Research paperCellulose acetate butyrate–pH/thermosensitive polymer microcapsules containing aminated poly(vinyl alcohol) microspheres for oral administration of DNA
Introduction
Oral gene delivery is a main goal for numerous biotechnological companies. The main advantages presented by oral gene delivery are the ease of target accessibility, the enhanced patient compliance owing to the non-invasive delivery method, and the possibility of local and systemic gene therapy. To date, most gene delivery strategies have concentrated on the parenteral route of delivery [1], [2], [3], [4], and oral administration has been largely ignored with few exceptions [5], [6]. The lacking of using oral route for gene delivery is mainly due to the large number of hurdles that need to be overcome, such as the acidic pH in the stomach, the presence of nucleases, lipases, and peptidases in the GI tract, and the poor permeability of both genes and gene vectors across the intestinal epithelium.
DNA delivery systems can be classified generally within two main classes: the viral mediated systems (e.g., adeno- and retroviral vectors) [7], [8] and the non-viral delivery systems (e.g., neutral or cationic polymers, micro- and nanospheres) [9], [10], [11]. Among non-viral transfection agents an attractive class to consider is represented by bioadhesive microspheres. The main advantage of these microspheres is the possibility to design and synthesize microparticles starting from polymers carrying different structural elements that can self-assemble with DNA by electrostatic interactions thus producing vectors with a range of properties [12]. In this view, the most important benefit is the possibility of not exposing nucleic acid molecules to chemical, thermal, and mechanical stresses during microparticle preparation.
In addition, bioadhesive microspheres have been reported to increase the peroral bioavailability of insulin and have been investigated for peroral gene delivery [13]. The increased bioactivity of insulin and of the plasmid DNA can be accounted to the uptake of microspheres by cells lining the small and large intestine epithelium. Bioadhesive microspheres by keeping the drug in the region proximal to its adsorption window allow targeting and localization of the drug at a specific site. However, the major problem of this alternative is to transport the loaded bioadhesive microspheres to small and large intestine avoiding the contact with gastric fluids. The literature presents different strategies for specific drug delivery to the various regions of gastro-intestinal tract and, in particular to the colon [14]. The most part of works are based on microencapsulation with enteric polymers, which are able to release the drug at a particular pH [15], [16]. However, in our hands, this method failed because of microsphere swelling during preparation process. We recently reported the encapsulation of cationic-exchange microspheres loaded with tetracycline in cellulose acetate butyrate (CAB) microcapsules [17]. The drug release was possible owing to the higher swelling degree of sulfopropylated dextran resins in intestinal than in gastric fluids, causing the rupture of CAB shell, and the escape of loaded microspheres. The present paper reports a new approach for the preparation of microspheres with cationic surface to improve the intestinal delivery of DNA. In particular, we propose the encapsulation of aminated PVA microspheres loaded with plasmid DNA in CAB microcapsules for intestinal delivery of the nucleic acid. Since aminated PVA resins, contrary to sulfopropylated microspheres, do not swell enough in intestine to produce the rupture of CAB shell, we incorporate in microcapsule composition different amount of pH/thermosensitive polymers (as enteric materials). These polymers weaken the resistance of the CAB shell by their dissolution in the intestinal fluids causing halls in CAB microcapsules and allowing the release of loaded PVA microspheres. In other words, the use of CAB/pH/thermo-responsible shell instead of conventional enteric capsules is more advantageous since the encapsulated PVA microspheres are progressively released through the halls of CAB microcapsules created by dissolution of pH/thermosensitive polymer. Also, the bioadhesive properties of aminated PVA microspheres with and without DNA were tested.
Section snippets
Materials
PVA (Mw = 18,000 g/mol; hydrolysis mole = 98.4%) was purchased from Air Products and Chemicals, Inc. (Utrecht, The Netherlands). Cellulose acetate butyrate (CAB), low viscosity, was obtained from Eastman Inc. (Kingsport, Tennesse, USA). Glutaraldehyde (GA) (2.6 M aqueous solution) was supplied by Fluka AG (Seelze, Germany). 3-Dimethylamino-1-propyl amine and N,N-carbonyldiimidazole (CDI) were purchased from Fluka AG (Buchs, Switzerland). N-isopropylacrylamide (NIPAAm), supplied by Sigma-Aldrich
Preparation and characterization of aminated PVA microspheres
PVA is a biocompatible and non-toxic polymer that is frequently used in biomedical applications, such as implants [22], soft contact lenses [23], artificial organs [24], and protein immobilization [25]. In addition, PVA is able to form hydrogels branded by high degree of swelling in reason of its hydrophilic character. Therefore, PVA is one of the synthetic polymers used in bioadhesive formulations [26] improving the mucosal adsorption of DNA.
Moreover, the presence of hydroxyl groups capable of
Conclusions
Aminated PVA microspheres loaded with plasmid DNA by electrostatic interactions were successfully encapsulated in CAB/SP microcapsules by the oil/water solvent evaporation method. The entrapped PVA microspheres do not have enough force by swelling to produce the rupture of the CAB shell, therefore the resistance of microcapsules was weakened by incorporating different amounts of SP. This copolymer is insoluble in the gastric juice at pH 1.2 and 36 °C, but quickly solubilized in the intestinal
Acknowledgements
This study was partly supported by grants from the Ministry for Foreign Affairs of Italy and the Ministry for Education and Research of Romania.
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