During the COVID-19 pandemic, the success of mRNA vaccines has greatly propelled the development of mRNA therapeutics. mRNA is a negatively charged nucleic acid that serves as a template for protein synthesis in ribosomes. Despite its utility, the instability of mRNA necessitates appropriate carriers for in vivo delivery. Currently, lipid nanoparticles (LNPs) are the most mature approach for protecting mRNA from degradation and enhancing its intracellular delivery. To further optimize the therapeutic efficacy of mRNA, researchers have developed a series of site-specific LNPs. Through local or systemic administration, these site-specific LNPs can accumulate in specific organs, tissues, or cells, allowing mRNA to be delivered to specific cells and enabling local or systemic therapeutic effects. These methods not only improve the efficiency of mRNA therapy but also reduce off-target adverse reactions.
LNPs are composed of different lipids, making quality control easier compared to other types of carriers such as macromolecules or viruses. Additionally, it is easy to develop new LNPs by altering lipid structures or compositions, increasing the versatility of LNPs. Typically, LNPs consist of four types of lipids, including ionizable lipids, helper lipids, cholesterol, and PEG lipids. Ionizable lipids are the most crucial component, responsible for the encapsulation of nucleic acids during formulation and endosomal escape after cellular uptake. The head groups of ionizable lipids carry a positive charge under acidic conditions, allowing for electrostatic interactions with negatively charged nucleic acids and enhancing encapsulation efficiency. The hydrophobic alkyl chains of ionizable lipids are unsaturated, forming a hexagonal lipid phase and enhancing the escape of mRNA from the LNP after cell entry. Apart from ionizable lipids, the other three components are also essential for LNPs. Helper lipids and cholesterol increase LNP stability and enhance cellular internalization. PEGylated lipids are crucial for improving LNP stability and prolonging circulation time, enhancing delivery efficiency after intravenous injection.
LNP for Localized Delivery of Specific Locations
Localized administration of LNP to specific sites is crucial for the delivery of mRNA. Various administration routes have been used to achieve site-specific delivery of LNP, including oral administration, inhalation, and local injection (intramuscular, intratumoral, and intracerebral injection).
1) Oral administration is a widely used, convenient, and well-established route of administration. However, it poses significant challenges for mRNA delivery due to the susceptibility of mRNA molecules to nucleases and the harsh acidic environment of the gastrointestinal tract. In this regard, researchers at the Georgia State University Center for Diagnostics and Therapeutics prepared LNPs loaded with IL-22 encoding mRNA for oral administration. The LNP consisted of phosphatidic acid, monoolein, and dioleoyltrimethylammonium propane. In a mouse model, oral administration of IL-22 mRNA-loaded LNP significantly increased IL-22 expression in the colonic mucosa and accelerated the healing of colitis. These results suggest that oral delivery of mRNA-loaded LNP is a viable strategy for treating gastrointestinal diseases by reestablishing the intestinal microenvironment.
2) Inhalation is also a preferred route of administration. Due to its large absorptive surface area and abundant pulmonary blood flow, inhaled drugs can rapidly transfer to the bloodstream, thereby increasing their bioavailability. However, achieving precise dosing through inhalation is challenging, and clearance in the airways adds to the difficulty. Inhaled aerosols undergo two types of clearance based on their size and deposition region. Aerosols larger than 5 μm are cleared by mucociliary clearance, resulting in the clearance of over 80% of the aerosols. In contrast, aerosols smaller than 5 μm are cleared by macrophages. Nebulization is the most common method of inhalation. Although advanced nebulization techniques can facilitate drug delivery to the lungs, shear forces may disrupt the structure of nanoparticles, and physical barriers in the airways can hinder their reach to the target. To address these issues, researchers at the Georgia Institute of Technology's Department of Biomedical Engineering reported a screening approach to identify optimal LNP components for mRNA delivery via nebulization. The results showed that a higher molar ratio of PEG lipids in LNPs improves the performance of cationic assist lipids. They prepared an mRNA-loaded LNP for pulmonary delivery, composed of a modified PEI compound 7C1, cholesterol, DMG-PEG 2000, and cationic lipid DOTAP. A high percentage (55%) of DMG-PEG2000 enhanced pulmonary delivery of the LNP. Subsequent studies found that this LNP, when loaded with mRNA encoding a broadly neutralizing antibody against coagulase, could protect mice from H1N1 influenza infection.
3) Local injection refers to the administration of drugs to a small area of the body. These drugs can affect not only the local site but also diffuse or transfer to the bloodstream, exerting systemic therapeutic effects. The COVID-19 vaccine is a typical example of a muscle injection that works systemically after administration. Currently, more mRNA-based research is focusing on local injections, which can provide targeted therapy at the injection site while minimizing the potential for off-target effects. Researchers at Tel Aviv University's Laboratory of Precision NanoMedicine developed an LNP-based CRISPR-Cas9 mRNA delivery system for the treatment of Duchenne Muscular Dystrophy (DMD) caused by functional loss-of-function mutations in the dystrophin gene. The CRISPR-Cas9 system can be used to restore dystrophin protein expression and has a persistent effect, requiring an appropriate delivery vehicle to deliver Cas9 mRNA and sgRNA to target cells. The researchers synthesized ionizable lipids with a triple hydrophobic alkyl tail and used them as components for formulating LNP vesicles to deliver Cas9 mRNA and specific sgRNA. This LNP showed good local therapeutic effects after intramuscular injection, while systemic therapeutic effects were observed after limb perfusion in a DMD mouse model, representing a promising carrier for delivering CRISPR-Cas9 gene editing tools.
Organ-Specific LNP for Administration via Vein
Intravenous injection is another standard route of administration, with a bioavailability of 100%. The bio-distribution of LNPs after intravenous injection is crucial, as off-target delivery of mRNA can lead to adverse reactions and greatly reduce therapeutic efficacy. In recent years, a lot of research has been focused on organ-specific LNPs.
1) Liver targeting: Commercially available LNPs carrying Onpattro siRNA primarily accumulate in the liver after intravenous injection. The targeting mechanism is achieved by using a 14-C lipid with DMG-PEG2000 in the LNP formulation. Due to the short 14-C chain and weak binding to the LNP surface, this lipid quickly dissociates from the LNP surface in circulation. Subsequently, apolipoprotein E (ApoE) binds with the LNP to form a corona, which is recognized by low-density lipoprotein receptors on liver cells and promotes LNP internalization. Many studies have followed the prescription of Onpattro to prepare liver-targeted LNPs. These mRNA-loaded LNPs deliver specific mRNA to the liver for the treatment of various liver-related diseases, including infectious diseases, liver fibrosis, cancer, and genetic disorders.
2) Spleen or lung targeting LNP: Researchers at the University of Texas Southwestern Medical Center reported that selective organ targeting capability can be achieved by altering the composition and components of LNPs. They named this passive targeting LNP as Selective Organ Targeting (SORT) nanoparticles. By adding a fifth lipid to the existing prescription of traditional four-component LNPs and adjusting the ratio of this lipid, selective targeting to different organs such as the liver, lung, and spleen can be achieved. After in vivo screening, they found that the traditional four-component LNPs mainly accumulate in the liver and partially in the spleen, while the introduction of the fifth lipid changes the distribution, which is dependent on the proportion of the fifth lipid. When using a cationic lipid as the fifth component, the distribution of LNPs in the liver decreases as the proportion of the fifth lipid increases. Regarding the distribution of LNPs in the lung and spleen, when the proportion of the cationic lipid is higher than 50%, more than 90% of the LNPs accumulate in the lung. If an anionic lipid is chosen as the fifth lipid, the amount of LNPs distributed to the liver decreases with an increase in the proportion of the anionic lipid, and there is almost no LNP distribution to the lung. For the distribution of LNPs in the spleen, the maximum distribution in the spleen is achieved when approximately 30% of anionic lipid is used. If the fifth lipid is an ionizable cationic lipid, the distribution of LNPs to the liver increases with an increase in the proportion of the fifth lipid and then decreases. Addition of 20% ionizable cationic lipid achieves the maximum distribution to the liver. In summary, the introduction of the fifth lipid greatly influences the bio-distribution of LNPs in major organs, and 50% cationic SORT lipid, 30% anionic SORT lipid, and 20% ionizable cationic SORT lipid promote the distribution of LNPs in the lung, spleen, and liver, respectively.
3) Bone targeting LNP: In recent years, there has been an increasing incidence of skeletal diseases and abnormalities, and the medical demand for novel biomaterials that can target the bone microenvironment remains unmet. It has been reported that LNPs loaded with siRNA can be systematically delivered to the bone marrow, but passive diffusion still presents a challenge for bone-targeted drug delivery. Inspired by the fact that ligand substitution can achieve targeted LNP delivery, researchers at the University of Pennsylvania's Department of Bioengineering synthesized a series of lipids based on bisphosphonates (BPs). These lipids can strongly bind to calcium ions on hydroxyapatite on the bone surface through chelation and achieve long-term retention in the bone microenvironment. They prepared LNPs loaded with luciferase mRNA using different types of BP lipids and screened them through cell experiments. The results showed that 490BPC14-LNP had the best transfection efficiency. Additionally, bone morphogenetic protein-2 (BMP-2) mRNA was loaded into the LNP, which exhibited excellent bone microenvironment targeting capability and high expression of BMP-2 in bone tissue after intravenous injection.
Cell-Specific LNP for Systemic Delivery
To achieve more specific mRNA delivery systems, cell-targeted LNPs have been developed that are designed to be taken up by specific cells and induce protein expression in these cells. Typically, this cell-specific uptake is mediated by ligand-receptor interactions, but recent research has found that this interaction can also be achieved by altering the lipid structure within the LNP.
1) White blood cell-targeted LNP
In order to achieve cell-specific mRNA therapy for inflammatory bowel disease (IBD), researchers prepared antibody-modified LNP to specifically deliver IL-10 mRNA to Ly6c+ inflammatory white blood cells. Ly6c+ cells serve as targets for treating IBD, and the immune-suppressive cytokine IL-10 has been reported to inhibit IBD. To induce long-term IL-10 production, IL-10 mRNA-loaded LNP was first prepared, followed by incubation with anchored secondary scFv enabling targeting (ASSET) micelles at 4°C for 48 hours and subsequent incubation with anti-Ly6c monoclonal antibody for 30 minutes. ASSET allows LNP to be bridged with targeting antibodies under mild conditions. Upon intravenous injection in mice with dextran sulfate sodium-induced colitis, this surface-modified LNP actively targeted Ly6c+ white blood cells and induced IL-10 production, significantly suppressing inflammation in the colon.
2) T cell-targeted LNP
Cardiac fibrosis is caused by excessive production of extracellular matrix by cardiac fibroblasts. Limiting fibrosis progression through anti-fibrotic therapy has been unsatisfactory for treating cardiac fibrosis. Recently, researchers at the University of Pennsylvania, developed a CAR-T therapy to remodel fibrosis by specifically delivering mRNA to T cells. Fibroblast activation protein cardiac fibroblast activation protein (FAP)-CAR, which recognizes FAP-positive cells and induces cell death, was encoded into mRNA and then loaded into LNP modified with anti-CD5 antibodies to achieve specific targeting of CD5+ T cells. FAP-CAR is expressed on the surface of T cells and specifically recognizes FAP-positive cells, reducing cardiac fibrosis. They also studied the therapeutic efficacy in a mouse model of angiotensin II/phenylephrine-induced cardiac injury. Systemic administration of CD5-targeted LNP loaded with FAP-CAR mRNA significantly reduced fibrosis and improved heart function. This represents a great success of mRNA-loaded LNP in treating heart disease.
3) Kupffer cell and liver sinusoidal endothelial cell (LSEC)-targeted LNP
Kupffer cells play an important role in liver inflammation and immune tolerance, primarily by engulfing and clearing particles. Increasing the size of LNP and modifying the surface with hydrophobic molecules enhances the cellular uptake by Kupffer cells and promotes immune regulation. LSECs are located in liver sinusoids and are responsible for blood filtration, metabolic regulation, antigen presentation, and lipid metabolism. To achieve cell-specific mRNA delivery to Kupffer cells or LSECs, researchers at the Georgia Institute of Technology applied various types of cholesterol in the formulation of LNP and found that the structure of cholesterol greatly affected the targeting ability of LNP.
1. X, Xiao.; et al. Recent Advances in Site-Specific Lipid Nanoparticles for mRNA Delivery. ACS Nanosci. Au. 2023, 3(3): 192-203.
2. W, Q, Li.; et al. Biomimetic Nanoparticles Deliver mRNAs Encoding Costimulatory Receptors And Enhance T Cell Mediated Cancer Immunotherapy. Nature Communications. 2021, 12: 7264.
3. J, G, Rurik.; et al. CAR T Cells Produced in vivo to Treat Cardiac Injury. Science. 2022, 375(6576): 91-96.