Regulation of Drug Release by Tuning Surface Textures of

Subscriber access provided by Fudan University

Article

Regulation of drug release by tuning surface textures of biodegradable polymer microparticles Mubashir Hussain, Jun Xie, Zaiyan Hou, Khurram Shezad, Jiangping Xu, Ke Wang, Yujie Gao, Lei Shen, and Jintao Zhu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b02002 • Publication Date (Web): 03 Apr 2017 Downloaded from http://pubs.acs.org on April 7, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Regulation of Drug Release by Tuning Surface Textures of Biodegradable Polymer Microparticles Mubashir Hussain,# Jun Xie,# Zaiyan Hou, Khurram Shezad, Jiangping Xu, Ke Wang, Yujie Gao, Lei Shen, and Jintao Zhu* Key Laboratory of Materials Chemistry for Energy Conversion and Storage (HUST), Ministry of Education, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China #

These authors contribute equally to this work

*Corresponding author. Email: [email protected]

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: Generally, size, uniformity, shape and surface chemistry of the biodegradable polymer particles will significantly affect the drug release behavior in vitro and in vivo. In this paper, uniform poly(D, L-lactic-co-glycolide) (PLGA) and PLGA-b-poly(ethylene glycol) (PLGA-b-PEG) microparticles with tunable surface textures were generated by combining the interfacial instabilities of emulsion droplet and polymer blending strategy. Monodisperse emulsion droplets containing polymers were generated through microfluidic flow focusing technique. Removal of organic solvent from the droplets triggered the interfacial instabilities (spontaneous increase in interfacial area), leading to the formation of uniform polymer particles with textured surfaces. With the introduction of homopolymer PLGA to PLGA-b-PEG, the hydrophobicity of the polymer system was tailored and qualitatively different interfacial behavior of the emulsion droplets during solvent removal was observed. Uniform polymer particles with tunable surface roughness were thus generated by changing the ratio of PLGA-bPEG in the polymer blends. More interestingly, surface textures of the particles determined the drug loading efficiency and release kinetics of the encapsulated hydrophobic paclitaxel (PTX), which followed a diffusion-directed drug release pattern. The polymer particles with different surface textures demonstrated good cell viability and biocompatibility, indicating the promising role of the particles in the fields of drug or gene delivery for tumor therapy, vaccine, biodiagnostics, and bioimaging.

KEYWORDS: Microfluidics, Microparticles, Polymer blends, Rough surface, Drug loading, Controlled release


Page 2 of 35

Page 3 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1. INTRODUCTION Polymeric microparticles with different sizes, shapes, and surface properties have attracted much interest in recent decades because of their wide range of applications in the fields of drug delivery,1 biotechnology,2 display,3 sensing4 and others. When acting as drug delivery vehicles, besides size and chemical composition, surface morphology of the polymer particles is of great importance for delivery and release of therapeutics. Surface properties provide protection to the encapsulated materials and escape from the macrophages.5, 6 Block copolymers (BCPs) can selfassemble into particles with variety of sizes, shapes and surface topologies. Among many of the polymers, poly (lactic acid)(PLA), poly(D, L-lactic-co-glycolide) (PLGA) and their BCPs (e.g., PLGA-b-poly(ethylene glycol) (PLGA-b-PEG) or PLA-PEG) have been widely used as drug 16

7-

or gene delivery carriers17-21 due to their biocompatibility and biodegradability. In general,

introduction of PEG into the biodegradable polymers will provide advantages for protection of the drug encapsulated in the particle core from macrophages and help the drug to be delivered to the site of action safely. Different strategies have been reported to produce polymer particles with various morphologies and surface textures. Various polymerization techniques have been employed to tune the morphologies of the polymer microparticles. For instance, by precise control of emulsification and photopolymerization, Nie et al. generated tripropyleneglycoldiacrylate (TPGDA) particles with different shapes in microfluidic reactors.22 In addition, suspension and swelling-assisted polymerization were used to tune the surface textures of polystyrene (PS) microparticles.23 Recently, modified seeded emulsion polymerization was applied by Huang et al. to tune the surface morphologies of PS microparticles.26 They used γ-irradiation at room temperature and ambient pressure to generate textured microparticles. In addition, poly(methyl



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

methacrylate) microparticles with textured surfaces have been produced through dispersion polymerization.27 By controlling interfacial properties of emulsion droplets containing polymers through selecting suitable surfactants, polymer particles with controllable internal structures as well as particle shapes can be produced.28-30 Moreover, polymer particles with various surface wrinkles or porous structures can be generated by controlling the internal phase separation of emulsion droplets, solvent evaporation rate, and amount of small amphiphiles which can form hydrogen bonds with one of the blocks.24, 25, 31 Release of the encapsulated species depends on the wrinkle features and shell thickness of the polymer particles. Recently, we have demonstrated the generation of amphiphilic PS-b-PEG particles with various surface textures by taking advantage of interfacial instabilities of emulsion droplets.32 In this case, the extraction of the solvent from emulsion droplets containing PS-b-PEG with different block ratios leads to the spontaneous expansion of the interfacial area and generation of polymer particles with porous, budding vesicular and dendritic structures on the surfaces.33, 34 Biodegradable polymer particles are highly desirable when acting as drug carriers. Pisani and co-workers investigated the generation of PLGA-b-PEG microcapsules with rough surfaces by applying emulsification-solvent evaporation technique.35 Recently, flower like PLA-b-PEG microparticles have been produced through rapid removal of oil phase from the emulsion system. Protrusion length on the polymer particles can be tuned by adjusting the block ratio and emulsification conditions (e.g., different surfactants, polymer and surfactant concentration).36 In addition, Milad et al. performed electro hydrodynamic atomization and manipulated experimental parameters (e.g., solvent vapor pressure, molecular weights of polymers or ratio of polymer blends) to generate PLGA microparticles with smooth or textured surfaces.37 In general, bulk emulsion technique gives rise to microparticles with polydispersity in size, and hence


Page 4 of 35

Page 5 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


release of entrapped bioactives is not well controlled. Uniformity of the polymer particles significantly influences the biodistribution of the particles in body, enhanced permeability and retention effect, release kinetics of the loaded drugs and resulting therapy efficiency.38-41 On the other hand, although Cremophor EL formulated paclitaxel (PTX) is still the most widely used formulation at clinical level,42 new formulations are required to circumvent the problems of its low solubility in aqueous solution and side effects (e.g., hypersensitivity and neurotoxicity).43, 44 Nowadays, it is still a challenge to generate biodegradable polymer particles with tunable sizes and surface textures in a facile and effective manner for controlled drug delivery and release. In this report, we will demonstrate the generation of uniform biodegradable polymer particles with tunable sizes and surface textures through the assistance of microfluidic technique and interfacial instabilities of emulsion droplets containing binary blends of PLGA-b-PEG and PLGA. In this case, hydrophobicities of the polymers can be tailored by just regulating the ratio of PLGA-b-PEG in the polymer blends without bothering chemical synthesis of BCPs with desirable block ratios. Furthermore, biodegradable polymer microparticles with varied roughness were used to encapsulate hydrophobic PTX with high loading efficiency. Interestingly, release profiles of these drug carriers demonstrate that surface roughness of the microparticles can control the drug release kinetics. Our results indicated that the formed uniform polymer particles with controllable sizes and surface textures are biocompatible and tunable in drug release kinetics and are potentially useful in drug and gene delivery, bioimaging, vaccines and biodiagnostics. 2. EXPERIMENTAL SECTION 2.1. Materials: PLGA100k (Mw=100,000) and PLGA-b-PEGs (PLGA50k-b-PEG5k, PLGA10k-bPEG20k and PLGA5k-b-PEG5k) were obtained from Daizheng Biotech., China. All PLGAs 5 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

consisted of lactide and glycolide with the ratio of 50:50. Poly(vinyl alcohol) (PVA, Mw = 13k– 23k, 87–89% hydrolyzed), Sodium Dodecyl Sulfate (SDS, Purity ≥ 98%) and dichloromethane (DCM) was purchased from Sigma-Aldrich. n-hexadecanol (HD) was supplied by Sinopharm Chemical Reagents, China. PTX (C47H51NO4) was obtained from Dalian Meilun Biotech., China. Nile Red (purity: 99%) was purchased from Arcos organics. Deionized water was produced using Heal Force Millipore Apparatus. All the chemicals were used as received without any further purification. 2.2. Generation of Uniform Microparticles: Glass capillary devices were used to generate uniform sized oil-in-water emulsion droplets containing polymers (Figure 1). Briefly, round glass capillary was tapered and nested inside a square capillary. Polymer blends at a concentration of 10 mg/mL in DCM were used as dispersed phase while aqueous solution containing 5 mg/mL PVA, acting as stabilizer to prevent the coalescence of formed emulsion droplets, was used as continuous phase. Oil and aqueous phases were injected into the capillary devices through syringe pumps to control the flow rates of both phases. The formed emulsion droplets were collected in a homemade evaporation device containing small amount of aqueous solution with same PVA concentration, where the organic solvent from the droplets was allowed to evaporate slowly at room temperature by diffusion through the aqueous phase to the air. The evaporation device was made by gluing a plastic tube with inner diameter of ~ 3 cm on a clean glass slide (Figure 1b). After complete evaporation of the solvent, the formed polymer particles were dialyzed against deionized water for a period of one week to remove residual organic solvent and/or PVA. The resulting microparticles were then freeze-dried and stored for further investigation. 2.3.Characterization 6 ACS Paragon Plus Environment

Page 6 of 35

Page 7 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Evolution of the emulsion droplets was observed under Olympus Microscope (IX71) equipped with high speed camera in bright field and fluorescence modes. Morphology of the polymer microparticles was studied by using Scanning Electron Microscope (Sirion 200 SEM) at an accelerating voltage of 10 kV. A dilute suspension of polymer particles was dropped on a clean silicon wafer and water was allowed to evaporate. A thin layer of gold was sprayed on particles before SEM analyses. Transmission Electron Microscope (TEM, Tecnai, G2-20) was employed to investigate the internal structures of the formed assemblies. For TEM sample preparation, a very dilute aqueous suspension of the polymer assemblies was transferred on a copper grid, where water was allowed to completely evaporate at room temperature before observation. Surface area of the polymer particles with various surface roughness was measured from Brunauer-Emmett-Teller (BET, NOVA 3200e surface area and porosity analyzer) by N2 adsorption and desorption. The above formed polymer particles were washed and freeze-dried for 72 h before analysis. Water contact angle on the polymer microparticles films was measured by JC2000C1 Contact Angle Measuring System (Shanghai Zhongchen Digital Technol. Instrument Ltd Co., China) at 25 oC. For sample preparation, concentrated suspensions of the polymer microparticles with different surface textures were deposited on clean glass slides. Removal of water from the samples at ambient temperature resulted in the formation of particles films. Before microparticle films preparation, polymer particles were washed with deionized water to remove the surfactant. 2.4. Determination of Encapsulation Efficiency (EE %) and Drug Loading (DL %): PTX was loaded in the polymer microparticles with different surface roughness. In general, PTX along with the polymer blends was dissolved in the oil phase (weight ratio of polymer/PTX = 10:1). Similar procedure was employed to produce PTX loaded microparticles, as described in section



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 35

2.2. The formed microparticles were subsequently washed for 3-5 times by centrifugation to remove PVA and unentrapped drug. The EE% and DL% were measured by UV-vis spectrophotometer (Rayleigh UV-1801). A standard curve for neat PTX solution was obtained at a detection wavelength of 227 nm (characteristics wavelength of PTX). EE% and DL% was calculated according to equation 1 and 2: EE% =

DL% =

!

"

× 100%

× 100%

(1) (2)

2.5. In vitro Drug Release: In vitro drug release was carried out in a dialysis tubing by suspending a weighed amount of PTX loaded polymer particles in phosphate buffer solution (PBS, pH 7.4). The samples were incubated at 37 oC and set on shaking at 200 rpm for 2 weeks. At intervals (0.5, 1, 2, 3, 4, 6, 8, 10, 12, 24, 48h,…), 1 mL of samples was withdrawn and replenished with 1 mL fresh PBS. The amount of PTX released was measured by UV-vis spectrophotometer at wavelength of 227 nm. The experiments were carried out for three times. 2.6. Cellular Uptake Experiments: Cell uptake and localization of Nile Red labelled microparticles was determined for A375 and B16-F10 cancer cell lines obtained from Shanghai Maisha Biotech., China. The cells were maintained in DMEM medium with 10% fetal bovine serum (FBS) (Gibco, Grand Island, USA). Both cell lines were cultured in an incubator humidified at 37 oC with 5% CO2 atmosphere. Qualitative determination of cellular uptake of Nile Red labelled polymer particles was carried on Confocal Laser Scanning Microscope (CLSM, Leica TCSNT1, Germany). A375 and B16-F10 cells were seeded at 5×105 cells per well in a 6 well plate. Polymer microparticles were fluorescence dye labelled by adding 0.1 wt% Nile Red relative to the polymer in oil phase before


Page 9 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


emulsification. These Nile Red labelled microparticles were then co-cultured with cells for 24 h. Finally, cells were washed with PBS for four times to remove any free microparticles outside the cells. Paraformaldehyde (4%, v/v) was used to fix the cells for 15 min, and again washed for three times with PBS. Nuclei and lysosomes of cells were stained with Hoechst 33342 and Lyso Tracker, Green DND-26 (Thermo Fisher, USA) respectively to get blue signal for nuclei and green signal for lysosomes under CLSM observation. By using glycerin, cover slips were sealed and imaged under CLSM. 2.8. In Vitro Cytotoxicity: In vitro cytotoxicity of the polymer particles was furnished by the CCK-8 Assay. B16-F10, A375 and RAW cells at a density of 104 were seeded in 96 well plates for 24 h. Polymer microparticles with final concentration of 100 µg/mL were also added in the well plate for 24 and 48 h. Control experiments were conducted for cells without polymer microparticles. Cells were washed for two times with PBS, and 10 µL of CCK8 reagent was added in 100 µL PBS in each well plate. After incubation for 2 h at 37 °C, proliferation of the cells was determined by absorption intensity at 450 nm with a microplate reader (Infinite®F50, Tecan Austria, Austria). All the experiments were performed for three times. 3. RESULTS AND DISCUSSION 3.1. Generation of Uniform Polymer Microparticles Generation of monodisperse emulsion droplets containing polymers was carried out by glass microcapillary device, and removal of organic solvent resulted in the formation of uniform polymer particles (Figure 1). PLGA-b-PEG with different block ratios, i.e., PLGA50k-b-PEG5k (PEG weight ratio WPEG = 10%), PLGA5k-b-PEG5k (WPEG= 50%), and PLGA10k-b-PEG20k (WPEG = 67%) were conducted to generate BCPs assemblies with various morphologies. Briefly, the BCPs at a concentration of 10 mg/mL were dissolved in DCM, acting as dispersed phase in 9 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 35

microcapillary device. 5 mg/mL PVA was added to the aqueous continuous phase to stabilize the formed emulsion droplets. The two phases were pumped into the microcapillary devices, and the dispersed organic phase was broken into monodisperse emulsion droplets due to the shearing force and interfacial tension. The formed droplets were collected in a small homemade evaporation device in a certain amount of PVA aqueous solution to prevent coalescence of the formed droplets (Figure 1b). By putting the evaporation device in open atmosphere, droplets were allowed to evaporate and shrink. Evolution of the emulsion droplets was investigated under an inverted optical microscope as the concentration of the dissolved polymers in the droplet increased during droplets shrinking. Ratio of the hydrophilic block (e.g., PEG) plays an important role in the interfacial behavior during solvent evaporation and surface morphology of the resulting polymer particles. When neat PLGA100k (WPEG = 0%) was employed, emulsion droplets were observed to shrink continuously during solvent evaporation process. Complete removal of the solvent led to the formation of spherical and smooth particles with uniform sizes (see Figure 2 a-f). By increasing PEG ratio to 10% (PLGA50k-b-PEG5k, WPEG=10%), different interfacial behavior was observed during solvent evaporation of the emulsion droplets. The droplets shrunk first, expanded, and interfacial area spontaneously increased when the concentration of the polymers in the droplets reached a critical value (Figure 2i and Supporting Information (SI), Movie S1). In this case, the droplet did not remain smooth, and bumpy appearance was observed (Figure 2i-2k). The protrusions would partially retract back, and further evaporation of solvent triggered the generation of tiny protrusions (sheet-like structures) on particle surface. Polymer particles with roughness were thus obtained (seen from SEM image in Figure 2l). Similar but qualitively different interfacial behaviors were observed when BCPs with higher PEG contents were


Page 11 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


employed (PLGA5k-b-PEG5k, WPEG=50% or PLGA10k-b-PEG20k, WPEG=67%). The emulsion droplets shrunk first, then broke into tiny droplets and finally disappeared, resulting in the formation of cylindrical and spherical micelles with nanoscale features (Figure S1 in the SI and Movie S2). The above observed interfacial roughness or spontaneous breakage of the emulsions into tiny droplets can be ascribed to the interfacial instabilities. Similar interfacial roughening and spontaneous droplet breakage have been found in oil-water-amphiphile systems, polymer blends and emulsion droplets containing amphiphile BCPs, and the driving force for the interfacial instabilities have been ascribed to the vanishing of interfacial tension.34, 45, 46 During solvent evaporation, concentration of the BCPs increases and the amphiphilic polymers start to move to the oil/water interface. Interfacial tension is reduced with the migration of hydrophilic PEG to the interface.47,

48

Subsequently, the droplets can not maintain their spherical shape, and

spontaneous droplets breakage or protrusion generation occurs (Movie S1, S2 in the SI). Similarly, in our previous report,32 we have observed that interfacial tension approached zero during solvent evaporation, triggering the interfacial instabilities of the emulsion droplets containing amphiphilic BCPs. We note that, besides block ratios, overall molecular weight (Mw) of the BCPs plays some role in the interfacial instabilities. Lower Mw (PLGA5k-b-PEG5k and PLGA10k-b-PEG20k) leads to lower viscosity in similar polymer concentration, which triggers the breakage of the emulsions into tiny droplets instead of protrusion generation. This rapid emulsification and ultimate breakage of the emulsion droplets leads to the generation of polymer nano-assemblies (e.g., spherical or cylindrical micelles). On the other hand, at high Mw, complete breakage and



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 35

dispersion of droplets is highly suppressed due to the enhanced viscosity of the system, and the protrusions on the droplets are frozen to form microparticles with rough surfaces. 3.2. Tuning Surface Textures of Polymer Particles by Binary blends As mentioned above, assemblies with different structures can be generated through interfacial instabilities of emulsions containing BCPs with different block ratios and Mw. Yet, tedious steps are needed to synthesize BCPs with precise and controllable structures. Alternatively, introduction of homopolymer into BCPs is a simple and effective route to tailor the interactions and thus morphologies of the assemblies. For example, we have previously reported the effect of hydrophobic PS on micellar diameter of PS-b-PEG by blending.46 In this work, PLGA-b-PEG microparticles with different surface textures can be produced when tuning block ratio by simply varying blending ratio of PLGA to PLGA-b-PEG. To do this, we started by blending PLGA50k-bPEG5k with PLGA100k before emulsification. PLGA50k-b-PEG5k/PLGA100k microparticles with varied surface roughness were generated through this facile strategy. When PLGA50k-b-PEG5k was blended with PLGA100k, similar interfacial instabilities were observed. For example, when the blend ratio of PLGA50k-b-PEG5k/PLGA100k was 70/30, solvent removal triggered the spontaneous increase of interfacial areas with the generation of small protrusions on the droplet interface (Figure 2o-q and Movie S3 in the SI). Polymer particles with textured surfaces were obtained with complete removal of organic solvent. Yet, when PLGA100k ratio approached high level (80%, for example), interfacial instabilities were highly suppressed due to high viscosity of the blended system and lower ratio of the PEG content. In this case, polymer particles with slight rough surfaces can be obtained. Overall size of polymer microparticles with varied surface textures was measured, and the results indicated that size of microparticles in each batch remained constant because of the microfluidic technique (Figure 3). Yet, variation of the surface


Page 13 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


textures on the particles results in a variation for particle diameter when employing initial emulsion droplets with same size (Figure 3a-e). In short, this strategy allows us to generate biodegradable polymer particles with tunable surface roughness and diameter by just simply varying content of PLGA50k-b-PEG5k in the blends (Figure 3). Surface area of the resulting polymer microparticles with varied roughness was characterized through BET measurement. The results shown in Figure 4 demonstrate that the surface area of the rough microparticles is much higher than that with lower roughness or smooth particles. At high ratio of PLGA50k-b-PEG5k in the blends, the expansion of PEG in the space is more as compared to the sample with low PLGA50k-b-PEG5k ratio. This expansion causes the increase in the surface area as compared to smooth and slight rough microparticles. For the polymer blends, the protrusions were frozen during solvent evaporation due to the enhanced viscosity with the addition of homopolymer, leading to the formation of particles with spiky and rough surfaces. These spiky protrusions increase the surface area of the formed microparticles. To test the generality of this technique, PLGA-b-PEG with different block ratio (e.g., PLGA50k-b-PEG5k and PLGA10k-b-PEG20k) were also employed to generate assemblies with varied morphologies through the binary blending strategy. Similarly, polymer microparticles with enhanced roughness were obtained with the increase of PLGA10k-b-PEG20k ratio up to 50%. When the ratio of PLGA10k-b-PEG20k was further increased to 60%, 70% and 80%, cylindrical assemblies instead of polymer particles were obtained (Figure S2 in the SI). Thus, our technique can be applied to different PLGA-b-PEG systems to produce polymer particles with tunable surface textures. 3.3. Regulation of Drug Loading and Release by Tuning Surface Textures



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 35

Surface textures of the polymer particles can significantly influence the drug loading and release kinetics. In this work, PTX was selected as a model drug, which is a hydrophobic anticancer drug and effective against different cancers (e.g., breast, ovarian, small lung cancer and Kaposi sarcoma). In general, PTX requires delivery vehicles for its transport to the tumor cells due to its hydrophobic nature. Typically, PTX with desirable amount was dissolved in DCM together with polymer (e.g., PLGA100k or PLGA50k-b-PEG5k/PLGA100k blends) before emulsification. Similar procedure was performed to produce PTX-loaded polymer particles. To make it consistence, concentration of the polymers in initial emulsion droplets and size of the droplets was maintained the same in all samples through microfluidics processing. In this case, amount of the polymer and drug in each microparticle was kept the same. Encapsulation efficiency (EE %) and drug loading (DL %) of PTX in different polymer particles were determined through UV-vis spectrophotometer measurements (Figure 5). Clearly, surface textures of the polymer particles significantly affect the EE % and DL %. Highest EE% was found in case of neat PLGA100k. With the increase of PLGA50k-b-PEG5k ratio in the binary blends, the EE% was observed to decrease from 92 ± 2% for neat PLGA100K to 64 ± 2% for neat PLGA50k-b-PEG5k. The reason for EE % variation can be attributed to the change in ratio of hydrophilic to hydrophobic component in the polymer microparticles. Increase in the PLGA amount will enhance the hydrophobic component which is benefit for the increase of PTX loading efficiency since hydrophobic PTX will be encapsulated in the hydrophobic domain of the polymer microparticles. Meanwhile, increase in PLGA50k-b-PEG5k will give rise to polymer microparticles with rough surface (e.g., enhanced surface area), which will also suppress the encapsulation efficiency of drug in the particles. To observe the effect of surface textures on release behavior, polymer microparticles with same DL% while with different surface roughness


Page 15 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


were studied in vitro. To do this, we first generated polymer particles with same DL % by manipulating the amount of drug in the initial emulsion droplets. Loading of PTX (8.02 wt %) in the PLGA50k-b-PEG5k/PLGA100k (ratio of PLGA50k-b-PEG5k=80%) polymer microparticles (overall size: 4.5 ± 0.33 µm) will give rise to the increase of the particle size (~ 0.75 µm) due to the entrapment of PTX in hydrophobic domain of particles while maintain the surface textures of the particles. Release of PTX was carried out at 37 oC in PBS (pH 7.4) to mimic the systemic conditions. The cumulative release profile for different samples is displayed in Figure 6. Clearly, initial burst release was observed for all the particle samples, which can be ascribed to the release of loosely incorporated drug from the microparticles’ matrix.49 After initial burst release, the release profile shows a gradual increase in the plots, indicating the uniform release of PTX due to the uniformity of particle size and drug distribution in microparticles. From Figure 6, we can see clearly that the highest release of PTX is from most rough particles (e.g., polymer binary blends with 80% PLGA50k-b-PEG5k content) while the slowest one was that with smooth microparticles (neat PLGA100k). Therefore, release of PTX was observed to be enhanced with the increase of surface roughness of the polymer microparticles. Water contact angle measurement reveals that increase of surface roughness (high PLGA-b-PEG content in the polymer blends, Figure S3 in the SI) triggers the enhanced hydrophilicity and interfacial area of the microparticles, which presumably increases the drug diffusion through hydrophobic domain. The higher surface area of the microparticles (Figure 4) makes more area of the microparticles to be available for water adsorption and hence enhanced wettability, leading to increased release as compared to those with less rough particles. It is worth noting that neat PLGA50k-b-PEG5k has relatively low release kinetics than that of binary blend particles with 80% PLGA50k-b-PEG5k. The reason can be attributed to the lower



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 35

surface area of neat PLGA50k-b-PEG5k (Figure 4). As mentioned above, the protrusions on the surface of the shrunk emulsion droplets containing neat PLGA50k-b-PEG5k would partially retract back and fuse together to form short sheet-like structures, leading to the reduction in surface area. To further clarify the role of surface textures on release kinetics of encapsulated drug, we prepared microparticles with smooth and textured surfaces while with same component (e.g., PLGA10k) following our previous report.50 In general, co-surfactant n-hexadecanol (HD) was added to emulsion droplets containing PLGA to trigger the interfacial instabilities and generate polymer particles with textured surface (Figure S4b and Movie S4 in the SI). The results indicated that textured PLGA10k particles showed enhanced PTX release as compared to smooth ones (Figure S4c in the SI). Therefore, we believe that surface area (e.g., surface roughness) and surface hydrophilicity of the polymer microparticles are the key factors in controlling the release of PTX, demonstrating the effective strategy to control drug loading and release kinetics by binary blending. 3.3. Modeling of Drug Release Drug release kinetics was modeled mathematically to assess the mass transport mechanism involved in drug release from microparticles with varied roughness. Polymer degradation for the microparticle matrix is not significant for the experimental period in this study (Figure S5 in the SI) because in vitro degradation from PLGA-based polymers usually starts after 5 weeks in previous report51-53; therefore it is believed that the release of the drug is diffusion controlled.51-54 Fick’s Second law of diffusion was thus employed to construct mathematical model for release of PTX from the polymer microparticles. Following assumptions were taken into consideration while drawing the model: i) Polymer microparticles are spherical in shape; ii) PTX distribution


Page 17 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


in microparticles is uniform; iii) Uniform initial drug concentration; iv) Constant diffusion coefficients and v) Perfect sink conditions. A mathematical model describing the release of drugs from spherical microparticles has been given by Siepmann and co-workers when diffusion is found to control the release process.55 This model follows Fick’s Second law of diffusion: #$ #%

= &'

#( $

#) (

+

+#$ )#)

,

(3)

Where c in equation (3) represents concentration of the drug, t is the time, D shows diffusion coefficient and r is radial coordinates. By applying boundary conditions, the solution of the above equation is given below:56 -.

-/

1

= 1 − 2( 3

6 574

4

5(

89: '−

5( 2 ( )(

&;, +

Regulation of Drug Release by Tuning Surface Textures of

Recommend Documents