Gene therapy stands out as one of the promising therapeutic strategies to treat various acute acquired and inherited diseases including sickle-cell anemia, hemophilia, neurodegenerative disorders, as well as different forms of cancers. Even though viral vectors have been widely used to deliver genes efficiently and for long-term expression, they may generate immune responses to limit their clinical applications. To overcome these disadvantages, non-viral vectors have been extensively explored to form complexes with nucleic acids and protect them from enzymatic degradation (1). Nanoparticles designed for nucleic acid delivery need to reach specific intracellular compartments following their uptake in the target cells to induce upregulation or downregulation of dysregulated proteins. More specifically, DNA loaded nanocarriers must translocate to the nucleus of the cells to achieve target protein expression (2). Lipid based nanocarriers have therefore been extensively developed to entrap DNA for its safe delivery and high transfection efficiency, ensuring stability, longer circulation, and avoiding renal clearance from the systemic circulation via PEGylation (3).
Cationic or ionizable lipid-DNA complexation based lipoplexes have been broadly utilized as DNA delivery systems by employing cationic or ionizable lipids to allow an easy complexation with negatively charged plasmid DNA (pDNA). These lipoplexes are generally prepared by bulk mixing process which results in non-uniform large-size vesicles and may require sonication or extrusion to achieve the desired particle size. Moreover, stability of lipoplexes against serum proteins and enzymes like nucleases is critical to maintain the integrity of pDNA and protect it from degradation in the systemic circulation. To overcome such limitations, lipid nanoparticles (LNPs) prepared by microfluidic mixing has emerged as a scalable and robust technology to encapsulate nucleic acids within the hydrophilic core (4). Currently, there are three FDA-approved LNP formulations designed for RNA delivery via microfluidic mixing approach. Amongst them, Onpattro® (or patisiran) developed by Alnylam Pharmaceuticals Inc. is a small-interfering RNA (siRNA) encapsulated in LNP for the treatment of transthyretin-mediated amyloidosis. This LNP system is composed of DLin-MC3-DMA ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)-butanoate) as the ionizable lipid, DSPC (disterarolyphosphatidychloline), cholesterol and a PEG-DMG to direct the LNPs towards the liver hepatocytes in vivo. The two most recently approved/authorized and broadly advertised LNP therapies are vaccines manufactured by Pfizer-BioNTech and Moderna Therapeutics Inc. in 2020 for prevention against severe coronavirus disease 2019 (COVID-19) infection and hospitalization initiated by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Both these formulations contain PEG and cholesterol, with Moderna including the ionizable lipid sphingomyelin-102 (SM-102) and Pfizer-BioNTech consisting of the ionizable lipid ALC-0315 from Acuitas to formulate the LNPs for successful delivery of Spike mRNA (5, 6). LNPs loaded with pDNA are being actively explored in preclinical settings to target various diseases and yet have to find their way in the market.
LNPs or lipoplexes possessing variable physicochemical characteristics can be fabricated by adjusting formulation components or process parameters. Amongst formulation parameters, type of cationic/ionizable lipid and the weight or charge ratio of cationic/ionizable lipid to pDNA (N/P ratio) are critical for efficient complexation of pDNA, its cellular uptake, endosomal escape, and successful transfection in the nucleus of the target cells. Chemical structure of cationic lipids like DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl) and DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) consist of quaternary amine groups that contribute to their high positive charge to condense and encapsulate negatively charged pDNA in liposomal formulations for intracellular delivery. Even though these quaternary ammonium lipids result in high transfection efficiency, they are found to be toxic rendering them incompatible for in vivo administration (7, 8). To overcome this challenge, arginine-based cationic surfactants including lauroyl arginine ethyl ester (LAE) and arginine–N-lauroyl amide dihydrochloride (ALA) were also explored by various researchers to develop cationic liposomes for charge mediated pDNA complexation and subsequent transfection in target cells in vitro (9, 10). However, cationic lipid or surfactant based lipoplexes may possesses in vivo barriers in terms of protecting the pDNA from enzymatic degradation and their permanent positive charge possibly leading to hemolysis making them unsafe for systemic administration. Therefore, pH-sensitive amino lipids were established as a series of ionizable lipids to address the challenges of systemic stability and subsequent cytosolic delivery of nucleic acids like pDNA. Different generations of ionizable lipids like DLin-DMA, DLin-MC3-DMA, C12-200, SM-102 and ALC-0315 have been synthesized by modifying the cationic head group, the length and unsaturation of hydrophobic chain or the introduction of ester bonds to promote transfection efficiency, endosomal escape, and biodegradability of resulting ionizable LNPs (11–13).
Selecting the right cationic or ionizable lipid is key to successful gene delivery given its importance in achieving complete nucleic acid complexation, cellular uptake, endosomal escape, and essentially high transfection efficiency to attain target protein expression. In this work, we have compared cationic lipid DOTAP, cationic surfactant LAE, and different generations of ionizable lipids with versatile chemical structures including DLin-DMA, DLin-MC3-DMA, and SM-102 to evaluate their N/P ratio required to complex pDNA, cellular uptake, transfection efficiency, biocompatibility and hemocompatibility for effective pDNA delivery. There are no previous reports illustrating the comparison of cationic lipid, cationic surfactant and ionizable lipids to identify the most effective cationic headgroup to deliver pDNA for preclinical and clinical applications. We selected the complexing lipids or surfactants based on their properties and previous use in approved nucleic acid formulations and formulated their cationic or ionizable liposomes in combination with phospholipids, cholesterol, and PEGylated lipid to further complex with pDNA resulting in lipoplexes. In addition to the formulation components, the technology used to prepare gene delivery nanocarriers is also of utmost importance. In this context, we evaluated if complexation of pDNA on exterior of liposomes was sufficient to ensure its systemic safety or there is a necessity to incorporate the pDNA in interior of lipid nanocarriers for in vivo applications. Therefore, we further prepared LNPs using microfluidic mixing with the optimized cationic or ionizable head group to incorporate the pDNA within its hydrophilic core. A systematic comparison of lipid nanocarrier with pDNA complexed on the interior versus exterior of the nanoparticles was executed in terms of physicochemical characterization, transfection efficiency and safety for parenteral administration. We assessed the in vitro transfection efficiency of each of the formulations using green fluorescent protein (GFP) as a reporter protein and characterized how well each of the formulations protect the pDNA from degradation by DNAse and fetal bovine serum (FBS). The following research is preliminary part of development of a novel non-viral gene therapy for BRAF inhibitor (BRAFi)-resistant melanoma. Thus, we have screened and optimized cationic head group and formulation technology to develop lipid nanocarrier for PTEN (Phosphatase and TENsin homolog deleted on chromosome 10) gene delivery for BRAFi-resistant melanoma, given it is often associated with decreased expression or loss of PTEN gene expression (14, 15). Therefore, all the cell culture-based assays were performed in developed BRAFi-resistant melanoma cell line, A375V, by using GFP-pDNA as a model gene. To our knowledge, there has been no previous work demonstrating a parallel comparison of both formulation components and technology for efficient pDNA delivery. We have demonstrated for the first time, the correlation between cellular uptake and transfection efficiency in 3D spheroid model for optimized formulation. Taken together, our study lays a foundation to opt for the right complexing lipid and technology to develop lipid nanocarriers for achieving target protein expression in oncology or other diseases.