RNA vaccines are a class of nucleic acid vaccines that encode the antigen proteins with mRNA technology. The main ingredient is an mRNA molecule with the gene sequence of a selected antigen protein. In general, the length is 1–15 kb and the molecular weight is about 104~106 Dalton. It has a negative charge. The natural intracellular half-life is about 7 hours. RNA vaccines do not contain whole pathogens or protein antigens in the formulation, but a "blueprint" that provides human cells with instructions to produce the target antigen protein. Presently, the RNA vaccines are divided into two categories: non-replicating mRNA and self-amplifying mRNA. The former has a simple structure and only retains the components essential for the expression of the antigen. The latter contains a replicase gene, which can replicate in cells to achieve higher antigen expression with a lower dose.
RNA vaccines need a series of exact steps to complete the activation of the immune system. After entering the target tissue, the vaccine is absorbed by cells through endocytosis to form endosomes. The mRNA needs to be released from the low pH endosomal environment to the cytoplasm. After release, the mRNA is recognized by the ribosome in the cytoplasm and translated into the protein encoded by the mRNA. The expressed antigen protein is recognized by the immune system in two ways to achieve activation. On the one hand, proteasomes cleave some antigen proteins into short peptides of 8–11 amino acids. The peptides are connected to major histocompatibility complex class I (MHC I) and presented on the cell surface. They are recognized by the cytotoxic T cell receptor to activate cytotoxic T cells, thereby establishing cellular immunity. On the other hand, some antigen proteins are secreted into the extracellular space in their entirety, where they can be directly recognized by B cells. This leads to B cell differentiation into plasma cells that secrete neutralizing antibodies and activation of helper T cells to establish humoral immunity. The mechanism above also reflects the advantages of RNA vaccines over traditional vaccines. Antigen proteins in RNA vaccines can activate both T cell-dependent and T cell-independent pathways to stimulate the immune system to produce neutralizing antibodies and cytotoxic T cells. As a result, RNA vaccines can establish humoral and cellular immunity at the same time, which can enhance the efficacy and reduce the escape of the pathogen.
RNA vaccine technology has several advantages:
Fast: Once the target antigen gene sequence is available, the design and synthesis of an mRNA vaccine take only a few weeks. This is one of its greatest strengths against emerging infectious diseases.
Fast production: The production of mRNA is performed in cell-free systems through enzymatic reactions. It is not dependent on cell cultures, which can significantly shorten the production cycle.
Safety: mRNA can only act in the cytoplasm and has no nucleophilic activity in the nucleus and no integration into the genome. The vaccine formulation does not contain any infectious pathogens and the risk of incomplete attenuation. In addition, the mRNA itself has a Toll-like receptor recognition, providing an adjuvant effect by itself.
Flexible: It is possible to change the antigen to be encoded quickly with the same system for different targets. The mRNA can also be chemically modified. For example, by substituting uridine with N1-methylpseudouridine, it is possible to reduce the immunogenicity of mRNA, increase its half-life and translation efficiency. This extreme modularity of the RNA vaccine system can be used to develop individualized medicine and therapeutic vaccines.
Fig.1 Diagram showing LNPs acting as bodyguards protecting RNA from enzymes (BOC Sciences Original).
Lipid nanoparticles (LNPs) are a delivery system for RNA vaccines. In which four basic components are included:
Ionizable lipids: They are the key functional component in LNPs. Ionizable lipids are near-neutral at physiological pH (~7.4) in order to minimize nonspecific interactions and toxicity and become positively charged at lower pH values (pH <6.0) in order to facilitate mRNA binding upon formulation and endosomal escape after uptake. The cationic lipid binds mRNA through electrostatic interactions.
Cholesterol: Aids in structure stabilization, modulates membrane fluidity and elasticity, and suppresses particle breakdown during storage and transport.
Phospholipids such as DSPC (distearoyl-phosphatidylcholine): Serve as a structural backbone, contribute to particle rigidity, and maintain particle morphology.
PEGylated lipids (PEG-Lipids): Used to modify the particle surface and prevent aggregation, prolong circulation time and decrease immunogenicity. The PEG chain length is usually 2000 Da. It is also important to control the amount of PEG-lipid with fine-tuning, as it has a significant impact on stability and delivery efficacy.
The four components self-assemble by microfluidics or ethanol injection into spherical particles 50–150 nm in diameter. They form an aqueous core with encapsulated mRNA at >90% efficiency.
Naked mRNA is extremely unstable and degradable outside of cells. RNases, which are ubiquitous enzymes in all organisms, can rapidly degrade unprotected RNA molecules. Physical protection against the external environment provided by LNPs is important in stabilizing and protecting mRNA. It has been demonstrated in preclinical studies that mRNA encapsulated by LNPs can be stable for as long as six months at 4℃ while free mRNA is degraded in hours. Particle size is a major factor affecting stability. LNPs in the range of 80–100 nm were found to be the most stable at different temperatures. After six months of storage at 4℃, the average mRNA integrity loss in 120–150 nm particles is 25–30%, while for 80–100 nm particles it is only ~15%. The smaller particles have a more compact structure with a reduced core-shell interfacial area, which limits exposure to water and results in a slower rate of hydrolytic degradation. Storage at frozen temperatures (-20℃) can also extend the shelf life of LNPs. For 80–100 nm LNPs, 95% of original bioactivity can be maintained for up to three months with only a slight decrease in activity over six months, while larger particles show a significant reduction in activity. LNPs prevent nonspecific interactions of encapsulated mRNA with other biomolecules that could affect its structure and function prior to administration.
Delivery using LNPs starts with recognition of the particle by cell surface and uptake, which is achieved mainly by clathrin-mediated endocytosis that forms early endosomes. As endosomes mature, the pH gradually decreases from 6.0–6.5 to 5.0–5.5, and this triggers a critical change in the functional properties of LNPs. Endosomal escape of mRNA determines the efficiency of delivery and is the rate-limiting step for most nanoparticle carriers. As endosomes acidify, the ionizable lipids become protonated and form positively charged species that can electrostatically interact with the anionic endosomal lipids. As a result, the membrane becomes destabilized due to the disruption of lipid packing. Endosomal escape may be caused by a number of different, but not mutually exclusive, mechanisms, including the "proton sponge effect" and the lipid phase transition. The former is the process by which the protonated lipids absorb H+ ions, leading to osmotic swelling and rupture of the endosomal membrane. In the latter, protonated lipids can trigger the conversion of the surrounding lipid bilayers into inverted hexagonal (HII) structures. These HII structures form transient hydrophilic channels through the endosomal membrane, which allow the release of the encapsulated mRNA into the cytoplasm. The process of endosomal escape is inefficient with only 2–3% of mRNA actually reaching the cytoplasm. Surface modifications of LNPs with PEG-lipids or ligands that target specific receptors on the cell membrane can redirect LNPs to certain cell types such as dendritic cells, thus enhancing uptake. The route of administration can also affect how LNPs are distributed in the body, which impacts uptake. Intramuscular injection allows the LNPs to be taken up by the antigen-presenting cells in the muscle tissue, which triggers a strong local immune response.
BOC Sciences provides customizable lipid nanoparticle formulations designed to enhance stability, delivery efficiency, and translational scalability for RNA vaccine applications.
The success of LNP technology in infectious disease prevention and control is well-established. COVID-19 RNA vaccines exemplify the platform's rapid response to emerging infectious diseases. Several mRNA vaccines were developed within months after receiving the viral genome sequence, made possible by the adaptability of the LNP delivery platform. By using four-component LNPs to deliver modified mRNA encoding the spike protein into human antigen-presenting cells, these vaccines induce strong neutralizing antibody titers and robust T cell activation. RNA vaccine technology is also being rapidly extended to other infectious disease targets. Multivalent mRNA influenza vaccines, which encode hemagglutinin proteins from multiple virus strains, aim to address the mismatch problem in traditional vaccines. RSV RNA vaccines deliver fusion protein mRNA within LNPs to induce long-lasting protective immunity in animal models. Zika virus, Ebola virus, and cytomegalovirus are other infectious diseases that have active RNA vaccine development. LNP-based vaccines are highly flexible for iterative design. Upon the emergence of a new variant, only the mRNA sequence needs to be modified while the LNP formulation can remain unchanged, greatly reducing vaccine iteration time. This plug-and-play nature of LNPs for RNA vaccines makes it a strategic platform for future pandemic preparedness.
Table 1. Key Infectious Disease RNA Vaccine Development Status.
| Disease Target | Antigen | LNP Formulation Features | Development Stage | Technical Advantage |
| COVID-19 | Spike protein | SM-102 or ALC-0315 ionizable lipids | Authorized use | Rapid development, high protection |
| Seasonal influenza | Multivalent hemagglutinin | Standard four-component LNP | Preclinical to Phase II | Multi-strain coverage, reduced mismatch |
| RSV | Fusion protein F | Optimized PEG-lipid ratio | Phase II | Induces neutralizing antibodies |
| Zika virus | PrM and envelope proteins | Spleen-targeted LNP modifications | Preclinical | Potential transplacental protection |
| Cytomegalovirus | Pentameric complex | Enhanced endosomal escape design | Phase I | Induces potent neutralizing antibodies |
Personalized mRNA vaccines represent a new paradigm in precision medicine. The fundamental concept is to target patient-specific tumor neoantigens for cancer immunotherapy. Tumor tissue samples are subjected to both whole-exome sequencing and RNA sequencing. Mutations unique to tumor cells are identified and used to predict immunogenic epitopes with bioinformatic algorithms. Tandem mRNA sequences encoding predicted neoantigens are then encapsulated into LNPs. The design of LNPs for cancer vaccines requires certain fine-tuning. As the key cellular targets are dendritic cells, LNPs need to be optimized for delivery and antigen presentation in these cells to initiate anti-tumor T cell response. Tuning surface charge and PEG density can promote lymph node accumulation and efficient dendritic cell uptake of LNPs. LNPs can also be designed to co-deliver adjuvant molecules, such as TLR agonists, for more potent antigen presentation and immune stimulation. LNPs that co-deliver neoantigen mRNA and adjuvants show strong inhibition of tumor growth and improved survival in preclinical melanoma models. LNPs can also be used for in situ CAR-T cell generation. In traditional CAR-T cell therapy, patient-derived T cells are extracted, then modified ex vivo to express CARs on their surface. These cells are expanded to large numbers and reinfused back to the patient. This process is highly laborious and expensive. LNPs can deliver CAR-encoding mRNA directly to T cells in vivo, leading to in situ generation of CAR-T cells. Antibody-modified LNPs can be used to selectively target T lymphocytes, enabling precise in situ reprogramming and simplifying the procedure, with promising potential for solid tumor therapy. The manufacturing time of personalized vaccines, from tumor sampling to LNP product generation, can be completed within 4–6 weeks. This meets the treatment time window for most patients. Standardized LNP production along with sequence-specific mRNA design has created a highly efficient and flexible platform for personalized vaccines.
The applications of RNA vaccine technology are also expanding to various disease areas beyond infectious diseases, including the following:
Cancer Immunotherapy: In addition to personalized neoantigen vaccines, shared antigen vaccines that target commonly overexpressed tumor antigens, such as carcinoembryonic antigen, can be developed without individualized sequencing. LNPs can also be used to deliver mRNA to lymph nodes enriched in antigen-presenting cells, inducing systemic anti-tumor immunity.
Protein Replacement Therapy: LNPs can be loaded with mRNA encoding functional proteins for transient supplementation in genetic diseases. VEGF mRNA delivery to fibroblasts can be used to induce angiogenesis, and CFTR mRNA delivery to lung epithelial cells to restore the function of cystic fibrosis transmembrane conductance regulator ion channels in cystic fibrosis. Organ-specific targeting of LNPs is crucial to avoid undesired systemic effects.
Gene Editing Support: LNPs can be designed to co-deliver mRNA encoding base editors or CRISPR-Cas systems with guide RNAs for precise gene modification in vivo. A number of studies have reported successful in vivo gene correction of pathogenic mutations in mouse models with LNP-mediated mRNA delivery.
Adjuvant Development: LNPs can also serve as carriers for adjuvant molecules, co-delivering immunomodulatory proteins such as STING or TLR agonists along with antigen mRNA to create self-adjuvanting vaccines, thereby reducing or even obviating the need for separate adjuvants.
Table 2. Functional LNPs for Drug Delivery at BOC Sciences.
| Product | Description | Price |
| Vaccine Delivery LNP | Designed for efficient delivery of mRNA or RNA vaccines, optimizing endosomal escape and antigen presentation to enhance immune responses. | Inquiry |
| Liver-Targeted LNP | Engineered with lipid composition or surface modifications to accumulate specifically in the liver, enabling gene therapy, protein replacement, or vaccine delivery. | Inquiry |
| Inhalable LNP | Formulated for pulmonary delivery via aerosol or inhalation, suitable for respiratory diseases or localized gene therapy. | Inquiry |
| Tumor Immunotherapy LNP | Developed for cancer mRNA vaccines or tumor immune modulation, targeting dendritic cells or lymph nodes to improve anti-tumor immunity. | Inquiry |
| Stimuli-Responsive Smart LNP | Responsive to pH, light, redox, or other environmental triggers, enabling spatiotemporally controlled mRNA release and improving delivery precision and efficiency. | Inquiry |
LNPs have been continuously optimized since their development. The first-generation ionizable lipids (DLin-MC3-DMA, MC3) enabled the first siRNA in 2018 to enter the clinic. MC3 with a pKa of ~6.44, is able to ionize in early endosomes for endosomal escape. However, further improvements in biodegradability and toxicity were required. The second-generation lipids were optimized to produce COVID-19 vaccines, SM-102 and ALC-0315. These lipids have improved ester linkage for higher biodegradability, more positive charges for higher delivery efficiency and lower cytotoxicity. Optimization of the four-component ratios remains critical. Cholesterol affects particle rigidity and stability. DSPC controls particle morphology. The PEG-lipid content needs to be optimized to balance particle stability and cellular uptake. A PEG molar fraction between 1.5 and 2.5% yields optimal in vivo behavior. Size control has been optimized. Microfluidic mixing enables precise size adjustment to 80–100 nm, balancing lymph node accumulation and cellular uptake. Particles smaller than 50 nm are less efficient in encapsulation, while those over 150 nm are rapidly cleared by the reticuloendothelial system. Microfluidics allow precise size control with polydispersity indices below 0.1. Storage stability has also improved through optimized freezing conditions and buffer systems, allowing long-term storage at -20 ℃ or -80 ℃. Lyophilization and novel buffers such as Tris-HCl are being developed for cold-chain-independent vaccines.
Table 3. Evolution of Ionizable Lipids in LNPs.
| Lipid | Generation | Key Feature | Example Application | Improvement Focus |
| DLin-MC3-DMA | 1st | pKa 6.44, efficient delivery | siRNA drug Onpattro | Baseline reference |
| SM-102 | 2nd | Improved ester structure, biodegradable | Moderna COVID-19 | Reduced toxicity, improved safety |
| ALC-0315 | 2nd | Optimized alkyl chain | Pfizer-BioNTech COVID-19 | Enhanced delivery efficiency |
| LP01 series | 3rd | Biodegradable groups | Preclinical | Improved biocompatibility |
| OF-02 | 4th | Targeting modifications | Experimental | Organ-specific delivery |
Targeted and organ-specific delivery are active research areas. Traditional LNPs accumulate mostly in the liver. Incorporation of SORT (Selective Organ Targeting) lipids allows organ-specific delivery to lungs or spleen, for example. By modifying the lipid charge, LNPs can be targeted to the lungs (permanent cationic lipid) or spleen (anionic lipid). Spleen-targeted LNPs can be used to efficiently deliver mRNA to immune cells, activating B and T cells for vaccine approaches. Modification of ligands on LNPs allows cell-specific targeting. Antibody or small-molecule ligands can be conjugated to LNPs to recognize specific cell surface markers. For example, anti-CD5-modified LNPs target T lymphocytes for in situ generation of CAR-T cells, anti-CD11c to target LNPs to dendritic cells, and anti-CD19 for B-cell targeting. These active targeting approaches can improve mRNA uptake and reduce off-target effects. The route of administration also has an impact on biodistribution. Intramuscular injection leads to a preference for local uptake in lymph nodes. Intradermal administration can target LNPs to Langerhans cells in the skin, intravenous administration enables systemic distribution, while inhalation can be used to achieve lung-specific delivery for lung-targeted respiratory disease.
Table 4. Organ-Specific LNP Delivery Strategies.
| Target Organ | Modification | LNP Surface Feature | Applicable Indication | Delivery Efficiency Gain |
| Liver | Standard four-component | Neutral to weakly negative | Metabolic disorders | Baseline |
| Lung | Permanent cationic lipids | Highly positive | Respiratory infections | 3–5× |
| Spleen | Anionic lipids/stearic acid | Enhanced negative | Cancer vaccines | 2–4× |
| T cells | Anti-CD5 antibody | IgG-enriched | In situ CAR-T | 5–8× |
| Dendritic cells | Anti-CD11c antibody | Complement-enriched | Cancer vaccines | 4–6× |
Endosomal Escape: Delivery to the cytoplasm is a bottleneck. Only 2–3% of mRNA reaches the cytoplasm. Strategies include lipids with a higher pKa for earlier protonation, fusogenic lipids that destabilize membranes, and external stimuli (light, ultrasound) to aid escape.
Immune Activation and Balance: LNPs can activate complement, produce PEG antibodies. Second-generation stealth lipids are being developed that should reduce off-target immunogenicity without loss of delivery efficiency.
Protein Corona: Adsorbed serum proteins affect biodistribution. Adjusting surface PEG density and charge or adding specific functional groups or materials can help guide LNP targeting.
Intracellular Degradation: mRNA faces enzymatic degradation by exonucleases. Cap structures, poly(A) tail, nucleotide modifications, and RNAse inhibitors can be used to extend half-life.
Repeated Administration: Repeated use of LNPs may be limited by anti-PEG antibodies. Biodegradable stealth lipids provide alternative strategies.
Table 5. RNA Vaccine Delivery Challenges and Solutions.
| Challenge | Specific Issue | Current Solutions | Emerging Strategies | Expected Improvement |
| Endosomal Escape | 2–3% mRNA release | Optimize pKa, add DOPE | External stimuli | 10–15% release |
| Immunogenicity | Complement activation, PEG antibodies | Develop non-PEG stealth lipids | Targeted modifications | Reduce side effects 30–50% |
| Protein Corona | Non-specific adsorption | Adjust PEG density and charge | Pre-coat specific proteins | 2–3× organ accumulation |
| Intracellular Degradation | Exonuclease activity | 5' cap, poly(A), nucleotide modification | Co-deliver inhibitors | Half-life up to 24 h |
| Repeat Dosing | Anti-PEG antibodies | PEG alternatives | Biodegradable stealth lipids | Enable multiple doses |
Chemical modification of the ionizable lipid structure has been identified as a strategy to help protect RNA. Newer lipids have biodegradable ester linkages that connect the hydrophobic tails, instead of non-degradable ether or amide linkages. The ester linkage would then allow the lipid to be rapidly cleaved by intracellular esterases upon release of the payload, limiting toxicity and accumulation. SM-102 and ALC-0315 have branched hydrophobic tails to better interface with the phospholipid bilayer and increase endosomal escape efficiency. Some studies have further modified lipid structures to incorporate disulfide linkages, leading to selective and tunable degradation of the lipid in high-glutathione environments, enabling a more controlled cytoplasmic release of the mRNA cargo. The ratio of the 4 major lipid components also plays a role in protecting RNA. Cholesterol is increased from the 35–40% previously used to 45–50% to increase transfection efficiency in mouse and non-human primate (NHP) studies by stabilizing the membrane and aiding in fusion with cellular membranes. The amount of phospholipid DSPC is tightly controlled around 10–15% because higher percentages dilute the ionizable lipid fraction and lead to a reduction in endosomal escape, while lower percentages weaken the structural integrity of the particle, leading to deformation of the structure. PEG-lipid is usually kept at 1.5–2.5% molar fraction, which gives rise to a 5–10 nm hydrated surface corona that shields from serum protein adsorption, but too much PEG may impede cellular uptake. Better understanding of the core–shell structure has also enabled formulation improvements. Cryo-electron microscopy (cryo-EM) has demonstrated that mRNA primarily resides in the LNP core in a disordered hydrated state, while DSPC and PEG-lipid are enriched in the outer shell (~2–3 nm). Some ionizable lipid and cholesterol are distributed between the core and shell to better stabilize the particle. This suggests that the mRNA core is not fully segregated from water, making hydrolysis a possible concern for long-term storage. The aqueous fraction of the core can be tuned (e.g., 30% to 20%) to make a significant improvement in mRNA integrity. Co-addition of lyoprotectants, such as trehalose or sucrose, during the freeze-drying process forms a glassy matrix that eliminates ice crystal damage, resulting in a stable solid formulation that is not reliant on cold-chain storage.
Table 6. LNP Development and Functionalization Service Offerings.
| Service Name | Description | Price |
| Lipid Nanoparticle for Vaccine | Formulate lipid nanoparticles as carriers for antigens to improve stability, immune response, and cellular uptake for effective vaccination strategies. | Inquiry |
| Custom LNP Formulation | Tailored design and preparation of LNPs encapsulating specific mRNA sequences for research or therapeutic applications. | Inquiry |
| LNP Targeting Modification | Surface modification of LNPs with ligands or antibodies to achieve organ- or cell-specific delivery. | Inquiry |
| LNP Stability Optimization | Optimization of lipid composition and formulation parameters to enhance mRNA protection and shelf-life. | Inquiry |
| In Vivo LNP Pharmacokinetics | Evaluation of LNP distribution, metabolism, and clearance in animal models to support preclinical studies. | Inquiry |
| LNP Process Development & Transfer | Development, scale-up, and technology transfer of LNP manufacturing processes for research or production. | Inquiry |
Surface charge: LNPs that have a nearly neutral surface charge at physiological pH have low non-specific interactions with biological molecules but suffer from a lack of attraction to the negatively charged cell membrane. Surface potential can be modulated by incorporating a small fraction of permanent cationic lipids, such as DOTAP, to shift the surface charge to +5–+10 mV. This will lead to better interactions with cell surface proteoglycans and endocytosis efficiency that is 2–3 times higher. The surface positive charge is partially shielded by serum proteins during circulation and is recovered when LNPs reach target tissues, which helps to balance the requirements of circulation stability and uptake.
Targeted ligand modification: Addition of an anti-CD11c single-chain antibody fragment to LNPs specifically delivers mRNA to dendritic cells, leading to 4- to 6-fold higher efficiency over non-targeted LNPs. Ligands are conjugated to the PEG-lipid terminus using maleimide-thiol or click chemistry to ensure surface presentation of the ligand. Targeting of other immune cell types has also been demonstrated, including cytotoxic T cells with anti-CD8 antibody modification and macrophages with mannose modification. The ligand surface density should be carefully controlled to 1–3 molecules per LNP, as higher ligand densities result in aggregation and lower densities do not provide sufficient targeting effect.
Stimuli-responsive LNPs: LNPs that are sensitive to pH have been developed to take advantage of the acidic tumor microenvironment (pH 6.5) to promote faster release of mRNA, which increases tissue transfection efficiency by 3- to 5-fold. Redox-responsive LNPs can also be used to provide a rapid release by disassembling in high-glutathione intracellular environments. Light-responsive LNPs that are triggered by localized laser irradiation have been developed to achieve spatial control of mRNA release. These stimuli-responsive systems shift the RNA delivery from passive diffusion to an actively controlled release.
Shape engineering: LNPs that are not perfectly spherical in shape, such as rod-shaped or disk-shaped particles, have unique hydrodynamic behavior that could be better tailored for certain applications. Rod-shaped LNPs (aspect ratio = 3: 1) with similar composition and size as spherical LNPs were observed to have double the accumulation in lymph nodes, attributed to better retention by the extracellular matrix. Disk-shaped LNPs have been shown to increase membrane curvature at the point of contact with the target membrane and promote fusion. By tuning the lipid ratios and processing conditions in a microfluidic system, it is possible to induce non-spherical morphologies and enable shape customization.
