静电纺丝纳米纤维的制备及其蛋白药物缓释体系

Preparation and Protein Sustained-release System of Electrospinning Nanofibers

细胞因子蛋白复杂脆弱的高级构象极易因组织工程支架制备过程中的苛刻条件变性而失活,甚至引起有害的抗体反应。目前有关组织工程支架材料的研究方法尚未实现以天然构象长期缓释细胞因子蛋白。针对这个难题,本研究运用水相-水相冷冻相分离蛋白颗粒制备法,将蛋白药物血管内皮生长因子(VEGF_(165))保护性载入葡聚糖纳米颗粒中,进而将载有蛋白的多糖颗粒混悬于作为缓释基质的聚乳酸-羟基乙酸共聚物(PLGA)中,通过高压静电纺丝,制得长效缓释VEGF_(165)的组织工程纤维膜。一系列物理化学表征,包括扫描电镜(SEM)、接触角、强度试验等,表明上述方法制得了表面形态与机械强度较理想的组织工程纤维。对制备过程中各个阶段的蛋白进行回收,并采用CCK-8法进行活性检测证明VEGF_(165)的生物活性在多糖颗粒制备阶段保持在97%以上,在最终的组织工程纤维膜阶段保持在87%以上,远大于w/o型乳液法制备得到的纤维(低于55%)。体外试验证明颗粒复合纤维能抑制蛋白突释且达到长效缓释效果,蛋白在初始5 d内的释放总量不到35%,到25 d时释放量超过92%。而w/o型乳液法制得的纤维在初始5 d内的释放总量超过53.12%,到25 d时释放量为84.52%。最后将纤维膜植入下肢缺血的大鼠体内,进行免疫组化观察,结果表明颗粒复合PLGA纤维的再生血管数量明显高于w/o型乳液法制备得到的纤维。

For both in situ or ex situ regeneration of tissues to be repaired, an ideal tissue engineering scaffold should create a local environment to facilitate homing, induced differentiation, and proliferation in addition to its molding function. For a tissue engineering scaffold to achieve this objective, releasing the relevant cell growth factors for sufficient period of time is more efficient and natural over that by material morphology. However, protein drugs are delicate and easily denatured by the harsh conditions of preparation for tissue engineering scaffolds, which may evoke undesirable antibody responses. None of the tissue engineering scaffolds reported to date have achieved long-term sustained-release of cell growth factor proteins in native conformation. To overcome this still-standing hurdle, the present study demonstrated a method to load cytokine proteins into tissue-engineering fibers with preserved native conformation for achieving long- term sustained-release. We first preloaded vascular endothelial growth factor （VEGFj65） as the model protein into dextran nanoparticles without contacting water-oil or water-gas interface （recognized protein conformation killer） by utilizing the freezing induced aqueous phase separation method developed in our lab. Then the VEGF165 protected in the matrix of dextran particles were suspended in a solution of poly （lactic-co-glycolic acid） （PLGA） prior to electrospinning to form fibers. In order to avoid burst release in the initial stage by the protein-preloaded particles exposed at the fiber surface, another layer of PLGA fiber which carried no VEGF^65 was spanned on the top of the already formed PLGA fiber membrane. A series ofphysicochemical characterizations comprising scanning electron microscopy （SEM）, contact angle, mechanical strength, confirmed that the tissue engineering fibers formed above possessed satisfied surface texture and mechanical strength. The VEGF165 was recovered after each of the preparation step and assayed using CCK-8 test for bioaetivity. The results indicated that the VEGF165 recovered from the dextran nanoparticles retained〉97 % bioactivity, and that from the final fiber was above 87 %, both were remarkably higher than that recovered from the fibers formed in which the protein was preloaded in solution form （〈55 %）. In vitro release test showed that nanoparticles composite fibers prepared as above achieved a sustained-release profile with minimal initial burst and un-released residue. The protein was released less than 35 % of the total load in the initial five days and over 92 % at day 25. For the fibers wherein VEGF165 was loaded in the solution form, the 53.12 % of protein load was released in the initial five days, and 84.52 % was released at day 25. The therapeutic efficacy of the VEGF165 loaded fiber membranes were examined on the rat model of lower- limb ischemia, followed immunohistochemistry observation. The results indicated that the regenerated blood vessels by dextran nanoparticles composite PLGA fibers were significantly more than that by the fibers to which VEGF165 was loaded in solution form. As conclusion, pre-loading proteins in polysaccharide nanoparticles prior to electrospinning was an effective method to prepare efficacious tissue engineere fibers for sustained-release cell growth factored with preserved bioactivity and improved release kinetics.