6
1. Introduction
Microsized 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 micron-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 analogs.[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 lithography[7], stretching techniques[8], and
sectioning[9], 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 and 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 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 typical degradation rates, which are compatible with clinical applications and result in lactic acid, a nontoxic degradation by-product.
[16, 17] However, as 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, for example, 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 micro- and nanoscale 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 an 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] 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 microsized 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.[30] 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 microsized 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 evaluation of the viability levels of human fibroblasts cultured in vitro.
6
2. Materials and methods
2.1. Electrospinning of PLLA
Medical-grade poly(L-lactic acid) (Purasorb PL65®; Corbion, The Netherlands) was dissolved at a concentration of 3% w/v in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP; Fluorochem, UK) or in a 4:1 v/v mixture of dichloromethane (DCM; Merck, Germany) and trichloromethane (TCM; Merck). A commercially available electrospinning apparatus (Advanced Surface Technology BV, 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; and voltage = 19.25 kV. Electrospinning of PLLA in a 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; and 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.
2.2. Preparation of PLLA microcylinders
PLLA membranes were subjected to an aminolysis process based on a previously described method, with some modifications.[11] Briefly, electrospun fibers were
immersed in a 5% v/v solution of ethylenediamine (EtDA; Sigma-Aldrich, USA) in isopropanol (IPA; Merck) for two different time points (3 and 5 hours). EtDA was chosen as a reactive amine donor, and IPA was used in order to promote the aminolysis reaction.[12] After aminolysis, four cycles of centrifugation/washing
with ddwater were performed to remove the diamine solution in excess. Samples were finally resuspended in ddwater, subjected to 40 seconds of ultrasonication at 100% amplitude (UP50H; Hielscher - Ultrasound Technology, Germany) to prevent aggregation of the microcylinders, freeze-dried, and stored at room temperature for further use.
2.3. Characterization of PLLA microcylinders
The morphology of the electrospun membranes and microcylinders was observed by SEM (SM3010; JEOL, Japan) under an acceleration voltage of 5 kV. Electrospun fibers as a mat and microcylinders as powder were fixed with carbon tape on aluminum holders and sputter-coated with gold (thickness ≈ 10 nm).
XRD (pw1830; Philips, The Netherlands) was performed to determine the crystallographic profile of the samples. Electrospun membranes as a thin planar layer or microcylinders in 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), and 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 the Scherrer equation:[35]