Quantification of cellular and nuclear uptake rates of polymeric gene delivery nanoparticles and DNA plasmids via flow cytometry

Abstract

Non-viral, biomaterial-mediated gene delivery has the potential to treat many diseases, but is limited by low efficacy. Elucidating the bottlenecks of plasmid mass transfer can enable an improved understanding of biomaterial structure-function relationships, leading to next-generation rationally designed non-viral gene delivery vectors. As proof of principle, we transfected human primary glioblastoma cells using a poly(beta-amino ester) complexed with eGFP plasmid DNA. The polyplexes transfected 70.6 ± 0.6% of the cells with 101 ± 3% viability. The amount of DNA within the cytoplasm, nuclear envelope, and nuclei was assessed at multiple time points using fluorescent dye conjugated plasmid up to 24 hours post-transfection using a quantitative multi-well plate-based flow cytometry assay. Conversion to plasmid counts and degradation kinetics were accounted for via quantitative PCR (plasmid degradation rate constants were determined to be 0.62 hr−1 and 0.084 hr−1 for fast and slow phases respectively). Quantitative cellular uptake, nuclear association, and nuclear uptake rate constants were determined by using a four-compartment first order mass-action model. The rate limiting step for these poly(beta-amino ester)/DNA polyplex nanoparticles was determined to be cellular uptake (7.5×10−4 hr−1) and only 0.1% of the added dose was taken up by the human brain cancer cells, whereas 12% of internalized DNA successfully entered the nucleus (the rate of nuclear internalization of nuclear associated plasmid was 1.1 hr−1). We describe an efficient new method for assessing cellular and nuclear uptake rates of non-viral gene delivery nanoparticles using flow cytometry to improve understanding and design of polymeric gene delivery nanoparticles.

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

“…PBAE 446 was synthesized in a Michael addition reaction as previously described (Fig. ) to create acrylate terminated polymer base structures, then dissolved in THF and reacted with small molecules to endcap the polymer . Specifically, monomers B4 (4‐butanediol‐diacrylate) and S4 (4‐Amino‐1‐butanol) were mixed at a ratio of 1.1:1 to form 1 g of base polymer and stirred for 24 hour at 90°C.…”

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

“…35 Cellular uptake efficacy was determined via flow cytometry by the fluorescence of the pH-insensitive fluorophore (Cy5) at 2 hr after adding nanoparticles to the cells (Figures 4A and 4B) as gated in Figure S5B. All nanoparticle formulations tested demonstrated similar levels of particle uptake within each cell type in terms of percent of uptake-positive cells as well as the normalized geometric mean uptake, which correlates to the average number of nanoparticles taken up by each cell.…”

“…Overall, PLL nanoparticles were approximately 50 nm in size with a zeta potential of +20 mV, and PEI nanoparticles were approximately 250 nm in size with a zeta potential of +25 mV. To test the influence of DNA labeling for the pH nanosensor on polymer/DNA binding strength, we performed a heparin competition binding assay using gel electrophoresis 35 and a Yo-Pro-1 iodide 4 competition binding assay that showed that polymer-DNA interaction was minimally affected by covalent labeling ( Figures 3C and 3D). …”

“…35 In brief, polyplex nanoparticles were formed between unlabeled or 100% labeled plasmid DNA and either bPEI or PLL at a w/w ratio of 2 and concentration of 0.03 mg/mL in 10 mM NaCl. The nanoparticles were then added to separate solutions of heparin sodium salt (Sigma H3393) diluted in 10 mM NaCl to give the specified amounts of heparin per well in nanograms.…”

Nanomaterials for convection-enhanced delivery of agents to treat brain tumors

“…For instance, computational modeling has been used to quantify rates of various cellular processes involved in nanoparticle uptake [100]. Combination of CED with nanotechnology and novel targeted therapies holds great potential for GBM treatment, but the challenges and shortcomings associated with local delivery should be addressed in order to improve chances for success in clinical trials.…”