values at the excitation wavelength (A x,530nm ) and the integral over the whole emission range (F x ) using a simplified relative method. , with rhodamine 101 in ethanol as reference (QY = 0.95, absorbances below 0.1))., according to the formula

𝑄𝑄𝑄𝑄 𝑁𝑁𝑁𝑁 = 𝑄𝑄𝑄𝑄 𝑟𝑟101 𝐹𝐹 𝑁𝑁𝑁𝑁 𝐴𝐴 𝑁𝑁𝑁𝑁,530𝑛𝑛𝑛𝑛 𝑛𝑛 𝐻𝐻 2 𝑂𝑂 2 𝐹𝐹 𝑟𝑟101 𝐴𝐴 𝑟𝑟101,530𝑛𝑛𝑛𝑛 𝑛𝑛 𝐸𝐸𝐸𝐸𝑂𝑂𝐻𝐻 2
Where NP corresponds to solutions of the nanoparticles in water and r101 to rhodamine 101 in ethanol and n corresponds to the respective refractive indices of the solvents (1.33 for water and 1.36 for ethanol).

Results and Discussion

Size

In a first step, PLGA and PMMA NPs were prepared "manually" by adding quickly solutions of the polymers in acetonitrile at 2 g.L -1 to a 9-fold excess of MilliQ water under shaking. This resulted in particles with sizes of 51 (±5) nm and 77 (±5) nm, respectively, for PLGA and PMMA, as determined by dynamic light scattering (DLS). DLS results showed relatively low polydispersity indices (PDI) of 0.14 (±0.03) and 0.07 (±0.02), in good agreement with results obtained previously. [START_REF] Reisch | Charge-Controlled Nanoprecipitation as a Modular Approach to Ultrasmall Polymer Nanocarriers: Making Bright and Stable Nanoparticles[END_REF] We then used the same solutions for the preparation of NPs using microfluidic mixers, where the organic to aqueous phase ratio was adjusted through the volume flow rates in a ratio 1:9. In the following, we first present the obtained sizes and their flow rate dependencies for the individual mixers, and will then compare and discuss the specific influences of the different mixers. It should be noted that the obtained NPs by manual and microfluidics assisted nanoprecipitation were stable over the time frame of the experiments, that is for at least one week.

The simplest mixer we used was a cross-shaped mixer, in which the two lateral inlets were fed with the aqueous phase, while the organic phase entered through the central inlet, with a long, mixing channel of 100 µm x 100 µm cross-section. This configuration leads to hydrodynamic flow focusing with the initial width of the organic phase being determined through the channel width and the ratios of flow rates. Here, this should correspond to a width of about 10 µm (as confirmed using confocal microscopy). The sizes of PLGA NPs obtained in this configuration were all larger than 100 nm, showed strong batch to batch variations, and had a tendency to become bigger with increasing flow rate (Figure 2 A). In the case of PMMA, frequent blocking of the mixer was observed, and no consistent results could be obtained. Observation of the mixer under the microscope showed indeed deposits inside the channel. A further decrease of the initial width of the organic layer can be achieved by employing the multilamination mixer. H ere, we used a multilamination mixer having 1 5 interdigitated microchannels with a width of 4 5 µ m each ( H PIMM) . The siz e of PLGA NPs obtained using this mixer decreased with increasing flow speed from around 1 2 0 nm at low speed until a plateau value is reached around 5 0 nm, which is slightly below the siz e of NPs obtained manually ( F igure 2 B ) . In the case of PMMA, again freq ue nt issues of block i ng occurred.

H owever, in the case of the multilamination mixer, it was possible to overcome these by reducing the concentration of the PMMA in the organic phase to 0 .5 g.L -1 . At low flow speed, very large NPs with siz es above 5 0 0 nm were obtained, but the siz e of the particles decreased q ui ck l y with increasing flow-speed to reach about 7 0 nm. Manual preparation of NPs using PMMA at 0 .5 g.L -1 yielded NPs of 6 3 nm, smaller than particles prepared from 2 g.L -1 ( 7 7 nm) , and also slightly smaller than the particles prepared using the multilamination mixer.

An approach to further improve the mixing is to split the flow in several sub-flows, to recombine the flows, and then to split and recombine them again several times. U sing such a split & ( re) combine mixer resulted for both, PLGA and PMMA, in NP siz es that were nearly independent of flow speed ( over the studied range, F igure 2 C ) . The resulting particle siz es were around 6 0 nm and 7 0 nm for PLGA and PMMA, respectively, and thus relatively close.

PLGA particles were slightly bigger and PMMA particles slightly smaller than corresponding particles prepared manually. A different way to improve mixing between the two phases in microfluidics is to induce a lateral flow component, which leads to vortices or " chaotic elements" within the flow that further decrease the thick ness of the phases. Such lateral flows can be achieved, for example, using ridges or grooves in the channel walls, giving rise to the so-called staggered herringbone mixer. [START_REF] Stroock | Chaotic Mixer for Microchannels[END_REF] Here, we used two versions of herringbone mixers: a plastic based system bearing 4 mixing-blocks and a channel cross-section of 600 µm x 200 µm, and a glass based systems bearing 30 mixing-blocks and a channel cross-section of 200 µm x 80 µm.

Interestingly, the dependence of the particle size on flow speed was very different in both cases (Figure 3): while in the case of the glass herringbone mixer the size was practically independent of flow speed over the studied range, there was a clear decrease in size with increasing flow speed for the plastic herringbone mixer. In the latter case, the particle sizes at low flow speeds were close to 200 nm for both polymers. At the highest flow speeds, the sizes decreased to around 50 nm and 110 nm for PLGA and PMMA, respectively, and thus to values close to those obtained with the glass herringbone mixer. Compared to manual preparation, the achievable sizes using herringbone mixers were hence slightly lower for PLGA and significantly bigger for PMMA. Another possibility to overcome laminar flow in microfluidics is to use so-called impact j et, impinging j et or vortex mixers, in which collision of the flows leads to turbulent behavior.

H ere, we tested two impact j et mixers ( K M-3 and K M-5 ) . F or both mixers, and for both polymers, the particle siz e depended strongly on the flow speed ( F igure 4 ) . In all cases, particle siz es obtained at low flow speeds were of the order of 1 7 0 nm, close to those observed for the plastic herringbone mixer, the cross-shaped mixer and, at least partially, for the multilamination mixer at low flow speed. W ith increasing flow speed, we observed a steep decrease in particle siz e, which was more expressed for PLGA than for PMMA, and for the former somewhat faster with the K M-3 mixer. In the case of PMMA, a minimum particle siz e of around 5 0 nm ( slightly bigger for the K M-5 mixer) was achieved at intermediate flow speed, followed by a slight increase in particle siz e. The latter was accompanied by increased batch to batch variations, which might be a sign of instabilities of the system. F or PLGA, the particle siz es decreased down to a plateau at 2 0 nm for the K M-3 mixer, a value that was also obtained for the PLGA NPs produced with the K M-5 mixer at the highest flow speeds. The siz es of PLGA and PMMA NPs prepared either at low ( 0 .2 4 m s -1 ) or high ( 2 .3 6 m s -1 ) flow speed were further measured as a function of time and compared to NPs prepared manually ( SI, Table S3 ) . No significant changes in siz e were observed, indicating that the preparation method had no influence on stability.

F or one of the impact-j et mixers ( K M-3 ) , we further determined the z -potential obtained at low and high flow rates ( SI, Table S2 ) . In all cases, the z -potentials were clearly negative, with higher absolute values ( around -3 0 mV ) for PLGA than for PMMA ( around -2 0 mV ) .

Interestingly, no significant influence of the flow rate was observed, and the values were close to those of NPs obtained through manual preparation.

Figure 5 . Analysis of PLGA NPs using TE M. Representative TE M images for different conditions of particle preparation are given above ( scale bar corresponds to 1 0 0 nm) , and the particle siz e distributions as obtained from > 2 0 0 p articles per condition are given below.

Selected NPs were then further analyzed using transmission electron microscopy (TEM), with respect to their size and their size distribution (Figure 5, Table 1, SI Figure S1). In all cases, the sizes obtained by TEM were smaller than those obtained by DLS, as observed previously. [START_REF] Egloff | Size-Dependent Electroporation of Dye-Loaded Polymer Nanoparticles for Efficient and Safe Intracellular Delivery[END_REF] This difference can be attributed first of all to the fact that DLS measures the hydrodynamic diameter, while TEM gives the hard sphere diameter. A second reason for this difference is that the intensity of scattering increases strongly with the size of the scattering particles. [START_REF] Stetefeld | Dynamic Light Scattering: A Practical Guide and Applications in Biomedical Sciences[END_REF] In consequence, larger particles contribute stronger to the results obtained in DLS.

Considering PLGA NPs, we observed, in general, a good agreement between the trends observed by TEM and DLS, notably the strong decrease in particle size for the impact jet mixers with flow speed. A closer look at the particle size distribution obtained from TEM indicates that two processes contribute to this decrease: (i) At low flow speed a significant amount of very large particles was obtained with sizes larger than 100 and even reaching 200 nm. The fraction and sizes of these large particles decreased in the flow speed range of 0.06 -0.12 m.s -1 , and at higher values they were practically entirely absent. (ii) At the same time, the size corresponding to the maximum (mode) of the distribution was relatively stable over this range of flow speeds, but decreased strongly when going to high flow speeds (here 0.6 m.s -1 ).

For the latter, a particularly narrow particle size distribution was observed and practically no particles larger than 30 nm were detected. In the case of PLGA NPs prepared using the multilamination mixer, the size distribution was similar to what was observed for manual preparation of NPs. For those prepared by the herringbone (glass) mixer the mean value of particle size (and the mode) were slightly higher.

In the case of PMMA NPs, both the mean sizes and the width of the size distributions, as obtained by TEM, were clearly larger than for PLGA NPs prepared in corresponding conditions. For example, in samples prepared manually or at low flow speed in the impact jet mixer, a large number of particles > 50 nm and > 100 nm, respectively, were detected. At high speed, the PMMA particles prepared with the impact jet mixer appeared often not clearly separated, but rather as chains in TEM. Taken into account the diameter of single "beads", a mean size of only 20 nm was obtained, compared to 50 nm measured by DLS. A possible explanation could be that smaller PMMA NPs are more prone to aggregation due to the higher hydrophobicity of PMMA. This is also in good agreement with the observed instabilities at the highest flow rates for PMMA (see above). a) Given are the mean values over > 200 particles ± the width of the distribution at half maximum.

A comparison of the smallest particles that could be assembled with each mixer for each polymer is given in Figure 6. A first observation is that in all cases PLGA particles were smaller than PMMA particles made under the same conditions. A second observation is that "manual" particle preparation performs quite well with respect to most of the microfluidic mixers that were tested. Indeed, in the case of PLGA, the smallest particle sizes that could be obtained using split & (re)combine, multilamination, and herringbone mixers are with 45 nm to 57 nm close to those obtained manually (51 ± 5 nm). However, two remarkable exceptions exist: the cross-shaped mixer gave much larger particles (> 100 nm), and the impact jet mixers gave clearly smaller particles with siz es going below 2 0 nm. In the case of PMMA, differences were more clearly mark ed: no reproducible particle preparation was achieved using the cross-shaped mixer. The herringbone mixers gave bigger particles than manual preparation, while the multilamination and the split & combine mixers gave slightly smaller NPs, approaching the siz e of the particles made from PLGA. A specific case for PMMA is the impact j et mixer. It gave again the smallest particles, however, there was a large discrepancy between the results obtained from DLS ( 5 0 nm ) and TE M ( 2 2 nm ) . In the case of relatively rapid mixing, nanoprecipitation is generally considered to occur via a nucleation and growth mechanism, in which supersaturation acts as a driving force that enables formation of nuclei, which then grow through incorporation of " monomers" ( single polymer, dye, or drug molecules) , but also through aggregation of particles. [START_REF] D'addio | Controlling Drug Nanoparticle Formation by Rapid Precipitation[END_REF][START_REF] Saad | Principles of Nanoparticle Formation by Flash Nanoprecipitation[END_REF][START_REF] Lepeltier | Nanoprecipitation and the "Ouzo Effect": Application to Drug Delivery Devices[END_REF][START_REF] Mora-Huertas | Influence of Process and Formulation Parameters on the Formation of Submicron Particles by Solvent Displacement and Emulsification-diffusion Methods: Critical Comparison[END_REF] The final particle siz e and siz e distribution then depends on the relative rates of nucleation, growth, and aggregation, with small particles being formed at high nucleation, low growth and very low aggregation rates. Stabilization from aggregation can typically be achieved through the implementation of charged groups on the particle surface, e.g. using polymers with a low percentage of charged groups as was the case here. [START_REF] Reisch | Charge-Controlled Nanoprecipitation as a Modular Approach to Ultrasmall Polymer Nanocarriers: Making Bright and Stable Nanoparticles[END_REF][START_REF] Zhang | Flash Nanoprecipitation of Polystyrene Nanoparticles[END_REF][START_REF] Morozova | On the Stability of Polymeric Nanoparticles Fabricated through Rapid Solvent Mixing[END_REF] Both, nucleation and growth depend on supersaturation, but the nucleation rate increases more rapidly with increasing supersaturation. [START_REF] D'addio | Controlling Drug Nanoparticle Formation by Rapid Precipitation[END_REF][START_REF] Mora-Huertas | Influence of Process and Formulation Parameters on the Formation of Submicron Particles by Solvent Displacement and Emulsification-diffusion Methods: Critical Comparison[END_REF] As the supersaturation depends on the state of mixing during particle formation, the mixing time (notably compared to the time scale of particle formation) is generally considered to be of high importance for the particle formation in nanoprecipitation. [START_REF] D'addio | Controlling Drug Nanoparticle Formation by Rapid Precipitation[END_REF][START_REF] Liu | Formulation of Nanoparticles Using Mixing-Induced Nanoprecipitation for Drug Delivery[END_REF] Let us leave aside the nature of the polymer for the moment and concentrate on one polymer (e.g. PLGA). In this case, supersaturation depends on the speed and quality of mixing of the two phases: the faster the mixing, the higher the water fraction and in consequence the supersaturation at the beginning of particle formation (in first approximation). The cross-shaped mixer, which gave the biggest particles can, indeed, be considered to have the lowest mixing speed, relying largely on diffusion over a relatively long distance. At the same time, the contact of the organic phase with the walls can also lead to adsorption of polymer and further perturb particle formation (in agreement with the strong batch to batch variation), an issue that can be addressed by using 3 dimensional hydrodynamic flow focusing. [START_REF] Khan | Production of Nanoparticle Drug Delivery Systems with Microfluidics Tools[END_REF][START_REF] Rhee | Synthesis of Size-Tunable Polymeric Nanoparticles Enabled by 3D Hydrodynamic Flow Focusing in Single-Layer Microchannels[END_REF] Particle sizes obtained with the multilamination and split & (re)combine mixers were markedly smaller, with relatively little dependence on flow speed, which can be attributed to a strong decrease of the diffusion length and so an increase in mixing speed.

However, especially at higher flow velocities, a contribution of turbulences to mixing is also possible. [START_REF] Bally | Improved Size-Tunable Preparation of Polymeric Nanoparticles by Microfluidic Nanoprecipitation[END_REF] The two herringbone mixers showed different dependencies of particle size on flow speed: while in the case of the plastic based mixer with 4 mixing elements particle sizes depended strongly on the flow speed, the glass based mixer with 30 mixing elements did not show such a dependence. This indicates that in the case of a lower number of mixing elements additional flow events (like turbulences at the entry or outlet) are important for efficient mixing that depend on the flow speed, while this is not the case if sufficient mixing elements are used. However, differences in the adsorption of the polymers on the wall materials could also have an influence. All these mixers gave particle sizes close to those obtained "manually", indicating that the achieved mixing times lie in a similar range. In the case of the impact jet mixers, the collision of the flows leads to very high shear rates that help break-up of the flow in small fluid segments. [START_REF] Nagasawa | Design of a New Micromixer for Instant Mixing Based on the Collision of Micro Segments[END_REF] (Reynolds numbers remain, however, below 2000 at the used flow parameters indicating that the flow does not become turbulent.) Previous studies have shown that for this type of mixer the mixing performance indeed increased with increasing fluid velocities, [START_REF] Nagasawa | Design of a New Micromixer for Instant Mixing Based on the Collision of Micro Segments[END_REF][START_REF] Anton | A New Microfluidic Setup for Precise Control of the Polymer Nanoprecipitation Process and Lipophilic Drug Encapsulation[END_REF] which should correspond to decreasing mixing times of the aqueous and organic phase with increasing flow speed, and to particular short mixing times for this kind of mixer. This can then explain the decrease in particle size observed here: as noted above, faster mixing times should lead to particle formation at higher water to solvent ratios (and so also higher supersaturation) and lower solid concentrations. Both conditions have been shown previously to lead to the formation of smaller particles. [START_REF] Egloff | Size-Dependent Electroporation of Dye-Loaded Polymer Nanoparticles for Efficient and Safe Intracellular Delivery[END_REF][START_REF] Saad | Principles of Nanoparticle Formation by Flash Nanoprecipitation[END_REF] At the same time, increasing nucleation speed should also narrow the size distribution, as it leads to distinct nucleation and growth phases, with all the particles growing at the same time. [START_REF] D'addio | Controlling Drug Nanoparticle Formation by Rapid Precipitation[END_REF][START_REF] Mora-Huertas | Influence of Process and Formulation Parameters on the Formation of Submicron Particles by Solvent Displacement and Emulsification-diffusion Methods: Critical Comparison[END_REF] Here, TEM images indicated a particular narrow size distribution for particles assembled at high flow speeds in impact jet mixers. Taken together, the previous studies and the results obtained here indicate that in the impact jet mixers, the fastest mixing can be reached among the microfluidic mixers tested here, probably close to those achieved in other types of impact jet or vortex mixers. [START_REF] Markwalter | Design of a Small-Scale Multi-Inlet Vortex Mixer for Scalable Nanoparticle Production and Application to the Encapsulation of Biologics by Inverse Flash NanoPrecipitation[END_REF][START_REF] Johnson | Chemical Processing and Micromixing in Confined Impinging Jets[END_REF][START_REF] Liu | Mixing in a Multi-Inlet Vortex Mixer (MIVM) for Flash Nano-Precipitation[END_REF] The bigger particle sizes observed for PMMA compared to PLGA can be attributed to its higher hydrophobicity. Indeed, in a systematic study we found that particle size in the case of manual nanoprecipitation correlated with hydrophobicity of the used polymers. [START_REF] Rosiuk | Controlling Size and Fluorescence of Dye-Loaded Polymer Nanoparticles through Polymer Design[END_REF] Possible reasons for this are a decrease in nucleation rate with increasing surface tension of the formed particles, [START_REF] D'addio | Controlling Drug Nanoparticle Formation by Rapid Precipitation[END_REF][START_REF] Mora-Huertas | Influence of Process and Formulation Parameters on the Formation of Submicron Particles by Solvent Displacement and Emulsification-diffusion Methods: Critical Comparison[END_REF] and a stronger tendency of particle aggregation. The higher hydrophobicity of PMMA is also thought to be related to its more pronounced tendency for clogging the microfluidic mixers through adsorption on the mixer surfaces, notably in the case of the crossshaped but also for the multilamination mixer. The case of the herringbone mixers requires a further comment: Indeed, the difference in size between PLGA and PMMA appeared to be particularly strong. However, it should be noted that for the cross-shaped and the multilamination mixer, synthesis of PMMA NPs was not feasible at standard conditions due to clogging (in the case of the multilamination mixer, PMMA NP assembly was possible at reduced concentration). This underlines the high difficulty to synthesize PMMA NPs using microfluidic mixers, probably due to a combination of increased aggregate formation and stronger adsorption of the polymer on the wall. The only two mixers performing quite well for PMMA were the split-and-recombine and the impact jet mixers. A particular case was observed for PMMA particles made using the impact jet mixers at high flow speeds: both DLS and TEM results showed that in this way the smallest PMMA NPs were achieved.

However, the obtained values, 50 nm by DLS and 22 nm by TEM were clearly different. At the same time, the TEM images revealed "chains" of very small PMMA NPs. A possible explanation for this discrepancy is that the impact jet mixer at high flow speeds yields very small NPs, both for PLGA and PMMA. In the case of PMMA, these very small NPs could show a stronger tendency to aggregate than in the case of PLGA, leading to an increase in the sizes observed by DLS. However, it cannot be excluded that the "aggregation" only occurs on the grids for TEM imaging. Here, MilliQ water was used for nanoprecipitation, however, it is known that ionic force and pH can also influence particle formation. [START_REF] Egloff | Size-Dependent Electroporation of Dye-Loaded Polymer Nanoparticles for Efficient and Safe Intracellular Delivery[END_REF][START_REF] Trofymchuk | Giant Light-Harvesting Nanoantenna for Single-Molecule Detection in Ambient Light[END_REF][START_REF] Reisch | Charge-Controlled Nanoprecipitation as a Modular Approach to Ultrasmall Polymer Nanocarriers: Making Bright and Stable Nanoparticles[END_REF][START_REF] Zhang | Flash Nanoprecipitation of Polystyrene Nanoparticles[END_REF] It would therefore be interesting to subsequently study the combined effects of precipitation medium and mixing in the future.

Fluorescence

In a next step, we then evaluated the microfluidic mixers for the assembly of fluorescent dye-loaded NPs. For this, 5 wt% (relative to the polymer) of the dye salt R18/F5-TPB were added to the polymer solution and nanoprecipitation was performed as before by mixing with a nine-fold excess of MilliQ water (Scheme 1). The association of the hydrophobic rhodamine derivative R18 with the bulky hydrophobic counterion F5-TPB has been shown previously to lead to very efficient encapsulation (>90% loading efficiency) in various polymer NPs. [START_REF] Reisch | Charge-Controlled Nanoprecipitation as a Modular Approach to Ultrasmall Polymer Nanocarriers: Making Bright and Stable Nanoparticles[END_REF][START_REF] Andreiuk | Fighting Aggregation-Caused Quenching and Leakage of Dyes in Fluorescent Polymer Nanoparticles: Universal Role of Counterion[END_REF] At the same time the counterion acts as spacer insulating the fluorophores and reducing aggregation caused quenching (ACQ), which allows achieving NPs of very high brightness.

Nanoprecipitation of solutions containing the polymer and the dye salt yielded pink, transparent solutions with no visible signs of aggregate formation. Absorbance spectra of the dye loaded NPs prepared manually and using different microfluidic mixers were, generally, very similar in intensity and close to the expected values (SI Figure S4, the details of the shape of the spectra are discussed below). Together with previous results obtained on similar systems this indicates efficient encapsulation of the dye salt in the polymer particles. [START_REF] Reisch | Tailoring Fluorescence Brightness and Switching of Nanoparticles through Dye Organization in the Polymer Matrix[END_REF][START_REF] Andreiuk | Fighting Aggregation-Caused Quenching and Leakage of Dyes in Fluorescent Polymer Nanoparticles: Universal Role of Counterion[END_REF] Here, we then further concentrated on the influence of the different mixers on the brightness of the obtained NPs, which is given by the product of the absorbance and the quantum yield (QY).

QY is the ratio of the photons emitted as fluorescence to the absorbed photons, and as the dye concentration is kept constant here, and so were the absorbances, the QY is the decisive factor determining brightness.

Manual preparation of dye-loaded NPs yielded PLGA NPs with a QY of 42 ± 6 % and PMMA NPs with a QY of 80 ± 5 %, in good agreement with previous results. [START_REF] Reisch | Tailoring Fluorescence Brightness and Switching of Nanoparticles through Dye Organization in the Polymer Matrix[END_REF] At the same time the sizes of these NPs increased to, respectively, 91 and 99 nm for PLGA and PMMA upon addition of R1 8 / F 5 -TPB ( compared to 5 5 and 7 7 nm, respectively, without dye salt) .

This increase could be related to the association of some of the R1 8 with negative charges on the particle surface, as observed previously. [START_REF] Andreiuk | Fighting Aggregation-Caused Quenching and Leakage of Dyes in Fluorescent Polymer Nanoparticles: Universal Role of Counterion[END_REF] W e then evaluated the Q Y s of PLGA particles obtained under the conditions giving the smallest NPs for the multilamination and herringbone mixers ( F igure S3 ) . Q Y s for all three were about 4 0 % , i.e. close to those obtained manually. The obtained particle siz es were also similar, except for the plastic herringbone mixer, which gave larger particles ( F igure S2 ) . In the case of the split & ( re) combine mixer both, siz e and Q Y , were of the order of those of the corresponding NPs obtained manually.

H owever, a slight increase in Q Y and decrease in siz e with flow speed was observed for both, PLGA and PMMA ( F igure S3 B ) . A still stronger influence of the mixing process was observed for impact j et mixers ( here we concentrated on K M-3 mixer, F igure 7 ) : for PLGA particles, we observed a continuous increase in the Q Y with increasing flow speed, going from less than 3 5 % at the lowest flow speed to over 7 0 % at the highest speed tested, where the increase seemed to level off ( F igure 7 ) . F or PMMA particles, on the other hand, we observed a slight decrease of the Q Y from 9 2 to 7 2 % over the same flow speed range. At the same time, the particle siz es decreased with flow speed, as observed for the pure polymer particles, reaching 2 3 nm and 5 2 nm for PLGA and PMMA NPs, respectively at the highest flow rates ( F igure S2 ) . In order to understand the striking increase in QY for PLGA NPs, a close observation of the corresponding absorption and emission spectra provides a better picture of the changes occurring with flow rate: in the case of PLGA, the absorption spectra showed only slight variations in the maximum absorbance (<10%, Figure S4). This also indicates that the preparation method did not influence significantly the efficiency of dye encapsulation. Indeed, the absorbance was very close to that observed for manual NP preparation, for which an encapsulation efficiency >90% was previously found. [START_REF] Andreiuk | Fighting Aggregation-Caused Quenching and Leakage of Dyes in Fluorescent Polymer Nanoparticles: Universal Role of Counterion[END_REF] However, a relative decrease of the intensity of the shoulder at 530 nm with respect to the maximum was clearly observed with increasing flow speed (Figure 7A). Such a spectral change indicates a decrease in aggregation of the dyes inside the NPs. [START_REF] Reisch | Tailoring Fluorescence Brightness and Switching of Nanoparticles through Dye Organization in the Polymer Matrix[END_REF][START_REF] Macdonald | Characteristics of Self-Quenching of the Fluorescence of Lipid-Conjugated Rhodamine in Membranes[END_REF] At the same time the intensity of fluorescence increased steadily with increasing flow speed (Figure 7B), corresponding to the observed strong increase in QY. In the case of PMMA (Figure S4, S5), no major changes of the intensity and shape of the absorption spectra were detected (except for the lowest flow speed, where absorbance was about 20% lower). Here, the absolute intensity of the maximum and the relative intensity of the shoulder at 530 nm of the dye loaded in NPs were close to what is observed for the same dye in solution, indicating optimal encapsulation and minimal aggregation, and only a slight decrease in the emission with flow speed was measured.

The smaller QY of R18 in PLGA compared to PMMA was attributed previously to a clustering of the dyes within PLGA in contrast to a more homogeneous distribution in PMMA due to the lower hydrophobicity of PLGA. [START_REF] Reisch | Tailoring Fluorescence Brightness and Switching of Nanoparticles through Dye Organization in the Polymer Matrix[END_REF] Here, we found that QY in PLGA NPs increased with increasing flow speed, suggesting that a better or faster mixing of the two phases leads to less clustering or aggregation of the dyes. The observed decreasing relative intensity of the shoulder in the absorbance spectra, attributed to dye dimers or aggregates, points indeed in this sense. A possible reason for such differences in organization could be that at faster mixing of the two phases, particle formation occurs at higher supersaturations, at which PLGA and dye salt precipitate faster, leaving no time for clustering of the dye salt. The opposite behavior in the case of the PMMA particles could be due to the fact that for faster mixing the dye salt is exposed to a medium with higher water fraction before being incorporated into particles, which could favor dissociation of the ion pair. Overall, faster mixing decreases the difference between the two polymers in terms of dye encapsulation and aggregation, so that the spectroscopic properties of the obtained NPs become similar.

Conclusions

Our evaluation of the use of microfluidics for the preparation of polymer NPs, and in particular fluorescent dye-loaded NPs, has shown that microfluidics is indeed a very powerful approach for reproducible synthesis of nanomaterials. Microfluidics offer notably the possibility to produce NPs in continuous flow, facilitating high quality synthesis of large amounts, and it could be further scaled-up through parallel fabrication. Comparing different microfluidic mixers, relying on very different mixing principles, showed that most of these performed at least as well as "manual" preparation in terms of achievable particle size and fluorescence properties. In particular, impact jet mixers allowed strongly decreasing the size of NPs made from both PLGA and PMMA, achieving even particles below 20 nm for the former.

This was attributed to a strongly enhanced mixing speed. At the same time the impact-jet mixer also allowed strongly increasing the fluorescence QY for PLGA NPs and, in consequence, the brightness of these particles, simply by tuning the mixing speed. Apart from the interest of achieving brighter fluorescence markers, this finding is of high importance for other fields using loaded polymer NPs. Indeed, the increase in fluorescence was attributed to changes in the encapsulation of the load. Tailoring the distribution of the load by simple variation of the flow conditions is expected to present important possibilities in the design of drug delivery systems.

Scheme 1 .

 1 Scheme 1. ( A) " Manual" preparation of NPs, showing the structures of the polymers ( PLGA: n = m; PMMA: x= 0 .0 1 3 ) , the dye with its counterion, and the principle of nanoprecipitation. ( B ) Microfluidics set-up used for the preparation of NPs ( except for Split and recombine) .

Figure 1 .

 1 Figure 1. O verview of mixers used here for nanoparticle preparation.

48

 48 Two impact-jet mixers (KM-3 and KM-5 respectively, Fujifilm Corporation, Kyoto, Japan) whose structure consists of three steel plates, namely the inlet, mixing and outlet plates. Both inlet fluid streams are split into 3 or 5 sub-streams thanks to microchannels of 150 or 100 µm, respectively. Then the alternated 6 or 10 sub-streams converge to a single pine hole of 300 or 350 µm, respectively, where they are mixed by frontal collision. Dynamic light scattering (DLS): The sizes of NPs were measured on a Zetasizer Nano series ZS for NPs prepared using the split & recombine mixer and on a Zetasizer Nano series ZSP (Malvern Instruments S.A.) for all other preparations. DLS measurements were performed directly after NP preparation without further dilution at concentrations of 0.05 -0.2 g.L -1 . For size determination, each sample was measured 10 times with a run length of 10 s each. The measurement position was fixed to 4.3 mm and the attenuation was adjusted automatically. The volume average values were used, which are determined by the Zetasizer software (Malvern) based on Mie theory. Mean values give the average over at least three independent preparations, error bars correspond to the standard deviation over the means from different preparations.

Figure 2 .

 2 Figure 2. Siz e of NPs obtained using ( A) the cross-shaped, ( B ) the multilamination, and ( C ) the split & ( re) combine mixers as a function of flow speed as measured by DLS. Polymer concentrations in the organic phase were 2 g.L -1 , except for PMMA in the case of the multilamination mixer ( and the corresponding manual preparation) , where the concentration was reduced to 0 .5 g.L -1 to avoid clogging. The values correspond to the mean of the volume average from at least three measurements. E rror bars give the standard deviation.

Figure 3 .

 3 Figure 3. Siz e of NPs obtained using ( A) the glass and ( B ) the plastic staggered herringbone mixer as a function of flow speed as measured by DLS. The values correspond to the mean of the volume average from at least three measurements. E rror bars give the standard deviation.

Figure 4 .

 4 Figure 4 . Siz e of NPs obtained using ( A) the K M-3 and ( B ) K M-5 impact j et mixers as a function of flow speed as measured by DLS. The values correspond to the mean of the volume average from at least three measurements. E rror bars give the standard deviation.

Figure 6 .

 6 Figure 6 . The siz es of the smallest PLGA and PMMA NPs obtained using the different mixers are given as measured by DLS. The values correspond to the mean of the volume average over at least three independent preparations. E rror bars give the standard deviation

Figure 7 .

 7 Figure 7 . Absorption ( A) and emission spectra ( B ) of PLGA NPs with 5 wt% of R1 8 / F 5 -TPB prepared manually or using the impact j et mixer K M-3 at different flow speeds. Absorption

Table 1 .

 1 Comparison of particle sizes obtained from DLS and TEM.

	Polymer	Mixer	Flow	Mean size
			speed
				DLS	TEM a
			[m s -1 ]	[nm]	[nm]
	PLGA	Manual	-	51 ± 5	37 ± 8
		Herringbone	0.31	46 ± 13	47 ± 12
		(glass)
		Multilamination	2.2	57 ± 9	39 ± 10
		Impact jet KM-3	0.24	141 ± 13	47 ± 15
			0.47	77 ± 8	43 ± 11
			0.71	44 ± 10	38 ± 8
			2.4	18 ± 5	20 ± 5
	PMMA	Manual		77 ± 5	63 ± 15
		Impact jet KM-3	0.24	138 ± 5	98 ± 25
			2.4	50 ± 3	22 ± 7

Author manuscript of article published in Langmuir (2022) and available at https://doi.org/10.1021/acs.langmuir.2c00534

Acknowledgements

This work was supported by the ANR JC/JC grant "Supertrack" ANR-16-CE09-0007, by an FRC Emerging Investigators grant, by the ERC Consolidator grant BrightSens 648528, and by the "Prix Espoirs de l'Université de Strasbourg". The authors acknowledge the support and the use of resources of the French Infrastructure for Integrated Structural Biology FRISBI ANR-10-INBS-05 and of Instruct-ERIC and especially C. Crucifix for help with electron microscopy.

Scheme S1. Top-view of some of the mixers used here for nanoparticle preparation.

Author manuscript of article published in Langmuir ( 2022) and available at https://doi.org/10.1021/acs.langmuir.2c00534

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally.

Conflicts of interest

There are no conflicts of interest to declare.

Supporting Information

Detailed results of dynamic light scattering, further electron microscopy images, additional spectra and figures on size and quantum yields of dye-loaded polymer nanoparticles.

Supplementary Information

Assembly of Fluorescent Polymer Nanoparticles using different Microfluidic Mixers

Huaiyou Chen, Ali Emre Celik, Angela Mutschler, Antoine Combes, Anne Runser, Andrey Klymchenko, Sébastien Lecommandoux,* Christophe Serra,* Andreas Reisch*