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 24h 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 0.62h(-1) and 0.084h(-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)h(-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 1.1h(-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.

Statement of significance: In this work, a quantitative high throughput flow cytometry-based assay and computational modeling approach was developed for assessing cellular and nuclear uptake rates of non-viral gene delivery nanoparticles. This method is significant as it can be used to elucidate structure-function relationships of gene delivery nanoparticles and improve their efficiency. This method was applied to a particular type of biodegradable polymer, a poly(beta-amino ester), that transfected human brain cancer cells with high efficacy and without cytotoxicity. A four-compartment first order mass-action kinetics model was found to model the experimental transport data well without requiring external fitting parameters. Quantitative rate constants were identified for the intracellular transport, including DNA degradation rate from polyplexes, cellular uptake rate, and nuclear uptake rate, with cellular uptake identified as the rate-limiting step.

Keywords: Brain cancer; Computational modeling; Gene delivery; Nanoparticle; Polymer.

Figure 1. Isolated nuclei

Isolated nuclei are obtained and stained with DAPI following the described nuclei isolation protocol.

Figure 2. Heparin washing procedure successfully removes all bound plasmid

Following an incubation for 1 hour at 4°C, 50 µg/mL heparin in PBS was used to eliminate all associated DNA. Cy3-labeled plasmid is removed from both cells (with added polyplex) and nuclei (with added plasmids). NGM indicated normalized geometric mean fluorescence (unitless).

Figure 3. Cellular and nuclear uptake of PBAE 447 polyplexes in human brain cancer cells

Percentages (left) and NGM (right) of Cy3-labeled DNA for both cells (top) and nuclei (bottom). P_cyto are plasmids which are in the cytoplasm, P_ne are plasmids which are nucleus-associated, and P_ni are plasmids which are intranuclear.

Figure 4. GFP transfection of human brain cancer cells with PBAE 447 polyplexes

Fluorescence microscopy images of human primary glioblastoma cells showing the eGFP channel (left) and the eGFP and phase contrast channels combined (right) for untreated cells (top) and for 447 30 w/w polyplex transfected cells (bottom) at 48 hours post transfection.

Figure 5. Fitting of the four-compartment first order mass-action model to the experimental data

The first order mass-action model is represented as dotted lines (bi-exponential degradation) and the experimental data as data points. Curves are shown for plasmid number within the cell (P_cyto), plasmid number associated with the nucleus (P_ne), and plasmid number within the nucleus (P_ni).

Scheme 1. Depiction of plasmid transfer rates between compartments of interest

The four compartments of interest are: the media (P_media), the cytoplasm (P_cyto), nuclear envelope-associated (P_ne), and nuclei-internalized (P_ni). The associated transfer rate constants between these four compartments are k_cell, k_ne, and k_ni respectively. k_bd is the rate to the cytoplasm from the nuclear envelope and from within the nucleus. The fast and slow degradation rate constants are k_deg1 and k_deg2 which are hypothesized to be associated with decomplexed and complexed plasmids, respectively.

Scheme 2. Reaction scheme of polymer B4-S4-E7 (447)

1,4-butanediol diacrylate (B4) was polymerized with 4-amino-1-butanol (S4) neat at 90°C for 24 hours and subsequently end-capped with 1-(3-aminopropyl)-4-methylpiperazine (E7) in THF at room temperature for 1 hour.

Scheme 3. Four-compartmental model depicting the direction of plasmid transfer