TY - JOUR
T1 - High-throughput evaluation of polymeric nanoparticles for tissue-targeted gene expression using barcoded plasmid DNA
AU - Kim, Jayoung
AU - Vaughan, Hannah J.
AU - Zamboni, Camila G.
AU - Sunshine, Joel C.
AU - Green, Jordan J.
N1 - Funding Information:
We thank Dr. Amy Anderson for her expertise and assistance with qPCR analysis. J.J.G. was supported by the Bloomberg–Kimmel Institute for Cancer Immunotherapy. The authors thank the NIH for support ( R01CA228133 , P41EB028239 , and R01EY031097 ). Dr. David Wilson is acknowledged for the assistance with gel permeation chromatography. J.K. received fellowship support from a Samsung Scholarship.
Publisher Copyright:
© 2021
PY - 2021/9/10
Y1 - 2021/9/10
N2 - 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.
AB - 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.
KW - Biodistribution
KW - Gene delivery
KW - High-throughput screening
KW - Polymeric nanoparticle
KW - Transfection
UR - http://www.scopus.com/inward/record.url?scp=85110527511&partnerID=8YFLogxK
U2 - 10.1016/j.jconrel.2021.05.047
DO - 10.1016/j.jconrel.2021.05.047
M3 - Article
C2 - 34097924
AN - SCOPUS:85110527511
SN - 0168-3659
VL - 337
SP - 105
EP - 116
JO - Journal of Controlled Release
JF - Journal of Controlled Release
ER -