Abstract 4428: Inhibition of tumor vascular protein laminin-411 by nanobioconjugate for glioma treatment

Abstract

Abstract New vascular marker, laminin 411 is overexpressed in 75% of patients with glioblastoma. Nanoconjugates for angiogenesis inhibition in vivo in glioma baring mice were designed and synthesized based on polymalic acid (PMLA) for efficient and specific delivering antisense oligonucleotides (AONs) into glioma cells. The mechanism for membrane binding and permeation of PMLA nanoconjuates was studied to understand their safety and efficiency for cytoplasmic delivery of AONs. Methods: PMLA nanoconjugates containing pH-dependent endosomal escape unit trileucine (LLL) or independent leucine ethylester (LOEt) were synthesized. Nanoconjugates were characterized by their composition, zeta potential, and size. Membranolysis of LLL and LOEt nanoconjugates was compared by liposome leakage. The mechanism for nanoconjugates binding to membrane was analyzed by fluorescence resonance energy transfer (FRET) and confocal fluorescence imaging using giant unilamellar vesicle. To compare their therapeutic difference, mice bearing human U87MG glioma were I.V. injected with both nanoconjugates with 8 injections in total at doses of 5 mg/kg AON against laminin-411, a tumor-specific vascular basement membrane protein. Results: The pH-dependence of membranolysis induced by LLL nanoconjugate paralleled the pH change in maturing endosomes suggesting its potential for endosome-routed cytoplasmic delivery. Mechanistically, LOEt nanoconjugate was found to bind to lipid membrane independent of pH, resulting in the pore-formation on cell membrane associated with cell cytotoxicity. Because LOEt nanoconjugate showed nonspecific binding to cell membrane, its specific targeting for tumor was diminished. Therefore, endosome routed cytoplasmic delivery by pH dependent LLL is superior to direct transmembrane delivery by pH independent LOEt in many aspects, including in vitro inhibition of laminin-411 synthesis, brain tumor targeting, and especially growth inhibition of human glioma in mice. After mice bearing U87MG glioma were treated with both nanoconjugates, they showed a smallest tumor volume of 4 mm3 of LLL group (p<0.001 vs. PBS), compared with 18 mm3 of LOEt group (p<0.01 vs. PBS), and with 47 mm3 of PBS group. On immunostained tumor sections, the signal for laminin diminished most after treatment by LLL nanoconjugate. Conclusion: The different mechanism for LLL and LOEt nanoconjugates interacting with membrane directly influenced their potential for cytoplasm delivery. pH-dependent LLL nanoconjugate excelled in every aspect over pH-independent LOEt counterpart, especially in suppression of glioma growth. Therefore, endosome-routed cytoplasm delivery of LLL nanoconjugate is regarded safe and efficient for the delivery of antisense oligonucelotide. Biodegradable, nontoxic and nonimmunogenic, PMLA based cytoplasm delivery systems can be designed for a wide array of safe and efficient applications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4428. doi:10.1158/1538-7445.AM2011-4428