High-throughput evaluation of polymeric nanoparticles for tissue-targeted gene expression using barcoded plasmid DNA

JOURNAL OF CONTROLLED RELEASE

卷:337

Article

Kim, Jayoung^1,2,3  Vaughan, Hannah J.^1,2,3  Zamboni, Camila G.^1,2,3  Sunshine, Joel C.^1,2,3,4  Green, Jordan J.^{1,2,3,5,6,7,8,9} 

[1] Johns Hopkins Univ, Sch Med, Dept Biomed Engn, Baltimore, MD 21205 USA

[2] Johns Hopkins Univ, Sch Med, Translat Tissue Engn Ctr, Baltimore, MD 21205 USA

[3] Johns Hopkins Univ, Inst Nanobiotechnol, Baltimore, MD USA

[4] Johns Hopkins Univ, Dept Pathol, Baltimore, MD USA

[5] Johns Hopkins Univ, Dept Mat Sci & Engn, Baltimore, MD 21218 USA

[6] Johns Hopkins Univ, Dept Chem & Biomol Engn, Baltimore, MD USA

[7] Johns Hopkins Univ, Sch Med, Dept Neurosurg, Baltimore, MD 21205 USA

[8] Johns Hopkins Univ, Sch Med, Dept Oncol, Baltimore, MD 21205 USA

[9] Johns Hopkins Univ, Sch Med, Dept Ophthalmol, Baltimore, MD 21205 USA

关键词: Gene delivery;    Polymeric nanoparticle;    High-throughput screening;    Biodistribution;    Transfection;   

DOI  :  10.1016/j.jconrel.2021.05.047

来源: Elsevier

【 摘 要 】

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.

【 授权许可】

Free