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首页> 外文学位 >Production of bioinspired and rationally designed polymer hydrogels for controlled delivery of therapeutic proteins.
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Production of bioinspired and rationally designed polymer hydrogels for controlled delivery of therapeutic proteins.

机译：具有生物启发性和合理设计的聚合物水凝胶的生产，可控制治疗性蛋白质的传递。

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摘要

Hydrogel systems for controlled delivery therapeutic growth factors have been developed in a wide spectrum of strategies: these systems aim for the release of growth factors via a passive diffusion, electrostatic interaction, degradation of hydrogels, and responsiveness to external stimuli. Heparin, a highly sulfated glycosaminoglycan (GAG), was employed for a targeted delivery system of vascular endothelial growth factor (VEGF) to endothelial cells overexpressing a relevant receptor VEGFR-2. Addition of dimeric VEGF to 4-arm star-shaped poly(ethylene glycol) (PEG) immobilized with low-molecular weight heparin (LMWH) afforded a non-covalently assembled hydrogel via interaction between heparin and VEGF, with storage modulus 10 Pa. The release of VEGF and hydrogel erosion reached maximum 100 % at day 4 in the presence of VEGFR-2 overexpressing pocine aortic endothelial cell (PAE/KDR), while those of 80% were achieved via passive release at day 5 in the presence of PAE cell lacking VEGFR-2 or in the absence of cell, indicating that the release of VEGF was in targeted manner toward cell receptor. The proliferation of PAE/KDR in the presence of [PEG-LMWH/VEGF] hydrogel was greater by ca. 30% at day 4 compared to that of PAE, confirming that the release of VEGF was in response to the cellular demand. The phosphorylation fraction of VEGFR-2 on PAE/KDR was greater in the presence of [PEG-LMWH/VEGF] hydrogel, increasing from 0.568 at day 1 to 0.790 at day 4, whereas it was maintained at 0.230 at day 4 in the presence of [PEG-LMWH] hydrogel. This study has proven that this hydrogel, assembled via bio-inspired non-covalent interaction, liberating VEGFon celluar demand to target cell, eroding upon VEGF release, and triggering endothelial cell proliferation, could be used in multiple applications including targeted delivery and angiogenesis.;Heparin has been widely exploited in growth factor delivery systems owing to its ability to bind many growth factors through the flexible patterns of functional groups. However, heterogeneity in the composition and in the polydispersity of heparin has been problematic in controlled delivery system and thus motivated the development of homogeneous heparin mimics. Peptides of appropriate sequence and chemical function have therefore recently emerged as potential replacements for heparin in select applications. Studied was the assessment of the binding affinities of multiple sulfated peptides (SPs) for a set of heparin-binding peptides (HBPs) and for VEGF; these binding partners have application in the selective immobilization of proteins and in hydrogel formation through non-covalent interactions. Sulfated peptides were produced via solid-phase methods, and their affinity for the HBPs and VEGF was assessed via affinity liquid chromatography (ALC), surface plasmon resonance (SPR), and in select cases, isothermal titration calorimetry (ITC). The shortest peptide, SPa, showed the highest affinity binding of HBPs and VEGF165 in both ALC and SPR measurements, with slight exceptions. Of the investigated HBPs, a peptide based on the heparin-binding domain of human platelet factor 4 showed greatest binding affinities toward all of the SPs, consistent with its stronger binding to heparin. The affinity between SPa and PF4ZIP was indicated via SPR ( KD = 5.27 muM) and confirmed via ITC (KD = 8.09 muM). The binding by SPa of both VEGF and HBPs suggests its use as a binding partner to multiple species, and the use of these interactions in assembly of materials. Given that the peptide sequences can be varied to control binding affinity and selectivity, opportunities are also suggested for the production of a wider array of matrices with selective binding and release properties useful for biomaterials applications.;Hydrogel consisting of SPa was formed via a covalent Michael Addition reaction between maleimide- and thiol-terminated multi-arm PEGs and Cys-SPa. The mechanical property of hydrogel was tunable from ca. 186 to 1940 Pa. by varing the cross-linking density, suggesting its flexible applications depending on matrix needs. The non-anti-coagulative property of SPa, assessed via activated partial thromboplastin time (APTT) and HeptestRTM in comparison to LMWH, implied its usefulness in applications without excessive bleeding. The VEGF released from [PEG-SPa] hydrogel showed up to ca. 400% greater bioactivity on proliferation of human umbilical vein endothelical cell (HUVEC) compared to the VEGF incubated in solution for the same period: this was significantly higher than that of [PEG] hydrogel (ca. 280%), suggesting the SPa may protect the bioactivity of VEGF when bound. The release of dual growth factor, i.e. VEGF and fibroblast growth factor-2 (FGF-2), were investigated on [PEG-SPa] hydrogel: the release of bFGF was lower than that of VEGF due to weaker binding affinity to matrix-bound SPa. The HUVEC culture on dual growth factor loaded [PEG-SPa] showed that the synergistic effects of dual system in select concentrations, suggesting the opportunity of manipulating cell responses. Given that sulfated peptides for various binding targets with desired affinity can be identified, applications are suggested in multiple growth factors delivery where an integrated action of multiple growth factors is required, such as angiogenesis.
机译：用于控制递送的治疗性生长因子的水凝胶系统已经在多种策略中得到开发：这些系统旨在通过被动扩散，静电相互作用，水凝胶降解以及对外部刺激的反应来释放生长因子。肝素是一种高度硫酸化的糖胺聚糖（GAG），用于将血管内皮生长因子（VEGF）靶向递送至过表达相关受体VEGFR-2的内皮细胞。将二聚体VEGF添加到固定有低分子量肝素（LMWH）的4臂星形聚乙二醇（PEG）中，通过肝素和VEGF之间的相互作用提供了一个非共价组装的水凝胶，储能模量为10 Pa。在过度表达VEGFR-2的主动脉内皮细胞（PAE / KDR）存在的第4天，VEGF的释放和水凝胶侵蚀达到最大100％，而在存在PAE细胞的第5天通过被动释放实现了80％的释放缺乏VEGFR-2或细胞不存在，表明VEGF的释放是针对细胞受体的。在[PEG-LMWH / VEGF]水凝胶的存在下，PAE / KDR的增殖大约增加。与PAE相比，第4天的比例为30％，这证实VEGF的释放是对细胞需求的响应。 [PEG-LMWH / VEGF]水凝胶存在下，VEGFR-2在PAE / KDR上的磷酸化分数更高，从第1天的0.568增加到第4天的0.790，而在存在第4天时则保持在0.230 [PEG-LMWH]水凝胶的制备。这项研究已经证明，这种通过生物启发的非共价相互作用组装而成的水凝胶，可以释放对靶细胞的细胞需求的VEGF，侵蚀VEGF的释放，并触发内皮细胞的增殖，可用于多种应用，包括靶向递送和血管生成。肝素由于其通过功能性基团的灵活模式结合许多生长因子的能力而已在生长因子递送系统中得到广泛利用。然而，肝素的组成和多分散性中的异质性在受控的递送系统中是有问题的，并因此刺激了均质肝素模拟物的发展。因此，最近在适当的应用中出现了具有适当序列和化学功能的肽作为肝素的潜在替代品。研究了多种硫酸化肽（SP）对一组肝素结合肽（HBP）和VEGF的结合亲和力的评估；这些结合伴侣可用于蛋白质的选择性固定和通过非共价相互作用形成水凝胶。硫酸化的肽通过固相方法生产，其对HBP和VEGF的亲和力通过亲和液相色谱（ALC），表面等离振子共振（SPR）以及在某些情况下通过等温滴定量热（ITC）进行评估。最短的肽SPa在ALC和SPR测量中均显示出HBP和VEGF165的最高亲和力结合，只有少数例外。在研究的HBP中，基于人血小板因子4肝素结合结构域的肽对所有SP表现出最大的结合亲和力，与其对肝素的更强结合相一致。 SPa和PF4ZIP之间的亲和力通过SPR（KD = 5.27μM）表示，并通过ITC（KD = 8.09μM）确认。 SPa对VEGF和HBP的结合表明它可用作多种物质的结合伴侣，并在材料组装中利用这些相互作用。考虑到可以改变肽序列来控制结合亲和力和选择性，还提出了生产更多具有选择性结合和释放特性的基质的机会，这些基质对生物材料的应用很有用。由SPa组成的水凝胶是通过共价迈克尔形成的。马来酰亚胺和巯基封端的多臂PEG与Cys-SPa之间的加成反应。水凝胶的机械性能可从大约1调整。通过改变交联密度，可在186至1940 Pa。的范围内使用，这表明它可根据基质需求灵活应用。通过与LMWH相比，通过活化的部分凝血活酶时间（APTT）和HeptestRTM评估的SPa的非抗凝特性表明其在应用中没有过多出血的用途。从[PEG-SPa]水凝胶中释放的VEGF最高可显示约。与在同一时间在溶液中孵育的VEGF相比，对人脐静脉内皮细胞（HUVEC）增殖的生物活性高400％：这显着高于[PEG]水凝胶的生物活性（约280％），表明SPa可以保护结合时VEGF的生物活性。释放双重生长因子即VEGF和成纤维细胞生长因子2（FGF-2）在[PEG-SPa]水凝胶上进行了研究：由于与基质结合的SPa的结合亲和力较弱，因此bFGF的释放低于VEGF的释放。载有双重生长因子[PEG-SPa]的HUVEC培养物显示了在选定浓度下双重系统的协同作用，表明有操纵细胞应答的机会。考虑到可以鉴定出具有所需亲和力的各种结合靶标的硫酸化肽，建议在多种生长因子递送中应用，其中需要多种生长因子的整合作用，例如血管生成。

著录项

  • 作者

    Kim, Sung Hye.;

  • 作者单位

    University of Delaware.;

  • 授予单位 University of Delaware.;
  • 学科 Engineering Biomedical.;Engineering Materials Science.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 196 p.
  • 总页数 196
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物医学工程;工程材料学;
  • 关键词

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