Dataset Information

Plasmid DNA Mono-Ion Complex for in Vivo Sustainable Gene Expression.

ABSTRACT: To cleave biocompatible poly(ethylene glycol) (PEG) from the mono-ion complex (MIC) for sustainable cellular uptake in vivo, ω-amide-pentylimidazolium end-modified PEG with an ester bond, that is, APe-Im-E-PEG, has been synthesized. The hydrolysis of the resulting APe-Im-E-PEG proceeded during the incubation for 2 weeks under physiological conditions, which was confirmed by gel filtration chromatography. APe-Im-E-PEG formed the MIC with plasmid DNA (pDNA), assessed by agarose gel retardation assay. Furthermore, dynamic light scattering measurement and transmission electron microscopy observations have estimated that the particle size of the resulting MIC was approximately 30 nm, with a rather flexible structure. The APe-Im-E-PEG/pDNA MIC incubated for 2 weeks exhibited hemolytic activity at endosomal pH, presumably because the pH-sensitive carboxyl groups revealed after the hydrolysis of an ester bond of APe-Im-E-PEG. The APe-Im-E-PEG/pDNA MIC enhanced the gene expression 2 weeks after transfection in vivo by intramuscular administration in mice. Consequently, in vivo sustainable gene expression has been achieved by the molecular design of APe-Im-E-PEG for cellular uptake and endosomal escape proceeded by temporal hydrolysis of the ester bond.

SUBMITTER: Kobayashi Y

PROVIDER: S-EPMC6681980 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Plasmid DNA Mono-Ion Complex for in Vivo Sustainable Gene Expression.

Kobayashi Yuki Y Taneichi Sakura S Kawakami Hiroyoshi H Negishi Yoichi Y Asayama Shoichiro S

ACS omega 20190701 7

To cleave biocompatible poly(ethylene glycol) (PEG) from the mono-ion complex (MIC) for sustainable cellular uptake in vivo, ω-amide-pentylimidazolium end-modified PEG with an ester bond, that is, APe-Im-E-PEG, has been synthesized. The hydrolysis of the resulting APe-Im-E-PEG proceeded during the incubation for 2 weeks under physiological conditions, which was confirmed by gel filtration chromatography. APe-Im-E-PEG formed the MIC with plasmid DNA (pDNA), assessed by agarose gel retardation ass ...[more]

PMID: 31460251

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Project description:Successful systemic gene delivery requires specific tissue targeting as well as efficient intracellular transfection. Increasingly, research laboratories are fabricating libraries of novel nanoparticles, engineering both new biomaterial structures and composition ratios of multicomponent systems. Yet, methods for screening gene delivery vehicles directly in vivo are often low-throughout, limiting the number of candidate nanoparticles that can be investigated. Here, we report a comprehensive, high-throughput method to evaluate a library of polymeric nanoparticles in vivo for tissue-specific gene delivery. The method involves pairing each nanoparticle formulation with a plasmid DNA (pDNA) that harbors a unique nucleotide sequence serving as the identifying "barcode". Using real time quantitative PCR (qPCR) for detection of the barcoded pDNA and quantitative reverse transcription PCR (RT-qPCR) for transcribed barcoded mRNA, we can quantify accumulation and transfection in tissues of interest. The barcode pDNA and primers were designed with sufficient sensitivity and specificity to evaluate multiple nanoparticle formulations per mouse, improving screening efficiency. Using this platform, we evaluated the biodistribution and transfection of 8 intravenously administered poly(beta-amino ester; PBAE) nanoparticle formulations, each with a PBAE polymer of differential structure. Significant levels of nanoparticle accumulation and gene transfection were observed mainly in organs involved in clearance, including spleen, liver, and kidneys. Interestingly, higher levels of transfection of select organs did not necessarily correlate with higher levels of tissue accumulation, highlighting the importance of directly measuring in vivo transfection efficiency as the key barcoded parameter in gene delivery vector optimization. To validate this method, nanoparticle formulations were used individually for luciferase pDNA delivery in vivo. The distribution of luciferase expression in tissues matched the transfection analysis by the barcode qPCR method, confirming that this platform can be used to accurately evaluate systemic gene delivery.

Dataset Information

Plasmid DNA Mono-Ion Complex for in Vivo Sustainable Gene Expression.

Publications

Plasmid DNA Mono-Ion Complex for in Vivo Sustainable Gene Expression.

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