Traceable polymeric micelles for tumour targeted drug and siRNA delivery

摘要

Introduction: The objective of this study was to develop traceable polymeric micellar structures for targeted delivery of drugs and/or siRNA to the tumor following systemic administration. This approach provided means to understand the distribution pattern of polymeric micelles in tumor models and design core/shell architectures that can provide better tumor accumulation and delivery of their cargo. Methods: Block copolymers of poly(ethylene oxide)-block-poly(E-caprolactone) (PEO-b-PCL) and PEO-b-poly(a-benzyl-carboxylate-E-caprolactone) (PEO-b-PBCL) were synthesized. The polymers were end-capped at their core with a near infrared (NIR) probe cy5.5-azide through a linker using click chemistry. Abreast cancer targeting peptide P18-4, was attached to the shell of both polymers. PEO-b-PCL or PEO-b-PBCL (containing cy5.5 or P18-4) were mixed and self-associated to form micelles. The micelles were assessed for their thermodynamic as well as kinetic stability. The correlation between micellar stability and its tumor accumulation and bio distribution in nude mice bearing MDA-MB-231 primary breast tumors following systemic administration was investigated. We also assessed the effect of P18-4 peptide ligand modification on micellar surface on their biodistribution and tumor accumulation pattern for stable PEO-PBCL and relatively unstable PEO-PCL micelles. For siRNA delivery, block copolymers of PEO-b-PCL with grafted spermine (PEO-b-P(CL-g-SP)) were synthesized.The effect of micellar stabilization though lipid modificatior of the core on their targeted siRNA transfection was investigated. MCL-1 siRNA was complexed in PEO-b-P(CL-g-SP) (MCL-SP) and its cholesterol modified version, i.e., PEO-b-P(CL-g-SP-Chol) (MCL-SP-Chol) micelles. RGD4C peptide was attached to the shell of MCL-SP and MCL-SP-Chol. The formulations were assessed for their stability and siRNA transfection both in vitro and in nude mice bearing MDA-MB-435 xenografts following intratumoral and intravenous (iv) administration. Results: Traceable micelles were successfully developed through conjugation of Cy5.5 to the core of PEO-b-PCL and PEO-b-PBCL. PEO-b-PBCL micelles exhibited a 7-fold decrease in CMC compared to PEO-b-PCL indicating greater thermodynamic stability. These micelles also showed enhanced kinetic stability compared to PEO-b-PCL micelles. In tumor bearing mice, PEO-b-PCL micelles started accumulating in the liver, tumor, kidney and spleen whereas PEO-b-PBCL micelles were seen primarily in the tumor. P18-4 modification led higher accumulation of both micelles in the tumor at earlier time-points when compared to unmodified micelles (Figure 2). Ex vivo images showed higher tumor accumulation for PEO-b-PBCL micelles when compared to PEO-b-PCL micelles, as well. Lipid modification of the micellar core, enhanced the stability and transfection efficiency of complexed siRNA in vitro in MDA-MB-435 cells. In line with the in vitro results, MCL-SP and MCL-SP-Chol treated animals showed 31 % and 38% down-regulation in relative MCL-1 mRNA expression (Figure 3A) and 1.9- and 3.5-fold decrease in tumor volume compared to animals receiving vehicle, respectively, after intra-tumoral administration. Following iv injection, MCL-SP and MCL-SP-Chol with RGD4C showed 40 and 36% down-regulation in relative mRNA expression as compared to 20 and 12% for micelles without RGD4C, respectively (Figure 3B). The latter observation, implied a positive contribution from RGD4C targeting and no effect by cholesterol modification on tumor targeted silencing activity of complexed siRNA Ⅱ polymeric micelles. Despite better silencing of MCL-1 for RGD4C modified micelles, similar inhibition in tumor growth was observed for both siRNA complexes with or without RGD4C modification. Conclusion: These findings show the potential of core/shell modifications for the development of optimized tumor targeted drugs and/or siRNA delivery systems.