Lipid Nanoparticle(LNP) for RNA Delivery

Messenger RNA (mRNA) has flourished as a strategy to generate transient gene expression in immune cells to mitigate the disadvantages associated with viral vectors, including CAR therapy. Lipid nanoparticles for mRNA delivery (LNP) have been used in the pharmaceutical industry as vectors for the delivery of a variety of therapeutics. Here, BOC Sciences specializes in providing you with fully validated LNP delivery systems for nucleic acids, especially siRNA and mRNA, to induce functional protein expression and to study the potential of mRNA and LNP-based gene therapy and to facilitate the development of mRNA vaccines.

What are Lipid Nanoparticles?

Lipid nanoparticles (LNPs) are nano-sized lipid-based carriers designed to encapsulate and deliver various therapeutic cargoes, including nucleic acids, proteins, and small molecules. These nanoparticles typically consist of a lipid bilayer surrounding an aqueous core, providing stability and protection to the cargo molecules. The choice of lipid composition, size, and surface modifications can be tailored to optimize delivery efficiency and minimize immunogenicity.

Composition and Structure of LNP

• Lipid Bilayer: This is the outer shell of the nanoparticle and is mainly responsible for encapsulating the cargo molecules. These generally consist of phospholipids, cholesterol, and ionizable lipids.
• Aqueous Core: An inner compartment that solubilizes water-soluble cargo molecules, e.g. nucleic acids.
• Surface Modifications: Surface-functionalized lipids, such as PEGylated lipids, can be added to improve the stability and in vivo circulation half-life.

Lipid Nanoparticles for Drug Delivery

Lipid nanoparticles (LNPs) represent a versatile and effective platform for drug delivery, offering several advantages over traditional delivery systems. LNPs are composed of a lipid bilayer surrounding an aqueous core, creating a nano-sized vesicle capable of encapsulating a wide range of therapeutic agents, including small molecules, nucleic acids, and proteins.

What is mRNA LNP?

The LNP mRNA delivery system works by encapsulating the viral mRNA in lipid nanoparticles that function as transport vehicles to carry the mRNA to the target cells. Its encapsulation protects the mRNA from degradative mechanisms and allows uptake into cells for translation to functional proteins. The discovery of LNP mRNA technology has transformed the state of the art of gene therapy and vaccination. This is one of the key benefits of LNP mRNA technology, because it can transiently express a protein in the target cell without integrating into the host genome. Thus LNP-mRNA therapies have an inherent safety measure with respect to traditional gene therapy approaches, which typically involve irreversible modification of the host genome. In addition, LNP mRNA vaccines have become a formidable weapon against infections, including the current COVID-19 challenge.

BOC Sciences' mRNA LNP Delivery Service

As a leading provider of pharmaceutical research services, BOC Sciences offers comprehensive LNP mRNA delivery services tailored to meet the specific needs of our clients. Our expertise in nanoparticle formulation and mRNA chemistry enables us to design custom LNP formulations optimized for stability, efficacy, and safety.

Figure 1. Schematic diagram of the synthesis of GalNAc-siRNA conjugates. (L, Zhang.; et al, 2022)

Service Process

Design and Synthesis of mRNA by IVT

In vitro transcription (IVT) of mRNA for therapeutic or research applications requires a series of careful steps in the design and synthesis of the mRNA to generate mRNA molecules of high quality. The DNA-dependent RNA polymerase enzyme (for example T7, T3, or SP6 RNA polymerase) then is used to transcribe a DNA template that encodes a desired mRNA sequence to RNA. BOC Sciences adopts the in vitro transcription technology platform for the custom synthesis of mRNA, which means providing one-stop services from design and optimization of the sequence, to chemical modification, purification, and quality control.

* Of course, we also provide more customization of other loadable nucleic acid types besides mRNA as follows.

LNP Formulation

BOC Sciences has a full range of customized LNP formulation services for the pharmaceutical and biotechnology industries. Leveraging our experience in lipid chemistry, nanotechnology, and drug delivery, we offer tailored solutions for the formulation, process development, optimization, and scale-up of lipid nanoparticles (LNPs), designed to deliver a variety of nucleic acid-based therapeutics (e.g., mRNA, siRNA, and DNA). Our skilled team of scientists works directly with clients to develop tailor-made LNP formulations that are tailored to their specific therapeutic payloads and target applications. In this process, we anticipate that by selecting lipid components and adjusting formulation parameters, supplemented by feasibility tests, we would be able to develop LNP formulations that have improved stability, biocompatibility and delivery efficiency. Key highlights of our LNP formulation services are: custom formulation design, formulation optimization, scale-up and manufacturing, etc.

* A portion of the LNP formulation options for mRNA delivery available at BOC Sciences.

mRNA LNP Preparation and Encapsulation

Utilising cutting edge technologies and analytical approaches, we are able to develop highly optimised LNP formulations for highest encapsulation efficiency, loading capacity and controlled release of the nucleic acids. By careful titration of lipid composition, weighted average particle sizes, and physicochemical properties, we determine the parameters providing the best therapeutic effect.

Analytical Characterization

We provide comprehensive analytical characterization services to assess the quality, stability, and performance of LNP formulations throughout the development process. Our analytical capabilities include particle size analysis, zeta potential measurement, encapsulation efficiency determination, drug release kinetics profiling, and stability studies under various storage conditions.

Other Optional mRNA LNP Services

• mRNA-LNP Amplification
• In Vivo mRNA-LNP Validation LNP
• Antibody Production LNP
• Immune Cell Engineering

Advantages of LNP for mRNA Delivery

Enhanced Stability

LNPs shield mRNA molecules from extracellular nucleases, preventing their degradation, thus resulting in increased stability and a longer circulation time in the body.

Improved Cellular Uptake

The cationic or ionizable nature of LNPs results in the electrostatic interaction between the LNPs and the negatively charged cell membranes, which leads to the uptake of the lipids and encapsulated mRNA payloads by the cells. This is an important feature, particularly for correct and effective targeting of cells.

Facilitated Intracellular Delivery

LNPs are necessary for the optimal delivery of mRNA through a range of cellular passages, such as the cell membrane or endosomal compartments, allowing it to reach the cytoplasm, where the translation of an mRNA encoding a protein ultimately takes place.

Targeted Delivery

Advancements include the property based engineered LNPs and surface modification or ligand incorporation to target the desirable cells or tissues. This could result in very accurate delivery of mRNA therapeutics to specific locations, which could reduce off-target effects.

Scalability and Manufacturability

The ease of synthesis of LNPs and the fact they can be scaleable for large-scale production make them attractive for commercialization of mRNA therapeutics.

Low Immunogenicity

Being low-immunogenic, LNPs minimize the risk of eliciting undesired immunological reactions upon in vivo administration, thus conferring the mRNA delivery platforms a safer profile.

Applications of LNP-mRNA Delivery

• Vaccine Development
• In Vivo Antibody Delivery
• Chimeric Antigen Receptor (CAR) T/NK Generation
• Protein Replacement Therapy
• Immune Cell Engineering: Macrophages, T Cells, NK Cells, B Cells, Dendritic Cells, Gamma Delta T Cells

Case Study

Case Study 1 Tailored mRNA-LNP Vaccines for Enhanced Antitumor Immunity.

Summary of experiments investigating the relationship between nanoparticle compositions and their T cell stimulatory capacity. (B, Sanne.; et al, 2022)

mRNA-LNP vaccines have shown remarkable efficacy against SARS-CoV-2 due to their ability to induce potent antibody responses. However, for effective cancer vaccines, robust CD8 T cell responses are essential. Achieving this requires systemic immunization and optimized LNP compositions to engage antigen-presenting cells. This case focuses on the potent impact of systemically immunized mRNA-LNP vaccines on tumor immunity. Through optimization of LNP compositions, we achieved high-magnitude tumor-specific CD8 T cell responses within a single round of optimization. Enhanced mRNA uptake by various splenic immune cell populations was observed with optimized LNP compositions, highlighting the importance of type I interferon and phagocytes in T cell response. Surprisingly, researchers discovered a role for B cells in stimulating the vaccine-elicited CD8 T cell response. Ultimately, this findings pave the way for tailored mRNA-LNP vaccines for further clinical assessment in antitumor immunotherapy.

FAQ

1. Is RNA a lipid?

No, RNA is not a lipid. RNA (ribonucleic acid) is a nucleic acid, not a lipid. Lipids are a group of chemicals that include fats and fatty acids. RNA plays an important role in the transmission of genetic information and protein synthesis in cells.

2. What is solid lipid nanoparticles?

Solid Lipid Nanoparticles (SLNs) belong to a nanotechnology family that is based on colloidal carrier for drug delivery and comprise typically solid lipids (e.g. These nanoparticles are manufactured by using a solid lipid matrix with an average diameter ranging from 10 to 100 nm. From a drug delivery point of view, SLNs can be utilized as relevant drug delivery carriers able to ameliorate the bioavailability and therapeutic effects of a drug while reducing drug toxicity and side effects. They can change their surface properties for controlled drug release and targeted delivery as well.

3. Can lipid nanoparticles cross the blood brain barrier?

Yes, it is possible to design lipid nanoparticles so that they can navigate the blood-brain barrier. The blood-brain barrier is an incredibly selective barrier that generally keeps most drugs from entering brain tissue from the blood. However, the surface characteristics, size of lipid nanoparticles, and then with some special ligands or functional group addition they are enabled to cross the blood-brain barrier. The enhancement, according to the researchers, can be used to deliver drug-containing lipid nanoparticles in a brain-directed manner and consequently, open up a possibility to cure numerous neurological disorders.

4. How are lipid nanoparticles made?

Pharmaceutical lipid nanoparticles are generally prepared by using selection of the material, dissolving, emulsifying, solidification and stabilization. First, a lipid is chosen, and dissolved in an organic solvent, the solution of the lipid is then injected into the water phase, forming tiny droplets; finally, the mixture is exposed under cooling to slightly harden the lipid or added with a cross-linker, leading the lipid to form a solid core, and a surfactant or polymer to stabilize followed by the surface of the nanoparticles(shaderaps). The procedure results in self-assembled lipid nanoparticles which are suitable for application including drug delivery.

5. What is the principle of mRNA/LNP delivery?

In condensation process, cationic lipids can electrostatically complex with negatively charged mRNA molecules to form complexes, which can enhance the stability of mRNA molecules before cell entry. The cationic phospholipid present in the mRNA/LNP drives the fusion of the membrane where the mRNA comes into contact with, the negatively charged membrane with which it is in contact, destabilizing it and increasing the permeability for the entrance of mRNA molecules. Following endocytosis into the cell, as acidification of lysosomes containing all sorts of hydrolytic enzymes starts to degrade both exogenous as well as foreign macromolecules, a pH reduction appears generating an acidic microenvironment that protonates ionizable lipids disturbing the bilayer organization within the LNP, allowing release of the mRNA for binding to ribosomes reading the dogma of life and translating the viral proteins, i.e. antibodies neutralizing the virus.

6. What are the composition of mRNA LNP?

In addition to negatively charged mRNA, Lipid nanoparticles (LNP) loaded with mRNA have four other components: ionizable cationic phospholipids, neutral auxiliary phospholipids, cholesterol, and polyethylene glycol modification PEGylated lipid). Here are the proportion of LNP components.

(1) Neutral auxiliary phospholipids, generally saturated phospholipids, increase the phase transition temperature of cationic liposomes, support the formation of lamellar lipid bilayers and stabilize their structural arrangement.

(2) Cholesterol has strong membrane fusion properties and promotes intracellular uptake and cytoplasmic entry of mRNA.

(3) PEGylated phospholipids are located on the surface of the lipid nanoparticles, improving their hydrophilicity, avoiding rapid clearance by the immune system, preventing particle aggregation and increasing stability.

(4) The most critical excipient is the ionizable cationic phospholipid, which is a decisive factor in mRNA delivery and transfection efficiency.

Reference

1. B, Sanne.; et al. mRNA-LNP vaccines tuned for systemic immunization induce strong antitumor immunity by engaging splenic immune cells. Molecular Therapy. 2022, 30(9), 3078 - 3094.

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