Abstract：
Anti-angiogenesis has emerged a promising strategy against colorectal cancer (CRC). However, the efficacy of anti-angiogenic therapy is greatly compromised by the up-regulated autophagy levels resulting from the evolutionary resistance mechanism and the presence of Fusobacterium nucleatum (F. nucleatum) in CRC. Herein, we report a cationic polymer capable of blocking autophagic flux to deliver plasmid DNA (pDNA) encoding soluble FMS-like tyrosine kinase-1 (sFlt-1) for enhanced anti-angiogenic therapy against F. nucleatum-associated CRC. The autophagy-inhibiting cationic polymer, referred to as PNHCQ, is synthesized by conjugating hydroxychloroquine (HCQ) into 3,3\'-diaminodipropylamine-pendant poly(β-benzyl-L-aspartate) (PAsp(Nors)), which can be assembled and electrostatically interacted with sFlt-1 plasmid to form PNHCQ/sFlt-1 polyplexes. Hydrophobic HCQ modification not only boosts transfection efficiency but confers autophagy inhibition activity to the polymer. Hyaluronic acid (HA) coating is further introduced to afford PNHCQ/sFlt-1@HA for improved tumor targeting without compromising on transfection. Consequently, PNHCQ/sFlt-1@HA demonstrates significant anti-tumor efficacy in F. nucleatum-colocalized HT29 mouse xenograft model by simultaneously exerting anti-angiogenic effects through sFlt-1 expression and down-regulating autophagy levels exacerbated by F. nucleatum challenge. The combination of anti-angiogenic gene delivery and overall autophagy blockade effectively sensitizes CRC tumors to anti-angiogenesis, providing an innovative approach for enhanced anti-angiogenic therapy against F. nucleatum-resident CRC. STATEMENT OF SIGNIFICANCE: Up-regulated autophagy level within tumors is considered responsible for the impaired efficacy of clinic antiangiogenic therapy against CRC colonized with pathogenic F. nucleatum. To tackle this problem, an autophagy-inhibiting cationic polymer is developed to enable efficient intracellular delivery of plasmid DNA encoding soluble FMS-like tyrosine kinase-1 (sFlt-1) and enhance anti-angiogenic therapy against F. nucleatum-associated CRC. HA coating that can be degraded by tumor-enriching hyaluronidase is further introduced for improved tumor targeting without compromising transfection efficiency. The well-orchestrated polyplexes achieve considerable tumor accumulation, efficient in vivo transfection, and effectively reinforce the sensitivity of CRC to the sFlt-1-derived anti-angiogenic effects by significantly blocking overall autophagy flux exacerbated by F. nucleatum challenge, thus harvesting robust antitumor outcomes against F. nucleatum-resident CRC.
摘要：
抗血管生成已经出现了针对结直肠癌（CRC）的有希望的策略。然而,进化抗性机制和核梭杆菌的存在导致的上调的自噬水平极大地损害了抗血管生成疗法的功效(F.核仁）在CRC中。在这里，我们报道了一种阳离子聚合物,其能够阻断自噬通量以递送编码可溶性FMS样酪氨酸激酶-1(sFlt-1)的质粒DNA(pDNA),用于增强抗血管生成治疗核仁F.抑制自噬的阳离子聚合物，称为PNHCQ，通过将羟基氯喹（HCQ）缀合到3,3'-二氨基二丙胺-侧基聚（β-苄基-L-天冬氨酸）（PAsp（Nors））中合成，可以与sFlt-1质粒组装并静电相互作用以形成PNHCQ/sFlt-1复合物。疏水性HCQ修饰不仅提高转染效率，而且赋予聚合物自噬抑制活性。进一步引入透明质酸(HA)涂层以提供PNHCQ/sFlt-1@HA,用于改善肿瘤靶向而不损害转染。因此,PNHCQ/sFlt-1@HA通过同时通过sFlt-1表达发挥抗血管生成作用并下调由F.核仁攻击加剧的自噬水平，在F.核仁共定位的HT29小鼠异种移植模型中显示出显著的抗肿瘤功效。抗血管生成基因递送和整体自噬阻断的组合有效地使CRC肿瘤对抗血管生成敏感,提供了一种创新的方法来增强对F.核仁常驻CRC的抗血管生成治疗。重要声明：肿瘤内上调的自噬水平被认为是临床抗血管生成治疗对致病性核仁F.定植的CRC功效受损的原因。为了解决这个问题，开发了一种抑制自噬的阳离子聚合物，以使编码可溶性FMS样酪氨酸激酶-1（sFlt-1）的质粒DNA能够有效地在细胞内递送，并增强针对核仁F.相关CRC的抗血管生成治疗。可以通过肿瘤富集透明质酸酶降解的HA涂层被进一步引入以改善肿瘤靶向而不损害转染效率。精心策划的聚合复合物实现了相当大的肿瘤积累，有效的体内转染，并通过显著阻断核仁F.攻击加剧的总体自噬通量，有效增强CRC对sFlt-1来源的抗血管生成作用的敏感性，因此获得了针对核仁F.驻留CRC的稳健抗肿瘤结果。
