Multifunctional Nanoparticles Containing sRAGE Potentiated Bioactive Peptides for Wound Healing

摘要 尽管存在几种治疗慢性糖尿病伤口的治疗选择，从封闭敷料， 真空辅助闭合、皮肤移植、生物工程皮肤替代物，在许多情况下，伤口不能 对治疗有充分的反应。慢性伤口的特征是不能从亲- 炎症性的伤口愈合的增殖阶段。虽然有人建议提供外源性的 但是使用这种方法很少成功， 在实践中肽生长因子由于在这样的细胞中蛋白酶的过量而迅速降解。 伤口此外，最近的证据表明，晚期糖基化终产物水平的增加， 糖基化终产物（AGEs）在糖尿病环境中可能干扰信号通路，从而使靶细胞 响应于生物活性肽（如生长因子和趋化因子）。我们最近发现， 可以通过使用可溶性β-内酰胺酶（sNAR）阻断AGEs（β-内酰胺酶）的受体来恢复应答。我们 提出开发由弹性蛋白样肽的融合蛋白组成的多功能纳米颗粒系统 （ELP）与相关的生物活性肽和唾液酸。我们假设这些纳米颗粒可以排除 蛋白酶，保护附着的生物肽免于降解，并且同时释放的唾液酸， 可以恢复糖尿病伤口的信号。此外，这些纳米颗粒自发地和可逆地 在生理温度下自组装，从而能够快速和廉价地纯化融合物 蛋白质，并且具有小于1微米的尺寸，纳米颗粒足够小以容易地掺入到细胞中。 局部治疗方式，包括先进的方法（例如，皮肤替代品，通常具有孔径 超过50微米）。为了验证这一假设，我们将开发sRAGE-ELP融合蛋白， 联合收割机将其与三种不同的生物活性肽中的一种结合，这三种生物活性肽针对伤口愈合的不同方面 过程：KGF-ELP（表皮）、SDF-ELP（真皮）和ARA 290-ELP（组织保护反应）。我们 具体目标是：（1）开发可逆地形成纳米颗粒的sRAGE-ELP融合蛋白， 自身和其他肽-ELP融合蛋白。(2)评价ELP为基础的生物活性 纳米颗粒在体外模拟糖尿病环境中的作用。(3)为了测试sRAGE-ELP纳米颗粒在小鼠中的作用， 体内糖尿病伤口条件。

ABSTRACT Although several therapeutic options to treat chronic diabetic wounds exist, ranging from occlusive dressings, vacuum assisted closure, skin grafts, to bioengineered skin substitutes, in many instances the wounds fail to adequately respond to treatment. Chronic wounds are characterized by a failure to progress from the pro- inflammatory to the proliferative phases of wound healing. While it has been proposed to provide exogenous growth factors to the wound to help in this transition, there has been very little success using such an approach in practice. Peptide growth factors are rapidly degraded due to the overabundance of proteases in such wounds. Furthermore, recent evidence suggests that the increased levels of advanced glycation endproducts (AGEs) in the diabetic environment may interfere with signaling pathways thus making target cells poorly responsive to bioactive peptides (such as growth factors and chemokines). We have recently shown that these responses can be restored by blocking the receptor to AGEs (RAGE) using soluble RAGE (sRAGE). We propose to develop a multi-functional nanoparticle system consisting of fusion proteins of elastin-like peptides (ELPs) with relevant bioactive peptides and sRAGE. We hypothesize that these nanoparticles can exclude proteases, protecting the attached biopeptides from degradation, and that the simultaneous release of sRAGE can restore signaling in the diabetic wound. Furthermore, these nanoparticles spontaneously and reversibly self-assemble at physiological temperatures, thus enabling rapid and inexpensive purification of the fusion proteins, and with a size below 1 micrometer, nanoparticles are small enough to be easily incorporated into topical treatment modalities, including advanced methods (e.g. skin substitutes, which typically have pore sizes in excess of 50 micrometers). To test the hypothesis, we will develop a sRAGE-ELP fusion protein and combine it with one of three different bioactive peptides that target different aspects of the wound healing process: KGF-ELP (epidermis), SDF-ELP (dermis), and ARA290-ELP (tissue protective response). Our specific aims are: (1) To develop sRAGE-ELP fusion proteins that reversibly form nanoparticles with themselves and other peptide-ELP fusion proteins. (2) To evaluate the biological activity of ELP-based nanoparticles in a simulated diabetic environment in vitro. (3) To test the effect of sRAGE-ELP nanoparticles in in vivo diabetic wound conditions.