RGD-decorated cholesterol stabilized polyplexes for targeted siRNA delivery to glioblastoma cells

Bo Lou,Garry P Duffy,Enrico Mastrobattista,Eduardo Ruiz-Hernandez,Kate Connor,Wim E Hennink,Kieron Sweeney,Alice O’Farrell,Annette T Byrne,David M Murray,Alan Wolfe,Ian S Miller

doi:10.1007/s13346-019-00637-y

Abstract

The development of an effective and safe treatment for glioblastoma (GBM) represents a significant challenge in oncology today. Downregulation of key mediators of cell signal transduction by RNA interference is considered a promising treatment strategy but requires efficient, intracellular delivery of siRNA into GBM tumor cells. Here, we describe novel polymeric siRNA nanocarriers functionalized with cRGD peptide that mediates targeted and efficient reporter gene silencing in U87R invasive human GBM cells. The polymer was synthesized via RAFT copolymerization of N-(2-hydroxypropyl)-methacrylamide (HPMA) and N-acryloxysuccinimide (NAS), followed by post-polymerization modification with cholesterol for stabilization, cationic amines for siRNA complexation, and azides for copper-free click chemistry. The novel resultant cationic polymer harboring a terminal cholesterol group, self-assembled with siRNA to yield nanosized polyplexes (~ 40 nm) with good colloidal stability at physiological ionic strength. Post-modification of the preformed polyplexes with PEG-cRGD end-functionalized with bicyclo[6.1.0]nonyne (BCN) group resulted in enhanced cell uptake and increased luciferase gene silencing in U87R cells, compared to polyplexes lacking cRGD-targeting groups.

Highlights

GBM is the most common malignant brain tumor in adults and is an incurable disease characterized by rapid tumor growth, neovascularization, and aggressive invasion throughout the brain [1]
The RNA interference (RNAi) is an established therapeutic approach based on the delivery of small interfering RNAs into target cells to induce sequence-specific gene silencing
Azobis(isobutyronitrile) (AIBN), 4,4′-azobis (N,N′-cyanopentanoic acid) (V501), cholesterol, dicyclohexylcarbodiimide (DCC), 4-(dimethylamino)pyridine (DMAP), and all other reagents and solvents used in the synthesis were obtained from Sigma-Aldrich and were used without further purification. 2,5-Dioxopyrrolidin-1-yl 4-((((1R,8S,9s)bicyclo[6.1.0] non-4-yn-9-ylmethoxy)carbonyl)amino)butanoate (BCN, SX-A1036) was purchased from Synaffix BV (Oss, The Netherlands). Cyclic RGD (cRGD)-azide was provided by ChinaPeptides Co.,Ltd. (Shanghai, China)

Summary

Introduction

GBM is the most common malignant brain tumor in adults and is an incurable disease characterized by rapid tumor growth, neovascularization, and aggressive invasion throughout the brain [1]. The standard of care (SOC) for GBM is maximal safe surgical resection followed by radiation therapy with concomitant and adjuvant temozolomide (TMZ) delivered via the established BStupp protocol^ [2]. The RNA interference (RNAi) is an established therapeutic approach based on the delivery of small interfering RNAs (siRNAs) into target cells to induce sequence-specific gene silencing. While RNAi has significant potential as a therapeutic strategy in oncology [3, 4], the therapeutic utility of Bunpackaged^ siRNAs is limited due to their intrinsic instability and poor delivery into target tissues. Delivery in the setting of GBM is further limited by the poor penetration of drugs into the tumor due to the presence of the blood-brain and

Methods

Results

Conclusion