Biomacromolecules 2005, 6, 1484-1488
1484
Poly(vinyl alcohol) Nanofibers by Electrospinning as a Protein Delivery System and the Retardation of Enzyme Release by Additional Polymer Coatings Jun Zeng,† Achim Aigner,‡ Frank Czubayko,‡ Thomas Kissel,§ Joachim H. Wendorff,† and Andreas Greiner*,† Department of Chemistry, Institute of Physical, Nuclear and Macromolecular Chemistry and Scientific Center of Materials Science, Philipps-University Marburg, Hans-Meerwein-Str., D-35039 Marburg, Germany, School of Medicine, Department of Pharmacology and Toxicology, Philipps-University Marburg, Karl-von-Frisch-Str. 1, D-35033 Marburg, Germany, and Department of Pharmaceutical Technology and Biopharmacy, Philipps-University Marburg, D-35032 Marburg, Germany Received November 23, 2004; Revised Manuscript Received March 3, 2005
Protein-loaded (bovine serum albumin (BSA) or luciferase) poly(vinyl alcohol) (PVA) nanofibers were obtained by electrospinning. Poly(p-xylylene) (PPX, also coined as parylene) coated PVA/BSA nanofibers were prepared by chemical vapor deposition (CVD). The release of BSA from PVA nanofibers under physiological conditions was monitored by absorption spectroscopy. Burst release of BSA was noted with uncoated PVA nanofibers. In contrast, PPX-coated nanofibers exhibited a significantly retarded release of BSA depending on the coating thickness of PPX (ranging from 40 to 300 nm). Luciferase was used here as model enzyme, which after electrospinning retained its enzyme activity. This preservation of enzyme activity and the continuous release of the intact enzyme from the immersed fibers meets a fundamental prerequisite for the application of enzymes or other sensitive agents released from electrospun nanofibers under physiological conditions. Introduction Recently, polymer nanofiber formation was established for a wide range of polymeric systems, including drug erodible polymers by the so-called electrospinning process.1 Using this technique, nanofibers were obtained by applying a high electrical field to polymer solutions or melts, which were pumped through a metal capillary (the capillary acts as an electrode) of a standard syringe. The fibers were collected as a nonwoven mesh on any substrate acting as the corresponding counter electrode. This process is highly versatile and allows processing not only of many different polymers into polymeric nanofibers but also the co-processing of polymer mixtures,2 mixtures of polymers and low molecular weight nonvolatile materials,3 etc. simply by using ternary solutions of the components for electrospinning or by coelectrospinning resulting in core-shell fibers. Naturally, this approach to fibers of mixed composition is not restricted to binary materials systems but can be also applied to multicomponent systems depending on the composition of the “precursor solution”. Research in our group with respect to electrospinning is mainly focused on the impact of different processing parameters on electrospinning,3b,3f,4 on the investigation of * To whom correspondence should addressed. E-mail: greiner@ staff.uni-marburg.de. † Department of Chemistry, Institute of Physical, Nuclear and Macromolecular Chemistry and Scientific Center of Materials Science. ‡ School of Medicine, Department of Pharmacology and Toxicology. § Department of Pharmaceutical Technology and Biopharmacy.
structure formation of electrospun fibers,2a,5 and on the application of electrospun fibers for different purposes including templates for the preparation of nanotubes.3a,b,6 Due to their large surface-to-volume ratio, electrospun polymer nanofibers gain more and more attention for medical applications such as tissue engineering and drug release. It will be of fundamental interest not only to make drug loaded electrospun fibers but also to study their release kinetics as a function of fiber materials composition, fiber diameter, fiber shape, etc. Furthermore, it will be of interest to investigate systematically how to control drug release (or the release of other reagents) from such polymer nanofibers, to investigate the functionality of drugs after the electrospinning procedure, to compare release characteristics of differently shaped nanotubes or nanoparticles of corresponding materials, and finally to study the impact of such nanofibers on biological systems. To the best of our knowledge, so far only few studies have been published on the release of macromolecules from electrospun fibers. Wnek et al. studied the release of tetracycline hydrochloride from poly(ethylene-co-vinylacetate), poly(lactide), and a blend thereof.2b Poly(lactideco-glycolide)/DNA composite nanofibers were prepared by electrospinning and used as a plasmid DNA delivery system by Luu et al.7a Verreck et al. studied electrospinning of itraconazole and hydropropylmethyl cellulose from watery solution.7b Jia et al. studied the enzyme activity of R-chymotrypsin being chemically attached to polystyrene nanofibers after electrospinning and found much higher enzyme activi-
10.1021/bm0492576 CCC: $30.25 © 2005 American Chemical Society Published on Web 04/09/2005
PVA Nanofibers as a Protein Delivery System
ties of this modified enzyme in comparison to the native enzyme.7c Also higher enzyme activities for enzymes immobilized on polymer nanofibers were reported by Xie et al.7d Kim et al. reported on release of antibiotics from electrospun poly(lactide) copolymers.7e It should be also noted that a patent reported on the preservation of enzymes etc. in electrospun scaffolds7f and that formation of protein loaded microbeads was accomplished by an electrostatic process.7g Most of these studies nicely contribute to the field by new concepts and ideas; however, systematic studies on release characteristics in combinations with structural and morphological characteristics of the nanofibers used as drugcarrier will be major future task. In addition, the alteration of release kinetics by coating of such fibers has not been reported so far. The goal of this report is a feasibility study on the release of comparably large proteins from electrospun PVA nanofibers. We show the release of fluorescin isothiocyanate labeled bovine serum albumin (FITC-BSA) from electrospun PVA nanotubes, and, most importantly, the controlled release properties by subjecting the poly(vinyl alcohol)/ enzyme nanofiber webs to a post-electrospinning coating of highly hydrophilic PPX. Furthermore, using luciferase as a model enzyme, we show a post-electrospinning enzyme activity and the continuous release of the intact enzyme from the immersed fibers. Experimental Section Materials. PVA (56-98, molecular weight ) 195 000, hydrolysis 98%) was obtained from Clariant and used as received. FITC-BSA aqueous solution (1.03 mg/mL) was obtained from Sigma, Taufkirchen. Phosphate buffered saline (PBS) was obtained from PAA Laboratories (Co¨lbe). Recombinant monomeric luciferase (MW 61 707) was purchased from Sigma (Taufkirchen) and dissolved in PBS to a final concentration of 10 mg/mL. Deionized water was used if not specified (as in case of PVA/BSA/Luciferase solution, PBS instead of deionized water was used). The coating of enzyme loaded nanofibers by PPX was performed in an automatic coating machine. [2.2]Paracyclophane (dimer) was used as starting material as obtained from Specialty Coating Systems (SCS) for the preparation of PPX. The coating principle has been described before in the literature.8 Measurements and Methods. The morphology and dimension of electrospun fibers were determined by scanning electron microscopy (SEM) (CamScan 4 with an accelerating voltage of 15 kV). A small piece of fiber mats was placed on the sample holder and sputter-coated with gold (Edward Type Auto 306 sputter coater) under a vacuum of
