mRNA therapeutics is an emerging approach that delivers messenger RNA (mRNA) molecules into human cells, providing genetic instructions for producing specific proteins or antigens. This technology transforms the patient's own cells into "protein factories" capable of synthesizing molecules with therapeutic potential. mRNA therapeutics has demonstrated promise across multiple applications, including infectious disease prevention, cancer treatment, metabolic disorder correction, and intervention in genetic conditions. Naturally occurring mRNA is inherently fragile, easily degraded in the body, and can trigger strong immune responses. Therefore, therapeutic mRNA is often chemically modified to reduce immunogenicity. More importantly, mRNA requires advanced nanotechnology to ensure safe and efficient delivery into target cells. Lipid nanoparticles (LNPs) have emerged as the most mature delivery platform, providing critical technical support for translating mRNA from laboratory research to practical applications.
Lipid nanoparticles function as both a "protective shell" and a "delivery vehicle" for mRNA, performing multiple essential roles. They encapsulate negatively charged mRNA molecules, shielding them from degradation by nucleases in the bloodstream. The size and surface characteristics of LNPs enable circulation in the body and facilitate delivery to target tissues. Additionally, LNPs help mRNA cross cell membranes and escape from endosomal compartments, ultimately releasing the mRNA into the cytoplasm for protein translation.
A typical lipid nanoparticle consists of four key lipid components:
Ionizable cationic lipids: Carry a positive charge in acidic environments to bind mRNA but remain neutral at physiological pH, minimizing toxicity.
Helper lipids (e.g., DSPC, DOPE): Stabilize nanoparticle structure and assist membrane fusion.
Cholesterol: Enhances particle stability and membrane fluidity.
PEGylated lipids: Extend circulation time and reduce immune clearance.
mRNA therapeutics offers several unique benefits over conventional approaches:
Rapid development: mRNA sequences can be quickly designed based on target protein sequences without requiring complex cell culture or protein purification.
High versatility: The same delivery platform can encode completely different proteins by simply altering the mRNA sequence.
Controllable expression: Protein levels and duration can be precisely adjusted by modifying mRNA dose.
Recent innovations in RNA design further enhance mRNA therapeutics:
Self-amplifying RNA (saRNA): Uses viral replicase proteins to generate multiple mRNA copies within cells, achieving the desired effect at lower doses.
Circular RNA (circRNA): Forms covalently closed loops resistant to exonuclease degradation, significantly prolonging half-life and protein expression duration.
These innovations increase both the practicality and durability of mRNA therapeutics.
A key step in the preparation of LNP formulations is the encapsulation of mRNA. The most frequently used approach is rapid mixing, where lipids dissolved in ethanol are mixed with an mRNA solution in acidic buffer using T-junctions or microfluidic chips. Positively charged ionizable lipids can bind to the negatively charged mRNA backbone through electrostatic interactions and pack mRNA into the hydrophobic core of the lipid to form spherical nanoparticles of approximately 80–100 nm in diameter. By fine-tuning the flow rate ratio and total flow rate, rapid mixing also allows for precise control over particle size, homogeneity, and encapsulation efficiency. Homogeneous particles with a polydispersity index of less than 0.1 can be achieved by adjusting flow rates, and encapsulation efficiencies are typically greater than 95% (i.e. the vast majority of mRNA molecules are encapsulated in LNPs). The encapsulated mRNA is stable for hours to days in vitro and is efficiently protected against nuclease degradation in vivo. Functional molecules can be co-encapsulated with mRNA. For instance, co-delivery of the anti-inflammatory drug dexamethasone into the core of LNPs results in a reduced inflammatory response without compromising mRNA expression levels. Small-angle X-ray scattering confirmed successful loading of the drug and sustained release for up to 48 hours.
Table 1. Typical Lipid Nanoparticle Composition and Function.
| Lipid Type | Representative Lipids | Core Function |
| Ionizable cationic lipids | DLin-MC3-DMA, SM-102 | mRNA binding, endosomal escape |
| Helper phospholipids | DSPC, DOPE | Structural stability, membrane fusion |
| Structural lipids | Cholesterol | Particle stability, membrane fluidity |
| PEGylated lipids | PEG2000-DMG | Extended circulation, reduced immune clearance |
Delivery of mRNA to cells by LNPs is typically achieved through endocytosis, in which nanoparticles are rapidly engulfed by the cell membrane upon contact to form endosomes for intracellular transport. Uptake is generally dictated by nanoparticle surface properties, with size, charge, and surface functionalization playing a role in determining the uptake efficiency. A range of surface functionalization strategies have been developed to target uptake to specific cell types. For example, decoration of LNPs with mannose permits uptake by sinusoidal endothelial cells through CD206 receptors. More specific strategies are also possible and often rely on antibodies or antibody fragments tethered to the surface of the nanoparticle to deliver mRNA to target cells.
Example 1: LNPs decorated with anti-CD4 antibody were able to achieve efficient delivery of mRNA into T cells and effect efficient gene editing in the spleen and lymph nodes.
Example 2: Targeting c-kit with LNPs on hematopoietic stem cells in vivo allowed for ~90% gene editing efficiency.
These strategies lay the groundwork for personalized, targeted and precise mRNA therapeutics.
Following cellular uptake, for mRNA to function, it needs to be released from the endosome into the cytoplasm, which is often a key rate-limiting step. As endosomes mature, they undergo gradual acidification, reaching pH 5–6 which protonates the ionizable cationic lipids and imparts a positive charge. The protonated lipids can then bind to negatively charged lipids on the endosomal membrane, and reorganize to form non-lamellar structures like inverted hexagonal arrangements. These structures perturb the membrane and cause endosome rupture and release of mRNA into the cytoplasm. The efficiency of endosomal escape is affected by a number of factors including lipid tail structure, degree of unsaturation, and biodegradable linkages to improve biocompatibility and reduce long-term toxicity.
LNPs have been shown to have intrinsic immunostimulatory properties and can be used to activate the innate immune system. Innate immune activation is often desired and can be used for vaccines but is unwanted for many protein replacement therapies. In mouse models, LNPs are rapidly internalized and induce release of inflammatory cytokines, such as IL-1β, TNF-α, and MCP-1. Cytokine levels can be ameliorated through co-delivery of anti-inflammatory drugs with mRNA, which does not significantly impact protein expression. Biodistribution studies in mice reveal that LNPs accumulate primarily in the liver when delivered intramuscularly and has led to exploration of LNPs as a delivery system for treatment of metabolic disorders affecting the liver. The ribosome of the cell recognizes the 5′ cap and poly(A) tail of mRNA following release into the cytoplasm, and translation is initiated. Cells are then able to produce the encoded protein, antigen, or gene-editing molecules, depending on the cargo. The expression level and duration is dictated by chemical modifications to mRNA, dose, and type of RNA (conventional mRNA typically expresses for days, while circular RNA has been shown to provide sustained protein production for weeks, which provides an option for sustained protein replacement).
Fig.1 Step-by-step process of LNP-mediated mRNA delivery (BOC Sciences Original).
BOC Sciences delivers customizable lipid nanoparticle platforms designed to improve intracellular delivery, expression efficiency, and translational potential for mRNA therapeutics.
LNPs can be immunogenic, based on their composition and physicochemical features. PEGylated lipids may cause the production of anti-PEG antibodies and subsequent accelerated blood clearance, especially with repeated administration. Ionizable cationic lipids are also neutral at physiological pH; however, they can still activate the complement cascade and inflammatory responses upon metabolism, which may cause acute immune responses. Several strategies are being developed to reduce immunogenicity, through the optimization of components and surface modifications:
Biodegradable linkages: Replacing the commonly used ether linkage with an ester linkage results in faster clearance of the lipid in vivo, preventing accumulation and toxicity.
PEG engineering: Cleavable PEG chains maintain long circulation times in the blood, but detach in the tissues, to maintain stability with lower immunogenicity.
Surface biomimicry: Cloaking LNPs with fragments of cell membranes (e.g. red blood cell membranes) can provide natural immune evasion. Biomimetic LNPs were found to have 3–5-fold longer circulation times and over 50% reduction in the release of inflammatory cytokines.
Co-delivery of immunomodulators: The use of encapsulated anti-inflammatory mRNA that can be co-delivered with the therapeutic mRNA allows for the co-expression of both therapeutic proteins and local immune modulators.
Table 2. Strategies to Reduce LNP Immunogenicity.
| Strategy Type | Approach | Advantage | Challenge |
| Lipid chemistry modification | Biodegradable ester linkages | Rapid metabolism, low accumulation | May affect encapsulation efficiency |
| PEG engineering | Cleavable PEG chains | Balance circulation and immunogenicity | Precise control of cleavage kinetics required |
| Surface biomimicry | Cell membrane coatings | Natural immune evasion, biocompatibility | Complex manufacturing, batch variability |
| Immunomodulator co-delivery | Anti-inflammatory mRNA co-encapsulation | Self-regulated immune balance | Optimizing ratio of two mRNAs |
mRNA is not inherently stable. Naked mRNA would be degraded by nucleases in the bloodstream within minutes. Lipid encapsulation can protect mRNA; however, there may be premature leakage of mRNA if LNPs become destabilized during circulation or metabolism. In addition, the liver is the main organ of metabolism and clearance, so most intravenously administered nanoparticles are cleared by the liver, and have uneven biodistribution to tissues and organs.
Chemical modifications can improve mRNA stability:
Uridine can be replaced with N1-methylpseudouridine to avoid immune activation and allow for better translation.
The 5′ cap can be optimized by using an anti-reverse cap analog or enzymatic capping for better recognition by ribosomes.
The length of the poly(A) tail is usually 120–150 nucleotides for optimal stability and translation.
Advanced forms of RNA can further increase stability:
Circular RNA (circRNA) is a covalently closed loop structure that is more resistant to exonuclease degradation. The intracellular half-life of circRNA can be 7–10 days compared to 1–2 days for linear mRNA.
Self-amplifying RNA (saRNA) is engineered to encode multiple copies of itself and can lead to long-lasting protein expression at extremely low doses due to continued amplification.
Biodistribution can also be optimized, through the tuning of particle size and surface charge:
Particles<50 nm="" can="" penetrate="" tissues="" but="" have="" shorter="" circulation="" times.="" particles="">200 nm are readily taken up by the spleen. Particles ~80–100 nm have a good compromise between liver accumulation and tissue penetration.
Neutral or slightly negative particles have longer circulation times; positively charged particles have more efficient uptake, but also higher toxicity.
Accurately delivering mRNA to specific tissues or organs is still a significant hurdle. With intravenous injection, over 70% of LNPs end up in the liver, which has precluded its use for many other organs and tissues. Blood-brain barrier, tumor microenvironment, and immune cell-specific uptake present additional barriers to specific targeting. Strategies to target LNPs to specific tissues include:
Ligand-mediated active targeting: Active targeting of LNPs to specific cell types by modifying the surface with proteins or antibodies. For example, ApoE-modified LNPs can specifically bind to LDL receptors in the liver. LNPs can be targeted to specific immune cells in the spleen with CD68 as the targeting ligand.
Organ-selective targeting: Through surface chemistry tuning, certain organs can be targeted for accumulation preferentially, such as the lungs or spleen. Changing the ratio of auxiliary lipids and ionizable lipids with specific pKa values can be used to target LNPs to lung endothelial cells, even without a targeting ligand.
Local administration: For more accessible tissues, direct injection or inhalation of LNPs can be used to deliver directly to muscle, skin, or lungs.
Physical-assisted targeting: Tissue-specific uptake can also be facilitated through the temporary increase of membrane permeability using methods such as electroporation or focused ultrasound.
Table 3. LNP Tissue Targeting Strategies.
| Strategy | Mechanism | Target Tissue | Typical Application |
| Passive targeting | Enhanced permeability and retention | Tumors | Cancer therapy |
| Ligand-mediated targeting | Antibody/peptide-receptor interaction | Immune cells | Immune modulation |
| Organ-selective targeting | Surface chemistry modulation | Lungs, spleen | Pulmonary or immune diseases |
| Local administration | Injection or inhalation | Muscle, skin, lungs | Vaccines, local protein delivery |
| Physical-assisted targeting | Ultrasound, electroporation | Brain, deep tissues | Neurological therapy |
Personalized medicine relies on tailoring treatment to individual patient profiles. LNPs' modular design makes them ideal for customized mRNA therapies. Microfluidic chip technology enables rapid production of patient-specific mRNA and LNPs within hours, achieving true "on-demand" therapy. Integration of multi-omics data guides LNP design. Transcriptomic, proteomic, and metabolomic profiles identify disease-specific markers and immune characteristics. mRNA sequences, chemical modifications, and lipid formulations can be optimized accordingly—for example, increasing anti-inflammatory components for high-inflammation patients or using more stable circRNA for fast-metabolizing individuals. Smart LNPs represent the next-generation approach. pH-, enzyme-, or redox-responsive nanoparticles can release mRNA selectively in disease microenvironments, such as acidic tumors or oxidative atherosclerotic plaques.
COVID-19 pandemic has seen the first mRNA vaccines being used on a massive scale. LNP-formulated mRNA encoding SARS-CoV-2 spike protein is administered by intramuscular injection, leading to the induction of neutralizing antibodies and cellular immunity against the virus. This platform is poised to be used to respond to future pandemics as well, with a working vaccine potentially being designed within weeks of the release of a viral genome sequence. Furthermore, multivalent mRNA vaccines that can target multiple viral strains are being developed, with promising results being observed for influenza, RSV, and Zika virus. There are also efforts to design universal influenza vaccines based on mRNA that encode conserved antigens.
LNPs for cancer immunotherapy encode tumor-associated or neoantigens to drive immune recognition of tumor cells. Personalized vaccines are created by sequencing tumor tissue to identify mutations that can serve as targets, synthesizing mRNA corresponding to these mutations, and packaging the mRNA in LNPs in a process that can take as little as 4–6 weeks. In early clinical studies, these vaccines are observed to induce strong CD8+ T cell responses and a suppression of tumor growth. Smart LNPs that can respond to specific conditions in the tumor microenvironment have also been designed to improve the local delivery and efficacy of mRNA cancer vaccines.
mRNA therapeutics can also be used to correct deficiencies in enzymes or proteins due to genetic metabolic disorders. This is because this approach allows cells to produce the needed protein of interest for an extended period of time as opposed to traditional gene therapy. For instance, LNPs delivering mRNA that encode phenylalanine hydroxylase restore the enzyme's activity for several weeks in models of phenylketonuria. In the case of cystic fibrosis, aerosolized LNPs can be inhaled by patients and successfully diffuse through airway mucus to deliver mRNA that encode functional CFTR and restore pulmonary function.
In addition to infectious disease vaccines, mRNA therapeutics can also be designed for the treatment of chronic infections or for modulation of the immune system. Therapeutic vaccines based on mRNA are being designed for persistent viral infections that would elicit a strong cellular immune response. Broad-spectrum antiviral approaches are also being designed based on the use of mRNA to drive the production of neutralizing antibodies in vivo. Furthermore, simultaneous delivery of mRNA that encode antiviral proteins along with immunomodulators such as IL-10 can be used to drive pathogen neutralization as well as control of inflammation. This makes mRNA therapeutics a promising treatment for both acute and chronic infections.
LNPs are the delivery systems of choice for mRNA therapeutics, and ongoing improvements and innovations are primarily focused on further optimizing the lipid formulations. The first LNPs were developed using first-generation ionizable lipids, with recent research largely focused on next-generation lipid candidates with improved performance and safety profiles. Next-generation ionizable lipids have been developed with features enabling more efficient intracellular release ("endosomal escape") and reduced in vivo toxicity. Incorporation of biodegradable ester linkages or other chemical cleavable connectors enables the lipids to be rapidly metabolized and cleared following mRNA delivery, greatly reducing the potential for long-term lipid accumulation in the body. Tuning of the lipid pKa allows for precise control of ionization in a specific tissue microenvironment, leading to more efficient intracellular release of mRNA. In addition to next-generation ionizable lipids, other important innovations involve helper lipids and stealth coatings. Formulations have been created incorporating lipids with engineered phase transition properties to further optimize the membrane fusion process, and new polymers to replace PEG for stealth coatings while still providing nanoparticle stability without increasing immunogenicity.
Table 4. LNP Categories for Advanced mRNA Delivery at BOC Sciences.
| Product | Description | Price |
| Biodegradable LNP | Incorporates biodegradable lipids to reduce in vivo accumulation and toxicity, suitable for long-term or repeated administration. | Inquiry |
| Targeted LNP | Surface-modified with antibodies, peptides, or sugars to achieve precise delivery to specific cells or tissues. | Inquiry |
| Multifunctional LNP | Capable of co-delivering mRNA along with small-molecule drugs or immunomodulators for synergistic therapy. | Inquiry |
| Smart/Responsive LNP | Sensitive to pH, enzymes, or redox conditions, enabling disease microenvironment-specific release. | Inquiry |
| circRNA-Encapsulating LNP | Designed for circRNA delivery, enhancing RNA stability and sustaining protein expression. | Inquiry |
A major area of innovation for more precise mRNA delivery to specific cells and tissues is functionalization of the LNP surface with targeting ligands. This can be accomplished by attaching a ligand that can bind to specific cell-surface receptors. This can be done in either of two ways:
Covalent conjugation: Antibodies or antibody fragments, peptides, or small molecule ligands can be anchored directly or through a linker to lipid molecules on the LNP surface. Galactose moieties that target asialoglycoprotein receptors on hepatocytes have been conjugated to LNPs to increase liver accumulation.
Non-covalent assembly: Targeting modules are reversibly attached to the LNP surface through a biotin-streptavidin system, or by DNA hybridization.
Incorporating this "active targeting" functionality greatly broadens the potential of mRNA therapeutics. For example, ligands that target pulmonary endothelial or alveolar epithelial cells are combined with aerosolized delivery to enable inhalation-based, lung-specific mRNA delivery. Ligands targeting specific CNS cells could be combined with methods for transiently opening the blood-brain barrier such as focused ultrasound, potentially enabling mRNA delivery to neural tissues.
The in vivo fate of LNPs is largely determined by their physicochemical properties, including particle size, surface charge, and lipid composition. Systematic optimization of all these parameters is key to developing effective delivery systems.
Particle size is a primary determinant of biodistribution and cellular uptake mechanisms. 80–150 nm is an efficient size range for liver uptake, as these particles are efficiently captured by liver sinusoidal endothelial cells. Particles < 80 nm can penetrate dense tissues or the tumor microenvironment more effectively, while larger particles (>150 nm) may have greater capture by the spleen and other immune organs.
Zeta potential is a common measure of the particle's surface charge. Particles with near-neutral surfaces experience minimal nonspecific interactions with blood components, prolonging circulation times. Environment-responsive charge-switching lipids can be used to change the LNP surface from neutral/negative to strongly positive in specific microenvironments, leading to stronger interactions with negatively charged cell membranes and increased uptake.
The ratio and selection of the four core lipid types (ionizable, helper, PEG, and targeting) ultimately determine the LNP's encapsulation efficiency, stability, membrane fusion properties, immunogenicity, and metabolic kinetics. High-throughput screening methods and computational modeling are used to establish relationships between lipid composition, physicochemical properties, and biological performance to rationally engineer custom LNPs for different applications such as vaccines, systemic protein replacement, or local delivery.
Table 5. Key LNP Parameters and Their Impact on mRNA Delivery.
| Key Parameter | Major Impact | Optimization Goals & Strategies |
| Particle size | Biodistribution: Determines uptake by liver, spleen, or other tissues. Cellular uptake pathway: Influences endocytosis mechanism. | Liver targeting: optimize to 80–150 nm. Extrahepatic targeting/penetration: explore smaller sizes (<50 nm) or specific ranges to leverage physiological pathways. |
| Surface charge | Circulation time: Near-neutral or mildly negative reduces protein adsorption and prolongs half-life. Cellular interaction: Positive charge enhances membrane interaction. | Long circulation: initially near-neutral (PEGylated). Target uptake: design pH-sensitive or environment-responsive charge-switching systems to expose positive charge at target sites. |
| Lipid composition | Encapsulation & stability: Determines mRNA loading and storage stability. Endosomal escape efficiency: Ionizable lipids are critical. Immunogenicity & toxicity: Lipid type and metabolism affect safety. | High delivery efficiency: screen ionizable lipids with optimal pKa. Reduce toxicity: introduce biodegradable bonds, use biocompatible helper lipids. Enhance stability: optimize PEG type and ratio. |
| PEGylation | Stealth effect: Reduces clearance by phagocytes and extends half-life. Cellular uptake: Excess PEG may hinder interactions; a shedding mechanism is needed. Immunogenicity: Anti-PEG antibodies may impact repeated dosing. | Balance: use appropriate short-chain PEG lipids with controlled shedding dynamics; explore non-PEG stealth materials (e.g., natural polysaccharide-derived lipids). |
Scalable, efficient, and robust production technologies are required to translate LNP-mRNA therapeutics from academic laboratories to real-world large-scale applications. Key innovations are related to mixing technologies, process control, and continuous manufacturing. Microfluidic mixing systems are currently the preferred method for producing high-performance LNPs. mRNA-containing aqueous solutions and lipid-containing organic solutions are combined in microfluidic channels<1 mm in diameter at controlled flow rates. This enables the LNPs to self-assemble instantaneously in a uniform manner. Compared to bulk or ultrasonic mixing methods, microfluidics results in much narrower particle size distributions, greater batch-to-batch consistency, and straightforward linear scalability. Transitioning from traditional batch production to continuous manufacturing is another important trend. Continuous manufacturing lines have streamlined the workflow for feeding, mixing, and purifying components in a single integrated workflow, greatly improving efficiency, lowering costs, and reducing manual steps. Online monitoring allows tracking of critical parameters such as particle size and polydispersity in real time to ensure product quality and stability.
Table 6. LNP Development and Validation Services.
| Service Category | Description | Price |
| Lipid Nanoparticles for mRNA Delivery | Encapsulate and deliver mRNA molecules efficiently into target cells using lipid nanoparticles, ensuring translation, protection, and high functional expression. | Inquiry |
| LNP Formulation Services | Customized LNP design based on target cells or tissues, optimizing lipid composition, ratios, and physicochemical properties, and screening multiple high-performance candidate formulations. | Inquiry |
| Targeted LNP Validation | Comprehensive service from ligand selection, surface conjugation, to in vitro targeting efficiency and cell-specific uptake validation for precise mRNA delivery. | Inquiry |
| LNP-mRNA Process Development | Transform lab-scale formulations into stable, uniform pilot-scale products, supporting preclinical studies sample supply with reproducible quality. | Inquiry |
| LNP Safety Evaluation | Full biological assessment including in vitro transfection, endosomal escape, in vivo biodistribution imaging, protein expression kinetics, and preliminary immunogenicity and toxicity analysis. | Inquiry |
| Innovative LNP Delivery for Novel mRNA | Optimize LNP encapsulation for circRNA or saRNA, addressing unique structural and functional challenges to maximize delivery efficiency and therapeutic potential. | Inquiry |
The lipid nanoparticle technology platform is a critical enabler for mRNA therapeutics. Optimization of lipid composition, particle size, surface modification and smart responsive designs are enabling LNPs to not only successfully deliver mRNA, but also provide additional control to achieve target-specific, tunable, and sustained protein expression. With advanced RNA formats, personalized design, and continuous manufacturing technologies, LNPs will likely have extensive application in vaccine and cancer immunotherapy, as well as for genetic metabolic disorders. BOC Sciences has matured technical capabilities in LNP research and process development, which allows for form-to-pilot LNP process support and complete end-to-end services including formulation design, surface functionalization, and pilot scale production to help customers effectively actualize their mRNA delivery products.
