Direct Interrogation of DNA Content Distribution in Nanoparticles by a Novel Microfluidics-Based Single-Particle Analysis

Abstract

Nonviral gene delivery holds great promise not just as a safer alternative to viral vectors in traditional gene therapy applications, but also for regenerative medicine, induction of pluripotency in somatic cells, and RNA interference for gene silencing. Although it continues to be an active area of research, there remain many challenges to the rational design of vectors. Among these, the inability to characterize the composition of nanoparticles and its distribution has made it difficult to probe the mechanism of gene transfection process, since differences in the nanoparticle-mediated transfection exist even when the same vector is used. There is a lack of sensitive methods that allow for full characterization of DNA content in single nanoparticles and its distribution among particles in the same preparation. Here we report a novel spectroscopic approach that is capable of interrogating nanoparticles on a particle-by-particle basis. Using PEI/DNA and PEI-g-PEG/DNA nanoparticles as examples, we have shown that the distribution of DNA content among these nanoparticles was relatively narrow, with the average numbers of DNA of 4.8 and 6.7 per particle, respectively, in PEI/DNA and PEI-g-PEG/DNA nanoparticles. This analysis enables a more accurate description of DNA content in polycation/DNA nanoparticles. It paves the way toward comparative assessments of various types of gene carriers and provides insights into bridging the efficiency gap between viral and nonviral vehicles.

Continuous microfluidic assembly of biodegradable poly(beta‐amino ester)/DNA nanoparticles for enhanced gene delivery

“…We developed a new assay to evaluate plasmid‐containing polyplex nanoparticles utilizing new NTA hardware and software on the NS500 with the capability of discerning fluorescent nanoparticles that contain plasmid DNA from non‐fluorescent nanoparticles. The estimated number of plasmids per PBAE nanoparticle from this method agrees with those published for bPEI nanoparticles analyzed by single particle microfluidic analysis when scaled by particle volume, providing validation for our method . While NTA does require dilution of the sample for analysis, we hypothesize that the characteristics of the particles analyzed following dilution accurately represent the characteristics of the nanoparticles as they would exist following effective dilution for in vitro or in vivo use.…”

“…The estimated number of plasmids per PBAE nanoparticle from this method agrees with those published for bPEI nanoparticles analyzed by single particle microfluidic analysis when scaled by particle volume, providing validation for our method. 54 While NTA does require dilution of the sample for analysis, we hypothesize that the characteristics of the particles analyzed following dilution accurately represent the characteristics of the nanoparticles as they would exist following effective dilution for in vitro or in vivo use. In contrast to methods such as TEM 55 that have previously been used to estimate the number of plasmids contained in a nanoparticle, relatively little perturbation of the particles occurs with our methods utilizing NTA.…”

A Triple-Fluorophore-Labeled Nucleic Acid pH Nanosensor to Investigate Non-viral Gene Delivery

“…Although it is true that the fluorescent labeling method does prevent expression from labeled plasmids, this reduction in reporter gene expression was due to UV exposure that likely results in nicks in the DNA during the labeling process ( Figure S2) and not necessarily fluorophore covalent conjugation. Recent studies have shown that bPEI polyplexes have on the order of 10 plasmids per nanoparticle, 37 meaning that with 20% pH nanosensor DNA used to form polyplexes in uptake and expression experiments, each polyplex would statistically contain unmodified plasmids.…”

Analysis of single nucleic acid molecules in micro- and nano-fluidics

“…205 , copyright 2014), and x-dimension through microfabricated cross-section constriction (figure reproduced from ref. 206 ).…”