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Competitive binding and molecular crowding regulate the cytoplasmic interactome of non-viral polymeric gene delivery vectors

ABSTRACT: In contrast to the processes controlling the complexation, targeting and uptake of polycationic gene delivery vectors, the molecular mechanisms regulating their cytoplasmic dissociation remains poorly understood. Upon cytosolic entry, vectors become exposed to a complex, concentrated mixture of molecules and biomacromolecules. In this report, we characterise the cytoplasmic interactome associated with polycationic vectors based on poly(dimethylaminoethyl methacrylate) (PDMAEMA) and poly(2-methacrylolyloxyethyltrimethylammonium chloride) (PMETAC) brushes. To quantify the contribution of different classes of low molar mass molecules and biomacromolecules to RNA release, we develop a kinetics model based on competitive binding. Our results identify the importance of competition from highly charged biomacromolecules, such as cytosolic RNA, as a primary regulator of RNA release. Importantly, our data indicate the presence of ribosome associated proteins, proteins associated with translation and transcription factors that may underly a broader impact of polycationic vectors on translation. In addition, we bring evidence that molecular crowding modulates competitive binding and demonstrate how the modulation of such interactions, for example via quaternisation or the design of charge-shifting moieties, impacts on the long-term transfection efficiency of polycationic vectors. Understanding the mechanism regulating cytosolic dissociation will enable the improved design of cationic vectors for long term gene release and therapeutic efficacy. Factors controlling release of loaded cargo from polycationic gene delivery vectors are still poorly understood. Here, the authors report on a study of mechanisms of RNA release, highlighting the role of competitive binding, and characterise the interactome associated with vectors upon cytosolic entry.

SUBMITTER: Raynold A

PROVIDER: S-EPMC8576037 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Hepatocellular carcinoma (HCC) is the third most deadly cancer in the US, with a meager 5-year survival rate of <20%. Such unfavorable numbers are closely related to the heterogeneity of the disease and the unsatisfactory therapies currently used to manage patients with invasive HCC. Outside of the clinic, gene therapy research is evolving to overcome the poor responses and toxicity associated with standard treatments. The inadequacy of gene delivery vectors, including poor intracellular delivery and cell specificity, are major barriers in the gene therapy field. Herein, we described a non-viral strategy for effective and cancer-specific DNA delivery to human HCC using biodegradable poly(beta-amino ester) (PBAE) nanoparticles (NPs). Varied PBAE NP formulations were evaluated for transfection efficacy and cytotoxicity to a range of human HCC cells as well as healthy human hepatocytes. To address HCC heterogeneity, nine different sources of human HCC cells were utilized. The polymeric NPs composed of 2-((3-aminopropyl)amino) ethanol end-modified poly(1,5-pentanediol diacrylate-co-3-amino-1-propanol) ('536') at a 25 polymer-to-DNA weight-to-weight ratio led to high transfection efficacy to all of the liver cancer lines, but not to hepatocytes. Each individual HCC line had a significantly higher percentage of exogenous gene expression than the healthy liver cells (P<0.01). Notably, this biodegradable end-modified PBAE gene delivery vector was not cytotoxic and maintained the viability of hepatocytes above 80%. In a HCC/hepatocyte co-culture model, in which cancerous and healthy cells share the same micro-environment, 536 25 w/w NPs specifically transfected cancer cells. PBAE NP administration to a subcutaneous HCC mouse model, established with one of the human lines tested in vitro, confirmed effective DNA transfection in vivo. PBAE-based NPs enabled high and preferential DNA delivery to HCC cells, sparing healthy hepatocytes. These biodegradable and liver cancer-selective NPs are a promising technology to deliver therapeutic genes to liver cancer.

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Competitive binding and molecular crowding regulate the cytoplasmic interactome of non-viral polymeric gene delivery vectors

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