A new type of block copolymer micelles for pH-triggered delivery of poorly water-soluble anticancer drugs has been synthesized and characterized. The micelles were formed by the self-assembly of an amphiphilic diblock copolymer consisting of a hydrophilic poly(ethylene glycol) (PEG) block and a hydrophobic polymethacrylate block (PEYM) bearing acid-labile ortho ester side-chains. The diblock copolymer was synthesized by atom transfer radical polymerization (ATRP) from a PEG macro-initiator to obtain well-defined polymer chain-length. The PEG-b-PEYM micelles assumed a stable core-shell structure in aqueous buffer at physiological pH with a low critical micelle concentration as determined by proton NMR and pyrene fluorescence spectroscopy. The hydrolysis of the ortho ester side-chain at physiological pH was minimal yet much accelerated at mildly acidic pHs. Doxorubicin (Dox) was successfully loaded into the micelles at pH 7.4 and was released at much higher rate in response to slight acidification to pH 5. Interestingly, the release of Dox at pH 5 followed apparently a biphasic profile, consisting of an initial fast phase of several hours followed by a sustained release period of several days. Dox loaded in the micelles was rapidly taken up by human glioma (T98G) cells in vitro, accumulating in the endolysosome and subsequently in the nucleus in a few hours, in contrast to the very low uptake of free drug at the same dose. The dose-dependent cytotoxicity of the Dox-loaded micelles was determined by the MTT assay and compared with that of the free Dox. While the empty micelles themselves were not toxic, the IC50 values of the Dox-loaded micelles were approximately ten-times (by 24 hours) and three-times (by 48 hours) lower than the free drug. The much enhanced potency in killing the multi-drug-resistant human glioma cells by Dox loaded in the micelles could be attributed to high intracellular drug concentration and the subsequent pH-triggered drug release. These results establish the PEG-b-PEYM block copolymer with acid-labile ortho ester side-chains as a novel and effective pH-responsive nano-carrier for enhancing the delivery of drugs to cancer cells.

Poly(2-aminoethyl methacrylate) (PAEM) homopolymers with defined chain-length and narrow molecular weight distribution were synthesized using atom transfer radical polymerization (ATRP), and a comprehensive study was conducted to evaluate the colloidal properties of PAEM/plasmid DNA polyplexes, the uptake and subcellular trafficking of polyplexes in antigen-presenting dendritic cells (DCs), and the biological performance of PAEM as a potential DNA vaccine carrier. PAEM of different chain-length (45, 75 and 150 repeating units) showed varying strength in condensing plasmid DNA into narrowly dispersed nanoparticles with very low cytotoxicity. Longer polymer chain-length resulted in higher levels of overall cellular uptake and nuclear uptake of plasmid DNA, but shorter polymer chains favored intracellular and intra-nuclear release of free plasmid from the polyplexes. Despite its simple chemical structure, PAEM transfected DCs very efficiently in vitro in media with or without serum and led to phenotypic maturation of DCs. When a model antigen-encoding ovalbumin plasmid was used, transfected DCs stimulated the activation of naïve CD8+ T cells to produce high levels of interferon-γ. The efficiency of transfection, DC maturation, and CD8+ T cell activation showed varying degrees of polymer chain-length dependence. These structurally defined cationic polymers may have much potential as efficient DNA vaccine carriers and immunostimulatory adjuvants. They may also serve as a model material system for elucidating structural and intracellular mechanisms of polymer-mediated DNA vaccine delivery.

DNA vaccination using cationic polymers as carriers has the potential to be a very powerful method of immunotherapy, but typical immune responses generated have been less than robust. To better understand the details of DNA vaccine delivery in vivo, we prepared polymer/DNA complexes using three structurally distinct cationic polymers and fluorescently labeled plasmid DNA and injected them intradermally into mice. We analyzed transgene expression (luciferase) and the local tissue distribution of the labeled plasmid at the injection site at various time points (from hours to days). Comparable numbers of luciferase expressing cells were observed in the skin of mice receiving naked plasmid or polyplexes one day after transfection. At day 4, however, the polyplexes appeared to result in more transfected skin cells than naked plasmid. Live animal imaging revealed that naked plasmid dispersed quickly in the skin of mice after injection and had a wider distribution than any of the three types of polyplexes. However, naked plasmid level dropped to below detection limit after 24 h, whereas polyplexes persisted for up to 2 weeks. The PEGylated polyplexes had a significantly wider distribution in the tissue than the nonPEGylated polyplexes. PEGylated polyplexes also distributed more broadly among dermal fibroblasts and allowed greater interaction with antigen-presenting cells (APCs) (dendritic cells and macrophages) starting at around 24 h post-injection. By day 4, co-localization of polyplexes with APCs was observed at the injection site regardless of polymer structure, whereas small amounts of polyplexes were found in the draining lymph nodes. These in vivo findings demonstrate the superior stability of PEGylated polyplexes in physiological milieu and provide important insight on how cationic polymers could be optimized for DNA vaccine delivery.

Nonviral gene transfection mediated by cationic polymer/DNA polyplexes often imposes stress and toxicity to cells. To better understand the relationship between cellular stress responses and polyplex-mediated transfection, polyplex-induced early autophagy in mouse fibroblasts was characterized and the impact of autophagy modulation on transgene expression evaluated. Transmission electron microscopy revealed the formation of double-membraned autophagosome in the cytoplasm of polyplex-transfected cells. Immunofluorescence staining and microscopy revealed intracellular LC3 punctation that was characteristic of early autophagy activation. Elevated expression of autophagosome-associated LC3 II protein was also detected by Western blot. When cells were treated with small-molecule modulators of autophagy, polyplex-mediated gene transfection efficiency was significantly affected. 3-Methyladenine (3-MA), an early autophagy inhibitor, reduced transfection efficiency, whereas rapamycin, an autophagy inducer, enhanced transgene expression. Importantly, the observed functional impact on gene transfection by autophagy modulation was decoupled from that of other modes of cellular stress response (apoptosis/necrosis). Treatment of cells by 3-MA or rapamycin did not affect the level of intracellular reactive oxygen species (ROS) but did decrease or increase, respectively, nuclear localization of polyplex-delivered plasmid DNA. These findings suggest new possibilities of enhancing polyplex-mediated gene delivery by codelivery of small-molecule regulators of autophagy.