Fusion and Self-Assembly of Biodegradable Polymer Particles into

Article pubs.acs.org/molecularpharmaceutics

Fusion and Self-Assembly of Biodegradable Polymer Particles into Scaffoldlike and Membranelike Structures at Room Temperature for Regenerative Medicine G. Rajmohan, Prasad Admane, Chakkumkal Anish, and Amulya K. Panda* Product Development Cell, National Institute of Immunology Aruna Asaf Ali Marg, New Delhi-110067, India S Supporting Information *

ABSTRACT: We report here a novel surfactant mediated fusion of polylactide particles into scaffoldlike structures at room temperature. In the presence of ethanol, evenly spread surfactant coated polylactide particles fused immediately into membranelike structures. Polymer scaffolds of the desired shape and size could be fabricated from polylactide particles using this fusion process. Desorption of surfactant molecules from the surface of the particles during ethanol treatment and the degree of solubility of the polymer in alcohol were found to be the main reasons for the fusion of particles into a scaffold at room temperature. TGA and DSC studies of the polylactide particles showed that the particles were stable at room temperature, and FTIR studies showed that there was no change in characteristics of the polymer after the fusion of particles into a scaffold-type structure. These scaffolds supported three-dimensional growth of animal cells in vitro and release model protein in a sustained manner for a long period of time. In an experimental animal wound model, the polylactide membranes showed faster wound closure, indicating its use as a passive dressing material. This polymer particle fusion process thus provides a novel method of scaffold fabrication for various biomedical applications. KEYWORDS: polylactide particles, surfactant mediated fusion, scaffold, cell growth, controlled release, wound healing

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INTRODUCTION Polylactic acid (PLA) and polylactic-co-glycolic acid (PLGA) are biodegradable polymers being extensively used for various biomedical applications especially as scaffolds in the field of tissue engineering.1−3 Ideally, scaffolds formulated using biocompatible and bioresorbable polymers like PLA should possess a well-defined macrostructure and microstructure with controlled porous architecture to promote cell attachment and proliferation.4−6 Apart from this, release of appropriate growth factors from the scaffold may promote controlled vascularization and tissue growth in three dimensions.7,8 Various methods like particulate leaching, emulsion freeze-drying, phase inversion technique, solvent casting, electrospinning, and thermal sintering have been employed to formulate scaffolds using PLA and PLGA for tissue engineering applications.6,9−13 PLA and PLGA nanofibers have also been used extensively as scaffolds particularly for skin tissue engineering.14−16 The particulate leaching method is widely used to fabricate scaffolds, but there are problems of residual salts in the scaffolds, irregularly shaped pores, and poorly interconnected structures for three-dimensional cell cultures.17 Polylactide particles or wafers wetted transiently with an organic solvent to form scaffolds have been tried.18,19 One of the drawbacks of such scaffold fabrication processes is the exposure of polymer to organic solvents and heat. This results in loss of polymer © 2014 American Chemical Society

characteristics and denaturation of labile biomolecules entrapped in the scaffold. Such processes have limitations for designing of a biomimetic scaffold for entrapping or controlled release of drugs/biologicals for tissue engineering applications. Self-assembly is the organization of smaller units into regular three-dimensional higher order structures without human intervention or involvement of external energy.20−22 The classical example is the self-assembly of lipid molecules in nature into tubular microstructure.23 Surfactant mediated selfassembly and synthesis of novel materials has been extensively investigated for various biomedical applications including selfassembly of nanoparticles into higher order structure.24−26 Scaffold made from self-assembling peptide nanofiber as well as polylactide composite membrane has been reported to accelerate wound healing.27,28 However, the major limitations of self-assembly of molecules into scaffolds are their inability to control pore size and lack of stable morphology. Polylactide composite membrane (Suprathel) is currently used as skin Special Issue: Engineered Biomimetic Tissue Platforms for in Vitro Drug Evaluation Received: Revised: Accepted: Published: 2190

February 4, 2014 April 29, 2014 May 1, 2014 May 1, 2014 dx.doi.org/10.1021/mp500106u | Mol. Pharmaceutics 2014, 11, 2190−2202

Molecular Pharmaceutics

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above. For preparation of fluorescent particles, 50 μL of coumarin-6 (Polysciences cat no. 8037L, USA) dye (1 mg/mL in dichloromethane) was added to OP during primary emulsion formation. Particle size distribution was measured using “Malvern mastersizer 2000” particle size analyzer (Malvern, U.K.). Polymer particles were suspended in Milli Q water having 1% Tween 20 before measurement. Fusion of Polymer Particles into Membrane-Type Scaffolds. Surfactant coated PDLLA particles were uniformly spread in Petri dishes and wetted with 100% ethanol. This resulted in fusion of particles immediately into membrane-type structures. This scaffold was elastic in nature and flexible while still in the solvent but hardened after the solvent was washed off with water. The resultant scaffold was washed thrice with sterile Milli Q water. Membranes of different dimensions (desired size and thickness) and shapes can be fabricated depending on the cast used, provided the particles are in physical contact with each other. Fusion of microparticles did not occur by treatment with Milli Q water and resulted in dispersion. For fabricating three-dimensional structures, the PDLLA-CTAB particles were spread on Petri dishes with different sizes and wetted with ethanol to form membrane-type structure. Digital movies of PDLLA-CTAB particles undergoing fusion in the presence of ethanol were taken using Canon A 530 camera at 5× magnification. The details of the movies are given as follows. Movie 1: PDLLA-CTAB particles undergoing fusion in the presence of ethanol. Movie 2: PDLLA-CTAB particles in the presence of water forming dispersion. Particles formulated with different types of polymers like PDLLA, PLGA, and PLLA with CTAB in EAP were tested for fusion by ethanol and methanol. The ability to form scaffolds was assessed visually, and desorption of CTAB was checked by zeta potential measurements before and after alcohol treatment. Solubility of different polymers in methanol or ethanol was tested by treating them overnight with solvents and analyzing the nature of polymer pellets. Microscopy Analysis of Fused Polymer Particles and Scaffolds. Fluorescence and bright field images of PDLLACTAB particles were taken using a Nikon ECLIPSE Ti−S fluorescent microscope. For scanning electron microscopy (SEM), scaffolds formed by fusion of polymer particles were dehydrated and placed on aluminum stubs for gold−palladium sputter coating (Electron Microscopy Sciences SC 7620). Polymer particles, fused membrane, and fusion bridges between the particles were visualized by SEM, model Zeiss EVO LS10. For transmission electron microscopy (TEM), suspensions containing fused nanoparticles were placed onto copper grids (Polysciences, Warrington, PA, USA) and coated with 1% uranyl acetate. Imaging was carried out using CM 10, Philips, Holland, using digital imaging softwareAMT image capture engine (version 5.42.391). Optical images of the fused particles were also taken under bright light in monochrome mode. Atomic force microscopy (AFM) of the surfactant coated particle surface after ethanol treatment was carried out to study surface topology using NanoScope (Veeco, USA). The samples were deposited onto mica supports or glass slides, and images were recorded. Amplitude−distance curves were used to optimize resolution and contrast in the semicontact mode of the atomic force microscope. Estimation of Surfactant Concentrations on the Surface of the Polymer Particles. Concentration of CTAB on the surface of the polymer particles was estimated by measuring the zeta potential of the particles before and after

substitute for treatment of burn victims but has the limitation of not supporting dermal growth or entrapping bioactive growth factors.29 It will be ideal if scaffold from polylactide polymer can be fabricated without using high temperature or dissolving them in organic solvent. Self-assembly of polylactide polymer particles to higher order structure having desired size, shape, and topology at room temperature is thus an attractive approach over conventional methods of scaffold fabrication. Polymer particle entrapping suitable growth factor/biomolecules can be fused to form biomimetic scaffolds. These scaffolds, thus apart from providing suitable geometry and surface topology, can release suitable growth factors/ biomolecules for cell growth. In this paper, a novel method of PDLLA scaffold fabrication has been described where surfactant molecules mediate the fusion of polylactide particles at room temperature into membrane-type structures. The mechanism involved in such surfactant mediated fusion of polylactide particles into a membrane-type structure at room temperature was investigated in detail. Polylactide scaffold in the form of membrane was tested for its potential for the growth of animal cells in three dimensions, for controlled delivery of biomolecules, and as a passive dressing material for wound healing. This method provides a novel way of designing biomimetic scaffolds for tissue engineering and controlled drug delivery.

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EXPERIMENTAL SECTION Preparation of Surfactant Coated Polymer Particles. Poly D,L-lactic acid (PDLLA) (inherent viscosity (i.v.) 0.55− 0.75 dL/g in CHCl3), poly lactic glycolic acid (PLGA) (i.v. 0.26−0.54 dL/g), and poly L-lactic acid (PLLA) (50 kDa) were purchased from Lactel, Durect Corporation, USA. Polymer particles were prepared using a double emulsion solvent evaporation method.30 The primary emulsion was prepared by mixing 2 mL of internal aqueous phase (IAP) consisting of 10% sucrose and 5% bovine serum albumin (BSA) as an emulsifier in Milli Q water with 4 mL of organic phase (OP) consisting of 200 mg of polymer dissolved in dichloromethane (DCM) by sonication (20 W, 40% duty cycle, 20 cycles) (Bandelin, Germany) for 3 min. This primary emulsion was dripped slowly into 300 mL of external aqueous phase (EAP) containing different surfactants like cetyltrimethylammonium bromide (CTAB) (cationic) or sodium dodecyl sulfate (SDS) (anionic) or Tween 20 (neutral) (Amresco, USA) at a concentration of 1% w/v with continuous stirring on a magnetic stirrer. The secondary emulsion was continuously stirred overnight on a magnetic stirrer to evaporate dichloromethane. Surfactant molecules in the EAP get coated on the surface of polymer particles during the secondary emulsification step of the solvent evaporation method. Particles were collected by settling and washed with Milli Q water to remove excess surfactant. Particles were freeze-dried for 20 h and further used for characterization like size estimation and microscopic examination. Polymeric micro- and nanoparticles were prepared by varying the energy inputs during emulsion preparation and the OP to EAP volume ratio as described earlier.31 Surfactants with different charge properties were used to evaluate their ability to form scaffolds. Particles prepared with 1% (w/v) poly(vinyl alcohol) (PVA) (Sigma Chemicals, USA) in EAP were used as control. A high concentration of sucrose in IAP (10%) helped to generate pores in the particle.32 Surfactant coated PLLA, PLGA, and polystyrene (PS) particles were prepared using the solvent evaporation method as described 2191

dx.doi.org/10.1021/mp500106u | Mol. Pharmaceutics 2014, 11, 2190−2202


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measured. To confirm the role of solvents (methanol/ethanol) in the desorption process of CTAB, PDLLA-CTAB particles were suspended overnight in Milli Q water and tested for loss of zeta potential. Characterization of Polymer Scaffolds after Fusion. Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC). Thermogravimetric analysis of plain PDLLA polymer and PDLLA-CTAB particles was carried out on a PYRIS series TGA 7 (PerkinElmer, USA). Each sample was analyzed from 30 to 500 °C at a scan rate of 10 °C. Differential scanning calorimetric analysis of the surfactant coated particles was carried out using a PYRIS series DSC 1 (PerkinElmer, USA). Samples were weighed and heated from 40 to 200 °C at 10 °C/min to record the DSC curve. Fourier Transformed Infrared Spectroscopy. Infrared (IR) spectroscopy was carried out using a PerkinElmer FT-IR system spectrum BX. Lyophilized PDLLA-CTAB microparticles with CTAB as an excipient (5 mg) were mixed with KBr and were pressed into disks (0.5 mm in thickness) for measurement. For the PDLLA-CTAB membranes, they were broken into small bits and mixed well with KBr for recording IR spectra. Three-Dimensional Cell Cultures of Animal Cells on PDLLA Scaffolds. For culture of animal cells, PDLLA-CTAB scaffolds were sterilized with overnight treatment of 70% ethanol, after which ethanol was washed off with Milli-Q and scaffolds were soaked in DMEM with 10% fetal calf serum (Gibco, USA) for 12 hours before inoculation with cells. Human breast cancer cells like MCF-7 and B16 melanoma were maintained in DMEM containing 10% FCS (Gibco, USA) and 1% antibiotic (Sigma A5955). Each scaffold was seeded with 0.05 × 106 cells in 50 μL of complete medium. Cells were allowed to attach for 3 h at 37 °C in a CO2 incubator, after which DMEM medium was added. Media were changed every other day. Growth of cells was observed under bright field microscope (Nikon Eclipse Ti). The scheme below describes in detail the protocol for cell seeding and proliferation.

treatment with ethanol. Apart from this, a spectrophotometric method was also used to estimate the CTAB concentration on the particle surface before and after ethanol treatment.33 A standard curve was constructed using CTAB solutions with concentrations ranging from 0 to 2 mM. 0.1 mL of methyl orange, 0.5 mL of buffer solution (0.5 M citric acid and 0.2 M disodium hydrogen phosphate mixed in equal volume), and 2.5 mL of chloroform were added into each sample. The tubes were vigorously vortexed to separate the aqueous and chloroform layers, and CTAB bound methyl orange in the chloroform layer was read at 415 nm in a UV/spectrophotometer, Ultrospec 2100 pro (Amersham Biosciences, USA). Samples of PDLLA-CTAB particles (5 mg) were treated with 1 mL of water or ethanol for 1 min, and the supernatants were collected after centrifugation. Ethanol treated samples were kept in a water bath at 80 °C until the ethanol was completely evaporated, and CTAB was resuspended in 1 mL of water. The test samples were treated similarly as described above, and the CTAB concentrations in each sample were estimated using a standard curve. A colorimetric method was employed to check the concentration of PVA desorbed from PDLLA particles during ethanol treatment.34 Briefly, PDLLA-PVA particle samples pretreated with ethanol were lysed with 2 mL of 0.5 M NaOH for 15 min at 60 °C. Samples were neutralized with 900 μL of 1 N HCL, and the volume was adjusted to 5 mL with distilled water. To each sample, 3 mL of 0.65 M solution of boric acid, 0.5 mL of a solution of iodine/potassium iodide (0.05 M/0.15 M), and 1.5 mL of distilled water were added. Absorbance of the samples was measured at 690 nm after incubation for 15 min at room temperature. Colorimetric estimation of SDS from PDLLA-SDS particles was carried out before and after ethanol treatment.35 Particles were treated with water or ethanol, and the washes were used for estimation of desorbed surfactant molecules. The ethanol phase was evaporated as described above, and the SDS molecules were suspended in water. Test samples (0.3 mL) were mixed with an equal volume of the methylene blue reagent, and the mixture was extracted with 1.2 mL of chloroform by vortexing. After centrifugation, the lower organic phase was transferred to an eppendorf tube and the absorbance of the supernatant was taken at 651 nm with chloroform as reference. Concentration of SDS in the test samples was calculated using a standard curve. Leaching of CTAB from the particle surface after treatment with ethanol/methanol also was estimated by congo red fading reaction.36 Ethanol was evaporated at 37 °C, and residual CTAB was dissolved in Milli Q water. 100 μL of this sample was added to a reaction mixture consisting of 280 μL of 0.1 mM congo red solution in Milli Q, 100 μL of HCl−sodium acetate buffer pH 6, and 520 μL of Milli Q water. Absorbance was taken at 512 nm on a UV/spectrophotometer, Ultrospec 2100 pro (Amersham Biosciences, USA). Quantification was carried out on the basis of a CTAB standard plot. Zeta Potential Measurements of Polymer Particles and Scaffold. Zeta potential measurement was carried out to confirm differential adsorption of CTAB on particles with respect to CTAB concentration in EAP. Zeta potential measurements of polymer particles before and after treatment with different alcohols were carried out in phosphate buffered saline (PBS) using Nano Z (Malvern Instruments, U.K.). Particles were formulated with 1%, 0.1%, 0.03%, 0.016%, 0.008%, and 0.0016% w/v CTAB in EAP. These particles were also tested for fusion by treatment of methanol. Scaffolds were broken down into fine fragments, and the zeta potential was

Cell proliferation was monitored by MTT assay at different time points. Cell seeded scaffolds were treated with MTT dye (0.05 mg/mL) at 37 °C in a CO2 incubator for 4 h. Scaffolds were then dissolved in 1 mL of DMSO, 200 μL of this solution was transferred to 96-well microtiter plates, and absorbance was read at 570 nm with reference to 630 nm. Cellular attachment and spreading on the scaffold were analyzed by scanning 2192



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Figure 1. Size distribution and morphology of the surfactant coated polymer particles. (A) Size analysis of PDLLA-CTAB polymer particles by Malvern Mastersizer 2000. The particles have an average size of 300 μm. (B) Scanning electron micrograph (SEM) of PDLLA-CTAB particles. The particles were discrete and highly porous. (C) SEM of a single PDLLA-CTAB particle showing porous surface.

electron microscopy. Briefly, cell seeded scaffolds were fixed with fixative (4% paraformaldehyde, 2.5% gluteraldehyde in 0.1 M sodium cacodylate) for 3 h at room temperature. Lipids were fixed for 1 h with a 1% solution of osmium tetroxide in 0.1 M sodium cacodylate buffer. Dehydration was achieved by changes of serial gradations of ethanol concentrations (25%, 50%, 75%, and 90% for 5, 7, 10, and 20 min respectively followed by 3 changes of 100% ethanol for 30 min each). Samples were further dried, mounted on aluminum stubs by gold palladium sputter coating, and checked by SEM. For analysis of the internal structure of the scaffold, cell seeded scaffolds were fixed overnight with 2.5% glutaraldehyde in PBS pH 7.4. Scaffolds were embedded in tissue freezing medium (OCT fluid, Jung), and 5−8 μm thick sections were taken at −26 °C on a Cryotome (Shandon, Thermo Fisher Scientific, USA). Sections were collected on egg white coated glass slides and stained by hematoxylin or DAPI (nuclear stain) for observation of distribution of cells. Imaging was done using the fluorescent microscope Nikon Eclipse Ti−S. Expression of MMP-2 by Cells Grown on a Polymer Scaffold. Three-dimensional (3D) growth of MCF-7 cells was compared to two-dimensional (2D) growth by evaluating the expression of martix metalloproteases (MMP-2), which is a tumor progression marker.37 MMP-2 expression increases when tumor cells start growing in a spheroidlike condition and is a hallmark of 3D culture of tumor cells.37,38 MMP-2 expression was compared by detection of the protein in cell culture lysate of cells grown on scaffolds or on tissue culture plates by Western blotting. Lysates were probed with antihuman MMP-2 antibody (CST 1:1000 in 1% lactogen, overnight at 4 °C). New born calf serum was used as positive control. Beta actin was used as loading control. Controlled Release of Biomolecules from PDLLACTAB Scaffold. PDLLA-CTAB microparticles were used to formulate controlled drug delivery implants by entrapping fluorescent isothiocyanate conjugated bovine serum albumin (FITC-BSA). Microparticles were formulated using a solvent evaporation method as described earlier. Microparticles were prepared by adding 50 μL (IAP to OP = 1:40) or 100 μL (IAP

to OP = 1:20) of internal aqueous phase consisting of 10% sucrose and 2.5% model protein (FITC-BSA). A known amount of dried microparticles was treated with acetonitrile to dissolve the polymer and to extract the entrapped protein. The pellet obtained was dried and dissolved in 1 mL of 1% SDS, and FITC-BSA was quantified directly (Ex-340, Em- 450) in a microtiter plate on fluorimeter (Fluostar BMG Labtech, Germany). Concentrations were calculated from the respective standard graphs. Entrapment efficiency of BSA was calculated by using the ratio of the actual load divided by the theoretical load of BSA. PDLLA-CTAB microparticles and their respective scaffolds were evaluated for in vitro release of FITC-BSA. A weighed amount of PDLLA-CTAB microparticles entrapping FITC-BSA microparticles was resuspended in sterile PBS pH 7.4. Particles were kept for shaking in an orbital shaker at 37 °C, 200 rpm. Microparticles were centrifuged at 13000 rpm for 10 min at 4 °C, and supernatants were collected for estimation of released protein by taking fluorescence readings at excitation wavelength 485 nm and emission at 520 nm. Scaffolds fabricated from PDLLA-CTAB particles (10 mg) entrapping FITC-BSA were treated similarly to particles to study the in vitro release of FITC-BSA. Evaluation of Polymer Membrane As Skin Substitute in Animal Wound Model. Animals were maintained according to the guidelines established by the Institute Animal Ethics Committee (IAEC) of the National Institute of Immunology, New Delhi. Preliminary evaluation of the polymeric membrane for wound healing was tested on Wistar rats. All surgical procedures were carried out under anesthesia. For the initial experiments, the polymeric membrane was evaluated for noninfected full skin thickness wounds (2 × 3 cm) on the dorsum of rats. Animals were divided into treated and untreated groups (n = 6). In the treated group, polymeric membrane formed immediately after the fusion process was directly transferred onto the wounds. Control rats were treated with a conventional wound dressing of paraffin gauze and cotton bandage. Wound scoring was done at different time intervals until complete closure of the wounds was observed.39 2193



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Figure 2. Microscopic analysis of the polymer scaffold. (A, B) SEM images of PDLLA-CTAB membranes after fusion of particles by ethanol. Fusion bridges among the particles can be seen prominently after the treatment with ethanol. (C, D) Transmission electron microscopy (TEM) showing surfactant mediated fusion of PDLLA-CTAB nanoparticles. In panel D, nanoparticles can be seen to be fused to each other through the surface.

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RESULTS Formulation and Characterization of Polymer Particles. Polylactide particles of different sizes were prepared using the solvent evaporation method.31 The PDLLA-CTAB macroparticles were made porous by incorporating sucrose in both internal and external aqueous phases during the particle formulation.32 Surfactant coating was achieved by incorporation of CTAB in the external aqueous phase during the secondary emulsification step of the solvent evaporation method. Particles prepared with different polymers by the double emulsion solvent evaporation method were porous and spherical in nature. Particles made from PDLLA, PLGA, and PLLA having PVA or other surfactants (CTAB, SDS, Tween 20) in the EAP showed similar morphology and mean geometric size of 300 μm (Vd10 100 μm−Vd90 378 μm) (Figure 1A). In comparison to other polymers, macroparticles made of PDLLA and PLGA with CTAB as a surfactant were most uniform with better physical properties. PDLLA-CTAB macroparticles were of uniform size and have pore in their surface (Figures 1B and 1C). PDLLA microparticles and nanoparticles were prepared as described earlier31 using CTAB in the external aqueous phase. The PDLLA-CTAB microparticles had an average size of 3−20 μm, and nanoparticles had an average size of 200−400 nm. Fusion of Polymer Particles into Membrane Type Structures. Fusion of PDLLA-CTAB macroparticles was carried out at room temperature in the presence of ethanol. Uniformly spread PDLLA-CTAB microparticles upon treatment with ethanol fused instantaneously with each other to form scaffolds (Movie 1). PDLLA-CTAB particles when wetted with water did not fuse and remained as a dispersion (Movie 2). The scaffold formed was elastic and flexible in the presence of ethanol but hardened upon addition of water. Removal of ethanol and washing with water resulted in a stable polymeric membranelike structure. Fusion of microparticles was also observed to occur with treatment of methanol. Scaffolds and regions of fusion between the particles were examined by scanning electron microscopy (SEM). These regions consisted of links of polymer from surface of particle

attaching with each other forming a stable network structure (Figure 2A,B). Particles of different size were also tested and found to fuse by treatment with methanol. CTAB coated PDLLA microparticles and nanoparticles fused as efficiently as the surfactant coated macroparticles. Fused nanoparticles observed by TEM also show the regions of fusion (Figures 2C and 2D). Fusion of PDLLA-CTAB nanoparticles was also associated with point attachment of the particles with each other at the surface (Figure 2D). By spreading the polymer particles in different sized Petri dishes and wetting with ethanol, different sized membranes were fabricated at room temperature (Figures 3A and 3B). Ten milligrams of PDLLA-CTAB particles upon fusion yielded a polylactide membrane of around 2 cm2. For the fabrication of three-dimensional structures, of different thickness, the PDLLA-CTAB particles were spread in Petri dishes and wetted with ethanol to form different sizes of scaffolds. Coumarin-6 labeled PDLLA-CTAB macroparticles were fused to form scaffolds and visualized under a fluorescence microscope (Figure 3C). The connective junctions are indicative of regions formed by the desorbed surfactant molecules between particles, through which the polymer is hypothesized to have transiently solubilized and led to fusion of particles. Spherical morphology of the particles was observed to remain intact, and polymer extensions were observed at the point of contact between the particles (Figure 3D). This indicated that the fusion of polymer particle was confined to the surface of the polymer particles. PDLLA particles coated with PVA (PDLLA-PVA) did not fuse and maintained their discreteness both in water and in ethanol. When PDLLA polymer pellets without any surfactant were wetted with DCM, ethanol, and water respectively, PDLLA pellets got solubilized in DCM and fused, but they did not solubilize and fuse immediately in the presence of ethanol and water. Wetting of PDLLA polymer pellets in the presence of both CTAB and ethanol did not result in fusion of polymer. Only particles coated with surfactant molecules fused into membrane type structure in the presence of ethanol at room temperature. 2194



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molecules were desorbed from the surface of the particles during ethanol treatment. Different batches of polylactide particles were prepared with 1%, 0.1%, 0.03%, 0.016%, 0.008%, and 0.0016% w/v CTAB in the external aqueous phase (EAP). Particles with 1% CTAB showed a positive zeta potential of +45 ± 10 mV, which decreased while using lower concentrations of CTAB during particle formulation (Table 2). Particles with 0.016% CTAB had a positive zeta potential Table 2. Effect of CTAB Concentration in EAP on Zeta Potential and Fusion of PDLLA-CTAB Particles

Figure 3. Fabrication of polymeric membranes and three-dimensional structures using surfactant mediated fusion of PDLLA-CTAB particles. (A) Polymeric membranes of different sizes fabricated by fusion of PDLLA-CTAB particles by treatment of ethanol at room temperature using Petri dishes. (B) PDLLA-scaffolds of different thickness and shapes ready for three-dimensional growth of cells. (C) Formation of polymeric bridges between fluorescent PDLLA-CTAB particles after the fusion process. Fine polymeric bridges can be seen between the surfaces of adjacent particles. (D) Formation of polymeric bridges between particles after the fusion process as observed by optical microscopy.

Table 1. (a) Extraction of Surfactant from Particles after Treatment with Ethanol or Water and (b) Residual PVA Content in Particles and Scaffold (a) Extraction of Surfactant from Particlesa extraction in ethanol 12 μg/mL 1.55 mM (b) Residual PVA Content

SDS CTAB condition

a

extraction in water 4 μg/mL 0.15 mM

PVA content (μg/mg of particle)

PDLLA-PVA microparticles ethanol untreated ethanol treated (scaffold)

zeta potential (mV)

fusion

1 0.016 0.008 0.0016

+45 ± 10 +16 ± 5 −0.5 −20.8

+ + − −

value of +16 ± 5 mV, whereas particles with 0.008% and 0.0016% CTAB showed a negative zeta potential value of −0.5 and −20.8 mV respectively. This decrease in positive zeta potential value and the shift toward negative values indicated a decrease in the amounts of positively charged CTAB molecules on the surface of the polylactide particles. After ethanol treatment, PDLLA particles with 1% CTAB in EAP was broken down into smaller fragments and the zeta potential measured. These fragments showed a negative zeta potential value of −20.7 mV, which indicated desorption of CTAB molecules from the particles during ethanol treatment. For confirmation of CTAB desorbed from PDLLA-CTAB microparticles upon treatment with ethanol, CTAB was quantified by congo red fading reaction in subsequent ethanolic fractions after scaffold fabrication. Almost complete CTAB extraction took place after three rinses, and no CTAB was detected in the fourth eluates (Supplementary 1 (Figure 1) in the Supporting Information). Overnight treatment of the PDLLA-CTAB particles in Milli Q water did not substantially change the zeta potential of the particles, indicating that the surfactant molecules are stably adsorbed on the particle surface. Polylactide particles prepared with 1%, 0.1%, 0.03%, and 0.016% w/v of CTAB fused when wetted with ethanol, whereas particles prepared with 0.008% and 0.0016% CTAB did not fuse, indicating that the adsorbed surfactant molecules were not sufficient to mediate the process of fusion in the latter two cases. It was observed that the process of fusion was immediately initiated on wetting with ethanol and after the process of fusion; further fusion between two such scaffolds did not occur in the presence of ethanol. PDLLA particles with surface coating of cationic (CTAB), anionic (SDS), and neutral emulsifier (PVA) were evaluated for fusion process. Concentrations of CTAB, SDS, and PVA were measured by a colorimetric method to evaluate the effect of ethanol on the surfactant coated particles. When 5 mg of PDLLA-CTAB particles was treated with ethanol and water for 1 min to check rapid desorption of surfactant molecules, 1.55 mM of CTAB desorbed into the ethanol phase in comparison to only 0.15 mM of CTAB into the water phase. Polylactide particles prepared using SDS/Tween 20 in the external aqueous phase also fused into higher order structures in the presence of ethanol. In the case of SDS coated PDLLA particles, in the presence of ethanol SDS was desorbed into the ethanol phase at a concentration of 12 μg/mL, while 4 μg/mL of SDS was desorbed in the presence of water. PDLLA-PVA particles did not fuse in the presence of ethanol, and colorimetric estimation

Desorption of Surfactants from Particle during Ethanol Treatment. It was of interest to know the role of surfactant in such a fusion process as only surfactant coated polymer particles fused in the presence of ethanol. The presence of CTAB on the polymer particle surface during ethanol treatment was quantitated by measuring the surface charges of the particles as well as using a colorimetric method. Colorimetric assays show that ethanolic fractions utilized to form scaffolds from PDLLA microparticles extract almost 10fold higher levels of surfactant in the case of CTAB and 3-fold in the case of SDS (in comparison to treatment with water) (Table 1a). Colorimetric estimation of PVA for PDLLA-PVA

surfactant

percentage of CTAB in EAP

15 14

Particles taken = 5 mg. Treatment time, 1 min.

microparticles, both before and after ethanol treatment, showed that PVA is stably adsorbed on PDLLA-PVA particles during ethanol treatment. No difference was observed in residual levels of PVA between ethanol treated and untreated groups of PDLLA-PVA microparticles (Table 1b). Zeta potential measurement of PDLLA-CTAB microparticles before and after ethanol treatment indicated that CTAB 2195



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Figure 4. Thermogravimetric analysis (TGA) of PDLLA polymer and particles: (A) thermogram of plain PDLLA polymer and (B) thermogram of PDLLA-CTAB particles.

of PVA, both before and after ethanol treatment, showed that PVA is stably adsorbed on PDLLA-PVA particles during ethanol treatment. The PDLLA-PVA particles (5 mg) retained 14 μg of PVA per mg of particles after ethanol treatment, while untreated PDLLA-PVA particles had 15 μg of PVA per mg of particles. These observations suggested that the fusion of polylactide particles in ethanol was associated with desorption of surfactant molecules from particle surface and was independent of the charge and nature of the surfactant molecules. It was also observed that, in spite of more desorption of SDS molecules into the water phase in comparison to CTAB molecules, there was no fusion of PDLLA-SDS particles in the presence of water. This indicated

that, in addition to rapid desorption of surfactant molecules from the particle surface, solubility of the polymer in the solvent phase also plays an important role in the fusion process. Fusion of Different Polymer Particles Using Other Alcohols. To evaluate whether CTAB molecules can mediate the fusion of other related polymer particles in ethanol, poly Llactide (PLLA), poly D,L-lactide-co-glycolide (PLGA), and polystyrene (PS) particles were prepared with CTAB molecules. No fusion of particles was observed with PLLACTAB, PLGA-CTAB, and PS-CTAB particles in the presence of ethanol despite CTAB desorption from the surface of particles in all cases as checked by zeta potential measurements. However, in the presence of methanol, PLGA-CTAB particles 2196



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Figure 5. Surface analysis of the PDLLA-CTAB scaffolds by scanning electron micrograph (SEM) and atomic force microscopy (AFM). (A) SEM of the scaffold surface showing pores. These allow free flow of medium into the scaffold, which is beneficial for cell growth. (B) AFM image of the particle shows rough topology of the particle surface at the nanoscale level; visible pores are indicated by arrows. (C) SEM image shows topology of fused porous particles forming the scaffold. (D) Topology of the scaffold promotes attachment and proliferation of cells. Arrow indicates a single cell attached just after incubation of the scaffold with B16 melanoma cells.

fused well. PDLLA-CTAB particles also fused well with methanol, and it was also observed that the fusion process with methanol was more rapid as compared to ethanol. It seems that, in addition to the desorption of the surfactant molecules in the presence of alcohol, the degree of solubility of the polymer particles in the alcohol also influenced the process of fusion. To assess the solubility profile of the above polymers in ethanol and methanol, equal amounts of PDLLA, PLGA, PLLA, and PS pellets were taken and kept in 50 mL of ethanol and methanol and observed overnight. After overnight treatment, it was observed that the PDLLA pellets stuck to each other in ethanol, and almost fused to a single mass in methanol. In the case of PLGA, there was hardly any sticking of the pellets in ethanol, but with methanol the pellets stuck to each other. PLLA and PS pellets did not fuse and maintained their discreteness both in ethanol and in methanol even after overnight treatment. The comparative solubility profile of the polymers in ethanol and methanol correlated well with the process of fusion of these polymer particles. Solubility of PDLLA is higher in methanol as compared to ethanol, and the process of fusion of PDLLACTAB particles in the presence of methanol was more rapid and stronger than with ethanol. In the case of PLGA, its solubility in ethanol was poor as compared with PDLLA, but showed better solubility in methanol. PLGA-CTAB particles fused well with methanol, but not with ethanol. PLLA and PS have poor solubility in methanol and ethanol, and PLLA-CTAB and PS-CTAB particles did not fuse in the presence of either of the solvents. The process of fusion in PDLLA-CTAB particles with ethanol and methanol was inhibited by adding PLLA to PDLLA in the ratio of 40:60 respectively during particle formulation. Effect of Temperature in Surfactant Mediated Fusion of Polymer Particles. Temperature can play an important

role in fusion of polylactide particles, since raising the temperature above the glass transition temperature (Tg) of PDLLA (which is about 60 °C) can result in fusion of PDLLA material or particles. Residual water present in the polylactide particles prepared by the double emulsion solvent evaporation method has been reported to lower the Tg of the polylactide particles.40 Thermogravimetric analysis (TGA) of PDLLACTAB particles was carried out to assess moisture content in the particles and the effect of excipients used in the formulation process like CTAB, sucrose, and bovine serum albumin on the thermal stability of the particles. The thermogram of PDLLA polymer alone shows a single stage thermal degradation (Figure 4A). Thermogram of PDLLA-CTAB particles (Figure 4B) showed no weight loss around 100 °C, indicating that the porous macroparticles were well lyophilized with minimal presence of water in the particles. The TG curve of the particles shows thermal degradation in stages with weight loss of 97% at 384 °C as compared to 99% weight loss at 379 °C for PDLLA. This was due to the presence of excipients like the surfactant molecules in the polymer particles. The DSC thermogram of the PDLLA-CTAB particles was analyzed to substantiate the findings of TGA, and the DSC curve showed an endothermic peak at 59 °C (Supplementary 2 (Figure 2) in the Supporting Information) characteristic for PDLLA polymers.41,42 An endothermic peak between 100 and 110 °C characteristic of absorbed moisture was not observed for the particles in the DSC curve. In summary, TGA and DSC studies showed that the PDLLA-CTAB macroparticles were relatively free of moisture content and the polymer showed little or no interaction with the excipients used in the formulation process. To further check the stability of the PDLLA-CTAB particles at ambient temperature, one batch of dry particles was kept at 37 °C in an incubator. The particles were stable and no fusion 2197



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Figure 6. Three-dimensional growth of B16 melanoma and MCF-7 breast cancer cells on polymer scaffold. (A−D) Growth of B16 cells on days 2, 4, 6, and 8, respectively. (E−H) Growth of MCF-7 cells on polymer scaffold on days 2, 4, 6, and 8, respectively. In both cases, on day 2, the cells can be seen attached to the surface, and on day 4, the cells start growing as multilayer growth. By days 6 and 8, the cells can be seen growing as threedimensional culture.

the polymeric membranes formed from the surfactant coated particles, and the cells showed high viability (>90%) until 7 days, beyond which there was a gradual decrease in viability. This can be attributed to confluence or due to necrosis of cells in the inner region of growth due to hypoxia (Figure 6 D). MCF-7 cells grew in a similar way as B16 cells on polymer scaffold (Figure 6E−H). Figures 6E and 6F represent MCF-7 cell growth on the second and fourth days whereas the cells grew like tissue-type structure on the sixth and eighth days (Figures 6G and 6H respectively). Growth kinetics of MCF-7 and B16 cells on PDLLA scaffold was carried out, and it was observed that more than one log cell growth could be achieved in 7 days (Figures 7 A and 7B). Growth kinetics of B16 cells on polymer scaffold (Figure 7B) was faster than that of MCF-7 cells (Figure 7A). It is expected that optimization of growth in controlled environments could help to grow more cells per unit area of the scaffold so that they can be used for an in vitro model for drug testing. Cell growth on PDLLA scaffold was also analyzed using SEM, and it was observed that the scaffold supported growth of both B16 and MCF-7 cells (Figures 8A and 8B). Western blot analysis of lysate of MCF-7 cells showed MMP-2 expression only when grown on polymer scaffolds (Figure 8C). Cell seeded or unseeded scaffolds (5 μm thick sections) were cryosectioned to study the particle internal structure and distribution of growing cells. Sections showed the internal porous nature of the particles (Figures 9A, 9B, and 9C). Hematoxylin and DAPI staining showed that cells were seen attached to the scaffold surface as well as in the interior of the particles (Figures 9D_1−D_6). Cells were uniformly distributed on the surface and in the void spaces between the particles as indicated by DAPI staining as well differential interference contrast (DIC) images (Figure 9D_3 and Figure 9D_5). Studies have shown that three-dimensional cultures of cancer cells are a better model in comparison to monolayer cultures.46,47 Thus, these three-dimensional cultures of cancer cells can be used as a model system for in vitro evaluation of anticancer drugs. PDLLA-CTAB Scaffolds As Controlled Drug Delivery Implants. To fabricate drug/protein eluting scaffold, polymer particles entrapping protein could be used to form higher ordered structure. PDLLA-CTAB particles entrapping FITCBSA were formulated and fused to form membrane-type structure. Microparticles entrapping protein were made with

took place for 24 h inside the incubator. PDLLA-CTAB particles were wetted with chilled ethanol at different temperatures, and fusion of PDLLA-CTAB particles was observed in the presence of ethanol even at −20 °C. PDLLA-CTAB particles and PDLLA-CTAB scaffold were analyzed by FTIR spectroscopy. The FTIR spectra of PDLLACTAB particles showed characteristics of polylactic acid as reported in the literature43 (Supplementary 3 (Figure 3) in the Supporting Information). Bands at 1456 and 1383 cm−1 correspond to CH3 group vibrations. The intense sharp band at 1759 cm−1 is contributed by CO vibration, and the band at 2946 cm−1 corresponds to CH stretch. The broad band between 3200 and 3600 cm−1 represents the presence of absorbed moisture. The IR spectra of the PDLLA-CTAB membrane show similar characteristic peaks as PDLLA-CTAB particles (Supplementary 4 (Figure 4) in the Supporting Information). Three Dimensional Growth of Cancer Cells on the PDLLA-CTAB Scaffold. The topology of the PDLLA-CTAB scaffold at both the microscopic and macroscopic scales looks promising for three-dimensional cultures of animal cells. At the micro scale, it was observed that the porous nature of the particles allows free flow of medium thus supporting cell growth (Figure 5 A). AFM images of the particles showed that the surface had a rough topology at the nano scale (Figure 5B), which may be suitable for cell attachment and growth.44,45 Washes of the PDLLA-CTAB scaffold after 2 h of treatment with ethanol showed negligible presence of CTAB molecules. Removal of the surfactant after the fusion of polylactide particles in ethanol provides a surfactant free nontoxic environment for the growth of cells. Ethanol treated PDLLACTAB scaffolds were found to be suited for three-dimensional cultures of animal cells. Earlier, we had reported growth of cancer cells on individual PDLLA-PVA particles.32 Here we show that scaffolds composed of the fused PDLLA-CTAB particles (Figure 5C) are also suited for three-dimensional growth of animal cells. After the incubation of the scaffolds with the cancer cells (B16 melanoma cells), initially few cells preferentially attach to the crevices between the fused particles (Figure 5D). Within 2−4 days, the attached cells start spreading and proliferating, resulting in a multilayered growth on the scaffold (Figures 6A and 6B). By days 6−8, multilayered tissuelike structures are formed by the cancer cells on the scaffold (Figures 6C and 6D). Toxicity was not observed with 2198



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Figure 9. Analysis of the internal structure of PDLLA scaffolds. (A) Particles were found to be hollow inside or (B) semiporous. (C) Hematoxylin staining shows that cells were uniformly distributed throughout the scaffold. (D_1−6) Distribution of cells in PDLLA scaffolds as seen by DAPI staining. Cells were seen to be growing on the surface as well as in the interiors of the particles. D_5 shows a differential interference contrast (DIC) image of individual particles where cells have been penetrated. D_ 3 and D_6 are the overlap images of DAPI and DIC showing cell attachment in the surface and interior of the particles. Figure 7. Growth kinetics of cancer cells on polymer scaffolds. Cell growth is presented as OD values of MTT assay. (A) Growth kinetics of B16 melanoma cells on PDLLA scaffolds. (B) Growth kinetics of MCF-7 cells on polymer scaffolds. The results are average values of three independent experiments.

50 μL of IAP. (Figures 10A and 10B). In vitro release profiles of protein from scaffolds were also analyzed. The release rates correlated to IAP/OP ratios of particles and their corresponding scaffolds. However, the cumulative amount of released protein from scaffolds was lower, as compared to that observed with free microparticles. Microparticles of 1:20 IAP/OP ratio (100 μL of IAP, porous particles) released almost 99% protein in 30 days whereas corresponding scaffolds released only 23%. Microparticles of 1:40 ratio (50 μL of IAP, compact particles) released 40% protein, and corresponding scaffolds released only up to 7% of entrapped protein in the same duration. These differences in release rates of entrapped protein among particles and corresponding scaffolds can be attributed to the lower surface area available to scaffolds in contact with the surrounding medium compared to the particles. However,

different IAP to OP ratios of 1:40 and 1:20. Particles of 1:20 IAP/OP ratio (100 μL in IAP) resulted in porous particles with lower entrapment efficiency (18%), whereas particles formulated with 1:40 IAP/OP ratio (50 μL IAP) resulted in formation of less porous particles with high entrapment efficiency (55.5%). The difference in porosity also had an effect on the protein release rates from the microparticles and their corresponding scaffolds (Figures 10A and 10B). Release of FITC-BSA from porous microparticles (100 μL of IAP) was faster that that observed from less porous particles made with

Figure 8. Scanning electron microscopy of cells grown on PDLLA scaffold. (A) Growth of B16 cells on scaffold on day 4. (B) Growth MCF-7 cells on scaffold on day 4. (C) Comparison of MMP-2 expression for MCF-7 cells cultured on PDLLA scaffolds and tissue culture (TC) plate. MMP-2 expression was detected only in cells cultured on PDLLA scaffolds whereas cells cultured on tissue culture plate did not express MMP-2. MMP-2 present in serum sample was used as positive control. Actin was used as loading control. 2199



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Figure 11. Evaluation of polylactide polymeric membrane formed by fusion process for wound healing. (A) 2 × 3 cm uninfected open wound, day 5. (B) Open wound treated with the polymer membrane, day 5. (C, D) Rats after 21 days of treatment: (C) rat with open wound; (D) rat treated with polymeric membrane. The wound closure was better in the group treated with the polymeric membrane.

closure. Thus, the polymer membrane can be used as a passive dressing material for wound management. Antibacterial agents such as antibiotic and/or growth factor entrapped in polymer particles can be fused to form polymer membrane which may provide an alternative to artificial skin.

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DISCUSSION Self-assembly and fusion of polylactide polymer particles to scaffolds of desired size and shape and particularly to membrane-type structures at room temperature is an attractive procedure for scaffold fabrication. Normal scaffold fabrication techniques for PLA/PLGA polymers are cumbersome and detrimental to the entrapment of labile biological molecules. For design of biomimetic scaffolds it will be ideal if the polymer is biodegradable and can entrap and release biomolecules to promote particular cellular activity. In such a scenario, surfactant mediated scaffold fabrication at room temperature is an attractive alternative, since particles containing the desired biomolecules can be self-assembled into stable scaffolds of the desired design at room temperature. Surfactant (CTAB/Tween 20/SDS) coated polylactide particles, when treated with ethanol, rapidly fused to form a polymeric network resulting in membranelike structures. Fusion was associated with desorption of the surfactant from the particle surface along with formation of polymeric bridges between particles. These polymeric bridges were indicative of the transient regions formed by the polymer molecules which were solubilized to fuse into a membrane-type structure. Normally, surfactant free PDLLA fuse when wetted with organic solvents like dichloromethane (DCM), where it is soluble, but not with ethanol. Ethanol wets porous PLA/PLGA polymer scaffold better than water,49 but was not able to fuse PDLLA at room temperature. Wetting PDLLA pellets or PDLLA-PVA particles with ethanol containing 1% CTAB or even after vortexing did not resulted in fusion of the polymer. This inferred that the mode of action of the surfactant molecules in the rapid fusion of PDLLA-CTAB particles in ethanol is different from its usual way of solubilization of sparingly soluble substances in solvents through formation of micelles. Polymer scaffold made from the fused particles had all the characteristics of PDLLA polymer as indicated by FTIR analysis. This indicated that fusion is mostly a surface phenomenon not associated with changes in the bulk properties of the polymer. The exact mechanism by which the CTAB molecules mediate PDLLA particle fusion during ethanol treatment remains to be elucidated; however,

Figure 10. In vitro release profile of FITC-BSA from polymer particles and scaffold. (A) In vitro release of FITC-BSA from PDLLA-CTAB microparticles formulated with different volumes of internal aqueous phases [■, porous particles (IAP 100 of μL)] and [◆, less porous particles (IAP 50 of μL)]. (B) In vitro release of FITC-BSA from scaffolds made from particles formulated having different internal aqueous phase volume [■, scaffold made from porous particles (IAP 100 of μL)] and [◆, scaffold made from less porous particles (IAP 50 of μL)]. Particles and scaffold made from particle having high internal aqueous phase volume were more porous and thus released the FITCBSA at a faster rate in comparison to particles or scaffold made with low volume of internal aqueous phase. The results are average values of three independent experiments.

these results demonstrate that the scaffold promotes controlled release of the entrapped protein. This indicated that these scaffolds apart from promoting cell growth can be used to release suitable growth factor or biomolecules like drug delivery devices. Evaluation of PDLLA Membrane As Artificial Skin Substitute. Polylactide membrane formulated by the above fusion process was evaluated as an artificial skin substitute for wound healing in experimental animal models.39,48 The PDLLA membranes were directly transferred onto noninfected full thickness wounds of Wistar rats to evaluate whether the polylactide membranes were biocompatible and would not delay wound healing. The control rats were treated with traditional wound dressing of paraffin gauze and cotton bandage. Preliminary evaluation of the polymeric membrane showed that the scaffolds were biocompatible and do not delay the healing process. The polymeric membrane composed of the fused polylactide particles adhered to the wound bed and did not cause adverse reactions or wound infection during the entire closure of the wound (Figure 11A−D). It was observed that rats treated with PDLLA membrane showed better wound 2200



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dimensional in vitro models for cancer cells. Surfactant mediated self-assembly of polylactide particles described here can be used to fabricate scaffolds of desired dimensions in a convenient way, which can also release various bioactive factors needed for the proper differentiation of cells into the tissue microenvironment. This novel drug delivering scaffold design method will be best suitable for designing biomimetic scaffolds for various regenerative medicine applications.

the rapid desorption of the surfactant from the porous polymer particle in the presence of alcohol and polymer solubility in the solvent play an important role in fusion of polymer particles into higher order structure at room temperature. Polymer scaffolds made by fusion of particles were found to be sterile and well suited for the three-dimensional growth of animal cells. The highly porous nature of the particles allows for the free flow of medium, and the topology of the scaffold seems to be beneficial in promoting three-dimensional growth of cells. Both B16 and MCF-7 cells proliferated nicely on polymer scaffold. Use of polymer scaffold leads to formation of tissuelike structure on days 6−8 in vitro. We are presently evaluating the three-dimensional cultures of cancer cells on polymer scaffold for evaluation of anticancer drugs, as many studies have shown that they are a better model in comparison to monolayer cultures.50,51 Microparticles entrapping FITC-BSA were formulated having different release characteristics by controlling the porosity of the particle during formulation. Scaffold formed from these particles exhibited controlled release of entrapped protein. This indicated that these scaffolds can be used as an efficient drug delivery platform. This opens up new possibilities of designing biomimetic scaffolds entrapping suitable growth factor or drug molecules. Polylactide membrane formulated by the above method was also evaluated as an artificial skin substitute in the treatment of wounds in rats. Preliminary evaluation in rats showed better performance of the polylactide membrane as passive dressing material. Preformed composite polylactide membranes are already in use as an artificial skin substitute for burn wound treatment,29 but it only provides a passive template for wound regeneration. Since infections of open wound with microbes and subsequent delay in healing are major problems faced in burn cases,52,53 it would be advantageous if the scaffold also releases drugs as reported for silver sulfadiazine to combat wound infections.54 The advantage of our process is that surfactant coated polylactide particles encapsulated with antibiotics and growth factors which promote wound healing can be made and conveniently stored until needed. When a burn case arrives, the particles can be spread out into desired sizes and wetted with ethanol to immediately form the polymeric membranes to be used to cover the wounds. This step also removes the surfactant molecules and effectively sterilizes the membrane. The process of making scaffold from PDLLA is very simple and easy in comparison to methods used widely for scaffold design.6

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ASSOCIATED CONTENT

S Supporting Information *

Supporting Information S1−S4 is available that describes the CTAB estimation, DSC analysis of the scaffold and FTIR spectra of polymer particles and fused scaffold. Two movies present the fusion of particles in the presence and absence of alcohol. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +91-11-26703509. Fax: +91-11-26742125. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the Department of Biotechnology, Government of India Project (BT/PR/11201/MED/32/45/ 2008), to A.K.P. as well as core funding of the National Institute of Immunology (NII) to A.K.P.

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REFERENCES

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CONCLUSION In this paper, we demonstrated that rapid desorption of surfactant molecules and their collective motion can lead to fusion of polylactide particles at room temperature into stable structures in the presence of ethanol. Desorption of the surfactant from the particle surface in the presence of ethanol transiently increased the polymer solubility resulting in formation of connecting bridges between particles. This approach can be used to fabricate biodegradable polymeric scaffolds in an easy and convenient way for various biomedical applications. Suitability of the polymer scaffold in supporting three-dimensional culture of cancer cells, as a drug/biomolecule delivering platform, and finally as a passive dressing material for wound healing was demonstrated. Growth of cells on a scaffold and expression of MMP-2 indicated the tissue-type structural growth of cells and suitability of these scaffolds as three2201



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Fusion and Self-Assembly of Biodegradable Polymer Particles into

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