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

最近发表的研究表明，蛋白质与聚乙二醇的共价偶联，即“蛋白质聚乙二醇化”，显著增加了从聚乳酸-羟基乙酸酯（PLG）微球库中释放的被封装蛋白质的总量和缓释后生物活性的部分保留。本研究基于这样的假设，即这些有益的影响主要是由于PEG接枝对蛋白质吸附的影响，而PEGylation控制了与微球储存库制造和药物缓释应用相关的油/水和固/水界面的吸附亲和性、可逆性、表面诱导构象变化和聚集的严重程度。聚乙二醇化已经是一种成熟的技术，可以增加体内循环时间和许多注射蛋白疗法的功效。提出的研究涉及蛋白质聚乙二醇化的新应用，以尽量减少蛋白质从可生物降解仓库持续释放过程中的生物活性损失。虽然蛋白质从PLG仓库释放是一个涉及许多耦合现象的复杂过程，但蛋白质在界面上的吸附是释放过程中蛋白质活性丧失的一个众所周知的来源。这项研究将确定聚乙二醇化是否以及如何调节吸附对蛋白质释放的有害影响，并将指出在未来的配方工作中，复杂吸附过程的哪些方面是最重要的。通过最大限度地减少广泛使用PLG库进行蛋白质持续释放的主要障碍之一，本研究将代表着朝着完全、持续释放全活性蛋白质药物的最终目标取得的重要进展。为此，互补的光谱和光学技术将用于揭示聚乙二醇化对蛋白质吸附到通常使用的双乳液微球制造过程中产生的油/水界面和从降解微球释放过程中产生的固体PLG/水界面的影响。将强调PEG接枝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。分子水平的吸附现象研究将与使用相同材料的体外释放研究并行进行，以便将释放行为与潜在的界面现象联系起来。通过调节蛋白质/表面的相互作用，众所周知的聚乙二醇化对蛋白质治疗效果的有益作用将被通过缓释库给药的偶联物放大。PLG微球最初被设想为可植入的蛋白质递送仓库，在非蛋白质药物方面比蛋白质药物成功得多，这主要是因为蛋白质的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性影响。为了实现从可生物降解仓库中持续释放蛋白质的全部承诺，必须尽量减少表面引起的生物利用度损失。本项目将为两名博士生提供研究培训。本科生也将在这三年的每一年参与研究。该项目还通过开发动手练习来补充本科第一年生物医学工程导论课程中的生物材料和药物输送课程，从而提供更广泛的本科教育效益。一项扩展计划将惠及匹兹堡公立学区的初中和高中科学教育。一名初中或高中科学教师将在暑假期间由PI和Co-PI主持，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。本次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.