Continuous Production of Discrete Plasmid DNA-Polycation Nanoparticles Using Flash Nanocomplexation

Abstract

Despite successful demonstration of linear polyethyleneimine (lPEI) as an effective carrier for a wide range of gene medicine, including DNA plasmids, small interfering RNAs, mRNAs, etc., and continuous improvement of the physical properties and biological performance of the polyelectrolyte complex nanoparticles prepared from lPEI and nucleic acids, there still exist major challenges to produce these nanocomplexes in a scalable manner, particularly for lPEI/DNA nanoparticles. This has significantly hindered the progress toward clinical translation of these nanoparticle-based gene medicine. Here the authors report a flash nanocomplexation (FNC) method that achieves continuous production of lPEI/plasmid DNA nanoparticles with narrow size distribution using a confined impinging jet device. The method involves the complex coacervation of negatively charged DNA plasmid and positive charged lPEI under rapid, highly dynamic, and homogeneous mixing conditions, producing polyelectrolyte complex nanoparticles with narrow distribution of particle size and shape. The average number of plasmid DNA packaged per nanoparticles and its distribution are similar between the FNC method and the small-scale batch mixing method. In addition, the nanoparticles prepared by these two methods exhibit similar cell transfection efficiency. These results confirm that FNC is an effective and scalable method that can produce well-controlled lPEI/plasmid DNA nanoparticles.

Keywords: DNA nanoparticles; flash nanocomplexation; gene delivery; linear PEI; scalable production.

Figure 1

Schematic diagram showing the CIJ device used to fabricate polyelectrolyte complex nanoparticles under rapid mixing conditions. Streams are independently loaded with lPEI and plasmid DNA, and lPEI/DNA complex nanoparticles are formed in a small confined chamber before being collected.

Figure 2

Effect of the flow rate on particle size and distribution. lPEI/DNA complex nanoparticles were prepared at varying flow rates and analyzed by TEM. DNA concentration was 25 μg/mL, N/P= 8, and pH of lPEI solution was 3.5. TEM images of lPEI-DNA complex nanoparticles prepared at 5 mL/min (A), 10 mL/min (B), 20 mL/min (C), 30 mL/min (D), 40 mL/min (E), and 50 mL/min (F). Scale bar is 500 nm.

Figure 3

Effect of the flow rate on number average size distribution of lPEI/DNA nanoparticles (A); insert graph sows mean number average particle size for the flow rates tested. Inter-batch variability for lPEI/DNA nanoparticles prepared at a flow rate of 40 mL/min. lPEI/DNA nanoparticles were prepared at an initial DNA concentration of 100 μg/mL, N/P= 8, and pH of lPEI solution is 3.5.

Figure 4

TEM images of lPEI/DNA complex nanoparticles prepared under fast mixing conditions (A–C) in a CIJ and bulk mixing (D–F) using different DNA concentrations. For fast mixing conditions, a flow rate of 20 mL/min was used. The pH of lPEI solutions was adjusted to 3.5 and a N/P ratio of 8 was used. Concentration of DNA used for complexation is 25 μg/mL (A, D), 50 μg/mL (B, E), and 100 μg/mL (C, F). Scale bar is 500 nm.

Figure 5

TEM images of lPEI/DNA complex nanoparticles of various shapes prepared under fast mixing conditions in a CIJ using PEI solutions of different pH. A flow rate of 20 mL/min, DNA concentration of 25 μg/mL and N/P = 8 were used. (A) pH of lPEI solution is 5.5. (B) pH of lPEI solution is 4.5. (C) pH of lPEI solution is 3.5, and (D) pH of lPEI solution is 2.5. Scale bar is 500 nm.

Figure 6

Physico-chemical characterization of lPEI/DNA complex nanoparticles generated under fast and bulk mixing conditions. (A) Distribution of DNA content for particles prepared under fast and bulk mixing conditions using Cylindrical Illumination Correlation Spectroscopy. The average number of DNA per particle was found to be 4.2 and 4.7 for fast and bulk mixing, respectively. (B) Zeta-potential of lPEI/DNA complex nanoparticles prepared under fast and bulk mixing, and polymer solutions of several pH values. DNA concentration was 25 μg/mL and N/P ratio 8.

Figure 7

In-vitro transfection efficiency of lPEI/DNA complex nanoparticles HeLa cells. (A) DNA-dose dependent transfection efficiency of lPEI/DNA complex nanoparticles generated under fast and bulk mixing conditions in 10% serum and serum free conditions. (B) Transfection efficiency of lPEI/DNA complex nanoparticles generated under fast and bulk mixing conditions, and prepared with lPEI polymer solutions at different pH values. DNA concentration was 25 μg/mL and N/P ratio 8. Flow rate was 20 mL/min. Each bar represents mean ± standard deviation (n = 4).

Figure 8

Transfection efficiency of lPEI/DNA nanoparticles prepared by FNC and bulk mixing methods in different organs of Balb/c mice at 48 h following i.v. injection. lPEI/DNA complex nanoparticles were prepared with lPEI polymer solutions at pH 3.5 for both methods. DNA concentration was 25 μg/mL. The flow rate was 20 mL/min for FNC. Each bar represents mean ± standard deviation (n = 4).