The targeted transduction of MMP-overexpressing tumor cells by ACPP-HPMA copolymer-coated adenovirus conjugates.

Shuhua Li,Xiaobin Xie,Jie Long,Juanzhi Chen,Huiyong Xu,Yajie Zhang,Matthew Bogyo

doi:10.1371/journal.pone.0100670

Abstract

We have designed and tested a new way to selectively deliver HPMA polymer-coated adenovirus type 5 (Ad5) particles into matrix metalloproteinase (MMP)-overexpressing tumor cells. An activatable cell penetrating peptide (ACPP) was designed and attached to the reactive 4-nitrophenoxy groups of HPMA polymers by the C-terminal amino acid (asparagine, N). ACPPs are activatable cell penetrating peptides (CPPs) with a linker between polycationic and polyanionic domains, and MMP-mediated cleavage releases the CPP portion and its attached cargo to enable cell entry. Our data indicate that the transport of these HPMA polymer conjugates by a single ACPP molecule to the cytoplasm occurs via a nonendocytotic and concentration-independent process. The uptake was observed to finish within 20 minutes by inverted fluorescence microscopy. In contrast, HPMA polymer-coated Ad5 without ACPPs was internalized solely by endocytosis. The optimal formulation was not affected by the presence of Ad5 neutralizing antibodies during transduction, and ACPP/polymer-coated Ad5 also retained high targeting capability to several MMP-overexpressing tumor cell types. For the first time, ACPP-mediated cytoplasmic delivery of polymer-bound Ad5 to MMP-overexpressing tumor cells was demonstrated. These findings are significant, as they demonstrate the use of a polymer-based system for the targeted delivery into MMP-overexpressing solid tumors and highlight how to overcome major cellular obstacles to achieve intracellular macromolecular delivery.

Highlights

Adenovirus (AdV) is a widely used vector for cancer gene therapy because of its capacity for transgene expression in both dividing and nondividing cells [1–4]
These results indicated the expressions of MMP2 and MMP9 were different in these 4 cell lines: overexpressed in cancer cells while under- expressed in Human bronchial epithelial (HBE) cell line for both mRNA and protein
The use of activatable cell penetrating peptide (ACPP), which possess a targeting mechanism based on selective local separation from cell penetrating peptides (CPPs), is a flexible strategy that concentrates the therapeutic conjugates in the immediate vicinity of matrix metalloproteinase (MMP)-overexpressing solid tumors

Summary

Introduction

Adenovirus (AdV) is a widely used vector for cancer gene therapy because of its capacity for transgene expression in both dividing and nondividing cells [1–4] When they are to be delivered intravenously to treat primary tumor or metastatic disease, the wide tissue distribution of the coxsackie and adenovirus receptor (CAR, the primary receptor for adenovirus type 5) precludes target selectivity, and neutralization of adenovirus by pre-existing antibodies can ablate the delivery. The virus can provoke immune responses which prevents repeated dosing and limits the duration of therapeutic gene expression [5,6] These factors have largely limited therapeutic use of adenovirus to local or direct administration. A useful viral gene therapy vector should be protected from neutralizing antibodies and enable delivery to target cells Addressing these challenges necessitates alternative approaches to redirect AdV type 5 (Ad5) for CAR-independent cellular internalization. To the best of our knowledge, a detailed investigation of pHPMA modification used to potentiate Ad5 infection and target solid tumor cells has not been reported

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Results

Conclusion