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

Area of Science:

• Biomaterials Science
• Molecular Biology
• Nanotechnology

Background:

• Efficient systemic gene delivery necessitates targeted tissue accumulation and effective intracellular transfection.
• Current in vivo screening methods for gene delivery vehicles are often low-throughput, hindering the evaluation of novel nanoparticle candidates.
• Developing high-throughput screening platforms is crucial for accelerating the optimization of gene delivery systems.

Purpose of the Study:

• To develop and validate a comprehensive, high-throughput method for evaluating polymeric nanoparticles in vivo for tissue-specific gene delivery.
• To assess the biodistribution and transfection efficiency of various poly(beta-amino ester) (PBAE) nanoparticle formulations.
• To establish a reliable platform for optimizing gene delivery vector performance by directly measuring in vivo transfection.

Main Methods:

• A barcoding strategy was employed, where each nanoparticle formulation was paired with a unique DNA barcode (plasmid DNA, pDNA).
• Real-time quantitative PCR (qPCR) and quantitative reverse transcription PCR (RT-qPCR) were utilized to detect barcoded pDNA and mRNA, quantifying nanoparticle accumulation and gene transfection.
• The method was validated by comparing barcode-based transfection analysis with luciferase gene expression data from individual nanoparticle formulations.

Main Results:

• The high-throughput screening platform successfully evaluated 8 PBAE nanoparticle formulations for systemic gene delivery.
• Significant nanoparticle accumulation and gene transfection were primarily observed in clearance organs: spleen, liver, and kidneys.
• Direct measurement of in vivo transfection efficiency proved critical, as higher tissue accumulation did not always correlate with increased transfection levels.

Conclusions:

• The developed barcoding method provides a sensitive and specific platform for high-throughput in vivo evaluation of gene delivery nanoparticles.
• This approach enables accurate quantification of both nanoparticle biodistribution and gene transfection efficiency, crucial for vector optimization.
• The findings underscore the importance of assessing direct transfection rather than solely tissue accumulation for effective gene delivery vector design.