Stable and Active bFGF-Polymer Conjugates for Wound Healing

描述（由申请人提供）：急性和慢性伤口除了显著的发病率和感染风险外，每年还会给许多人带来疼痛和痛苦。因此，对诱导和加速伤口修复的新疗法有相当大的需求。碱性成纤维细胞生长因子（bFGF）刺激几种在伤口愈合中起关键作用的细胞类型的增殖和迁移。然而，蛋白质在运送或储存时迅速降解，迄今为止阻碍了其治疗用途。这项研究的一个目的是通过共价偶联一种合成聚合物来稳定bFGF，这种聚合物模仿了蛋白质的天然保护剂肝素。bFGF诱导其受体二聚化，肝素辅助受体活化。因此，本研究的第二个目标是生产bFGF的二聚体聚合物偶联物，以便相对于未修饰的bFGF增加蛋白质的有丝分裂和迁移细胞活性。第三个目的是测试bFGF偶联物的临床前愈合效果。为实现这些目标，提出了三个具体目标。第一个目的是评估bFGF-p (SS)-co-PEGMA聚合物偶联物与对照物的稳定性和聚合物的细胞毒性。假设bFGF-p (SS)-co- pegma将比bFGF- ppegma、bFGF- peg和未修饰的蛋白更稳定，并且在治疗有用的浓度下聚合物将无毒。为了实现这一目标，bFGF偶联物对胰蛋白酶、酸性pH、加热、搅拌和储存的稳定性将通过标准生化和细胞分析进行量化。第二个目的是确定bFGF-p (SS)-co-PEGMA-bFGF偶联物的体外生物活性。假设二聚体bFGF p (SS)-co-PEGMA偶联物在人真皮成纤维细胞（HDF）和人真皮角质形成细胞（HDK）细胞增殖和迁移试验中比单体偶联物和bFGF单独更活跃。为了研究这一点，将确定结合物诱导的细胞增殖和细胞迁移的体外刺激。受体激活将被验证。第三个目的是确定二聚体和单体bFGF磺化聚合物缀合物促进伤口愈合的能力。假设二聚体bFGF p(SS)-co-PEGMA偶联物在体外和体内对正常和糖尿病伤口的愈合比单体偶联物或单独的bFGF更有效。为了验证这一点，将采用小鼠的器官型培养和浅表损伤模型。这项研究的一个潜在结果是开发一种促进皮肤修复的活性剂。本研究的长期目标是研制出有效稳定的治疗急慢性伤口的药物。

DESCRIPTION (provided by applicant): Acute and chronic wounds cause pain and suffering, in addition to significant morbidity and risk of infection, to a large number of people each year. As a result, there is considerable need for new therapeutics that induces and accelerate wound repair. Basic fibroblast growth factor (bFGF) stimulates the proliferation and migration of several cell types that have crucial roles in wound healing. However, the protein is rapidly degraded when delivered or upon storage, thus far preventing its therapeutic use. One objective of this research is to stabilize bFGF by covalently conjugating a synthetic polymer that mimics a natural protector of the protein, heparin. bFGF induces dimerization of its receptors and heparin aids in receptor activation. Thus, a second objective on this research is to produce dimeric polymer conjugates of bFGF in order to increase the mitogenic and migratory cellular activity of the protein relative to unmodified bFGF. The third objective is to test preclinical efficacy of the bFGF conjugates to heal wounds. Three specific aims are proposed to reach these objectives. The first aim is to evaluate the stability of bFGF-p (SS)-co-PEGMA polymer conjugates compared to controls and cytotoxicity of the polymers. It is hypothesized that bFGF- p (SS)-co-PEGMA will be more stable than bFGF-pPEGMA, bFGF-PEG and the unmodified protein, and the polymers will be nontoxic at therapeutically useful concentrations. To accomplish this, stability of the bFGF conjugate against trypsin, acidic pH, heat, stirring, and storage will be quantified by standard biochemical and cellular assays. The second aim is to determine in vitro bioactivity of bFGF-p (SS)-co-PEGMA-bFGF conjugates. It is hypothesized that dimeric bFGF p (SS)-co-PEGMA conjugates will be more active in human dermal fibroblast (HDF) and human dermal keratinocyte (HDK) cell proliferation and migration assays than the monomeric conjugates and bFGF alone. To investigate this, conjugate-induced stimulation of cell proliferation and migration of cells in vitro will be ascertained. Receptor activation will be verified. The third aim is to determine the ability of dimeric and monomeric bFGF sulfonated polymer conjugates to enhance wound healing. It is hypothesized that dimeric bFGF p(SS)-co-PEGMA conjugates will be more effective at healing normal and diabetic wounds in vitro and in vivo than the monomeric conjugate or bFGF alone. To test this, organotypic cultures and superficial wounding models in mice will be employed. One potential outcome of this research is to develop an active agent that promotes skin repair. The long-term goal of this research is to produce efficacious and stable therapeutics to treat acute and chronic wounds.