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Ionizable, Cationic, Neutral Lipid and Polymeric Nanoparticles

COVID-19 mRNA vaccines proved the potential of lipid nanoparticles as a robust and effective delivery systems. There is a lot of room to grow to make these nanoparticle more effective for other vaccines, and therapeutics. COVID vaccines have certainly expedited the use of these nanoparticles in wide range of gene therapies, cell therapies, specially for cancer and infectious diseases, vaccines and therapeutics. T&T Scientific is offering all inclusive formulation and manufacturing platform to help pharmaceutical companies with formulation and manufacturing of these vaccines, and therapeutics from preclinical stage to clinical phases and commercial manufacturing. We have a team who specializes in this area, and have initiated and executed over 20 RNA LNP formulation and manufacturing in preclinical, clinical phases, and commercial.

Ionizable Lipid Nanoparticles: Ionizable lipid nanoparticles are composed of three different segments: 1) the amine head group, 2) the linker section, and 3) the hydrophobic tails. These type of lipid nanoparticles are widely used for siRNA delivery with lipids such as DLinDMA and DLin–KC2–DMA. The mixing of cationic and anionic lipids in these nanoparticles help with the transfection in the way that the more stable lamellar phase translates to the less stable hexagonal phase, therefore aiding fusion of the liposomal and endosomal membranes (1-4). Solvent or ethanol injection is the most common technique to make these therapeutics and vaccines, as these techniques are low cost, fast, and provide desired size, size distribution polydispersity index (PDI), and encapsulation efficiency. We offer these services at our CDMO and has commercial equipment that can be installed and qualified at your site (5, 6).

Cationic Lipid Nanoparticles: Cationic lipid nanoparticles are also key components of lipid nanoparticles (LNP) delivery vehicles, which serve as a platform for delivery of various RNA, and therapeutics, and vaccine agents. Cationic lipid nanoparticles are increasingly regarded as one of the most promising mRNA delivery systems because of their good biocompatibility and easy large-scale production. Cationic lipid nanoparticle protect mRNA from degradation by nucleases and deliver the therapeutics and vaccines to cells by electrostatic adsorption of the charged lipids and fusion with the human cell membrane (3, 4). Solvent or ethanol injection is the most common technique to make these therapeutics and vaccines, as these techniques are low cost, fast, and provide desired size, size distribution polydispersity index (PDI), and encapsulation efficiency. We offer these services at our CDMO and has commercial equipment that can be installed and qualified at your site (5, 6).

Calcium-Phosphate Lipid Nanoparticles (CaP):These lipid nanoparticles are made of inner leaflet of a cationic lipid to encapsulate negatively charged polymers or nucleic acids. In cases neutral lipid is located on the outer leaflet to reduce non-specific cellular uptake and toxicity. The final nanoparticle is generated by adding free lipids to the cores. The formation of asymmetric bilayer happens as the organic solvent is removed from the mixture and exposed to an aqueous solution (7, 8). Solvent or ethanol injection is the most common technique to make these therapeutics and vaccines, as these techniques are low cost, fast, and provide desired size, size distribution polydispersity index (PDI), and encapsulation efficiency. We offer these services at our CDMO and has commercial equipment that can be installed and qualified at your site (5, 6).

Lipoplexes: Lipoplexes are another form of lipid delivery system, they are also known as cationic liposomes, are nonviral lipid nanoparticle carriers mainly used to encapsulate and deliver DNAs targetedly. Because of their positive surface charge, they complex well with negatively charged DNA. Lipoplexes in general made of of three components: a synthetic cationic lipid, a synthetic neutral lipid, and a plasmid DNA (10-15).

Liposomes, Lipid Nanoparticles (LNPs), and other lipid-based formulations have been clinically proven to improve the therapeutic index of a wide variety of active pharmaceutical ingredients (API). Lipids are an amphipathic class of biological molecules that exhibit ideal safety and pharmacokinetic profiles when incorporated into pharmaceutical drug products. Recently, LNPs have gained widespread attention across the globe due to their incorporation into messenger RNA (mRNA)-based vaccine formulations aimed at preventing the spread of the COVID-19 pandemic. LNP nanotechnology has quickly become the preferred drug delivery system (DDS) for gene therapeutics and other complex parenteral drug products and represents the future of nanomedicine .

Solid lipid nanoparticles (SLN) are lipid-based nanocarriers made with high phase transition lipids that are solid at body temperature and stabilized by emulsifiers. The SLN has a different morphology from liposomes in that it contains a solid lipid interior that appears as an electron- dense core in electron micrographs. SLN can be manufactured in the nanoscale size range and have good long-term stability with nonpolar API. Unfortunately, SLN shows poor drug loading efficiency and exhibits difficult-to-control drug release characteristics. Recently, advanced methods to produce nanostructured lipid carriers (NLC) with solid and liquid phase lipid components were designed to overcome the limitations of SLN. NLCs show improved drug loading capacity in the lipid matrix core and demonstrate ideal and predictable drug release profiles (10-25).

The ability to treat rare and previously undruggable diseases by expressing therapeutic or mutated proteins, silencing pathological genes, or editing the native genome of patients has become a clinical reality. Current examples of nucleic acid therapeutics that have been approved or are in late-stage clinical trials include antisense oligonucleotides (ASO), small interfering RNA (siRNA), messenger RNA (mRNA), and plasmid DNA (pDNA). The emergence of mRNA vaccines, whereby viral antigens are expressed by host cell machinery before immunization, has allowed researchers to develop solutions to the COVID-19 pandemic with unprecedented speed. This is largely due to the 20+ years of proven clinical and commercial success of LNPs as an effective and safe delivery agent for genetic payloads like mRNA and DNA. Considering any gene in the human genome is druggable, gene therapeutics is poised to become the future of modern medicine and allow researchers to conquer rare, incorrigible ailments. Alas, nucleic acids are volatile and require a delivery vehicle to protect the genetic cargo and facilitate entry into the target cells in vivo. Initial efforts to encapsulate and deliver DNA and RNA with lipid-based formulations involved passive encapsulation strategies with neutral, zwitterionic lipid formulations. To improve the loading efficiency of often expensive nucleic acid payloads, cationic lipids (e.g., DOTAP, DOTMA) were incorporated into liposomal formulations to boost encapsulation through electrostatic lipid/DNA lipoplexes. Lipoplex-mediated delivery of gene therapy has shown considerable utility for in vitro transfection experiments (e.g., Lipofectamine®). However, the complexation process parameters are spontaneous and difficult to control, resulting in particles characterized by wide size distributions ranging from the nanoscale up to several microns. According to FDA guidance for GMP Drug Manufacturers, control over particle size and particle distribution are important for any lipid-based drug delivery system. As manufacturing methods for lipid-based solutions in nanomedicine have advanced, the focus has moved toward LNP morphology for the delivery of nucleic acids in the pharmaceutical industry (16-31).

The development of mRNA vaccines in the fight against COVID-19 is one of the most important medical discoveries of the 21st century. Predictably, LNP formulations are quickly becoming the gold standard for nucleic acid delivery. Initial investigations with LNP formulations began with the spontaneous assembly of lipid-nucleic acid complexes internalized and stabilized in a lipid core, analogous to NLC morphology discussed herein (16-31).

The first LNP formulations employed a detergent dialysis method for production, often employed to encapsulate hydrophilic API in hybrid NLC formulations. The introduction of ionizable cationic lipids combined with ethanol injection and microfluidic techniques has provided a scalable manufacturing method to achieve high loading efficiencies of nucleic acids in monodisperse LNP less than 100 nm in diameter. These LNP systems exhibit low surface charge, which helps overcome noted toxicity and pharmacokinetic issues in vivo (16-31).

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