pH-Sensitive polymers that enhance intracellular drug delivery in vivo

Abstract

Cytosolic delivery from endosomes is critical for those drugs that are susceptible to attack by lysosomal enzymes, such as DNA, RNA, oligonucleotides, proteins and peptides. Therefore, we have designed pH-sensitive, membrane-disruptive polymers to enhance the release of drugs from the acidic endosomal compartment to the cytoplasm. We have found that one polymer in particular, poly(propylacrylic acid) (PPAA), is very effective at membrane disruption at pHs below 6.5, based on hemolysis studies. PPAA also significantly enhances in vitro transfections of lipoplex formulations in cell culture, and does so in the presence of as much as 50% serum. In this study, we have extended our in vitro hemolysis and cell culture studies to an in vivo murine excisional wound healing model. A pilot study with a green fluorescent protein (GFP)-encoding plasmid indicated that injection of formulations containing PPAA into healing wounds resulted in increased GFP expression. Subsequently, by administering sense and antisense DNA for the angiogenesis inhibitor thrombospondin-2 (TSP2), we were able to alter the wound healing response in TSP2-null and wild type mice, respectively. Our findings showed that when PPAA was added to lipoplex formulations, expression of TSP2 was enhanced in TSP2-null mice compared to control formulations. These results show that PPAA can enhance in vivo transfections and that inhibition of TSP2 expression may lead to improved wound healing. These results suggest that PPAA can provide significant improvements in the in vivo efficacy of drugs such as DNA.

Introduction

Cytosolic delivery from endosomes is critical for many biomolecular drugs, in order to avoid degradation by lysosomal enzymes. This is particularly important for gene therapy, where viral delivery vectors have been very efficient at DNA delivery, but whose clinical use is potentially limited by their immunogenicity and toxicity [1]. Non-viral DNA carriers utilize polycationic systems, such as polycationic polymers or cationic liposomes, and these delivery systems have shown considerable clinical potential; however, they suffer from low transfection efficiencies. Efficient non-viral delivery systems for cell transfections depend on enhancement of several key steps, including: (a) endocytosis; (b) release of endocytosed DNA from the endosome into the cytoplasm; and (c) transport of the DNA into the nucleus. Among these barriers, a major cause of the low transfection efficiencies is the trafficking of DNA-polycation complexes to lysosomes, where they are degraded. Since the pH of an endosome is lower than that of the cytosol by 1–2 pH units, one approach taken to enhance cytosolic delivery of DNA has been to mimic fusogenic viral peptides by designing synthetic pH-sensitive peptides that can enhance the transport of endocytosed drugs from the endosomal compartment to the cytoplasm [2], [3]. However, such peptides could stimulate antigenic responses, and they are also costly. Our approach has been to design and synthesize totally synthetic, pH-sensitive polymers that become membrane-disruptive at the pH of the endosome. These polymers should disrupt lipid bilayer membranes at pH 6.5 and below, but should be non-lytic at pH 7.4. We expect that these polymers should also exhibit minimal antigenicity, although we have not tested this yet.

One of the most effective pH-sensitive polymers that we have synthesized is poly(propylacrylic acid) (PPAA) (Fig. 1), based on its ability to hemolyse red blood cells (RBC) suspended in acidic buffers. PPAA was found to hemolyse completely a 1 ml suspension of ca. 10⁸ red blood cells at pH 6.1 in 1 h, using PPAA concentrations as low as 3–5 μg/ml [4]. PPAA was not hemolytic at pH 7.4 and displayed a pH-dependent hemolysis that rose sharply as pH dropped below 6.5. We also found that PPAA similarly enhanced hemolysis of RBC at acidic pHs when it was complexed via biotin to the protein streptavidin [5]. Based on these promising results, we have added this polymer to lipoplex–DNA vectors in cell culture and observed significant enhancements of transfections, especially in serum, where non-viral carriers are usually unstable and ineffective [14].

In the study reported here, we have investigated the ability of these lipoplex formulations to transfect cells in an in vivo mouse wound healing model. This model is based on previous studies which have shown that healing is accelerated and resolves with reduced scarring in thrombospondin 2 (TSP2)-null animals [6]. These changes are associated with prolonged and enhanced neovascularization and irregular deposition of extracellular matrix (ECM). These findings are consistent with the proposed role of TSP2 as a modulator of cell–matrix interactions [7], [8], [9]. We postulated that if we were able to block TSP2 expression in WT mice, we might observe improved healing. This approach has been successful in altering the foreign body response to implanted biomaterials [15]. To do this, we designed a strategy to inhibit TSP2 expression during healing by the introduction of antisense TSP2 cDNA. We similarly postulated that if we were able to induce TSP2 expression in TSP2-null wounds by transfecting with sense TSP2 cDNA, we might observe a partial reversal of the TSP2-null phenotype, and a less effective healing process.