Liposomes have long been established as an effective drug delivery vehicle for API with poor pharmacokinetics, limited bioavailability or solubility, and high toxicity. Discovered in the 1960s by Alec D. Bangham, liposomes were originally utilized as an artificial bilayer to study membranes’ biophysical properties. The liposome morphology can be described as an aqueous core surrounded by one or many lipid bilayers, a lipid phase preferred by cylindrical space-filling lipid molecules. The amphiphilic bilayer, typically comprised of phospholipids and sterols, resembles biological cell membranes and facilitates cellular endocytosis and subsequent cellular uptake. Led by the efforts of Gregory Gregoriadis in the 1970s, liposomes would be revealed as ideal DDS solutions for pharmaceutical applications. This research would provide the first knowledge of the complex relationship between the physical properties of nanomedicines and the biological milieu in vivo. By 1995, a liposomal formulation of doxorubicin (Doxil®) would become the first FDA-approved nano DDS.

Whether comprised of synthetic or natural lipid components, conventional liposomes are highly versatile and have been extensively investigated as multifunctional excipients in targeted drug delivery, imaging, and diagnostics. In pursuit of an alternative assembly method for lipid-based DDS, DemetriosPapahadjopoulos would champion reverse-phase evaporation (RPE), a highly reproducible process encapsulating API using a combination of aqueous and organic solvents during production. Eventually, Batzri and Korn would establish the ethanol injection method, whereby lipids, dissolved in ethanol, are injected in excess aqueous buffer forming a nanoscale colloidal dispersion. The incorporation of next-gen microfluidic techniques with ethanol injection has provided a scalable manufacturing process that can expedite the clinical translation of lipid-based pharmaceutical solutions, as seen in the recent breakthrough of mRNA-LNP vaccines for COVID-19.

SOLID LIPID NANOPARTICLES AND NANOSTRUCTURED LIPID CARRIERS

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 electrondense 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.

Figure 2. Structure of LNP, from lipid nano-emulsions to solid lipid nanoparticles (SLN). Nanostructured lipid nano-carriers (NLC) contain a blend of solid and liquid-lipid phases ideal for increased-loading capacity and controlled drug release profiles.

GENE THERAPY AND MRNA VACCINES:

GENE THERAPY AND MRNA VACCINES:

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 Lipid Nanoparticle Delivery Nucleic Acids mRNA, the focus has moved toward LNP morphology for the delivery of nucleic acids in the pharmaceutical industry.

LIPID NANOPARTICLES:

SOLID LIPID NANOPARTICLES (SLN) AND NANOSTRUCTURE LIPID CARRIERS (NLC)

The development of mRNA vaccines in Ionizable Lipid NanoparticleCationic Liposomes the fight against COVID-19 is one of the most important medical discoveries of PEG the 21st century. Predictably, LNP formulations are quickly becoming the gold standard for nucleic acid delivery. Initial investigations with LNP formulations began with the SensitiLipipHdveNucleAcidsicNucleAcidsicCatioLipidHelpen +ircspontaneous assembly of lipid-nucleic

acid complexes internalized and stabilized in a lipid core, analogous to NLC morphology discussed herein. Figure 3.Comparison of lipoplex and LNP morphology with encapsulated nucleic acid cargo

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.

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