Studies On Micro-/Nano-Structured Drug Controlled Release Systems Based On Biodegradable Polymers: Preparation, Characterization And In Vitro/In Vivo Evaluation

Although the controlled release system based on biodegradable microspheres has made great progress. The application of this formulation in clinical is still very limited. There are still many drawbacks, such as crystallized drug in the microsphere surface and severe burst release of small molecular drug in initial stage, as well as the strong tendency of bioactivity loss for bioactive substances. All these crucial problems maintain unsolved so far. In this paper, we are aimed to explore and overcome above shortcomings. We designed and prepared the micro-/nano-structured drug controlled release systems based on biodegradable materials with different compositions, which corresponded to microspheres and electrospun nanofibers respectively. In addition, their in vitro/in vivo characters were thoroughly evaluated. The main research content and results are as follows.1. A series of ibuprofen-loaded microspheres based on biodegradable polymers, such as Polylactide (PLA), Poly(ε-caprolactone) (PCL), as well as their block copolymer (P(LA-b-CL)) have been prepared by O/W emulsion solvent evaporation method. For PLA and P(LA-b-CL) microspheres, The mean diameter was about 40 ～ 50 μm, and the drug entrapment efficiency could be up to 80% under an optimized condition, but it was very low for PCL microspheres (<30%). The crystallization characteristics of polymer matrices and ibuprofen dispersion state in the microspheres were studied by DSC, SEM and POM. It was found that the drug release behavior was significantly affected by their dispersion in each materials. PLA and P(LA-b-CL) microspheres showed better effect of sustained release tendency, in terms of the introduction of e-CL to PLA, ibuprofen release rate can be advisably modulated to meet different requests.2. A hydrophobic peptide, cyclosporine A (CyA), was respectively incorporated in microspheres based on Poly(LA-b-CL) (CL content: 21.3 and 51.9%) and Poly(lactide-co-glycolide) (PLGA, LA/GA: 80/20) using oil-in-water (O/W) emulsionsolvent evaporation method. All microspheres showed a mean diameter at around 35 urn, and CyA entrapment efficiency exceeded 96%. The results from DSC and X-ray diffraction indicated that was evenly dispersed in all microspheres. CyA release rate decreased in the order of P(LA-b-CL) (48.1/51.9), Poly(LA-b-CL) (78.7/21.3) and PLGA (80/20). Compared to PLGA microspheres, Poly(LA-b-CL) microspheres exhibited more significant burst release degree in initial stage. The in vivo results showed good correlation with in vitro results. P(LA-b-CL) microspheres provided a higher blood level of CyA than PLGA microspheres post 2 days administration. And then P(LA-b-CL) microspheres showed a more constant CyA level for extended periods of time. Given the fact that CyA hold a very narrow therapy window and dose-depended immune effect, our results indicated that such CyA-loaded P(LA-b-CL) microspheres showed more advantages, than those prepared with PLGA, such as improved bioavailability and reduced side effects, accordingly could meet clinical needs more efficiently.3. A high hydrophilic and basic peptide, a-cobrotoxin (a-CT), was successfully incorporated into the microspheres based on PLGA (50/50) using a modified W/O/O method. The mixture of acetonitrile and dichloromethane was used as polymer solvent, and liquid paraffin was used as outer oil phase. The microspheres were solidified through the coinstantaneous solvent extraction and evaporation. The obtained PLGA microspheres with an average diameter of about 25 urn showed smooth and compact surface. Moreover, the bioactivity of a-CT was not destroyed. The addition of sodium alginate (SA) into the inner aqueous phase enhanced the entrapment efficiency of a-CT (> 85%), resulting from the increase of the viscosity of the inner aqueous phase. For the same reason, as well as the formation of water soluble complex between SA and a-CT, a significantly decreased initial burst release was obtained. In addition, the blend of fluorescent Poly[1,3-bis(p-carboxy-phenoxy) propane-co-p-(carboxyethylformamido) benzoic anhydride] (P(CPP:CEFB)) played a considerable role on the morphology and degradation rate of the microspheres, on the other hand, it greatly enhanced the mucosa adsorption of the microspheres. When its content accounted for 30% in the loading materials, the microspheres still exhibitedsmooth surface and strong fluorescence. The LD50 of this microsphere formulation was 78.4 mg/kg after intro-peritoneal injection to mice, which contained about 0.348 mg/kg cx-CT, and it was equivalent to 2.32 times that of free a-CT. These results proved that this blend microspheres could significantly enhance the safety of a-CT in clinical. In addition, the analgesic response by the incorporated a-CT could be extended to 16 days with a considerable efficiency after nasal administration (80 ug/kg body weight) to rat. That was due to the improved adherence of PLGA/P(CPP:CEFB) microspheres to the nasal mucosa. The toxicity of a-CT in association with the microspheres to nasal mucosa was proved to be mild and reversible.4. The concept of hydrophobic ion pairing was adopted for incorporating lysozyme into PCL/Poly(ethylene glycol) (PEG) electrospun non-woven membranes. The solubility and stability of lysozyme in DMSO was enhanced through the formation of lysozyme-oleate complexes, which could be directly loaded into PCL/PEG membranes with 2.5% loading using electrospinning technique. The mixture of DMSO and chloroform was used as polymer solvent to obtain 15% w/v concentration. The resultant PCL/PEG nanofibers have a compact structure with an average diameter ranged from about 0.4 to 0.9 urn. There was no conglutination between each other. The addition of PEG into the PCL nanofibers not only improved the hydrophilicity of the membrane, but also played an important role on in vitro lysozyme release rate. It was found that the in vitro release rate of lysozyme was enhanced with the increase of PEG content, as well as the salt concentration in the release medium. In addition, the released lysozyme retained most of enzymatic bioactivity through activity measurement. This method provided a useful reference for the entrapment of some hydrophilic peptides and proteins into electrospun nanofiber membranes.5. A one-step, mild procedure based on coaxial electrospinning was developed to incorporate two model proteins, BSA and lysozyme, into biodegradable nanofibers. Wherein protein-containing PEG was selected as core liquid and PCL dissolved in DMSO/chloroform as shell liquid. The resultant nanofibers showed clear core-shell character, which was proved by SEM and TEM. The thickness of the core and shellcould be adjusted by the feed rate of the inner dope, which in turn affected the release profiles of the incorporated proteins. Such system effectively avoided the initial burst release and bioavailability loss, furthermore, it could also produced a stable release rate. The implication is that bioactive agents such as growth factors and DNA could readily be integrated into the nanofibers scaffolds using the method we described, and they showed great potential of application to gene therapy and tissue engineering.