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

CBET-0755284Tilton最近发表的工作表明，蛋白质与聚乙二醇的共价偶联，即蛋白质的聚乙二醇化，显著增加了从聚丙交酯-乙交酯(PLG)微球中释放的微囊化蛋白质的总量，以及在持续释放后生物活性的部分保留率。这项研究的基础假设是，这些有益的效果主要是由于聚乙二醇接枝对蛋白质吸附的影响，并且聚乙二醇化控制着与微球库制造和药物缓释应用相关的油/水和固体/水界面上的吸附亲和力、可逆性、表面诱导的构象变化和聚集的严重程度。聚乙二醇化已经是一种被证明可以增加体内循环时间和许多注射的蛋白质疗法的有效性的技术。这项拟议的研究涉及一种新的应用，即蛋白质聚乙二醇化，以减少从可生物降解的储存库中持续释放过程中蛋白质的生物活性损失。虽然蛋白质从PLG库中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众所周知的在释放过程中失去蛋白质活性的一个来源。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，并将指明复杂吸附过程的哪些方面在未来的配方工作中最需要控制。通过最大限度地减少广泛使用PLG仓库进行持续蛋白质释放的主要障碍之一，这项研究将代表着朝着完全、持续释放完全有效的蛋白质药物的最终目标取得的重要进展。为此，将使用互补的光谱和光学技术来揭示聚乙二醇化对蛋白质在通常使用的复乳微球制造过程中产生的油/水界面以及从降解微球释放过程中产生的固体PLG/水界面的吸附的影响。重点讨论了聚乙二醇接枝对蛋白质表面诱导的二级和三级构象变化和聚集的易感性以及吸附的程度和可逆性的影响。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并行进行，以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，通过缓释库给药的结合物将放大聚乙二醇化对蛋白质治疗效果的众所周知的有益影响。PLG微球最初被设想为可植入的蛋白质传递库，但在非蛋白质药物中的成功程度远远高于蛋白质，这在很大程度上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。为了实现蛋白质从可生物降解库中持续释放的全部承诺，必须将表面引起的生物利用度损失降至最低。拟议的项目将为两名博士生提供研究培训。本科生也将在三年中的每一年参与这项研究。该项目还通过开发实践练习来补充一年级本科生生物医学工程概论课程中关于生物材料和药物输送的讲座，从而提供更广泛的本科教育益处。一项外展计划将使匹兹堡公立学校区的初中和高中科学教育受益。一名初中或高中科学教师将在暑假期间由PI和Co-PI接待，以开发适当版本的动手练习，以及相关的教学材料，他们将纳入自己的课堂。这项活动的成果将通过共同撰写一篇论文提交给一家领先的科学教师期刊来广泛传播。

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.