How To Master Lipid Nanoparticle Formulation

GUIDE Lipid Nanoparticle Formulation Basic Concepts & Preparation Procedures The success of mRNA-based COVID-19 vaccines could not have been possible without decades of research on lipid-based drug delivery (LBDD). LBDD systems are highly versatile and have been used to deliver various bioactive molecules to targeted cells and tissues. LBDD has several advantages over conventional drug delivery methods, including increased drug stability, bioavailability, and distribution. Lipid nanoparticles (LNPs) are a significant advancement for the delivery of nucleic acid-based therapeutics. Nucleic acids encapsulated within LNPs are protected from enzymatic degradation during the delivery process and are efficiently delivered to cells, where the therapeutic cargo is released. Use this guide to learn about LBDD systems, the cargoes they deliver, and to explore basic concepts and procedures for the preparation of LNPs. Contents LBDD Basic Concepts LNP Formulation 2 3-5 6 7-8 9-11 12 13 14 Types of LBDD Systems Structural Components Cargo Design Procedures Stability & Storage Characterization Assessment www.caymanchem.com LBDD Basic Concepts 2 Lipid Nanoparticle Liposome Solid Lipid Nanoparticle Nanostructured Lipid Carrier Lipid Shell Monolayer Bilayer Surfactant Surfactant Internal Core Reverse micelles Aqueous Solid lipids Solid and liquid lipids Cargo Nucleic acids Hydrophobic and/or hydrophilic small molecules Hydrophobic and/or hydrophilic small molecules Hydrophobic and/or hydrophilic small molecules Size ~50-150 nm ~50-1,000 nm ~40-1,000 nm ~40-1,000 nm Types of LBDD Systems Lipid nanoparticles (LNPs): a lipid shell surrounding an internal core composed of reverse micelles that encapsulate and deliver nucleic acids, like siRNA and mRNA, and plasmid DNA (pDNA). Liposomes: contain one or more lipid bilayers and an aqueous core. They are further classified by lamellarity and size. Liposomes are used for hydrophobic and/or hydrophilic small molecules. Solid lipid nanoparticles (SLNs): a surfactant shell surrounding a core matrix composed of solid lipids. They are used for hydrophobic and/or hydrophilic cargo. Nanostructured lipid carriers (NLCs): a surfactant shell surrounding a core matrix composed of solid and liquid lipids. They are used for hydrophobic and/or hydrophilic cargo. Micelles: self-assemblies of lipid monolayers in aqueous solutions. They have a hydrophobic core, where the phospholipid tails are oriented towards the interior, and can be used for small hydrophobic cargo. Reverse micelles: an inverted structure compared with traditional micelles. They form a hydrophilic core, with the phospholipid tails oriented towards the exterior, and can be used for small hydrophilic cargo, like nucleic acids in LNPs. Cationic Lipid PEGylated Lipid Phospholipid Surfactant Nucleic Acid Sterol Lipid Solid Lipid Liquid Lipid Aqueous Phase Hydrophilic Drug Hydrophobic Drug LNPs compared to other LBDD systems 3 LBDD Basic Concepts www.caymanchem.com Structural Components LNPs are composed of ionizable cationic lipids, helper lipids, which include glycerophospholipids, sterol lipids, and PEGylated lipids to protect nucleic acids, which are contained within an aqueous phase. Many of the same structural components used in LNPs are components of other LBDD particles. Lipids and molecules that contain them, like surfactants, can be used to tailor the behavior and properties of LBDD particles. Examples of lipid components are listed below for each class. Ionizable cationic lipids circumvent the untoward cytotoxicity associated with cationic lipids. These lipids possess a transient cationic charge that is acquired at low pH (typically <7), forming reverse micelles that encapsulate nucleic acids in the LNP core. As these lipids have near-neutral charge at physiological pH, they deliver nucleic acid cargo without cytotoxicity. N+ DODMA pKa Value of Tertiary Amine: 6.59 O O N DODAP pKa Value of Tertiary Amine: 5.59 O O N O O SM-102 pKa Value of Tertiary Amine: 6.68 O O N OH O O ALC-0315 pKa Value of Tertiary Amine: 6.09 HO N O O O O DLin-KC2-DMA pKa Value of Tertiary Amine: 6.70 O O N DLin-MC3-DMA pKa Value of Tertiary Amine: 6.44 O O N Sterol lipids, such as cholesterol, fill lipid membrane packing defects and provide structural integrity. They also aid in membrane fusion of the LNP and target cell, and some cholesterol derivatives, like 7α-hydroxy cholesterol, have been used to improve the delivery of nucleic acid cargo. N+ H H H HO H OH Cholesterol 7α-hydroxy Cholesterol H H H HO H www.caymanchem.com LBDD Basic Concepts 4 1,2-DOPE 1,2-DSPC 1,2-DOPC 1,2-POPC Glycerophospholipids, colloquially known as helper lipids, are a class of phospholipid that contains a hydrophilic head group and two hydrophobic fatty acyl tails attached to a glycerol backbone. The hydrophilic head determines the surface charge of the LBDD particle, which can be neutral, anionic (negative), or cationic (positive). N+ Neutral phospholipids improve the efficacy of membrane fusion and can distribute or modify the net surface charge of the lipid particle. The phospholipid head groups with an overall neutral charge are phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Anionic lipids are typically used for the delivery of small molecules and are incorporated into neutral LBDD systems to prevent storage aggregation. They can modify the net surface charge of the lipid particle and influence cellular targeting. Phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidic acid (PA) are phospholipids with anionic head groups. O O O O O P O O ONH3 + O O O O O P O O ON+ O O O O P O O ON+ O PEGylated lipids prevent serum protein adsorption, inhibiting uptake by the mononuclear phagocyte system (MPS), a major obstacle in the delivery of LBDD systems. Some PEGylated lipids also contain terminal functional groups, such as amine or maleimide, which can be used to conjugate other molecules that improve cellular targeting and uptake. N+ DMG-PEG(2000) 1,2-POPG ALC-0159 1,2-DOPS O n O O O O O O n O O N O O O O O P O O ON+ O O O O P O O OO OH OH O O O O O P O O OO OH NH2 5 LBDD Basic Concepts www.caymanchem.com Read our article The Heads and Tails of Lipid-Based Drug Delivery to dive deeper into the biophysical properties of lipids used in LBDD systems. The aqueous phase solubilizes hydrophilic molecules, like nucleic acids in LNPs and hydrophilic drugs in liposomes, whereas hydrophobic cargo can be solubilized in the lipid phase. Surfactants are a major component of the lipid shell of SLNs and NLCs. They reduce the interfacial tension between the lipophilic core and aqueous phase due to their amphipathic nature and improve stability during storage. Lecithin, which contains phosphatidylcholines, is one surfactant that has been used in SLNs and NLCs. Solid lipids, colloquially known as fats, are solid at ambient temperature and used in the preparation of both SLNs and NLCs. Solid lipids used to prepare SLNs or NLCs are typically saturated and include glycerolipids, such as triacylglycerols, and fatty acids, including stearic acid, as well as fatty alcohols and fatty esters. Liquid lipids, also referred to as oils, are fluid at ambient temperature. NLCs are formulated with a mixture of solid and liquid lipids, which increases drug loading capacity and prevents drug leakage. Liquid lipids used in NLCs are typically unsaturated and include oleic acid, α-tocopherol, and squalene. Custom synthesis of high-purity lipids from smaller batch sizes to larger scale CGMP quantities. · Ionizable Cationic Lipids · Helper Lipids · Sterol Lipids · Novel or Proprietary Lipids Custom Lipid Synthesis Services Learn more at www.caymanchem.com/custom-synthesis www.caymanchem.com/lipid-properties www.caymanchem.com LBDD Basic Concepts 6 Cargo Small Molecules Hydrophobic and/or hydrophilic small molecules can be solubilized in the lipophilic or aqueous compartments, respectively, of liposomes, SLNs, and NLCs. Hydrophobic drugs are dispersed in the lipophilic compartments of LBDD systems. Amphotericin B, an antifungal agent, and verteporfin, a photosensitizing agent, are examples of FDA-approved hydrophobic drugs that have been formulated in liposomes. Hydrophilic drugs are solubilized in the aqueous compartments of LBDD systems. Doxorubicin, an antitumor antibiotic, is a hydrophilic drug. Doxil® is a form of doxorubicin encapsulated in liposomes and was the first LBDD formulation to be approved by the FDA. mRNA, siRNA, and pDNA are common nucleic acid cargo. Nucleic acids are negatively charged and best encapsulated within LNPs using ionizable cationic lipids. mRNAcontaining LNPs are the basis for most COVID-19 vaccines, and LNPs containing transthyretin-targeting siRNA are used to treat hereditary amyloidogenic transthyretin (ATTRv) amyloidosis. LNPs are superior for the encapsulation of nucleic acids, whereas other LBDD systems are preferable for the delivery of small molecule inhibitors or lipids. The requisite LBDD system for your application depends on the cargo, and the localization of the cargo within the LBDD particle depends on its physicochemical properties. Nucleic Acids From new researchers to experienced scientists, our collection of products and resources is your go-to source for LNP research and development. · Lipids for LNP Formulation · LipidLaunch™ Research Tools · LNP Development Services · Articles, Webinars, & Application Notes Lipid Nanoparticle Resource Center Explore all at www.caymanchem.com/lipid-nanoparticles 7 LNP Formulation www.caymanchem.com Design Several factors should be considered when selecting lipids and how they are formulated into LNPs. Below, we outline these factors and how each of them influences the physiological behavior of LNP particles. The lipid:nucleic acid weight ratio influences the encapsulation efficiency. Most LNPs are formulated with a lipid:nucleic acid weight ratio of 10-30:1. The ionizable lipid nitrogen:nucleic acid phosphate (N:P) molar ratio represents the charge balance between the cationic tertiary amine of the ionizable cationic lipid and the anionic phosphate group in the nucleic acid backbone. This property is the basis for the complexation of ionizable cationic lipids with nucleic acids. LNPs commonly have an N:P ratio around six. The lipid molar ratio determines the lipid composition of the particles and influences their size, polydispersity, and efficacy. We recommend starting with a literature review to identify lipid molar ratios that have been previously developed for similar applications. N+ N+ N+ a Ionizable cationic lipid:neutral phospholipid:cholesterol:PEGylated lipid Patisiran BNT162b2 mRNA-1273 Cargo siRNA mRNA mRNA Ionizable Cationic Lipid DLin-MC3-DMA ALC-0315 SM-102 Neutral Phospholipid 1,2-DSPC 1,2-DSPC 1,2-DSPC Sterol Lipid Cholesterol Cholesterol Cholesterol PEGylated Lipid C-DMG-PEG(2000) ALC-0159 DMG-PEG(2000) Lipid Molar Ratioa 50:10:38.5:1.5 46.3:9.4:42.7:1.6 50:10:38.5:1.5 Lipid molar ratios for LNPs in FDA-approved agents Lipid Nanoparticle Cationic Lipid PEGylated Lipid Phospholipid Sterol Lipid Nucleic Acid Aqueous Phase www.caymanchem.com LNP Formulation 8 Three important parameters for an aqueous buffer are its composition, ionic strength, and pH. Buffers stabilize nucleic acids in solution, and ionizable cationic lipids become protonated and positively charged in the acidic aqueous buffer upon mixing. Commonly used buffers in LNP preparations are 25-50 mM sodium acetate or sodium citrate, pH 4-5. After preparation, LNPs are dialyzed into a neutral buffer, such as PBS, pH 7.4, for storage and use. The particle size alters the pharmacokinetics of the administered particle. Smaller particles typically have longer circulation half-lives, as they evade elimination by the MPS. Particles less than 100 nm can easily pass through fenestrated endothelium to penetrate target tissues. The particle size is dependent on the preparation method. Depending on the LNP preparation method, extrusion can be used to achieve smaller, more uniform particle sizes. The preparation method determines the properties of LNPs, including size, homogeneity, and encapsulation efficiency. When selecting a preparation method, cost, scalability, reproducibility, and time commitment should also be considered. The two most commonly used routes of administration for LNPs are intravenous and intramuscular injection. Of note, formulations optimized for a given route of administration are generally not applicable for other routes of administration. Intravenously administered LNPs with net positive, neutral, and negative charges can be targeted to the lungs, liver, and spleen, respectively. The inclusion of cholesterol or PEGylated lipids in the formulation, as well as increasing the LNP size, increases distribution to the spleen. Intramuscular administration is commonly used for vaccines, as it facilitates lymph node targeting and activation of the immune response. When a vaccine is administered, antigen-presenting cells (APCs), like macrophages and dendritic cells, are recruited to the delivery site, where they can encounter vaccine antigens. They then migrate to lymph nodes where they stimulate adaptive immune responses. The lipid acid dissociation constant (lipid pKa ) is the pH at which the ionized and nonionized forms of a lipid exist in equal concentrations. Lipid pKa impacts the LNP encapsulation efficiency, efficacy, delivery, and toxicity. For RNA delivery, the lipid pKa generally ranges from 6-7. 9 LNP Formulation www.caymanchem.com Procedures In the next sections, we give a series of simple procedures to follow for producing LNPs. The entire range of the LNP life cycle is covered, starting with LNP preparation at the bench and ending with how to use and what to expect when testing LNPs in your in vitro or in vivo experiments. LNP Preparation Mixing Before beginning, ensure that all supplies, reagents, and working environments are RNase-free. siRNA and mRNA are chemically labile to RNases, which are enzymes that degrade RNA-based nucleic acids. The steps in LNP formulation are summarized below. LNPs are prepared by mixing an ethanolic lipid mixture with an acidic aqueous solution containing nucleic acids. A 1:3 ratio of ethanolic lipid mixture to aqueous buffer is generally used. Several methods are suitable for laboratory-scale, small-volume LNP production. Four of these methods, applicable for a range of specialized-to-basic equipment, are compared briefly on page 10. Nucleic Acids in Aqueous Acidic Buffer Rapid Mixing Lipids in Ethanol Cationic Lipid PEGylated Lipid Phospholipid Sterol Lipid Nucleic Acid Aqueous Phase LNP preparation workflow Schematic of nucleic acid-containing LNP formation Lipid Nanoparticle (LNP-102) Exploration Kit Workflow Prepare ethanolic lipid mixture with ionizable cationic lipid, helper lipid, sterol lipid, and PEGylated lipid Prepare acidic aqueous nucleic acid solution Mix using a range of specialized-to-basic equipment methods Optional: Perform size extrusion Uniformly sized lipid nanoparticles www.caymanchem.com LNP Formulation 10 Microfluidic Mixing Devices: Automated microfluidic devices or microfluidic chips are fast and efficient methods to prepare LNPs. These devices enable rapid mixing in a highly controllable, reproducible manner that achieves homogeneous LNPs and high encapsulation efficiency. In these devices, individual streams of the ethanolic lipid mixture and aqueous nucleic acid solution are rapidly combined. LNPs form as the two streams mix and are collected into a single collection tube. Parameters such as the flow rate ratio (FRR) and total flow rate (TFR) can be altered to fine-tune LNPs. Hand-mixing: This is a simpler alternative method to ethanol-injection. Transfer the ethanolic lipid mixture into the aqueous acidic nucleic acid solution and mix for 15 seconds by rapid pipetting. Leave the mixture undisturbed for 10 minutes. As with the ethanol-injection method, hand-mixing of LNPs results in heterogeneous LNPs with low encapsulation efficiency and can yield variable results. LipidLaunch™ LNPs and reagent kits support LNP research, offering simple and cost-effective solutions from discovery to bioanalysis. · Preloaded LNPs · Loadable LNPs · LNP Exploration Kits · LNP Uptake Kits LipidLaunch™ Research Tools View the guide at www.caymanchem.com/lipidlaunch-guide Cost Scalability Encapsulation Efficiency Reproducibility Polydispersity Index Microfluidic Mixing Devices High High High High Low Hand Mixing Low Low Low Low High Feature comparison of LNP preparation methods 11 LNP Formulation www.caymanchem.com Final Preparation The final preparation of LNPs is performed after they have been formed during the mixing step. These steps help ensure that the LNPs are homogeneous, stable during storage and use, and free of any residual chemical or biological contaminants. Extrusion: Extrusion reduces and unifies particle size. This step is generally performed with preparation methods that yield LNPs with variable particle sizes. N+ Filter-sterilize: Filtration is the recommended method for LNP sterilization. Filter-sterilize LNPs with a 0.22 µm filter before storage. Dialysis: Dialyze the LNPs in storage buffer using appropriate molecular weight cut-off (MWCO) tubing. This step removes unencapsulated cargo, excess lipids, and ethanol from the final preparation. Dialysis also adjusts the pH of the LNPs from the acidic preparation buffer to the neutral storage solution. For larger volumes, tangential flow filtration is the optimal method for neutralizing pH and removing ethanol. N+ N+ Customize an LNP development program from our suite of services to meet your project goals. · Custom Lipid Synthesis · Formulation & Characterization · Bioanalysis & Screening LNP Development Services Learn more at www.caymanchem.com/custom-synthesis www.caymanchem.com LNP Formulation 12 Stability & Storage After LNPs have been prepared, they may either be used immediately or stored for later use. Below, we discuss factors that can compromise LNP integrity during storage and provide tips to limit storage instability. Biological stability relates to the capacity of LNPs to avoid early degradation. Factors that contribute to biological stability include lipid composition, particle size, and surface charge. Reduce serum protein opsonization: · Include PEGylated lipids · Decrease particle size · Achieve near-neutral zeta potential · Increase LNP hydrophilicity Chemical stability defines the resistance of LNP lipid and cargo components to modifications in their molecular structure. Hydrolysis, oxidation, and transesterification can lead to nucleic acid and lipid degradation or the formation of lipid-nucleic acid adducts and loss of efficacy. Limit cargo degradation: · Use RNase-free reagents and supplies · Consider nucleic acid cargo with backbone modifications Prevent lipid oxidation: · Include antioxidants or cryoprotectants during storage Physical stability describes the structural integrity of LNPs during storage. Particle fusion or aggregation and leakage of encapsulated cargo are examples of physical instability. Ensure size distribution remains small and homogeneous: · Use anionic or PEGylated lipids to prevent particle fusion/aggregation Prevent cargo leakage: · Include cholesterol N+ Storage is a critical parameter in the stability of LNP formulations. Generally, LNPs may be stored at 4°C for up to one week or, for long-term storage, lyophilized and held at -80°C. Storage temperature, buffers, and pH may need to be optimized. The inclusion of cryoprotectants is recommended when freezing with or without lyophilization. Follow storage requirements: · Adjust temperature, buffers, and pH · Avoid freeze-thaw cycles Follow storage requirements: · Adjust temperature, buffers, and pH · Avoid freeze-thaw cycles Prevent cargo leakage: · Follow storage requirements · Increase LNP stability with cholesterol or helper lipids 13 LNP Formulation www.caymanchem.com Characterization Characterization of LNP attributes prior to in vitro or in vivo use is critical for reproducibility. Lipid and cargo integrity are essential for the efficacy and stability of LNPs. Refer to the stability and storage section on page 12 for more information. Our Chemical Synthesis team offers lipid characterization services. The apparent/global pKa of formulated LNPs determines the effect of the ionizable lipid on the apparent pKa of LNPs. This parameter influences the LNP ionization and surface charge, stability, potency, and toxicity. Encapsulation efficiency is the final amount of nucleic acid contained within the LNP compared to nucleic acid not encapsulated within the LNP. Microfluidic mixing yields the highest encapsulation efficiencies. Encapsulation efficiency can be measured using nucleic acid-binding fluorescent dyes in the presence and absence of detergent, such as 1% Triton X-100. This technique can also serve to report the encapsulated nucleic acid concentration. The zeta potential is the electrostatic potential surrounding the LNP. In general, a nearneutral zeta potential is desirable. Anionic LNPs may be electrostatically repelled from negatively charged plasma membranes, and cationic LNPs can be cytotoxic. The zeta potential can be adjusted by altering the N:P ratio. Zeta potential is measured by a specialized instrument. The LNP size describes the average diameter of LNPs and influences their biodistribution and cellular uptake. The polydispersity index (PDI) is a measure of the LNP size distribution. Homogeneous, uniformly sized samples have small PDIs, and samples with heterogeneous size distributions have large PDIs. The LNP size and PDI of an LNP preparation can be reduced by optimizing lipid components, increasing the mixing rate, selecting a different preparation method, or by adding an extrusion step. These attributes can be measured by dynamic light scattering (DLS) using a specialized instrument. · Reagents to determine ionizable lipid effect on the apparent pKa of LNPs Item No. 702680 LipidLaunch™ LNP Apparent pKa Assay Kit (TNS Method) Learn more at www.caymanchem.com/pka-assay www.caymanchem.com LNP Formulation 14 Assessment LNPs are internalized by target cells via endocytosis. Endosomal escape is the process by which the LNP cargo is delivered to the cytosol. Ionizable cationic lipids become positively charged in the acidic environment of the endosomal lumen, which disrupts the negatively charged endosomal membrane and promotes release of the encapsulated nucleic acid cargo into the cytosol, where translation occurs. Cellular models and simple molecular biology techniques can be used to test the efficacy of LNPs in vitro. Measurement of knockdown or expression of the gene or protein of interest can be accomplished via qPCR or Western blot, respectively. Cell-based reporter or luciferase assays are also used to determine LNP efficacy. Animal models provide valuable insight into the efficacy of LNP-based therapeutics in vivo. Protein expression of nucleic acid products in vivo follows a target-dependent time course. Nucleic acids encoding functional proteins produce changes in protein concentrations within hours, whereas those designed to elicit an antibody response can occur between several days to a couple of weeks. It is often necessary to use repeated dosing regimens to achieve sustained protein expression. ELISAs and multiplexed assays can be used to measure target protein responses, and flow cytometry can determine changes in cellular phenotype. The fate of LNPs after administration depends on the lipid composition, LNP design, and the route of administration. Lipids used in LNPs are detectable in the tissues that LNPs are distributed to after administration. These lipids are biocompatible and rapidly degraded, and they are generally eliminated within 24 to 48 hours after administration. To determine lipid tissue concentrations, mass spectrometry-based approaches can be used. Our Analytical Chemistry team can assist you in the detection of lipids in various tissues and sample matrices. LNP Endocytosis Protein Endosomal escape Translation Technical Support at any step of your research Our technical support and product development scientists are here to help answer your questions about products, applications, and protocols, and to help you find additional resources. Contact our Technical Support specialists. 1180 East Ellsworth Road Ann Arbor, MI 48108 www.caymanchem.com Phone: (800) 364-9897 (toll free) Fax: (734) 971-3640 Connect with Us: