Microfluidic Controlled Self-Assembly of Polylactide (PLA)-Based Linear and Graft Copolymers into Nanoparticles with Diverse Morphologies

This study outlines the microfluidic (MF) controlled self-assembly of polylactide (PLA)-based linear and graft copolymers. The PLA-based copolymers (PLA-Cs) were synthesized through a convenient one-pot/one-step ROP/RAFT technique. Three distinct vinyl monomers—triethylene glycol methacrylate (TEGMA), 2-hydroxypropyl methacrylate (HPMA), and N-(2-hydroxypropyl) methacrylamide (HPMAA) were employed to prepare various copolymers: linear thermoresponsive polylactide-b-poly(triethylene glycol methacrylate) (PLA-b-PTEGMA), graft pseudothermoresponsive poly[N-(2-hydroxypropyl)] methacrylate-g-polylactide (PHPMA-g-PLA), and graft amphiphilic poly[N-(2-hydroxypropyl)] methacrylamide-g-polylactide (PHPMAA-g-PLA). The MF technology was utilized for the controlled self-assembly of these PLA-based BCs in a solution, resulting in a range of nanoparticle (NP) morphologies. The thermoresponsive PLA-b-PTEGMA diblock copolymer formed thermodynamically stable micelles (Ms) through kinetically controlled assemblies. Similarly, employing MF channels led to the self-assembly of PHPMA-g-PLA, yielding polymersomes (PSs) with adjustable sizes under the same solution conditions. Conversely, the PHPMAA-g-PLA copolymer generated worm-like particles (Ws). The analysis of resulting nano-objects involves techniques such as transmission electron microscopy, dynamic light scattering investigations (DLS), and small-angle X-ray scattering (SAXS). More specifically, the thermoresponsive behavior of PLA-b-PTEGMA and PHPMA-g-PLA nano-objects is validated through variable-temperature DLS, TEM, and SAXS methods. Furthermore, the study explored the specific interactions between the formed Ms, PSs, and/or Ws with proteins in human blood plasma, utilizing isothermal titration calorimetry.

The bis(ethyl trithiocarbonate) was dissolved in ethyl acetate (100 mL) and 4,4'-azobis(4cyanopentanoic acid) (V-501, 4.57 g, 0.0169 mol) was added.This solution was refluxed under nitrogen at 90-100 o C for 20 hours.The solution was concentrated and purified by flash silica chromatography (30.0 × 5.0 cm) using n-pentane/ethyl acetate/acetic acid (1/1/0.03v/v/v).After rotary evaporation of the appropriate fractions (Rf = 0.65 in n-pentane/ethyl acetate/acetic acid 1/1/0.03v/v/v), a viscous yellow liquid was obtained.The yellow liquid was placed at -20 o C, which yielded yellow crystals that were subsequently filtered, washed with ice-cold pentane and dried in vacuo.Yield: 5.76 g.  with a magnetic stirrer, sealed, subjected to three freeze-thaw cycles, and then purged with argon gas.
The reaction mixture was placed in a preheated oil bath at 75°C.Conversion was determined by comparing the remaining monomer concentration with the initial monomer feed.The final copolymer was purified by dialysis against acetone using a molecular weight cut-off (MWCO) of 2 kDa.The block copolymer was recovered by evaporating the solvent, dried under vacuum for approximately 24 hours, and then analyzed by 1 H-NMR spectroscopy (refer to Figure S3 in ESI) and SEC analysis (refer to Figure S6, black curve).The results are presented in Table 1.HPMA (1.17 mL, 8.4 mmol), LA (408 mg, 2.8 mmol), CEPA (30.0 mg, 0.081 mmol), AIBN (3.321 mg, 0.020 mmol), DMAP (40.0 mg, 0.32 mmol), and 1.0 mL of methylene chloride, were mixed together in a dry 10 mL Schlenk flask.The graft copolymer was analyzed using 1 H-NMR spectroscopy (refer to Figure S4 in ESI) and SEC analysis (refer to Figure S6, red curve).The results are presented in Table 1.proceeded similarly to the previous two copolymers.Subsequently, the crude product was dissolved in a small amount of 1,4-dioxane, and the graft copolymer was purified using Sephadex LH-20 with 1,4-dioxane as the mobile phase.The copolymer was then precipitated in cold diethyl ether and vacuum-dried to yield a solid.The obtained graft copolymer was characterized using 1 H-NMR spectroscopy (see Figure S5 in ESI) and SEC analysis (see Figure S6, green curve).The results are presented in Table 1.

Hydrodynamic flow focusing nanoprecipitation microfluidics (MF)
Microfluidics is an emerging field for the production of particles, droplets, capsules and multiple emulsions.
Among other common techniques such as nanoprecipitation, film-rehydration and electroformation, microfluidics allows more controlled and precise way for the production of highly monodisperse polymeric structures including micelles, worms and vesicles.
Microfluidics nanoparticle preparation systems with high shear micromixing and hydrodynamic focusing methods give precise particle sizes in the range of ~20 nm to 500 nm.The microfluidics use continuous and controllable laminar flow for the production of high-yield and high-quality nanoparticles.The superior control over the size, shape and morphology of particles enables greater reproducibility and scalability.These substantial improvements in nanoparticle generation are highly valued in pharmaceutical industry compared to conventional batch methods.In this thesis, for production of well-defined nanosized polymersomes was used the commercial microfluidic chip from Dolomite company (Figure S6).The mixing time ( mix ) for the hydrodynamic flow focusing using a two-dimensional model is estimated according to equation 1: where D is diffusivity of the solvent, w is channel width, and R is the ratio of flow rate of the polymeric stream to the total flow rate of water.For D ~ 10 -9 m 2 s -1 and w = 125 µm equation 1 predicts a mixing time in the range of 0.6 to 7 ms for typical flow ratios (R = 0.04 -0.8) in this device.Particles sizes can be finely tuned by changing the flow ratios between the organic and the aqueous phase, the slowest flow gives the smallest particles which was explained by the decrease in the  mix .Furthermore, besides the lower particle sizes a decrease in the width of the particle size distribution is observed compared with the bulk nanoprecipitation process.The NPs produced using this microfluidic device always presented size distributions with dispersity below 0.1 when measured by dynamic light scattering.

Size exclusion chromatography (SEC)
The apparent average molecular weights (i.

The dynamic light scattering (DLS)
The DLS measurements were performed using an ALV CGE laser goniometer consisting of a 22 mW HeNe linear polarized laser operating at a wavelength (λ = 632.8nm), an ALV 6010 correlator, and a pair of avalanche photodiodes operating in pseudo-cross-correlation mode.The samples were loaded into 10 mm diameter glass cells and maintained at 37 ± 1 °C.The data were collected using the ALV Correlator Control The hydrodynamic radius (R H ) or the distributions of R H were calculated by using the Stokes-Einstein relation: being k B the Boltzmann constant, T the absolute temperature, and η the viscosity of the solvent.The transition temperature (T tr ), characterizing the polymer chain conformation changes, was evaluated from the temperature dependence of the hydrodynamic diameter (D H ) and was determined from the intersection point of two lines formed by the linear regression of a lower horizontal asymptote and a vertical section of the Sshaped curve (sigmoidal curve) fit.

The small-angle X-ray scattering (SAXS)
The SAXS experiments were recorded by using a pinhole camera (MolMet, Rigaku, Japan, modified by In the core-shell model, the scattering intensity is defined by equation 3: where R core is the radius of the core, ΔR shell is the shell thickness, and Δη is the scattering length density difference between core/shell and the solvent.
The log-normal distribution is defined by equation 5: where Norm is a normalization factor determined during numerical calculation, μ=ln(x med ), x med is the median value of the distribution and σ (=PD or polydispersity ratio) is a parameter describing the width of the underlying normal distribution.The values of these parameters obtained by fitting for PLA-b-PTEGMA and PHPMA-g-PLA NPs from this work are summarized in Table S1.
Table S1.Parameters of the core-shell model for PLA-b-PTEGMA and PHPMA-g-PLA NPs at various temperatures with a log-normal distribution of core and shell radii to account for the polydispersity of the samples.
a scattering length density of water was fixed at 1E-4 Å -2 .
The WormLikeChainEXV model is characterized by the structure factor F S (q,R,L) which consists of a crosssection term F cs (q,R) and a longitudinal term F L (q,L), so that where B 1 (x) is the first order Bessel function of the first kind and where The parameter R describes the cross-section radius of the worm, L is the total length of the worm and b is the Kuhn length which describes the stiffness of the chain and corresponds to twice the length of locally stiff segments.The exponents of the Bessel function are strongly dependent on the L/b ratio and the behavior is quite complex, so we refer the reader to the original text (reference 72 in the main manusript) for details.
The extended Guinier law model defines scattering intensity as where Rg is the radius of gyration and α is the dimensionality factor which is 0 for a spheroid, 1 for a rodlike shape and 2 for a plane.The parameter obtained by fitting for PHPMAA-g-PLA worms from this work are summarized in Table S2.
Table S2.Parameters of the WormLikeChainEXV and extended Guinier law models PHPMAA-g-PLA worms.

The transmission electron microscopy (TEM)
Observations were studied using a transmission electron microscope, specifically the Tecnai G2 Twin 120kV (FEI, Czech Republic), operated at an acceleration voltage of 120kV.For the procedure, 2 μL of the aqueous solutions were dropped onto a copper TEM grid (400 mesh) coated with a thin, electron-transparent carbon film.Excess solution was removed by touching the bottom of the grid with filtering paper.This rapid removal of the solution occurred after 1 minute of sedimentation to minimize oversaturation during the drying process.Nanoparticles were negatively stained with uranyl acetate (2 µL of 2 wt.% solution dropped onto a not completely dried grid and removed after 30 s using the same method described above).The samples were then left to dry completely at ambient temperature and subsequently observed in the TEM microscope.Under these conditions, the micrographs displayed a negatively stained background with bright nanoparticles.
WormlikeChainEXV extended Guinier law Before sample preparation at different temperatures, solutions and microscopic grids were tempered for 24 hours either in a refrigerator (5°C) or in an oven (60°C).Approximately 2.0 μL of the tempered aqueous solution was directly applied to the tempered microscopic grid, following the same procedure as described above, either in the refrigerator or in the oven.
After complete drying, the particles were negatively stained at room temperature with uranyl acetate, following the procedure described above.

Isothermal titration calorimetry (ITC)
The nanoparticles, from linear PLA-b-PTEGMA, PHPMA-g-PLA and PHPMAA-g-PLA graft copolymers 5.0 mg mL -1 each, in ultrapure water were titrated with human blood plasma, diluted with PBS to 10% vol/vol on ITC 200 titration calorimeter (Malvern Panalytical, Malvern, UK) at 20, 37 and 54°C.The ultrapure water was selected as a dispersion medium for polymer particles because the undiluted PBS triggered the gelation of the particles.Diluted human plasma was added in 20 consecutive injections, the first injection was always 0.4 µL followed by 19 injections of 2.0 µL.Additionally, the diluted plasma was titrated similarly to ultrapure water at all experimental temperatures to account for its heat of dilution.The calorimetry data were plotted as heat per injection, normalized to the injection volume (µJ/µL) vs the ratio of the injected volume of diluted human blood plasma (cumulative) to the mass of particles (µL/mg).

Figure S6 .
Figure S6.Dolomite setup of the micromixing chip with two water inputs and one polymer solution input.
e., number-average molecular weight (M n ) and weight-average molecular weight (M w ) and the molecular weight distribution or dispersity (Đ) of the PLA-b-PTEGMA, PHPMA-g-PLA, and PHPMAA-g-PLA copolymers were obtained by SEC performed at 25 ºC with two PLgel MIXED-C columns (300 x 7.5 mm, SDV gel with particle size 5 μm; Polymer Laboratories, USA) and with UV (UVD 305; Watrex, Czech Republic) and RI (RI-101; Shodex, Japan) detectors.N,N-Dimethylformamide (Sigma Aldrich, Czech Republic) with LiBr (0.01 % v/v) was used as mobile phase at flow rate of 1 mL min -1 .The M n values were calculated using the Clarity software (Dataapex, Czech Republic).Calibration with poly(methyl methacrylate) (PMMA) standards was used.
software and the counting time was 45 s.The measured intensity correlation functions g 2 (t) were analyzed using the algorithm REPES (incorporated in the GENDIST program) 57 , resulting in the distributions of relaxation times shown in equal area representation as τA(τ).The mean relaxation time or relaxation frequency ( = τ −1 ) is related to the diffusion coefficient (D) of the nanoparticles as  =  vector being n the refractive index of the solvent and θ the scattering angle.
SAXSLAB/Xenocs) attached to a microfocused X-ray beam generator (Rigaku MicroMax 003) operating at 50 kV and 0.6 mA (30 W).The samples were sealed into boro-silicate capillaries (∼2 mm diameter) and the scattering intensities were recorded by using a Pilatus 300 K detector with an exposure time of 480 min.The scattering profiles were fitted by using the SASfit software.The PLA-b-PTEGMA and PHPMA-g-PLA nanoparticles were fitted by the core-shell model (with the core representing the hydrophobic PLA component and shell representing the amphiphilic component of the graft copolymer).Polydispersity of the particles was accounted for in the form factors by using the log-normal distribution.The PHPMAA-g-PLA worms were fitted by the combination of the WormLikeChainEXV model (to describe the worm-like chains) and the extended Guinier law model (to describe the spherical PHPMAA corona block).The effect of solvent (water) was approximated by the constant function I H2O = 3.41.