Matrix regenerative nanotherapeutics for small abdominal aortic aneurysm repair

摘要 我们的目标是研究一种新的，微创再生纳米疗法，以阻止或消退 小（直径<5.5 cm）腹主动脉瘤（AAA）生长。AAAs是局部扩展的 最终破裂的腹主动脉小AAA的早期手术没有治疗获益， 存在其它经证实的疗法。同时恢复结构性细胞外基质（ECM; 胶原蛋白和弹性纤维）对于阻止或逆转AAA生长至关重要，这受到以下因素的阻碍：a）它们的 通过上调的基质金属蛋白酶（MMPs）酶在主动脉壁中的慢性破坏和B）缺乏 克服成人血管固有缺陷和有缺陷的弹性纤维再生修复的方法 平滑肌细胞（SMC）。口服强力霉素（DOX）可抑制AAA中的MMP。 壁，以减缓AAA的增长，但有全身性的副作用，并抑制弹性蛋白的生物合成在高剂量。到 为了避免这种情况，我们已经配制了可生物降解的聚乳酸-羟基乙酸共聚物（PLGA）纳米颗粒（NPs）， 稳定，持续释放强力霉素（DOX），一种MMP抑制剂在AAA壁内， 经导管输注至短暂血流闭塞的AAA节段。在低得多的释放水平（<10 发现DOX维持其MMP抑制作用，但也有益地刺激弹性基质， 新组装（弹性发生）。我们还用阳离子两亲物独特地表面官能化了我们的NP， 具有促弹性蛋白生成和抗蛋白水解作用，与释放的DOX的作用分开。在此基础上 有希望的初步数据，我们现在建议确认独特的亲， 基质再生和抗MMP作用，鉴定DOX-NP制剂， 一个显著刺激仿生和稳定的弹性纤维组装，设计和测试磁导向 用于有效NP递送到AAA壁的系统，并证明DOX-NP在消退中的功效。 已经在临床前（大鼠）模型中形成了小的AAA。我们的目标将测试假设，1）前弹性 DOX的作用是由JNK降低介导的，JNK降低触发TGF-β1的增加，2） DOX可通过调节JNK抑制的程度来调节弹性纤维的组装; 3）DOX对JNK的抑制程度越大， 在刺激弹性蛋白方面比SP 600125（在其JNK的IC 50剂量下）有效，因为它也直接失活 MMP，和4）由于DOX-NP的再生刺激将恢复AAA壁中的基质稳态，以阻止其生长。 增长目的1将JNK的DOX抑制的严重性与下游弹性蛋白生成和抗MMP 大鼠AAA SMC培养物的结果。目标2将产生具有上级促弹性蛋白生成和抗弹性蛋白生成的DOX-NP制剂。 基质修复性能。目标3将开发一种磁性系统，将DOX-NPs靶向大鼠AAA壁 模型目的4将评估磁响应性DOX-NP在大鼠AAA中的治疗功效。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 前瞻性减少或延迟高风险老年AAA患者未来手术的需求。

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.