多肽/胶原蛋白互穿杂化水凝胶用于化药/蛋白共载及瘤内控释的研究

Reducing the burst release and stabilizing the protein are essentially important for the development of the co-delivery of chemotherapeutic drugs and protein in designing in situ forming hydrogel. Our previous results suggested that the burst release of the in situ forming depot could be significantly reduced by encapsulating drug-loaded microparticles in the depot. Accordingly, in the present study, a nanoparticle encapsulated peptide/collagen interpenetrating network hybrid hydrogels is designed to provide a long-term sustained release intratumoral drug delivery system that can not only co-deliver chemotherapeutic drugs and proteins, but also reduce the burst release. Firstly, the model chemotherapeutic drug (doxorubicin, DOX) and the model protein (interferon-γ, IFN-γ) are co-encapsulated in heparin- cholesterol nanoparticles (NPs) that are functionalized with collagen mimetic peptide (CMP). IFN-γ is then encapsulated in the heparin layer via electrostatic interaction, while DOX is encapsulated in the lipid core. The functionalization of heparin can not only improve the stability of protein, but also sustain the protein release. Secondly, the CMP-NPs are dispersed in collagen solution to prepare the CMP/collagen interpenetrating network hybrid hydrogels. The CMP-NPs are anchored in the hydrogels via the strand exchange of short CMPs into the triple-helical collagen. The diffusion of the drugs or NPs can therefore be delayed, which may further reduce the burst release. The drug release behavior from the hybrid hydrogels can be totally assessed and further modified by investigating the effects of CMP length and density on the drug release from hydrogels. The in vitro drug release, in vivo drug release in normal muscle and in vivo drug release in tumor are studied to understand the drug release mechanism of hybrid hydrogels in tumor. The CMP-NPs/collagen interpenetrating network hybrid hydrogels may provide an excellent biodegradable sustained release injection drug delivery system for intratumoral co-delivery of chemotherapeutic drugs and proteins.

实现化药/蛋白的稳定化共载、无突释化释放是构建瘤内注射用化药/蛋白共载原位水凝胶的重要课题之一。前期研究显示微粒/凝胶杂化原位贮库可显著降低药物突释。基于此，本课题以阿霉素和IFN-γ为模型药，预先将二者共载于胶原模拟多肽修饰的肝素-胆固醇纳米粒中，利用肝素强负电荷性担载并稳定蛋白药，同时肝素本身可延缓蛋白药释放，利用胆固醇疏水性担载化药，使二者稳定共载于同一纳米释药单元中；再将纳米粒分散至胶原蛋白中，利用胶原模拟多肽可与胶原蛋白肽链形成杂化三螺旋结构的特点，将纳米粒锚定于胶原蛋白凝胶中，构建多肽/胶原蛋白互穿型杂化水凝胶，实现无突释化缓慢释药。通过研究胶原模拟多肽的肽链长度及修饰密度对水凝胶释药行为的影响，评价其释药可调控性；通过体外释药、健康组织及瘤内释药研究，阐明该凝胶体系肿瘤部位释药机制。该体系将为化药/蛋白瘤内给药提供一种载药可控、释药可调控、无突释的瘤内给药体系。

原位贮库型长效注射给药系统已被广泛用于小分子药物和生物大分子药物的缓释给药。但是药物突释是原位贮库体系普遍存在的问题，并且现阶段影响药物释放的关键因素尚不明确。以上两方面因素直接限制了原位贮库体系的广泛应用。因此，本项目设计了一种微粒/凝胶复合型原位贮库体系，延缓药物扩散速率，以降低药物突释，获得平稳释药行为。此外，本项目还分别系统阐述了影响小分子药物和生物大分子药物释放行为的关键因素。. 研究显示，将阿霉素（DOX）和干扰素-γ（IFN-γ）共载于微粒/凝胶复合型原位贮库体系中，可显著降低药物突释，体内外药物释放速率较为平稳，符合零级释药特点。微粒粒径是影响生物大分子药物体内外药物释放和降解的重要因素之一，与大粒径微粒（~20μm）相比，小粒径微粒（~5μm）由于比表面积大，初期药物释放速度较快；但其内部结构致密，后期降解速率慢，因此，后期药物释放缓慢。由此可见，小粒径微粒是导致前期突释和后期释药过慢，血药浓度过低的主要因素，因此，应严格控制微粒中小粒径微粒比例。对于阿霉素类弱碱性药物，微粒制备过程中微pH环境是影响药物释放的重要因素之一。通过提高制备环境pH值，可使部分PLGA降解，暴露出更多的游离羧基，增加PLGA与药物分子间相互作用，提高包封率；同时由于PLGA部分降解，可诱导微粒呈多孔结构，加速药物释放，避免释药延滞期，从而使药物释放速度更平稳，呈现零级释药特征。. 本项目构建的微粒/凝胶复合型原位贮库有效解决了药物突释问题，为无突释化平稳释药长效注射给药系统的设计提供了新思路。同时，本项目阐明了影响小分子药物和生物大分子药物释放行为的关键因素，这为长效注射给药系统的研究应用奠定基础并提供参考。