Microfluidics-driven templating preparation of polymer vesicles with tailorable dimensions and rapid cellular internalization

Abstract

Polymer vesicles hold immense potential in biomedicine and nanotechnology, yet conventional rehydration methods face critical limitations in controlling the vesicle architecture due to stochastic block copolymer (BCP) self-assembly. Here, we present a first-reported microsphere-templated strategy that synergizes microfluidic precision with BCP assembly to overcome these constraints. By engineering emulsion templates via flow rate, BCP concentration and collection distance optimization, we established a method based on the radius-square law governing the evolution of uniform vesicles (size range diameter: 70–170 nm, PDI: 0.16), enabling on-demand size tuning, a capability unattainable with traditional approaches. Multi-scale characterization (DLS, OM, SEM and TEM) elucidates the non-equilibrium templating-to-vesicle transition, revealing critical dynamics of BCP film reorganization. The resultant nano-scale vesicles exhibit rapid cellular uptake (>95% in 3 h) by HUVECs and 4T1 cells with exceptional biocompatibility (>85% viability, 36 h), outperforming many cytotoxic counterparts. This work not only provides a scalable platform for precision vesicle fabrication but also establishes foundational principles for templated self-assembly, bridging microfluidics and soft matter science. Our methodology opens avenues for tailored vesicles in drug delivery, nanoreactors and synthetic biology, addressing the persistent demand for functionally adaptive polymeric nanostructures.

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1. Introduction

Polymer vesicles are self-assembled structures derived from amphiphilic block copolymers (BCPs).^1–4 They have attracted considerable attention due to their tunable bilayer structure, exceptional mechanical properties and robust colloidal stability.^5,6 These polymer vesicles have found extensive applications across the biomedical and life sciences fields as gene delivery systems,^7,8 artificial cellular membranes,^9–12 nanoreactors,^13,14 and vehicles for drug delivery.^15–18 While these functional capabilities are promising, a critical bottleneck persists in translating laboratory successes to commercial applications: the precise control over vesicle size and the scalability of fabrication processes.¹⁹ To address this challenge, researchers have developed several conventional preparation methods, including film hydration,^20,21 solvent exchange,^10,22 and electroformation.²³ For these approaches, the vesicles are often generated with a varied size distribution due to the size selection mechanism inherent in the formation process.^24–26 For example, the formation of polymer vesicles from the solvent exchange method involves growth and the bending and closure of the bilayer membrane driven by hydrophobic interactions.^1,2 The size of end-state vesicles is determined by the interplay between membrane growth and closure, indicating a vital kinetic requirement rather than a thermodynamic one.^27–29 Hence, in order to better control the size of polymer vesicles, a method that can kinetically control the growth of polymer vesicles will show a strong size selection mechanism.

Film rehydration, one of the most common formation methods of polymer vesicles, has demonstrated the potential for controlling the size of vesicles.²⁰ Since the exchange between the BCP chain and polymer membrane is so slow, the unbinding rehydration process is contingent upon membrane variations in the film rehydration process.³⁰ So, by employing templates to limit variations of BCP films, the rehydration method can kinetically control the formation of vesicles.^15,23,24,31 Howse and his co-workers have reported a method to produce a controlled size distribution of micro-scale polymer vesicles by photolithography and dewetting with the ‘bottom-up’ control of micro-scale polymer vesicles.²⁴ Due to the limited surface area of polymer patterning, this method tends to prepare micro-scale polymer vesicles. Therefore, Li and his co-workers have introduced a straightforward and efficient method for the preparation of nano-scale polymer vesicles through electrospray particle templates.¹⁵ However, electrospray particles tend to form less controlled disc-like microparticles due to a low degree of entanglement between BCP chains and the rapid evaporation of volatile solvents.^32,33 To enhance the control of kinetic growth of polymer vesicles, it is necessary to develop a facile and continuous manner method for template production.

Microfluidics has been receiving great attention for the continuous production of micro-scale vesicles based on different templates, including double emulsion,^34,35 droplet emulsion transfer,³⁶ pulsed-jetting³⁷ and jet-flow methods,³⁸ and flow-focusing.^39,40 These methods are able to affect the kinetic formation of polymer vesicles to alter the size characteristics by changing the supply of BCPs.^31,41 The use of double emulsion templates, the pulsed-jetting method and the droplet emulsion transfer method are classical methods to produce polymer vesicles under the emulsion droplet template.^37,42,43 Since they are limited to the size of the microchannel, they typically produce micro-scale polymer vesicles.^42,44 Single-emulsion templates provided a robust way to control the polymer vesicles by regulating the concentration of BCPs and the feedstock supply in the emulsion template. It broke through the limitation of the emulsion template size to prepare homogeneous nanoscale polymer vesicles.⁴⁵ However, despite these advancements, there is still a lack of a method to prepare vesicles in a more facile and continuous manner using microfluids. Specifically, the time for emulsion template formation is prolonged by the rate of solvent evaporation in the single emulsion template method.⁴⁵ There is a need to find a biocompatible and greener way to efficiently prepare templates continuously.

Herein, we present a simple and efficient method for preparing narrowly dispersed vesicles using BCP microsphere templates produced through microfluidics based on the radius-square law. We utilized poly-(ethylene glycol)-block-poly-(lactic acid) (PEG-b-PLA), a biocompatible material, to prepare polymer vesicles. Initially, microsphere templates were created through microfluidics, where we investigated the effects of a series of parameters, including the flow rate, BCP concentration and collection distance, on template formation. Subsequently, polymer vesicles were formed from these microsphere templates through a rehydration process, allowing us to observe the morphological transitions during vesicle formation using multi-scale characterization techniques. Furthermore, coumarin 6 (Cour-6) served as a model drug to evaluate drug-loading efficiency and biological cell studies were conducted to assess the biocompatibility of nanoscale polymer vesicles.

2. Materials and methods

2.1 Materials

PEG-b-PLA, PEG₁₁₃-b-PLA₁₆₇ (M_w = 18 200 g mol⁻¹, Đ(M_w/M_n) = 1.20) was attained from Jinan Dai Gang Biomaterial Co., Ltd (Jinan, China). Ethyl acetate (EA) and sodium phosphotungstate were sourced from Sigma-Aldrich (St Louis, Missouri). Polyvinyl alcohol (PVA, M_w = 22 000–24 000 g mol⁻¹) was obtained from a commercial supplier – Shanghai YingJia Co., Ltd (Shanghai, China). Deionized water was produced using a laboratory water purification system (Shanghai, China). Cour-6 and Nile red were purchased from Aladdin Industrial Corporation (Shanghai, China). All chemicals were utilized as received unless otherwise specified.

2.2 Microfluidic equipment

The microfluidic device consisted of a coaxial capillary (inner diameter: 300 μm) and a steel needle (inner diameter: 100 μm). The needles were precisely assembled using three-way fitting with a seamless connection. Microfluidic systems need to be purged of air before use. Therefore, the microfluidic system was constructed as described in our previous study to prepare microsphere templates.⁴⁵

2.3 Microsphere template and polymer vesicle preparation

The BCP EA solution served as the dispersed phase (oil phase) with concentrations ranging from 0.008 to 10% w/w. The oil phase was introduced through the inlet under flow rates ranging from 1 mL h⁻¹ to 7 mL h⁻¹. Concurrently, a 0.5% w/w PVA aqueous solution was pumped from the outlets as the continuous phase with flow rates ranging from 10 mL h⁻¹ to 70 mL h⁻¹. The conditions were consistent with those reported by a previous study.⁴⁵ The different collection distances between the inlet capillary and glass needle were 80 μm, 110 μm, 250 μm, 310 μm, 390 μm and 480 μm, respectively. The emulsion was generated and evaporated unrestrictedly at room temperature for 6 hours. After evaporation, the microsphere samples were washed 5 times and vacuum dried for 24 hours. The parameters were chosen.

Polymer vesicles were self-assembled from the pre-prepared microspheres via rehydration and stirred for 12 h at 45 °C. The polymer vesicles’ concentration ranged from 0.25% w/w to 10% w/w.

2.4 Characterization of microsphere templates and polymer vesicles

The morphology of emulsion and microsphere templates was measured by optical microscopy (OM), confocal laser scanning microscopy (CLSM, Zeiss LSM 880 NLO, Oberkochen, Germany), transmission electron microscopy (TEM, JEOL, JEM-1400 Flash, Tokyo, Japan) and scanning electron microscopy (SEM JEOL, JSM-7900F, Tokyo, Japan). SEM samples were sputtered with platinum for 3 min with an accelerating voltage of 5 kV. The diameters of the emulsion and microspheres were quantitatively analyzed by using ImageJ software 1.48 V.

One 10 μL droplet of the polymer vesicle solution was placed on copper TEM grids (Beijing Zhongjingkeyi, Co., Ltd, Beijing, China) and the sample was allowed to sit for 1 minute before blotting to eliminate any excess liquid. The polymer vesicle samples were then stained with a 2% w/w solution of sodium phosphotungstate for 10 s before blotting to remove any excess stain. The self-assembly of BCPs was analyzed by dynamic light scattering (DLS, Brookhaven Omni, New York, USA) for size, size polydispersity index (PDI) and zeta potential. The measurements were performed at a test angle of 90 degrees at 25 °C. The data were averaged across three consecutive measurements.

Fluorescence emission intensity was measured by using Spectra Max iDx (Molecular Devices, San Jose, USA) at 0, 10, 20, 30, 60, 120, 240 and 360 min after rehydration.

2.5 Storage stability of polymer vesicles

The polymer vesicles were preserved at 4 °C and 25 °C, respectively. DLS measurements were conducted on 0, 1, 2, 4, 5, 8 and 10 days, respectively. The auto correlation function exhibited a monodisperse characteristic when it adhered to a mono-exponential function, indicating uniformity in size distribution. Conversely, a disperse profile was observed when the auto correlation function did not conform to this behavior, suggesting a broader variation in particle sizes within the sample. This systematic approach facilitated the assessment of stability under different storage conditions.

2.6 Fabrication of Cour-6@polymer vesicles

To prove the approach for the application of hydrophobic drug encapsulation and delivery, Cour-6 was poured into an EA solution and stirred for at least 12 h. Cour-6 was capsulated via this method (microsphere rehydration). Cour-6-loaded polymer vesicles were incorporated into the oil phase to prepare microsphere templates. Cour-6-loaded polymer vesicles were prepared via microsphere template rehydration. Cour-6-loaded polymer vesicles were purified by dialysis in DI water (Spectra Pro 6, Spectrum Labs 3500 Da). After dialysis, the polymer vesicle solution was collected and lyophilized. The resulting lyophilized powder was weighed and then re-dissolved in dichloromethane. Drug loading efficiency (DLE) was calculated using the following equation:where W_loading is the Cour-6 weight in the Cour-6-loaded polymer vesicles and W_total is the total Cour-6 weight in the oil solution. Cour-6 concentrations were measured by UV-vis (Shimadzu, UV-3600Plus, Kyoto, Japan).

2.7 Cell line culture

In this work, 4T1 and HUVEC cell lines purchased from the American Type Culture Collection (ATCC, Manassas, VA) were cultured in DMEM containing 10% FBS and antibiotics under a 5% CO₂ atmosphere.

2.8 Cell viability and cellular uptake assay

Polymer vesicles were incubated with 4T1 and HUVEC cell lines for 24 h and 36 h with different concentration-treated 96 well plates. HUVECs were selected to evaluate biocompatibility due to their widespread applicability in vascular biology and drug development, while 4T1 cells were chosen to explore the therapeutic potential in a disease model, specifically for cancer research. After the incubation, 4T1 and HUVEC cell lines used a CCK-8 kit to test the cell viability at 450 nm. After testing the cell viability, 1 mg mL⁻¹ polymer vesicles were incubated with a cell line for 1 h, 3 h, 6 h and 12 h. The cells were washed with PBS and CLSM images were acquired. Uptake efficiency was measured via the analysis of the blue channel in flow cytometry (BD FACSVerse^TM). Cell viability was calculated using the following equation:

Here, OD_N, OD_P and OD_t are the absorbance values of the samples, negative control (PBS) and positive control (water), respectively.

3. Results and discussion

3.1 Morphological characterization of emulsion and microsphere templates

Microfluidic systems allow for precise control over various parameters that can influence the formation of droplet templates, making them an ideal choice for a wide range of applications.^46,47 Typically, surface tension, inertial force and viscous force will directly influence the preparation of the droplet template formation. Therefore, the flow rate of oil/aqueous solution, collection distance and concentration of BCPs are tested in a microfluidic system. From Fig. 1, it can be seen that emulsion templates were generated with diameters ranging from 20 μm to 250 μm. The emulsion's coefficient of variation (CV) was maintained below 5%, demonstrating excellent control over the size of emulsion templates. The OM results showed that at a fixed flow rate of the disperse phase, the size of the droplets decreased from 82 ± 15 μm to 39 ± 20 μm as the continued flow rate increased (Fig. 1a). And the droplets’ size increased as the dispersed phase's flow rate increased (Fig. S1† and Fig. 1e). This phenomenon can be attributed to the balance between the squeezing force of the continuous phase and the viscous drag acting on the droplets. As the viscous drag force can overcome the interfacial tension force, the stream will form smaller droplets.⁴⁸ The expansion process of the discrete phase was shortened, resulting in a smaller droplet size.⁴⁹ The collection distance was also an important factor affecting the diameter of the emulsion template. As collection distances increase from 80 μm to 470 μm, the size of the droplets increases from 64 ± 16 μm to 197 ± 42 μm (Fig. S2† and Fig. 1c). The size increase can be explained by the hydrodynamic effects that change as the droplets travel further away from the intersection of the inner capillaries, allowing more time for coalescence and facilitating a transition from dripping to jetting modes.⁵⁰ The droplet size changes resulting from variations in flow rates and collection distances reveal the complex interactions between hydrodynamic forces and fluid properties. This complexity necessitates investigating how polymer concentration affects the characteristics of emulsion templates.

Fig. 1 OM images of O/W single emulsion templates and statistical data of the diameters of emulsion templates. (a) OM images of a single emulsion template with different aqueous solutions – 10, 15, 20, 30, 35, 40, 50 and 60 mL h⁻¹, respectively. The concentration of PVA aqueous solution was 0.5% w/w. The scale bar is 300 μm. (b–e) Statistical data of the emulsion template diameter with different oil flow rates, collection distances and BCP concentrations.

To investigate the relationship between BCP emulsion and microsphere templates, specific PEG₁₁₃-b-PLA₁₆₇ emulsion concentrations were deliberately selected for emulsion templates. These concentrations included 0.008%, 0.05%, 0.125%, 0.25%, 1%, 2%, 5% and 10% w/w. The observed droplet diameter varied from 131 ± 25 μm to 34 ± 7 μm (Fig. S3† and Fig. 1b). The BCP concentration will also influence the structure, shape and self-assembly behavior of the microsphere.⁴⁵ When the concentrations ranged from 10% w/w to 0.025% w/w, the diameter of the microsphere varied from 117 ± 5 μm and 35 ± 4 μm. As the concentration decreased to 0.008% w/w, the microspheres collapsed due to insufficient BCPs supporting them (Fig. 2a). It indicates that higher BCP concentrations promote a larger microsphere formation, while lower concentrations result in smaller sizes.

Fig. 2 SEM images of the emulsion templates with different concentrations. (a) SEM images of emulsion templates with different concentrations: 10%, 5%, 2%, 1%, 0.5%, 0.25%, 0.125%, 0.05% and 0.008% w/w, respectively. (b) Schematic picture of the microsphere shape transformation.

The investigation of the morphological changes from the emulsion template to the microsphere was conducted using OM images. The EA emulsion templates (evaporation time: 50 s) reached stability faster than the DCM emulsion (829 s) (Fig. S4†). This may be because of the low density of EA (0.90 g cm⁻³, 20 °C); the PEG-b-PLA emulsions tended to float on the surface between air and the aqueous solution. Therefore, EA molecules could diffuse into air directly rather than into a PVA aqueous solution. This indicates that the evaporation of EA occurs more rapidly, allowing it to achieve a stable state much sooner than DCM. Additionally, the evolution of emulsion evaporation follows the radius-square law.⁵¹ This law states that the square of emulsion radius tends to evolve linearly with the evaporation time.

Building on the understanding of the evaporation time of emulsion, the morphological characteristics of the microspheres were analyzed. As the concentration of BCPs increases, the shape of the microsphere templates changed from red blood cell-like to the spherical microsphere templates (Fig. 2a). The OM images illustrated the transformation of the emulsion during evaporation (Fig. S5†). The shape of the evaporation structures was influenced by at least four processes: the dissolution of the polymer, the radial flow of the solvent driven by evaporation, the diffusion of the dissolved polymer molecules and the swelling of the polymer.⁵² This change in morphology may be attributed to the unbalanced extraction ratio, which caused the aqueous extractant to rapidly remove the organic solvent within the droplet, allowing the BCPs to gradually deposit into a red blood cell-like morphology.⁵³ In summary, BCP concentration critically dictates microsphere morphology, with evaporation kinetics serving as a key structural determinant.

3.2 Morphological characterization of polymer vesicles

Building on the understanding of the microsphere templates, the transformation characteristics from the microspheres to polymer vesicles were systematically analyzed from 0.075% w/w to 0.300% w/w. At the BCP concentration of 0.075% w/w, micro-scale polymer vesicles began to bud from emulsion templates during evaporation (Fig. 3a and c). OM and CLSM observations further confirmed that micro-scale vesicles emerged after 10 minutes of evaporation (Fig. S6 and S7†). Compared to the emulsion template method for the preparation of polymer vesicles, the microsphere template exhibited a lower ‘budding’ concentration.⁴⁵ It can be inferred that EA emulsion has a faster evaporation rate (Fig. S4†). The rapid evaporation likely restricted the time available for the block copolymers to assemble into well-defined molecular membranes, while the insufficient solvent lubrication caused the system to solidify into a microsphere state.

Fig. 3 OM, CLSM and SEM images of PEG₁₁₃-b-PLA₁₆₇ O/W single emulsion templates. (a) OM and CLSM of PEG₁₁₃-b-PLA₁₆₇ O/W single emulsion templates with different concentrations – 0.037%, 0.075%, 0.150% and 0.300% w/w, respectively. The fluorescent agent was Cour-6. (b) Diameter distribution of microsphere template self-assembly in PVA aqueous solution. (c) SEM image of the microsphere templates.

To further investigate the budding phenomenon of these micro-scale vesicles, observations were conducted on the changes in the membrane thickness and size of polymer vesicles. The CLSM image (Fig. 4) indicated that the membrane containing Nile red was confined and hydrophobic with a diameter range from 10–60 μm. Moreover, the SEM image suggested that the average membrane thickness of micro-scale vesicles gradually reduced from 1000 ± 217 nm to 208 ± 62 nm after evaporation for 12 h and 24 h (Fig. 4c). This decrease in membrane thickness may be attributed to the presence of residual organic solvent in the hydrophobic layer of the membrane.³¹ This also indicates that the budding of micro-scale vesicles was facilitated by organic solvents and hydration effects.

Fig. 4 CLSM and SEM images of PEG₁₁₃-b-PLA₁₆₇ O/W microsphere templates and micro-scale polymer vesicles. (a) CLSM and OM of PEG₁₁₃-b-PLA₁₆₇ O/W microsphere templates after evaporating for 12 h. The fluorescent agent was Nile red. N@MSP was equal to microsphere templates containing Nile red. (b and c) Diameter distribution of micro-scale polymer vesicles and the membrane thickness of micro polymer vesicles (0.075% w/w) in PVA aqueous solution. (d) SEM image of the microsphere template and micro-scale polymer vesicle. The red arrows point at the edge of the vesicle's membrane.

It has been demonstrated that mass concentration (w/w) significantly influences the formation of micro-scale polymer vesicles.^1,29 In this study, polymer vesicles were prepared by rehydrating microsphere templates, with concentrations ranging from 0.25% w/w to 10% w/w. And the microsphere template BCP concentration was 5% w/w for its uniform diameter with a CV < 5% (Fig. 3a). TEM analysis (Fig. 5) revealed a distinct morphological transition from micelles to polymer vesicles as the concentration increased from 0.25% to 10% w/w. Analysis of the DLS data (Fig. 5a) revealed that the diameter PDI of polymer vesicles from the film rehydration (size range diameter: 60–1200 nm, PDI: 0.28) was broader than that from microsphere template rehydration (MSP rehydration) (size range diameter: 70–170 nm, PDI: 0.16) at 10% w/w. The PDI of hydrodynamic diameters was determined through the Gaussian analysis of the intensity-weighted particle size distribution.⁵⁴ Therefore, it indicated that polymer vesicles from film rehydration were highly heterogeneous in size. This may be because during the hydration stage; the stirring may help to detach the extra BCP films from the internal surface, making the size distribution broader.⁵⁵ Additionally, TEM micrographs quantitatively supported this finding, showing film-rehydrated vesicles with an average diameter of 300 nm, whereas MSP rehydration yielded 150 nm vesicles, consistent with the DLS results.

Fig. 5 Diameter distribution and TEM image of PEG₁₁₃-b-PLA₁₆₇ polymer vesicles prepared by microsphere template rehydration and film rehydration. (a) Diameter distribution of PEG₁₁₃-b-PLA₁₆₇ polymer vesicles produced from film rehydration and MSP rehydration. The data have been normalized. MSP rehydration means microsphere template rehydration. TEM image of PEG₁₁₃-b-PLA₁₆₇ self-assemblies. (b) TEM image of 10% w/w polymer vesicles prepared by film rehydration. (c–f) Self-assemblies of PEG₁₁₃-b-PLA₁₆₇ prepared by microsphere templates at (c) 10% w/w, (d) 2% w/w, (e) 1% w/w, and (f) 0.25% w/w, respectively.

3.3 Morphological characterization of rehydration

The microsphere template rehydration method is essentially an extension of the film rehydration technique. The mechanism of film rehydration of polymer vesicles originates from membrane unbinding under water attraction forces.²⁶ As the water content increases by steps, the repulsive forces overwhelm the attractive interaction between BCP layers. The system transforms from a bound to an unbound state and the final morphology depends on how the membrane undulations evolve, including the sponge phase or onion-like phase.⁵⁶ This mechanism between microsphere template methods is influenced by water attraction forces and the plasticization of residues of the solvent.³¹ Therefore, the mechanism of the microsphere templates may affect the kinetic formation process of polymer vesicles. Further analysis is needed to understand the reasons behind the varied PDIs observed in the different methodologies. Observations from SEM revealed that the microsphere templates maintained partial stability in shape (Fig. 6). The surface of microsphere templates progressively transitioned from a relatively flat state (0 h) to nano-scale intermediate pores (72 h) as time progressed. Budding is a common mechanism for vesicle generation during rehydration. Typically, the shape of BCP templates restricts both the size of the lamella bilayer and the direction of hydration.^24,31 Consequently, vesicles typically form in layers, leading to a gradual detachment of polymer vesicles from the microsphere templates, resulting in the transformation from a smooth surface to a porous one. However, since SEM has limitations in observing the vesicle budding process in situ, DLS was employed for ongoing monitoring. The number of scattered photons, a parameter measured in DLS, can qualitatively reflect particle concentration. An increase in the number of scattered photons was observed over time, while maintaining concentration and applying mechanical stirring further enhanced the vesicle dissolution rate (Fig. 6 and Fig. S8†). This phenomenon is attributed to the nano-scale vesicle outgrowth, as evidenced by the change in the scattered photon number.

Fig. 6 SEM image of microsphere templates during rehydration. (a–d) SEM image of PEG₁₁₃-b-PLA₁₆₇ O/W microsphere templates at different rehydration times – 0 h, 15 h, 48 h and 72 h, respectively.

To support the aforementioned dynamics of vesicle budding, Cour-6 was loaded into microsphere templates and rehydrated in an aqueous solution. The fluorescence emission intensity of the vesicles exhibited an increasing trend at progressive time intervals (0, 10, 20, 30, 60, 120, 240 and 360 minutes) after rehydration, indicating that as rehydration time increased, an increasing number of vesicles entered the aqueous solution (Fig. S9†). This observation is consistent with the scattering photon number observed in the DLS and SEM analyses. The observed phenomenon can be attributed to the low solubility of Cour-6 in water, which means it could only disperse into the aqueous solution through the dissolution of the polymer vesicles. Moreover, PEG-b-PLA (as a copolymer) has a low critical micelle concentration (CMC)³⁰ and low chain exchange between the BCP chain and the microsphere.^45,57 Amphiphile diffusion manifests not through individual molecules but through the collective diffusion of these nonergodic assemblies.^26,30 The increase in scattered photon numbers and fluorescence emission intensity indicates that the nano-scale vesicles are at least liberated through budding. In summary, the microsphere template rehydration method facilitates vesicle generation through significant morphological changes, while DLS analysis demonstrates the effects of time, concentration and stirring on vesicle formation dynamics. These techniques are crucial for elucidating the dynamics of vesicle formation.

3.4 Stability and biocompatibility

It is crucial to emphasize the necessity of investigating the stability of the vesicles, given the heterogeneous nature of the preparation process, which subjects their formation to kinetic trapping. Polymer vesicles were collected and subjected to preservation experiments at room temperature and 4 °C, followed by size characterization through DLS. For the auto correlation function of polymer vesicles kept at 4 °C (Fig. 7), a delay time of τ ranged from 503 μs to 462 μs. In the context of nanoparticle dispersion, the auto correlation function exhibits a more rapid decay for smaller particles compared to larger particles.⁵⁸ It indicates that it may generate small particles after storing for 8 days. These findings suggest that the vesicles can be preserved for up to 4 days at room temperature and up to 8 days at 4 °C. The fitting of the auto correlation function was analyzed (eqn (3) and eqn (S1)–(S6) and Fig. S10):†^58,59

Fig. 7 Stability of nano-scale polymer vesicles prepared by microsphere template rehydration in PVA aqueous solution. (a–c) Auto correlation function C(τ) of polymer vesicles kept at 0 d, 1 d, 2 d, 4 d, 5 d and 10 d, respectively. Polymer vesicles were saved at 4 °C. (d–f) Auto correlation function C(τ) of polymer vesicles kept at 0 d, 1 d, 2 d, 4 d, 5 d and 10 d, respectively. Polymer vesicles were saved at 25 °C.

where k_B is the Boltzmann's constant, η is the viscosity of the DI water solution, R_h is the rehydration radius and T is the temperature.

Data could be analyzed by fitting the normalized auto correlation function of the scattered intensity g₂(τ) shown in Fig. 7. According to eqn (S1)–(S4),† the auto correlation function fitting results indicated that the diameter of polymer vesicles has changed from 80 nm (storage for 5 days, 4 °C) to 40 and 640 nm (storage for 8 days, 4 °C). And at room temperature, the diameter of polymer vesicles changed from 70 nm (storage for 2 days, 25 °C) to 50 and 270 nm (storage for 4 days, 25 °C). This indicated that the polymer vesicles prepared under these conditions are kinetically limited in their formation and are unstable at conventional temperatures. However, lowering the temperature can extend the time before vesicle instability occurs, which has significant implications for the preparation, drug loading and transport of vesicles.

According to our results, it is evident that microsphere templates made by microfluidics provide a facile and continuous route, enabling high-throughput fabrication of nano-scale polymer vesicles. Therefore, the general applicability of this fabrication approach has been further explored for drug delivery applications. In this study, the model drug Cour-6 was added to an oil solution containing PEG₁₁₃-b-PLA₁₆₇ beads, resulting in the formation of drug-loaded vesicles through microsphere rehydration. The drug loading efficiency of Cour-6 was 91% (the mass of Cour-6 was measured using a UV-Vis spectrophotometer and calculated based on the ultraviolet absorbance concentration–intensity calibration curve, Fig. S9†), indicating that Cour-6 has been effectively incorporated into the polymer vesicles. Cour-6@polymer vesicles were subsequently used to deliver payloads into healthy 4T1 cells and HUVECs in vitro, as evidenced by various assay methods.

To assess the cytotoxicity of our polymer vesicle delivery systems, normal polymer vesicles and Cour-6@polymer vesicles were incubated with 4T1 cells and HUVECs for 24 h and 36 h. Cell viability was subsequently evaluated using an MTT assay. The results demonstrated that after 24 hours of incubation with 4T1 cells and HUVECs, cell compatibility exceeded 95%. After 36 hours, cell viability still remained at 85%, indicating that polymer vesicles made from PEG₁₁₃-b-PLA₁₆₇ and Cour-6@polymer vesicles had excellent biocompatibility (Fig. 8 and Fig. S11 and S12†). These results highlighted the cell biocompatibility of the polymer vesicles derived from the microsphere templates.

Fig. 8 Cellular uptake assay of HUVECs and 4T1 cells. (a) CLSM image of HUVEC cell lines after incubation for 1 h, 3 h, 6 h and 12 h, respectively. Actin = Actin-Tracker Red-555. (b) Time-dependent cellular uptake profile of Cour-6@polymer vesicles with HUVECs. (c) Time-dependent cellular uptake profile of Cour-6@polymer vesicles with 4T1 cells.

The cellular uptake and intracellular distribution of a drug delivery system are vital to ensuring effective drug action. To assess the effectiveness of Cour-6 encapsulated in polymer vesicles, flow cytometry was used to evaluate its impact on 4T1 cells and HUVECs. The increased accumulation of Cour-6 was further investigated by measuring and comparing fluorescence emission intensity levels under different incubation conditions. As shown in Fig. 8b and c, after the endocytosis experiment 3 h later, both 4T1 cells and HUVECs achieved maximum drug uptake. The cellular uptake and intracellular distribution of Cour-6@polymer vesicles were further examined and visualized using CLSM. This approach enabled a detailed examination of how each formulation was internalized and distributed within both 4T1 cells and HUVECs. The Cour-6@polymer vesicles showed a significant increase in the fluorescence intensity from Cour-6 as the incubation time progressed, indicating heightened cellular uptake and effective localization within the cells. These results indicated that polymer vesicles created by microsphere template rehydration exhibited good cellular uptake efficiency and potential for biological applications.

4. Conclusion

In summary, we developed monodisperse microsphere templates using microfluidic control techniques to streamline the preparation of polymer vesicles. A systematic investigation was conducted on the effects of the flow rate, acceptance distance and block concentration on the microsphere templates, which ranged in size from 20 to 250 μm with a CV < 5%. The experimental results demonstrated that BCP concentration in the emulsion template is crucial for generating both micro-scale and nano-scale polymer vesicles. Specifically, BCP concentrations below 0.075% w/w produced micro-scale vesicles, while a concentration of 5% w/w enabled the fabrication of nano-scale vesicles. Various morphological characterization studies successfully showed the morphological transformation of polymer vesicles, indicating that both nano-scale vesicles may form from the microsphere templates. Further results demonstrated that uniform polymer vesicles were produced at 10% w/w with a diameter range of 70–170 nm (PDI 0.16). Additionally, drug loading experiments with the polymer vesicles demonstrated a high drug loading efficiency of 91% for Cour-6. Furthermore, the DLS results revealed that the nano-scale polymer vesicles exhibited excellent stability (5 d, 4 °C). Cell safety and endocytosis tests demonstrated remarkable biocompatibility of the polymer vesicles with rapid cellular uptake (>95% within 3 hours) by HUVEC and 4T1 cells and cell viability exceeding 85% after 36 h. This innovative approach could pave the way for simple and efficient processes and methodologies in the preparation of polymer vesicles. This method has great potential for large-scale production of nano-scale vesicles for drug delivery, nanoreactors and synthetic biology, addressing the demand for adaptable polymeric nanostructures.

Author contributions

Donghua Dong: conceptualization, methodology, data curation, formal analysis, investigation, validation, and writing – original draft. Tong Zhu: biological experiments, data curation and formal analysis. Guoxing Liao: formal analysis and writing – review & editing. Fangrong Tan: biological experiments, data curation and formal analysis. Lei Chen: data curation, formal analysis and validation. Qianqian Yu: data curation, formal analysis, methodology, and writing – review & editing. LinGe Wang: conceptualization, methodology, data curation, funding acquisition, resources, supervision, and writing – review & editing.

Data availability

The data supporting the findings of this study are provided in the ESI† and are supposed to be published alongside the main article. Readers can access the ESI† for detailed datasets and additional information related to this manuscript.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgements

The authors are thankful for the financial support from the Guangdong Province Basic and Applied Basic Research Fund Project (no. 2023A1515012013), the National Natural Science Foundation of China (no. U22A20316) and the GJYC Program of Guangzhou (no. 2024D02J0004).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5bm00377f

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