Exploring Critical Vehicle Parameters for the Design of Multitargeted Nanoparticles for Cancer Specific Gene Delivery

Abstract

The greatest obstacle to clinical application of cancer gene therapy is lack of effective delivery tools. Gene delivery vehicles must protect against degradation, avoid immunogenic effects and prevent off target delivery which can cause harmful side effects. The stealth liposomes has greatly improved tumor localization of small molecule drugs and is a promising tool for nucleic acid delivery as the polyethylene glycol coating its surface protects against immune recognition and blood clearance. In this study, DNA was fully encapsulated within in stealth liposomes by complexing the macromolecule with a cationic polymer before encapsulation. Formation methods and material compositions were then investigated for their effects on encapsulation. This technology was translated for protective delivery of siRNA designed for HPV viral gene silencing and cervical cancer treatment. Stealth liposomes encapsulating siRNA were functionalized with a targeting peptide which binds to the alpha6 integrin, a cervical cancer biomarker. It was found that both targeting and polymer complexation before encapsulation were critical components to effective transfection. Varying the siRNA:polymer ratio revealed an optimal concentration for enhanced transfection, but no improvement to internalization, suggesting polymer complexation enhances transfection through an intracellular mechanism. Nanoparticles functionalized with cancer-targeting ligands have shown promise but are still limited by off-tumor binding to healthy tissues that nevertheless express low levels of the molecular target. Targeting two unique cancer biomarkers using dual-targeted heteromultivalent nanoparticles presents a solution to this challenge by requiring overexpression of two separate ligands for localization. In order to guide experimental design, a kinetic model was built to explore how the affinity and valency of dual-ligand liposomes affect the binding and selectivity of delivery to cells with various receptor expression. α5β1 and α6β4 integrin expression levels were then quantified on 20 different cell lines to identify appropriate model cells for in vitro investigation. Dual-targeting heteromultivalent liposomes were synthesized using the alpha6 targeting peptide and an alpha5beta1 targeting peptide. Heteromultivalent liposomes with varying peptide ratios were delivered to cells with varying integrin concentrations. Binding and internalization was then evaluated to understand the effect of valency and avidity on binding kinetics and delivery. Dual-ligand liposomes with equal valencies of each targeting peptide achieved enhanced binding efficiency and selectivity for cells expressing equal and high receptor levels. This liposome formulation was used as a gene delivery vehicle to achieve improved transfection to dual-receptor expressing cells. The insights gained from this study inform rational design of modular heteromultivalent nanoparticles for enhanced specificity to target tissue, for the creation of more effective cancer treatments.