Purpose: Vascular endothelial growth factor receptor 2 (VEGFR-2) and neuropilin-1 (NRP-1) are two prominent synergistic receptors overexpressed on new blood vessels in glioma and may be promising targets for antiglioma therapy. The aim of this study was to design a dual receptor targeting and blood-brain barrier (BBB) penetrating peptide-modified polyethyleneimine (PEI) nanocomplex that can efficiently deliver the angiogenesis-inhibiting secretory endostatin gene (pVAXI-En) to treat glioma.
Materials and Methods: We first constructed the tandem peptide TAT-AT7 by conjugating AT7 to TAT and evaluated its binding affinity to VEGFR-2 and NRP-1, vasculature-targeting ability and BBB crossing capacity. Then, TAT-AT7-modified PEI polymer (PPTA) was synthesized, and a pVAXI-En-loaded PPTA nanocomplex (PPTA/pVAXI-En) was prepared. The physicochemical properties, cytotoxicity, transfection efficiency, capacities to cross the BBB and BTB (blood-tumor barrier) and glioma-targeting properties of PPTA/pVAXI-En were investigated. Moreover, the in vivo anti-angiogenic behaviors and anti-glioma effects of PPTA/pVAXI-En were evaluated in nude mice.
Results: The binding affinity of TAT-AT7 to VEGFR-2 and NRP-1 was approximately 3 to 10 times greater than that of AT7 or TAT. The cellular uptake of TAT-AT7 in endothelial cells was 5-fold and 119-fold greater than that of TAT and AT7 alone, respectively. TAT-AT7 also displayed remarkable efficiency in penetrating the BBB and glioma tissue in vivo. PPTA/pVAXI-En exhibited lower cytotoxicity, stronger BBB and BTB traversing abilities, higher selective glioma targeting and better gene transfection efficiency than PEI/pVAXI-En. More importantly, PPTA/pVAXI-En significantly suppressed the tube formation and migration of endothelial cells, inhibited glioma growth, and reduced the microvasculature in orthotopic U87 glioma-bearing nude mice.
Conclusion: Our study demonstrates that PPTA/pVAXI-En can be exploited as an efficient dual-targeting nanocomplex to cross the BBB and BTB, and hence it represents a feasible and promising nonviral gene delivery system for effective glioma therapy.

Keywords: VEGFR-2 and NRP-1 targeting, glioma penetration, multifunctional peptide, anti-angiogenesis, gene delivery system


中文翻译：

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双受体靶向和 BBB 穿透肽功能化聚乙烯亚胺纳米复合物，用于分泌性内皮抑素基因递送至恶性胶质瘤


目的：血管内皮生长因子受体 2 (VEGFR-2) 和神经毡蛋白-1 (NRP-1) 是在神经胶质瘤新生血管上过表达的两种重要协同受体，可能是抗神经胶质瘤治疗的有希望的靶点。本研究的目的是设计一种双受体靶向和血脑屏障（BBB）穿透肽修饰的聚乙烯亚胺（PEI）纳米复合物，可以有效地递送抑制血管生成的分泌性内皮抑素基因（pVAXI-En）来治疗神经胶质瘤。

材料和方法：我们首先通过将 AT7 与 TAT 缀合构建串联肽 TAT-AT7，并评估其与 VEGFR-2 和 NRP-1 的结合亲和力、血管靶向能力和 BBB 穿越能力。然后，合成TAT-AT7修饰的PEI聚合物(PPTA)，并制备负载pVAXI-En的PPTA纳米复合物(PPTA/pVAXI-En)。研究了 PPTA/pVAXI-En 的理化特性、细胞毒性、转染效率、穿过 BBB 和 BTB（血肿瘤屏障）的能力以及神经胶质瘤靶向特性。此外，在裸鼠中评估了 PPTA/pVAXI-En 的体内抗血管生成行为和抗神经胶质瘤作用。

结果： TAT-AT7与VEGFR-2和NRP-1的结合亲和力比AT7或TAT大约高3至10倍。内皮细胞对 TAT-AT7 的细胞摄取分别是单独 TAT 和 AT7 的 5 倍和 119 倍。 TAT-AT7 在体内穿透 BBB 和神经胶质瘤组织方面也表现出显着的效率。与PEI/pVAXI-En相比，PPTA/pVAXI-En表现出更低的细胞毒性、更强的BBB和BTB穿越能力、更高的选择性胶质瘤靶向性和更好的基因转染效率。更重要的是，PPTA/pVAXI-En显着抑制内皮细胞的管形成和迁移，抑制神经胶质瘤生长，并减少原位U87神经胶质瘤裸鼠的微血管。

结论：我们的研究表明，PPTA/pVAXI-En 可以作为一种有效的双靶向纳米复合物来穿过 BBB 和 BTB，因此它代表了一种可行且有前途的有效神经胶质瘤治疗的非病毒基因递送系统。


关键词： VEGFR-2和NRP-1靶向，胶质瘤渗透，多功能肽，抗血管生成，基因递送系统