Interfacial Activity of PEG-modified Proteins with Application to Sustained Release

最近发表的工作表明，蛋白质与聚（乙二醇）的共价缀合，即，通过“蛋白质PEG化”，显著增加了从聚（丙交酯-共-乙交酯）（PLG）微球贮库释放的包封蛋白质的总量和持续释放后生物活性的保留分数。 本研究基于以下假设：这些有益效果主要是由于PEG接枝物对蛋白质吸附的影响，并且PEG化控制吸附亲和力、可逆性、表面诱导的构象变化和聚集的严重性，在油/水和固体/聚乙二醇化已经是一种经过验证的技术，用于微球贮库制造和持续药物释放应用。增加体内循环时间和许多注射的蛋白质治疗剂的功效。拟议的研究涉及一种新的应用蛋白质聚乙二醇化，以尽量减少蛋白质生物活性的损失，从可生物降解的仓库持续释放。虽然蛋白质从PLG贮库中释放是一个涉及许多耦合现象的复杂过程，但蛋白质吸附到界面是释放期间蛋白质活性损失的众所周知的来源。这项研究将确定聚乙二醇化是否以及如何缓和吸附对蛋白质释放的有害影响，并将表明在未来的制剂工作中，复合吸附过程的哪些方面是最重要的控制。 通过最大限度地减少广泛使用PLG库持续释放蛋白质的主要障碍之一，这项研究将代表朝着完全，持续释放完全活性蛋白质药物的最终目标的重要进展。 为此，将使用互补的光谱和光学技术来揭示PEG化对蛋白质吸附到油/水界面的影响，所述油/水界面是在常用的双乳液微球制造过程中产生的，所述固体PLG/水界面是在从降解微球释放期间产生的。 PEG接枝物对蛋白质易受表面诱导的二级和三级构象变化和聚集的影响，以及吸附的程度和可逆性将被强调。 吸附现象的分子水平研究将与使用相同材料的体外释放研究平行进行，以便将释放行为与潜在的界面现象相关联。通过调节蛋白质/表面相互作用，聚乙二醇化对蛋白质治疗功效的众所周知的有益作用将被放大，用于通过缓释贮库施用的缀合物。PLG微球，最初设想为可植入的蛋白质递送仓库，对于非蛋白质药物比对于蛋白质更成功，主要是因为蛋白质构象和溶解度对局部环境高度敏感，特别是对蛋白质-表面相互作用的破坏作用。 为了实现从可生物降解的贮库中持续释放蛋白质的充分承诺，必须使表面诱导的生物利用度损失最小化。 拟议项目将为两名博士提供研究培训。学生本科生也将在三年中的每一年参与研究。 该项目还提供了更广泛的本科教育的好处，通过动手练习的发展，以补充在第一年本科生介绍生物医学工程课程的生物材料和药物输送讲座。一个推广计划将有利于匹兹堡公立学校区的初中和高中科学教育。PI和Co-PI将在暑假期间主持一名中学或高中科学教师，以开发适当版本的实践练习，以及相关的教学材料，并将其纳入自己的课堂。这项活动的成果将通过共同撰写一篇论文提交给科学教师的主要期刊来广泛传播。

CBET-0755284TiltonRecently published work indicates that covalent conjugation of proteins with poly(ethylene glycol), i.e., "protein PEGylation", significantly increases both the total amount of encapsulated protein released from poly(lactide-co-glycolide) (PLG) microsphere depots and the fractional retention of biological activity after sustained release. This research is based on the hypothesis that these beneficial effects are due mainly to the effects of the PEG grafts on protein adsorption and that PEGylation controls the adsorption affinity, reversibility, severity of surface induced conformational changes and aggregation, at both the oil/water and solid/water interfaces that are relevant to microsphere depot manufacture and sustained drug release application.PEGylation is already a proven technology for increasing in vivo circulation times and efficacy of many injected protein therapeutics. The proposed research concerns a new application of protein PEGylation to minimize protein bioactivity loss during sustained release from biodegradable depots. While protein release from PLG depots is a complex process involving many coupled phenomena, protein adsorption to interfaces is a well known source of lost protein activity during release. This research will determine whether and how PEGylation moderates the deleterious effects of adsorption on protein release, and will indicate just which aspects of the complex adsorption processes are most important to control in future formulation efforts. By minimizing one of the major obstacles to widespread use of PLG depots for sustained protein release, this research will represent important progress toward the ultimate goal of complete, sustained release of fully active protein drugs. To that end, complementary spectroscopic and optical techniques will be used to reveal the effects of PEGylation on protein adsorption to the oil/water interfaces that are generated during the commonly used double emulsion microsphere manufacturing process and the solid PLG/water interfaces that are generated during release from the degrading microspheres. The effects of PEG grafts on protein vulnerability to surface-induced secondary and tertiary conformational change and aggregation, as well as the extent and reversibility of adsorption will be emphasized. Molecular level studies of adsorption phenomena will be conducted in parallel with in vitro release studies using the same materials in order to correlate release behaviors with the underlying interfacial phenomena.By modulating protein/surface interactions, the well-known beneficial effects of PEGylation on protein therapeutic efficacy will be amplified for conjugates administered via sustained release depots. PLG microspheres, originally envisioned as implantable protein delivery depots, have been far more successful for non-protein pharmaceuticals than for proteins largely because protein conformation and solubility are highly sensitive to local environment, particularly to the damaging effects of protein-surface interactions. To achieve the full promise of sustained protein release from biodegradable depots, surface-induced loss of bioavailability must be minimized. The proposed project will provide research training for two Ph.D. students. Undergraduate students will participate in the research during each of the three years as well. The project also provides broader undergraduate educational benefits through the development of hands-on exercises to complement lectures on biomaterials and drug delivery in the first year undergraduate Introduction to Biomedical Engineering course. An outreach program will benefit middle school and high school science education in the Pittsburgh Public Schools district. One middle school or high school science teacher will be hosted by the PI and Co-PI during a summer break to develop appropriate versions of the hands-on exercises, plus associated teaching materials, that they will incorporate into their own classrooms. Products of this activity will be broadly disseminated by co-authoring a paper to submit to a leading journal for science teachers.