Scalable Purification of Plasmid DNA Nanoparticles by Tangential Flow Filtration for Systemic Delivery

Abstract

Plasmid DNA (pDNA) nanoparticles synthesized by complexation with linear polyethylenimine (lPEI) are one of the most effective non-viral gene delivery vehicles. However, the lack of scalable and reproducible production methods and the high toxicity have hindered their clinical translation. Previously, we have developed a scalable flash nanocomplexation (FNC) technique to formulate pDNA/lPEI nanoparticles using a continuous flow process. Here, we report a tangential flow filtration (TFF)-based scalable purification method to reduce the uncomplexed lPEI concentration in the nanoparticle formulation and improve its biocompatibility. The optimized procedures achieved a 60% reduction of the uncomplexed lPEI with preservation of the nanoparticle size and morphology. Both in vitro and in vivo studies showed that the purified nanoparticles significantly reduced toxicity while maintaining transfection efficiency. TFF also allows for gradual exchange of solvents to isotonic solutions and further concentrating the nanoparticles for injection. Combining FNC production and TFF purification, we validated the purified pDNA/lPEI nanoparticles for future clinical translation of this gene nanomedicine.

Keywords: DNA nanoparticles; biocompatibility; gene delivery; linear PEI; tangential flow filtration.

Figure 1.

(a) Schematic diagram of pDNA/lPEI nanoparticle purification by TFF. Comparison of (b) the z-average hydrodynamic size (c) DNA recovery and (d) free lPEI fraction of the nanoparticles before purification (FNC original nanoparticles) vs. following purification by TFF and ultrafiltration methods using the 5-volume washing (5×) and 10-volume washing (10×) protocols, respectively (n = 3). The 5× and 10× washing refer to 5 and 10 times of the volume of the solvent used, respectively, in reference to the volume of nanoparticles. This is measured by the volume of the permeate collected during the TFF process.

Figure 2.

The pDNA/lPEI nanoparticle characteristics and in vitro transfection activities after TFF purification with optimized conditions. (a) Percentage of the free lPEI, (b) DNA recovery, and (c) z-average size of the nanoparticles before and after TFF purification; FNC/TFF 5× and FNC/TFF 10× (see Fig. 1 for group description). (d) TEM images of the nanoparticles prepared by (i) bulk-mixed: Polyplus protocol (ii) FNC original (unpurified), and (iii) FNC/TFF 10×. Scale bar is 500 nm. (e) The intensity average size distribution of the nanoparticles before and after TFF. (f) In vitro cellular viability of PC3 cell line in bulk-mixed: Polyplus protocol, FNC original, FNC/TFF 5×, FNC/TFF 10× nanoparticles and naked DNA with different DNA concentrations (nanoparticle concentration was titrated to match the DNA concentration for comparison purpose). For statistical analysis, the comparison is against DNA group and bulk-mixed group, respectively. (g) In vitro transfection efficiency with different incubation time of bulk-mixed: Polyplus protocol, FNC original, FNC/TFF 5×, FNC/TFF 10× purified pDNA/lPEI nanoparticles by PC3 cell line. (h) In vitro transfection efficiency of bulk-mixed: Polyplus protocol, FNC original, FNC/TFF 5×, FNC/TFF 10× purified nanoparticles by NCI-H1299 cell line. For statistical analysis, n.s. denotes no statistical significance with p > 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001 from one-way ANOVA test.

Figure 3.

(a) In vivo transfection efficiency of the injected Polyplus bulk-mixed, FNC original and FNC/TFF 10× pDNA/lPEI nanoparticles, respectively in Balb/c mice. The injection dose is 40 μg DNA/mice. IVIS whole-body images were taken at 24 h post injection of particles. Scale bar: local radiance with unit of 10⁶ photon/s/sr/cm² (b) 1: The necrosis at the tail injection site of the unpurified nanoparticles. The images of harvested livers in each group. 2: Polyplus bulk-mixed, 3: FNC original, 4: FNC/TFF 10× pDNA/lPEI nanoparticles, 5: Naked DNA. (c) The IVIS ROI quantitative analysis results at 24 h time point from Balb/c mice. Each bar represents mean ± standard deviation (n = 5). (d) AST assessments of Balb/c mice injected with Polyplus bulk-mixed, FNC original, and FNC/TFF 10× pDNA/lPEI nanoparticles, respectively. (e) Percentage of necrotic area in liver of Balb/c mice injected with Polyplus bulk-mixed, FNC original, and FNC/TFF 10× pDNA/lPEI nanoparticles, respectively. (f) H&E-stained liver tissue sections of Balb/c mice injected with Polyplus bulk-mixed, FNC original, and FNC/TFF 10× pDNA/lPEI nanoparticles, respectively. Scale bar 1000 μm (Left) and 200 μm (Right). For statistical analysis, n.s. denotes no statistical significance with p > 0.05 and *p < 0.05 from one-way ANOVA test.

Figure 4.

(a) In vivo transfection efficiency of CD-1 mice. The dose is 40 μg DNA/mice. IVIS whole-body images were taken at 24 h post injection of particles. Scale bar: local radiance with unit of 10⁶ photon/s/sr/cm² (b) The IVIS ROI quantitative analysis results at 24-h time point from CD-1 mice. Each bar represents mean ± standard deviation (n = 5). (c) AST assessments of CD-1 mice injected with Polyplus bulk-mixed, FNC original, FNC/TFF 5×, and FNC/TFF 10× pDNA/lPEI nanoparticles, respectively. For statistical analysis, n.s. denotes no statistical significance with p > 0.05 from one-way ANOVA test.