(W1230-11-73) Lipid Nanoparticles versus Lipoplexes: Optimizing Complexing Head Group and Formulation Technology for Efficient Gene Delivery

Purpose: Gene therapy using plasmid DNA (pDNA) is well-explored for variety of genetic diseases. However, numerous in vivo biological barriers demand an optimal delivery system. Non-viral vectors like lipid nanoparticles (LNPs) and lipoplexes have gained traction but there is no comparative evaluation of these lipid nanocarriers to deliver pDNA. LNPs or lipoplexes possessing variable physicochemical characteristics can be fabricated by altering formulation components. Amongst them, type of complexing head group is critical for efficient complexation of pDNA, cellular uptake, endosomal escape, and subsequent transfection in target cells. We have primarily compared cationic lipid (DOTAP), cationic surfactant (LAE) and ionizable lipids with versatile chemical structures (DLinDMA, DLin-MC3-DMA, SM-102) to identify the most effective complexing headgroup to deliver pDNA. In addition to the formulation components, the technology used to prepare gene delivery nanocarriers is also of utmost importance. We therefore prepared LNPs using microfluidic mixing with the optimized complexing head group to incorporate pDNA. Systematic comparison of lipid nanocarrier was performed with pDNA complexed on the interior versus exterior of the nanoparticles in terms of physicochemical characterization, transfection efficiency, and safety for potential parenteral administration. Hence, we have executed parallel comparison of both complexing head group and formulation technology for efficient pDNA delivery (Fig. 1a). This research was carried out as a preliminary part of developing a novel non-viral gene therapy to deliver antitumor genes for BRAF inhibitor (BRAFi)-resistant melanoma.

Methods: Cationic/ionizable liposomes composed of cationic/ionizable lipid or surfactant, DOPC, DOPE, cholesterol and DSPE-PEG2000 were prepared by ethanol injection method followed by physicochemical characterization. Fluorescence-labeled liposomes were prepared by incorporating NBD-PC lipid to evaluate their cellular uptake in developed BRAFi-resistant melanoma cell line, A375R. Biocompatibility of liposomes was analyzed in A375R cells by MTT assay, while their hemocompatibility was tested in mice RBCs. Lipoplexes were prepared with GFP-pDNA at N/P ratios of 5, 10 and 20, and their transfection efficiency was evaluated in A375R cells. The capability of five screened cationic/ionizable liposomes to form electrostatic complexes with GFP-pDNA was assessed by agarose gel electrophoresis. Next, GFP-pDNA loaded LNPs were prepared with optimized complexing head group, DOPC, DOPE, cholesterol and DSPE-PEG2000 by employing staggered herringbone micromixer at total flow rate of 5 mL/min to obtain various N/P ratios. Fabricated LNPs were subjected to physicochemical characterization and their transfection efficiency was evaluated in A375R cells in comparison to commercial transfection reagent TransIT^®-LT1. A Mirus Label IT^® Nucleic Acid Labeling Kit was used to conjugate Rhodamine to GFP-pDNA and labeled plasmid was incorporated in optimized LNPs to simultaneously evaluate their penetration and transfection in A375R 3D tumor spheroid model. To identify and compare the systemic safety of LNPs and lipoplexes, heparin displacement and serum stability assays were performed.

Results: All liposomal formulations exhibited particle size of 80-120 nm, PDI of < 0.3, and zeta potential of +35-45 mV, indicating formation of positively charged nanosized liposomes to complex negatively charged GFP-pDNA via electrostatic interaction (Fig. 1b-c). SM-102 liposomes exhibited highest cellular uptake in A375R cells with maximum fluorescence intensity as compared to other cationic or ionizable liposomes (Fig. 1d). Subsequently, lipoplexes formed with SM-102 liposomes demonstrated highest transfection efficiency of 60-70% at all tested N/P ratios in comparison to other cationic/ionizable lipoplexes (Fig. 2a). The order of transfection efficiency was: SM-102 > DLin-MC3-DMA > DLinDMA > DOTAP > LAE. Agarose gel electrophoresis results denoted that SM-102 was the most effective in binding GFP-pDNA at lowest N/P ratio of 5 with no DNA migration bands (Fig. 2b). Also, in terms of biocompatibility and hemocompatibility, SM-102 liposomes were found to be the safest (IC₅₀ >200 μM, IC₉₀ >1800 μM) in A375R cells with negligible hemolysis (4.8%) (Fig. 2c-d). Therefore, LNPs containing SM-102 as complexing head group were fabricated at optimized N/P charge ratio of 10 to yield particle size of 96.15 ± 4.7 nm, PDI of 0.133, neutral surface charge and >99% entrapment efficiency of GFP-pDNA as determined by PicoGreen assay (Fig. 3a). LNPs exhibited 65-75% transfection efficiency in A375R cells, which was comparable to TransIT^®-LT1 (Fig. 3b). Treatment in A375R 3D spheroids signified substantial penetration (red fluorescence) and high transfection (green fluorescence) of Rhodamine labeled LNPs (Fig. 3c). Heparin-induced dissociation of DNA was observed from lipoplex while DNA was well retained within LNPs (Fig. 3d). DNA complexed to lipoplexes was found to be completely degraded in presence of serum while that incorporated in LNPs demonstrated high serum protein-resistance capability (Fig. 3e). Therefore, LNPs employing SM-102 as ionizable lipid were found to be systemically safer with acceptable transfection efficiency and will now be investigated to incorporate an anticancer gene for BRAFi-resistant melanoma therapy.

Conclusion: Our study lays a foundation to opt for the right complexing head group and formulation technology for development of lipid nanocarriers mediating gene delivery. It has opened the doors to designing “state-of-the-art” LNP based therapies by entrapping any functional plasmid that targets life-threatening ailments.

Fig. 1. (a) Schematic representation demonstrating parallel comparison of complexing head group and formulation technology for efficient pDNA delivery. (b) Dynamic light scattering graph and cryo-TEM image representing unimodal particle size distribution. (c) Dynamic light scattering graph demonstrating positive zeta potential of liposomal formulation containing SM-102 as ionizable lipid. (d) Fluorescence microscopy images and quantification of fluorescence intensity demonstrating cellular uptake of cationic/ionizable liposomes in A375R cells. Scale bar: 200 μm. *p < 0.05, **p < 0.01 and ****p < 0.0001.

Fig. 2. (a) Fluorescence microscopy images and quantification of transfection efficiency of various lipoplexes at N/P ratio 5 following 48 h in A375R cells. Scale bar: 200 μm. (b) Agarose gel electrophoresis image illustrates binding efficiency of various cationic/ionizable liposomes for GFP-pDNA at N/P ratio of 5. (c) Cell viability curve of A375R cell line following treatment with cationic/ionizable liposomes as determined by MTT assay. (d) In vitro hemolysis study results indicating negligible hemolysis by most liposomal formulations except LAE. *p < 0.05, **p < 0.01 and ****p < 0.0001.

Fig. 3. (a) Dynamic light scattering graphs and cryo-TEM image demonstrating unimodal particle size distribution and neutral zeta potential of optimized LNPs. (b) Representative fluorescence microscopy images demonstrating transfection efficiency of LNPs following 48 h in A375R cells, comparable to TransIT®-LT1. Scale bar: 400 μm. (c) Fluorescence images of A375R 3D tumor spheroids illustrating significant uptake and penetration of Rhodamine labeled-LNPs and simultaneous transfection of GFP-pDNA following treatment. Scale bar: 1000 μm. (d). Representative agarose gel electrophoresis image illustrating higher binding stability of GFP-pDNA to LNPs when compared to lipoplexes as demonstrated by heparin displacement assay. (e) Representative agarose gel electrophoresis image showing stability of GFP-pDNA in presence of serum when complexed in form of LNPs as compared to lipoplexes.