Polyplex micelle installing intracellular self-processing functionalities without free catiomers for safe and efficient systemic gene therapy through tumor vasculature targeting

Abstract

Both efficiency and safety profiles are crucial for promotion of gene delivery systems towards practical applications. A promising template system was previously developed based on block catiomer of poly(ethylene glycol) (PEG)-b-poly{N'-[N-(2-aminoethyl)-2-aminoehtyl]aspartamide}-cholesteryl [PEG-PAsp(DET)-cholesteryl] with strategies of ligand conjugation at the α-terminus for specific affinity to the targeted cells and cholesteryl conjugation at the ω-terminus for structural stabilization to obtain systemic retention. Aiming for advocating this formulation towards practical applications, in the current study, the binding profile of this polymer to plasmid DNA (pDNA) was carefully studied to address an issue of toxicity origin. Quantification of free polymer composition confirmed that the toxicity mainly results from unbound polymer and polyplex micelle itself has negligible toxicity. This evaluation allowed for identifying an optimal condition to prepare safe polyplex micelles for systemic application that possess maximal polymer-binding but exclude free polymers. The identified polyplex micelles then faced a drawback of limited transfection efficiency due to the absence of free polymer, which is an acknowledged tendency found in various synthetic gene carriers. Thus, series of functional components was strategically compiled to improve the transfection efficiency such as attachment of cyclic (Arg-Gly-Asp) (cRGD) peptide as a ligand onto the polyplex micelles to facilitate cellular uptake, use of endosome membrane disruptive catiomer of PAsp(DET) for facilitating endosome escape along with use of the conjugated cholesteryl group to amplify the effect of PAsp(DET) on membrane disruption, so as to obtain efficient transfection. The mechanistic investigation respecting the appreciated pH dependent protonation behavior of PAsp(DET) permitted to depict an intriguing scenario how the block catiomers manage to escape from the endosome entrapment in response to the pH gradient. Subsequent systemic application to the pancreatic tumor demonstrated a capability of vascular targeting mediated by the cRGD ligand, which was directly confirmed based on in situ confocal laser scanning microscopy observation. Encouraging this result, the vascular targeting to transfect a secretable anti-angiogenic gene was attempted to treat the intractable pancreatic tumor with anticipation that the strategy could circumvent the intrinsic physiological barriers derived from hypovascular and fibrotic characters. The obtained therapeutic efficiency demonstrates promising utilities of the proposed formulation as a safe systemic gene delivery carrier in practical use.

Zwitterionic Nanocarrier Surface Chemistry Improves siRNA Tumor Delivery and Silencing Activity Relative to Polyethylene Glycol

“…26 Most top-performing polyplex systems are PEGylated and frequently suffer from low tumor uptake without addition of targeting ligands or cholesteryl stabilizing moieties. 44,60 Our PMPC-based polyplexes, on the other hand, without active targeting, increased average tumor cell uptake 3-fold compared to 5k PEG and 2-fold compared to 20k PEG and achieved up to 90% Cy5-positive tumor cells after only a single, relatively low-dose administration. The 20k PMPC polyplexes preserve the stability advantages of high molecular weight stealth coronas while also producing higher cell uptake than high molecular weight PEGylated polyplexes.…”

Design concepts of polyplex micelles for in vivo therapeutic delivery of plasmid DNA and messenger RNA

“…In vivo delivery of pDNA to target cells is relatively easier than that of mRNA due to the higher resistance of pDNA to nucleases . Indeed, various reports including ours, have showed successful treatment outcomes using pDNA especially in the field of cancer therapy even using systemic delivery, which is technically more challenging than local delivery . Recently, clinical trials using liposomal pDNA carriers containing the anti‐transferrin receptor single chain antibody exhibited promising results in cancer treatment after systemic injection .…”

“…pDNA polyplex micelles containing cancer targeting cRGD ligands, PAsp(DET) polycation with endosomal escape capability, and Chol moieties for stabilization showed a high pDNA transfection capability even in the absence of free polycations. 119 As a result, systemic introduction of antiangiogenic factor pDNA using this system led to successful cancer treatment with minimal toxicological responses.…”

“…31 Indeed, various reports including ours, have showed successful treatment outcomes using pDNA especially in the field of cancer therapy even using systemic delivery, which is technically more challenging than local delivery. 61,63,88,119,138,139 Recently, clinical trials using liposomal pDNA carriers containing the anti-transferrin receptor single chain antibody exhibited promising results in cancer treatment after systemic injection. 140,141 However, pDNA transport to cell nuclei is inefficient, except for dividing cells in which the reduced integrity of the nuclear membrane allows for pDNA diffusion to nuclei, and thus the pDNA transfection efficiency in nondividing cells is very low.…”

Polymer‐mediated gene therapy: Recent advances and merging of delivery techniques

“…Transfection efficiencies were evaluated in vitro in HeLa or HUVEC cells using luciferase as the reporter gene, by measuring its activity in lysed cells 48 hr post‐transfection (Q. Chen et al, ; Ge et al, ). Addition of a cyclic oligopeptide (cRGD) allowed targeting of ανβ3 and ανβ5 integrins (Oba et al, ), facilitated accumulation of micelles at the tumor site (Q. Chen et al, ; Ge et al, ), and correlated with effective antitumor activity relative to untargeted control micelles (Q. Chen et al, ; Ge et al, ). This copolymer assembly also enabled delivery of pDNA encoding the sFlt‐1 gene in a BxPC3 pancreatic adenocarcinoma xenograft tumor model following intravenous injection (Q. Chen et al, ).…”