摘要
粒细胞-巨噬细胞集落刺激因子(Granulocyte-Macrophage Colony-Stimulating Factor,以下简称GM-CSF),是一种具有刺激粒系、巨噬细胞系细胞分化和成熟的糖蛋白,被临床广泛用于对骨髓移植等治疗手段或HIV感染等疾病造成的粒细胞减少的治疗。此外,它在各类疫苗中作为免疫佐剂的功效也被广泛的研究。
然而,与一般的蛋白治疗药物相似,人的GM-CSF的体内半衰期短,同时相关疾病的治疗周期长,病人必须接受频繁而长期的注射。
为了减少其注射频率、延长蛋白作用的时间,国际上报道了各种尝试,然而,它们各自仍然存在许多问题。总结起来,如何在尽可能保持蛋白自然状态,即既不对蛋白本身进行结构的改变,如化学修饰,也不因为制备等过程伤害蛋白结构的稳定性的情况下,制备能够抑制突释和不完全释放等问题,具有良好释放形态的缓释系统已成为大分子缓释领域一大挑战,也成为我们的研究目标。
本研究中我们采用了新型的高分子水相-水相乳化技术,利用两种水溶性高分子通过表面活性剂的作用,在温和的、无油相存在的条件下乳化形成稳定的乳液,冻干后形成多糖玻璃体颗粒包封蛋白。由于乳化过程中无有机溶剂的存在,避免了水-油界面和界面张力的产生,保护了蛋白活性。该法制成粒径在1μm～5μm的多糖玻璃体微粒,大小均匀、表面圆滑、不粘连。第二步,我们将含有蛋白的多糖微粒通过水包油包固的方法担载于PLGA缓释微球中。由于蛋白担载于含有多糖高分子的玻璃体颗粒内,高分子玻璃体颗粒对于有机溶剂具有一定的抗性,再次避免了在传统W/O/W法中蛋白直接与水-油界面相接触造成的活性损失,在该步骤内进一步保护蛋白。该步骤制得的含颗粒微球粒径在60-120μm左右,微球外观性质良好。
对该法制备过程的考察结果显示,对GM-CSF其具有较高的包封率,达到80%以上;对回收率的检测结果显示,第一步颗粒的制备过程中,几乎没有物理损失,而第二步微球制备中,回收率在60%-70%。同时,该法在制备过程中较好的保护了蛋白活性,颗粒和微球的活性都在85%以上。而对体外释放和活性的进一步考察表明,该方法可以通过改变微球中多糖与蛋白的比例等参数条件调节优化蛋白的释放动力学特征,改善初期严重的突释和不完全释放;并且在释放过程中较好的保护蛋白活性。
Human granulocyte-macrophage colony-stimulating factor (GM-CSF), by exerting it’s proliferate and differentiation effects on granulocytes, macrophages and eosinophilic precursors, has been in clinical trials in accelerating the recovery of neutrophil levels and also widely studied as the adjuvant in varied kinds of vaccine.
However, as many as protein therapeutics, the plasma half-life of hGM-CSF is considerably short and frequent injection is required, causing problems of patients compliance.
Research effort to develop long acting and sustained release systems have not yet resulted in a successful commercial product. Activity loss in formulation processes or the release period is the major technical hurdle in development of sustained release depot.
The primary objective is to exam a new microencapsulation process using a unique Aqueous-Aqueous emulsion system. Stucture delicate GM-CSF was mixed with a Dextran solution then emulsified into a PEG solution containing sodium alginate. The proteins preferentially partitioned into the dextran depersed phase and lyophilized to solid particles, 1-5μm in diameter. The particles were then encapsulated into PLGA microspheres, 60-120μm in diameter.
A TF-1 cell proliferation assay and a SEC-HPLC analysis after each formulation step indicated that bioactivity and interrity of rhGM-CSF was preserved during the formulation process. The dextran glassy particles also played a role in improvement of protein release kinetics by minimizing initial burst and incomplete release. For a given protein loading, increasing dextran content ensured complete release, while limiting dextran content minizing intial burst. GM-CSF released from the microsphere formulation in PBS buffer at 37℃showed preserved activity based on TF-1 cell proliferation assay.
This thesis study demonstrated a promising microencapsulation process for formulating structurally delicated protein theraputics.
引文
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