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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.

SUBMITTER: Liu HW

PROVIDER: S-EPMC9701136 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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

Liu Heng-Wen HW Hu Yizong Y Ren Yong Y Nam Hwanhee H Santos Jose Luis JL Ng Shirley S Gong Like L Brummet Mary M Carrington Christine A CA Ullman Christopher G CG Pomper Martin G MG Minn Il I Mao Hai-Quan HQ

ACS applied materials & interfaces 20210623 26

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 sc ...[more]

PMID: 34162211

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Project description:Background and objectivesRed blood cell (RBC) units in hypothermic storage degrade over time, commonly known as the RBC storage lesion. These older RBC units can cause adverse clinical effects when transfused, as older RBCs in the unit lyse and release cell-free haemoglobin (Hb), a potent vasodilator that can elicit vasoconstriction, systemic hypertension and oxidative tissue injury after transfusion. In this study, we examined a novel method of washing ex vivo stored single RBC units to remove accumulated cellular waste, specifically cell-free Hb, using tangential flow filtration (TFF) driven by a centrifugal pump.Materials and methodsThe TFF RBC washing system was run under hypothermic conditions at 4°C, at a constant system volume with 0.9 wt% saline as the wash solution. The RBC washing process was conducted on 10 separate RBC units. For this proof-of-concept study, RBC units were expired at the time of washing (60-70 days old). Cell-free Hb was quantified by UV-visible absorbance spectroscopy and analysed via the Winterbourn equations. Pre- and post-wash RBC samples were analysed by Hemox Analyser, Coulter counter and Brookfield rheometer. The RBC volume fraction in solution was measured throughout the wash process by standard haematocrit (HCT) analysis.ResultsNo substantial decrease in the HCT was observed during the TFF RBC washing process. However, there was a significant decrease in RBC concentration in the first half of the TFF RBC wash process, with no significant change in RBC concentration during the second half of the TFF cell wash process with an 87% overall cell recovery compared with the total number of cells before initiation of cell washing. Utilization of the extinction coefficients and characteristic peaks of each Hb species potentially present in solution was quantified by Winterbourn analysis on retentate and permeate samples for each diacycle to quantify Hb concentration during the washing process. Significant cell-free Hb reduction was observed within the first four diacycles with a starting cell-free Hb concentration in the RBC unit of 0.105 mM, which plateaus to a constant Hb concentration of 0.01 mM or a total extracellular Hb mass of 0.2 g in the resultant washed unit. The oxygen equilibrium curve showed a significant decrease in P50 between the initial and final RBC sample cell wash with an initial P50 of 15.6 ± 1.8 mm Hg and a final P50 of 14 ± 1.62 mm Hg. Cooperativity increased after washing from an initial Hill coefficient of 2.37 ± 0.19 compared with a final value of 2.52 ± 0.12.ConclusionOverall, this study investigated the proof-of-concept use of TFF for washing single RBC units with an emphasis on the removal of cell-free Hb from the unit. Compared with traditional cell washing procedures, the designed system was able to more efficiently remove extracellular Hb but resulted in longer wash times. For a more complete investigation of the TFF RBC washing process, further work should be done to investigate the effects of RBC unit storage after washing. The designed system is lightweight and transportable with the ability to maintain sterility between uses, providing a potential option for bedside ex vivo transfusion in clinical applications.

Dataset Information

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

Publications

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

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