(T1030-04-24) Intracellular Delivery of Plasmid DNA Using TPGS Formulated Lipid Nanoparticles

Purpose: Lipid nanoparticles have emerged as promising carriers of genetic material such as mRNA, siRNA, and plasmid DNA. One of the most significant challenges in manufacturing gene-loaded lipid nanoparticles is their poor water stability during storage, where they undergo physical and chemical property changes. Synthetic PEG lipids have been frequently utilized to coat lipid nanoparticles to prevent aggregation and fusion. However, lengthy polymeric acyl chains of synthetic PEGs have been linked to immunogenicity and toxicity. D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS), a water-soluble natural vitamin E derivative, is an amphiphilic macromolecule that can be used as an alternative to the synthetic PEG molecules. Here, we report an attempt to formulate a non-toxic and stable LNP using a vitamin E-modified PEG lipid called D-α-tocopherol polyethylene glycol succinate (TPGS) to deliver plasmid DNA.

Methods: A 3-way microfluidic micromixer chip was used to formulate pDNA-LNPs having 50/11.5/34.5/4 mol ratio of DOTAP/DPPC/cholesterol/TPGS. Lipid solution in ethanol flowed through the inner channel of the chip whilst the DNA and sterile water flowed through the outer channels. The samples were then subsequently dialyzed against 1X PBS pH 7.4 buffer overnight to remove ethanol. The particle size, PDI, and zeta potential were determined using NanoBrook 90Plus (Brookhaven Instruments Corporation, USA). HeLa cells (human transformed cervix epithelial cells) were used to study the transfection of the pDNA-loaded lipid nanoparticles at various time points. Cells were transfected for 24 hours, and media was replaced to complete media for the incubation period of 48hrs. Cells were imaged using Keyence microscope at 20X focus. The cytotoxicity of pDNA-loaded TPGS-LNPs and DMG-PEG2000-LNPs to HeLa cells was evaluated using the MTS assay.

Results: A clear colloidal solution of pDNA LNPs was achieved using microfluidic mixing. The particle size and PDI of TPGS pDNA-LNPs were 82nm and 0.19 respectively. Fluorescence microscope images revealed that the LNPs were able to transfect HeLa cells efficiently. TPGS-LNP, on the other hand, transfected the cells significantly greater than lipofectamine and DMG-PEG 2000 LNPs at 1000ng, 2000ng, and 4000ng pDNA doses. TPGS-LNPs were able to transfect HeLa cells till 15 days, indicating the formulation was stable for at least 15 days. TPGS-containing LNPs exhibited higher than 95% viability of HeLa cells at all lipid doses; whereas dose-dependent toxicity was observed in cells treated with DMG-PEG 200-containing LNPs.

Conclusion: TPGS-modified LNP effectively delivers plasmid DNA to HeLa cells. TPGS-LNPs did not demonstrate toxicity in HeLa cells when compared to the commonly used synthetic PEG analog DMG-PEG2000. TPGS-LNPs in liquid formulation demonstrated stability for at least 15 days.

References: Clement, J., et al., Large-scale production of lipoplexes with long shelf-life. Eur J Pharm Biopharm, 2005. 59(1): p. 35-43
Webster, R., et al. PEG and PEG conjugates toxicity: towards an understanding of the toxicity of PEG and its relevance to PEGylated biologicals. 2009.
Stone, C.A., Jr., et al., Immediate Hypersensitivity to Polyethylene Glycols and
Polysorbates: More Common Than We Have Recognized. J Allergy Clin
Immunol Pract, 2019. 7(5): p. 1533-1540.e8.
Sellaturay, P., S. Nasser, and P. Ewan, Polyethylene Glycol-Induced Systemic
Allergic Reactions (Anaphylaxis). J Allergy Clin Immunol Pract, 2021. 9(2): p.
670-675.
Zhang, Z., S. Tan, and S.S. Feng, Vitamin E TPGS as a molecular biomaterial
for drug delivery. Biomaterials, 2012. 33(19): p. 4889-906.

Figure 1. Transfection ability of pDNA loaded lipid nanoparticles for stability testing.
The pDNA-LNP systems were formulated using the lipid ratio 50:11.5:34.5:4 of DOTAP:DPPC:Chol:TPGS. Fluorescent microscopy of HeLa cells after transfection with TPGS modified pDNA-LNPs at various time points. The time points for characterizations and transfections were freshly made, 15 and 30 days. Lipofectamine was used as a positive control. pDNA solution was used as a negative control. Data are expressed as mean ± SD (n = 3).>



Figure 2. Higher transfection ability of TPGS-LNPs against synthetic DMG-PEG containing LNPs
Fluorescent microscopy of HeLa cells after transfection with TPGS containing pDNA-NPs were compared with DMG-PEG containing pDNA-LNPs at varying pDNA doses (500, 1000, 2000 and 4000 ng). Lipofectamine was used as a positive control.pDNA solution was used as a negative control. Data are expressed as mean ± SD (n = 3).



Figure 3. TPGS containing LNP-pDNA systems are less toxic to HeLa cells than DMG-PEG containing LNP-pDNA systems.
Evaluation of cytotoxicity of Lipofectamine, TPGS-LNP, and DMG-PEG LNP toward Hela cells. Cells were treated with a total lipid dose of 4.6, 9.37, 18.75, 37.5, and 75 ng of LNPs for 24 hrs. Data are expressed as mean ± SD (n = 3). ****p < 0.0001, **p < 0.01, ANOVA.