Stable and Active bFGF-Polymer Conjugates for Wound Healing

描述(由申请人提供)：每年，除了严重的发病率和感染风险外，急性和慢性伤口还会给许多人带来疼痛和痛苦。因此，有相当大的需要新的治疗方法，以诱导和加速伤口修复。碱性成纤维细胞生长因子(BFGF)刺激多种细胞的增殖和迁移，这些细胞在伤口愈合中起关键作用。然而，这种蛋白质在运送或储存时会迅速降解，从而阻碍了其治疗用途。这项研究的一个目标是通过共价连接一种模拟蛋白质天然保护剂肝素的合成聚合物来稳定bFGF。碱性成纤维细胞生长因子诱导其受体的二聚化，而肝素有助于受体的激活。因此，这项研究的第二个目标是生产碱性成纤维细胞生长因子的二聚体聚合物偶联物，以增加该蛋白相对于未修饰的碱性成纤维细胞生长因子的有丝分裂和迁移细胞活性。第三个目标是测试碱性成纤维细胞生长因子结合物修复伤口的临床前疗效。为实现这些目标，提出了三个具体目标。第一个目的是评价bFGF-p(SS)-co-PEGMA聚合物偶联物的稳定性和聚合物的细胞毒性。据推测，bFGFp(SS)-co-PEGMA将比bFGFp(SS)-co-PEGMA、bFGGMA-PEGMA和未修饰的蛋白质更稳定，并且聚合物在治疗有用的浓度下将是无毒的。为了实现这一点，bFGF结合物对胰酶、酸性pH、热、搅拌和储存的稳定性将通过标准的生化和细胞分析来量化。第二个目的是测定bFGFp(SS)-co-PEGMA-bFGF偶联物的体外生物活性。推测二聚bFGFp(SS)-co-PEGMA结合物在人真皮成纤维细胞(HDF)和人真皮角质形成细胞(HDK)的增殖和迁移实验中将比单体结合物和bFGF单独结合物更活跃。为了研究这一点，我们将确定结合物在体外对细胞增殖和迁移的刺激作用。受体的激活将得到验证。第三个目的是确定二聚体和单体碱性成纤维细胞生长因子磺化聚合物结合物促进伤口愈合的能力。二聚体bFGFp(SS)-co-PEGMA结合物在体内外修复正常创面和糖尿病创面方面可能比单体结合物或单独的bFGFp(SS)结合物更有效。为了测试这一点，将使用器官类型培养和小鼠的表浅创伤模型。这项研究的一个潜在结果是开发出一种促进皮肤修复的活性物质。这项研究的长期目标是产生有效和稳定的治疗方法来治疗急慢性伤口。

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.