Top-Down Approach for the Preparation of Highly Porous PLLA

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A Top-down Approach for the Preparation of Highly Porous PLLA Micro-cylinders Antonio Castro, Maria Cristina Lo Giudice, Tina Vermonden, Sander C.G. Leeuwenburgh, John A. Jansen, Jeroen JJP van den Beucken, and Fang Yang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00522 • Publication Date (Web): 20 Sep 2016 Downloaded from http://pubs.acs.org on September 23, 2016

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ACS Biomaterials Science & Engineering

Article type: Article

A Top-down Approach for the Preparation of Highly Porous PLLA Micro-cylinders

Antonio G. B. Castro, Maria Cristina Lo Giudice, Tina Vermonden, Sander C. G. Leeuwenburgh, John A. Jansen, Jeroen J. J. P. van den Beucken, and Fang Yang*

A. G. B. Castro, M. C. Lo Giudice, Dr. S. C. G. Leeuwenburgh, Prof. J. A. Jansen, Dr. J. J. J. P. van den Beucken Department of Biomaterials, Radboud University Medical Center, Philips van Leydenlaan 25, Nijmegen, 6525 EX, The Netherlands

Dr. T. Vermonden Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Faculty of Science, Utrecht University, P. O. Box 80082, 3508 TB Utrecht, The Netherlands

*Dr. F. Yang E-mail: [email protected] Department of Biomaterials, Radboud University Medical Center, Philips van Leydenlaan 25, Nijmegen, 6525 EX, The Netherlands

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Abstract A wide range of particles have been developed for different applications in drug-delivery, tissue engineering, or regenerative medicine. In contrast to traditional spherical particles, nonspherical (e.g. cylindrical) particles possess several structural and morphological advantages that make them attractive for specific applications. Here, we developed a top-down approach to process electrospun fibers into micro-sized cylinders (i.e. micro-cylinders) with high specific surface area and with or without surface porosity. To obtain these micro-cylinders, poly(L-lactic acid) (PLLA) solutions were subjected to electrospinning, followed by an aminolysis-based chemical scission procedure. The morphology, structure, and chemical composition of the micro-cylinders were then characterized. Specific surface area and surface porosity of the micro-cylinders were controlled by the volatility of the solvents and their length was dependent on the duration of the aminolysis reaction. During aminolysis, the micro-cylinders became functionalized with amine groups, enabling potential further modifications by grafting with compounds containing desired chemical groups, e.g. carboxyl, carbonyl or hydroxyl functional groups. Additionally, the micro-cylinders showed in vitro biocompatible properties related to cell viability. These data demonstrate that PLLA microcylinders with high specific surface area, optional surface porosity, amine-based functional handles granting additional functionalization, and cytocompatible properties are candidate materials for future biomedical applications.

Keywords: poly(L-lactic acid), microparticles, electrospinning, aminolysis, micro-cylinders


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1. Introduction Micro-sized particles have been widely used in the development of drug delivery systems. These particles can be fabricated from organic compounds like natural and synthetic polymers or inorganic minerals and metallic oxides. Each composition presents unique characteristics and opportunities to create particles of different sizes and shapes. Particles with a spherical geometry have been most commonly studied, especially in the development of systems that require high levels of cellular internalization and long circulation times in vivo.1-2 Compared to spherical structures, non-spherical particles such as micro-sized cylinders present several advantages for the development of drug or biomolecule delivery systems. First, higher drug loading levels can be achieved using non-spherical particles due to a higher surface area to volume ratio for particles with similar dimensions.3 Second, non-spherical particles are cleared less rapidly from the body.2, 4 This property is related to the different hydrodynamic forces to which these non-spherical particles are subjected in the body, leading them to stumble and move across the vessel walls and to adhere to endothelial cells, rather than following the more linear pattern in the circulatory system shown by spherical analogues.1,4 Finally, non-spherical micrometric particles have shown to be less susceptible to phagocytosis in vitro because their internalization is highly dependent on the particles’ geometry and length-normalized curvature (Ω).5-6 Although several physical methods have been developed to fabricate non-spherical structures, such as lithography7, stretching techniques8 and sectioning9, chemical-based strategies present appealing advantages, including lower costs and mild experimental conditions. Also, when compared, for example, to mechanical drawing techniques, a better control of the fiber diameter is achieved.10 In 2008, Kim & Park showed that poly(L-lactic acid) (PLLA) electrospun fibers could be shortened by a chemical transverse fragmentation method, leading to the development of cylindrical microstructures.11 A wet-chemical process called aminolysis



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was used to induce this transverse fragmentation. Aminolysis comprises a reaction between a reactive species and an amine or ammonia group donor, resulting in chemical scission of the reactive species and the incorporation of amine or amide groups to its molecular structure.12-13 Alternatively, aminolysis is commonly used for the functionalization of polymers with amine groups, which can increase the material’s biocompatibility or facilitate the conjugation of other compounds to the polymer.13-15 PLLA is one of the most extensively studied polymers for the development of new biomaterials, which is justified by its high biocompatibility, biodegradability, good mechanical properties, and its typical degradation rates, which are compatible with clinical applications and which result into lactic acid, a non-toxic degradation by-product.16-17 However, as the other polyesters, PLLA is in its pristine form hydrophobic, having a reduced wettability and consequently cellular adhesion. This drawback can be overcome by chemically modifying the surface or the molecular backbone of the polymer with specific functional groups, e.g. amine groups, hydroxyl groups, or peptidic sequences.18,19 As mentioned above, aminolysis renders the polymer with amine groups and can be used to functionalize PLLA. The use of electrospinning has been well established in the production of fibers with microand nano-scale diameters.20 Established by Formhals, this technique produces polymeric fibers by the application of an electric current to a polymeric solution. An electrospinning apparatus is essentially constituted by an electrical power source, a nozzle and a collector. When a electrical potential between the nozzle and the collector is generated, the polymeric solution’s droplet is stretched, creating a jet. Due to solvent evaporation polymeric fibers are then generated and collected.21,22 Electrospun scaffolds of biodegradable polymers, such as polylactic acid (PLA), poly(ε-caprolactone) (PCL), polyglycolic acid (PGA), and their copolymers have found widespread applications in various biomedical fields.23-25


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Furthermore, the high surface area to volume ratio and the possibility to incorporate biomolecules within and/or on the surface of the electrospun fibers encourages the use of these structures as a platform for the delivery of therapeutic drugs and growth factors.26 Importantly, electrospun fibers with different morphologies and surface porosity can be obtained in a simple manner using solvents with different volatilities.27-29 Surface porosity has an important effect on the functional properties of micro-sized particles, especially in the development of drug-delivery systems. Porous particles have shown higher loading efficiencies compared to dense particles, largely due to their increased surface-area-to-volume ratio.28 Further, the porosity of a particle affects the drug release kinetics: by creating macroporosity a higher initial release can be achieved,31,32 while the presence of interconnected micropores leads to larger diffusion pathways, resulting in a more sustained release and diminished burst release profile.33,34 Considering the advantages of electrospinning and aminolysis for the preparation of microsized particles, we combined both techniques in a top-down approach for (i) production of PLLA-based electrospun fibers, and (ii) generation of PLLA-based micro-sized cylinders that feature high specific surface area and with or without surface porosity. To this end, PLLA polymeric fibers were electrospun and subsequently subjected to an aminolysis-based scission procedure. The morphology and chemical composition of the resulting dense or porous microcylinders were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and Infrared Spectroscopy. Additionally, their biocompatibility was assessed by an evaluation of the viability levels of human fibroblasts cultured in vitro.



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2. Materials and Methods Electrospinning of PLLA: Medical grade poly(L-lactic acid) (Purasorb PL65®; Corbion, Gorinchem, The Netherlands) was dissolved at a concentration of 3% w/v in 1,1,1,3,3,3hexafluoroisopropanol (HFIP; Fluorochem, Hadfield, UK) or in a 4:1 v/v mixture of dichloromethane (DCM; Merck, Darmstadt, Germany) and trichloromethane (TCM; Merck). A commercially available electrospinning apparatus (Advanced Surface Technology BV, Bleiswijk, The Netherlands) was used for the preparation of PLLA membranes. Electrospinning of PLLA in HFIP was performed using the following parameters: volume = 8 ml; needle diameter = 1.2 mm; feeding rate = 2.0 mL h-1; distance between the needle and the collector = 35 cm; voltage = 19.25 kV. Electrospinning of PLLA in DCM:TCM mixture was performed using the following parameters: volume = 8 ml; needle diameter = 1.2 mm; feeding rate = 2.0 mL h-1; distance between the needle and the collector = 35 cm; voltage = 35.00 kV. Fibers were collected in a stationary flat collector, covered with aluminum foil. Electrospinning terminated after 4 hours. The obtained membranes of about 0.2 mm thick were left overnight in a fume hood and freeze-dried for 3 days to remove the residual solvent. Preparation of PLLA micro-cylinders: PLLA membranes were subjected to an aminolysis process based in a previously described method, with some modifications.11 Briefly, electrospun fibers were immersed in a 5% v/v solution of ethylenediamine (EtDA; SigmaAldrich, Saint Louis, MO, USA) in isopropanol (IPA; Merck) for two different time points (3 hours and 5 hours). Ethylenediamine was chosen as a reactive amine donor and isopropanol was used in order to promote the aminolysis reaction.12 After aminolysis, 4 cycles of centrifugation/washing with ultrapure water were performed to remove the diamine solution in excess. Samples were finally resuspended in ultrapure water, subjected to 40s of ultrasonication at 100% amplitude (UP50H; Hielscher - Ultrasound Technology, Teltow,


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Germany) to prevent aggregation of the micro-cylinders, freeze-dried and stored at room temperature for further use. Characterization of PLLA micro-cylinders: The morphology of the electrospun membranes and micro-cylinders was observed by scanning electron microscopy (SEM; JEOL-SM3010, Japan) under an acceleration voltage of 5 kV. Electrospun fibers as a mat and micro-cylinders as powder were fixed with carbon tape on aluminum holders and sputter-coated with gold (thickness ≈10 nm). X-ray diffraction (XRD; Philips pw1830, The Netherlands) was performed to determine the crystallographic profile of the samples. Electrospun membranes as a thin planar layer or micro-cylinders in the powder form were placed in a copper holder and scanned. XRD spectra were registered at 40 kV, 30 mA (Cu-Kα radiation with a wavelength of 1.54 Å), 2θ between 10 and 30˚, at a step size of 0.005°. The crystallite sizes of PLLA for the main (110) and (203) reflections were determined according to Scherrer equation35: =

(1)

Where is a dimensionless shape factor (0.9 for the spherulite structure), the wavelength of Cu-Kα radiation, and the full width at half maximum value of the diffraction peak at 16.7° for PLLA given in radians. Accordingly, is 16.7° for all calculations in this study. The degree of crystallinity (χc) of the PLLA as received and after electrospinning and aminolysis processes was determined by Differential Scanning Calorimetry (DSC; Discovery®, TA instruments; The Netherlands). For DSC analysis, samples were sealed in an aluminum pan and scanned from 20 to 200 ˚C at a heating rate of 5 ˚C min-1. Degree of Crystallinity (χc) was calculated considering the ratio between the experimental enthalpy of fusion (∆H ) and the enthalpy of fusion of 100% crystalline PLLA (∆H = 93.7 J g-1), according to the following equation35:



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χ c =

∆ ∆

× 100%

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(2)

Thermogravimetric analysis (TGA; TA instruments), was effectuated in a nitrogen atmosphere with a temperature range from 20 to 400 ˚C and a heating rate of 5 ºC min-1. The specific surface area of the different samples was determined by nitrogen gas adsorption/desorption isotherms (Gemini V, Micrometrics; USA) and based on the BrunauerEmmett-Teller (BET) model36: 1) ! ⁄! # − 1%& = 1⁄m ' + ' − 1#⁄m ' ! ⁄! #

(3)

where is the weight of the gas adsorbed, m is the weight of the adsorbate as a monolayer, ! ⁄! is the relative pressure and ' is the BET constant. Specific surface area (*) can be obtained from the following equation36: * = m + ,cs⁄-

(4)

where + is the Avogadro’s number, ,cs is adsorbate cross-sectional area (16.2 Å2/ molecule for N2) and - is the weight of the sample. The presence of amine groups was determined by attenuated total reflectance-infrared spectroscopy (ATR-IR; UATR two, PerkinElmer, The Netherlands) with a resolution of 4.0 cm-1 and a scanning range from 400 cm-1 to 4000 cm-1. A fluorescamine assay was used in order to qualitatively detect the presence of amine groups. Briefly, the materials were incubated for 1 hour in a 1:1 (v/v) mixture of ultrapure water and a 1 mg ml-1 fluorescamine (Sigma-Aldrich) solution in acetone (Lab-Scan, Gilwice, Poland) at room temperature. Afterwards, 3 centrifugation steps with ultrapure water were performed to remove the excess dye. Fluorescence was assessed by confocal microscopy. Fluorescence intensity heatmaps were created using ImageJ software (National Institutes of Health, Bethesda, MD, USA). In order to quantify the amount of amine groups in the samples, a 2,4,6trinitrobenzenesulfonic acid (TNBS) colorimetric assay was performed. Briefly, a known 8 ACS Paragon Plus Environment

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amount of sample was incubated with 400 µL of a 0.5% w/v picrylsulfonic acid solution (Sigma Aldrich) for 2 hours at 40 ˚C. Afterwards, 700 µL of pure hydrochloric acid (HCl, 37% w/v, 12.1M) was added in order to dissolve the samples and the samples were incubated at 60 ˚C until the solution was completely clear. Absorbance at λ = 405 nm was determined with a UV-VIS microplate reader (Synergy® HTX, Biotek, The Netherlands). The amines concentration was calculated assuming a 1:1 stoichiometry of the complex between the amine groups and the dye. Values have been normalized for the weight of each sample. In vitro cytotoxicity of PLLA micro-cylinders: Adherent human foreskin fibroblasts were grown and maintained in DMEM medium (Gibco®, Life Technologies, Grand Island, USA) with 10% Fetal bovine serum (FBS; Gibco) and a 2% mixture of penicillin/streptomycin (Gibco). Cells were cultured in an incubator with 5% CO2 at 37°C. For cytotoxicity analyses, fibroblasts were seeded at a density of 30000 cells cm-2. A LIVE/DEAD assay (Molecular ProbesTM, Invitrogen®, Thermo Fisher Scientific, Bleiswik, The Netherlands) was performed at 72 hours, according to the instructions of the manufacturer. A suspension of porous microcylinders in culture medium was prepared (0.5 mg ml-1) and added to the cells 24 hours after seeding. Prior to LIVE/DEAD assay samples containing the micro-cylinders were placed in an incubator overnight, in order for the micro-cylinders to settle in the bottom of the wells. Cells cultured in the same conditions without micro-cylinders were served as a positive control. For the negative control group, cells were maintained at the same condition as the positive control except that the culture medium was changed to 70% v/v methanol in ultrapure water 30 minutes prior to perform the LIVE/DEAD assay. Additionally, cells were cultured with PLLA porous micro-cylinders (0.1, 0.5 and 1.0 mg ml-1) in 24-well culture plates and cell viability was evaluated by performing an AlamarBlue® viability test (Molecular ProbesTM, Invitrogen®, Thermo Fisher Scientific, Bleiswijk, The Netherlands) per the manufacturer’s



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instructions to obtain quantitative data on cellular activity. Positive and negative controls were prepared as described for LIVE/DEAD assay. Statistical analysis: Prism 6.01 (GraphPad software, Inc.; San Diego; CA, USA) was used to perform all statistical calculations. Fiber diameter and micro-cylinders dimensions (length and diameter) were estimated from the corresponding SEM images using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Twenty different measurements were taken from 5 different regions of each electrospun membrane or each micro-cylinders group (n = 100 for fiber diameter and n = 100 for micro-cylinders’ length and diameter). Data was reported as mean ± standard deviation (SD). Statistical analysis was effectuated by using twoway analysis of variance (ANOVA) combined with a Sidak’s multiple comparison post-hoc test. The time of aminolysis and the morphology of fibers or micro-cylinders were considered as independent variables. Crystallite size data obtained by XRD from 3 samples (n = 3) and TNBS amines quantification data in triplicate (n = 3) were analyzed by a two-way ANOVA test combined with Sidak’s multiple comparison post-hoc test, using the same variables. AlamarBlue® data was assayed in triplicate (n≈3) and analyzed using a one-way ANOVA test combined with a Tukey’s post-hoc comparison test. Differences were considered significant at p

Top-Down Approach for the Preparation of Highly Porous PLLA

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