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之外，每年还对大量的人造成疼痛和痛苦。因此，对诱导和加速伤口修复的新治疗剂存在相当大的需求。碱性成纤维细胞生长因子（bFGF）刺激在伤口愈合中起关键作用的几种细胞类型的增殖和迁移。然而，该蛋白质在递送或储存时迅速降解，因此阻碍了其治疗用途。本研究的一个目的是通过共价结合模拟蛋白质的天然保护剂肝素的合成聚合物来稳定bFGF。bFGF诱导其受体的二聚化，肝素有助于受体活化。因此，本研究的第二个目的是产生bFGF的二聚体聚合物缀合物，以相对于未修饰的bFGF增加蛋白质的促有丝分裂和迁移细胞活性。第三个目的是测试bFGF缀合物愈合伤口的临床前功效。为实现这些目标，提出了三个具体目标。第一个目的是评价与对照相比bFGF-p（SS）-co-PEGMA聚合物缀合物的稳定性和聚合物的细胞毒性。假设BGF-p（SS）-co-PEGMA将比bFGF-pPEGMA、bFGF-PEG和未修饰的蛋白质更稳定，并且聚合物在治疗上有用的浓度下将是无毒的。为了实现这一点，bFGF缀合物对胰蛋白酶、酸性pH、加热、搅拌和储存的稳定性将通过标准生物化学和细胞测定来定量。第二个目的是确定bFGF-p（SS）-co-PEGMA-bFGF缀合物的体外生物活性。假设二聚体bFGF β（SS）-co-PEGMA缀合物在人真皮成纤维细胞（HDF）和人真皮角质形成细胞（HDK）细胞增殖和迁移测定中比单体缀合物和单独的bFGF更有活性。为了研究这一点，将确定体外偶联物诱导的细胞增殖和细胞迁移刺激。将验证受体激活。第三个目的是确定二聚体和单体bFGF磺化聚合物缀合物增强伤口愈合的能力。假设二聚体bFGF β（SS）-co-PEGMA缀合物在体外和体内愈合正常和糖尿病伤口方面比单体缀合物或单独的bFGF更有效。为了测试这一点，将采用小鼠中的器官型培养物和浅表创伤模型。这项研究的一个潜在成果是开发一种促进皮肤修复的活性剂。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性和慢性伤口。

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.