Matrix regenerative nanotherapeutics for small abdominal aortic aneurysm repair

摘要 我们的目标是研究一种新的、微创的再生纳米疗法，以阻止或逆转 小(直径5.5厘米)腹主动脉瘤(AAA)的生长。AAA是对 最终破裂的腹主动脉。早期手术治疗小AAA没有治疗益处，也没有 还有其他已被证实的治疗方法。同时恢复结构细胞外基质(ECM； AAA壁中的胶原和弹性纤维)对于阻止或逆转AAA的生长至关重要，这受到a)它们的阻碍 基质金属蛋白酶(MMPs)酶上调导致的主动脉壁慢性破坏和b)缺乏 成人血管弹性纤维再生修复克服先天缺陷的途径 平滑肌细胞(SMC)。口服多西环素(DOX)可抑制AAA中的MMPs 可减缓AAA的生长，但有全身副作用，并在高剂量时抑制弹性蛋白的生物合成。至 为了避免这种情况，我们已经研制了可生物降解的聚乳酸-羟基乙酸(PLGA)纳米颗粒(NPs)，用于 多西环素(DOX)是一种在AAA管壁内稳定、持续释放的基质金属蛋白酶抑制剂， 经导管输注一过性血流闭塞的AAA节段。在低得多的版本级别(&lt；10 μg/ml)，可以维持其对基质金属蛋白酶的抑制作用，但也有利于刺激弹性基质 新组装(弹性形成)。我们还用阳离子两亲性对我们的NPs进行了独特的表面官能化 从释放的DOX的影响中分离出促弹性和抗蛋白水解性。以此为基础 有希望的初步数据，我们现在建议确认信号机制背后的独特的PRO- 口腔以下剂量DOX的基质再生和抗基质金属蛋白酶作用，确定DOX-NP配方 显著刺激仿生和稳定的弹性纤维组装，设计并测试一种磁性导向器 有效地将NP递送到AAA壁的系统，并展示了DOX-NPs在退变中的有效性 已经在临床前(大鼠)模型中形成了小的AAA。我们的目标将检验以下假设：1)支持弹性增长 阿昔洛韦的作用是通过Jnk的减少触发转化生长因子-β的增加1)数量和质量 弹性纤维组装可通过调节DOX对JNK抑制的严重程度来调节，3)DOX越多 在刺激弹性蛋白方面比SP600125(JNK的IC50剂量)更有效，因为它还直接使弹性蛋白失活 MMPs，以及4)DOX-NPs的再生刺激将恢复AAA壁上的基质稳态，以阻止其 成长。目的1将多柔比星抑制JNK的严重程度与下游弹力形成和抗基质金属蛋白酶相关联 大鼠AAA系膜细胞培养的结果。AIM 2将生产具有卓越的促弹性和 矩阵修复性质。AIM 3将开发一种磁性系统，将DOX-NPs靶向大鼠的AAA壁上 模特。目的4将评估磁响应型DOX-NPs对大鼠AAA的治疗效果。如果成功， 我们的方法将在更大的动物模型中得到验证，以使未来的临床试验合理化。我们的方法可以 前瞻性地减少或推迟高危老年AAA患者未来的手术需求。

Abstract Our objective is to investigate a new, minimally-invasive regenerative nanotherapy to arrest or regress growth of small (<5.5 cm diameter) abdominal aortic aneurysms (AAAs). AAAs are localized expansions of the abdominal aorta that ultimately rupture. Early surgery on small AAAs provides no treatment benefit and no other proven therapies exist. While reinstating homeostasis of the structural extracellular matrix (ECM; collagen and elastic fibers) in the AAA wall is critical to stop or reverse AAA growth, this is impeded by a) their chronic breakdown in the aorta wall by upregulated matrix metalloprotease (MMPs) enzymes and b) lack of approaches to overcome intrinsically deficient and defective elastic fiber regenerative repair by adult vascular smooth muscle cells (SMCs). Oral dosing of doxycycline (DOX) has been shown to inhibit MMPs in the AAA wall to slow AAA growth, but has systemic side effects and inhibits elastin biosynthesis at the high doses. To avoid this, we have formulated biodegradable polylactic-co- glycolic acid (PLGA) nanoparticles (NPs) for steady, sustained release of doxycycline (DOX), an MMP inhibitor within the AAA wall following one-time, catheter-wise infusion to a transiently flow-occluded AAA segment. At the much lower release levels (<10 μg/ml), DOX was found to maintain its MMP inhibitory effects, but also to beneficially stimulate elastic matrix neoassembly (elastogenesis). We also uniquely surface-functionalized our NPs with cationic amphiphiles that have pro-elastogenic & anti-proteolytic separate from the effects of the released DOX. Building on this promising preliminary data, we now propose to confirm the signaling mechanisms underlying the unique pro- matrix regenerative and anti-MMP effects of DOX at sub-oral doses, identify DOX-NP formulations that provide a significant stimulus to biomimetic and stable elastic fiber assembly, design and test a magnetic guidance system for efficient NP delivery to the AAA wall, and demonstrate efficacy of the DOX-NPs in regressing already formed small AAAs in a preclinical (rat) model. Our aims will test hypotheses that 1) pro-elastogenic effects of DOX are mediated by JNK decreases which trigger increases in TGF-β1, 2) quantity and quality of elastic fiber assembly can be regulated by modulating severity of JNK inhibition by DOX, 3) DOX is more effective than SP600125 (at their IC50 doses for JNK) in stimulating elastin since it also directly inactivates MMPs, and 4) regenerative stimuli due to DOX-NPs will restore matrix homeostasis in the AAA wall to arrest its growth. Aim 1 will correlate severity of DOX inhibition of JNK to downstream elastogenesis and anti-MMP outcomes in rat AAA SMC cultures. Aim 2 will generate DOX-NP formulations with superior pro-elastogenic & matrix reparative properties. Aim 3 will develop a magnetic system to target DOX-NPs to the AAA wall in a rat model. Aim 4 will assess therapeutic efficacy of magnetically-responsive DOX-NPs in rat AAAs. If successful, our approach will be validated in larger animal models to rationalize future clinical trials. Our approach can prospectively reduce or delay need for future surgery in high risk elderly AAA patients.