Lipid nanoparticles (LNPs) are colloidal carriers that self-assemble from lipids at the nanometer scale. These particles typically comprise four major components: ionizable amino lipids, cholesterol, helper phospholipids, and PEG-modified lipids. The ionizable amino lipid is the key constituent of LNPs. At low pH, the ionizable amino groups are protonated and bear a positive charge to form strong electrostatic interactions with negatively charged nucleic acids such as siRNA. At near-neutral pH, the lipids are largely uncharged, which helps improve particle stability in circulation and reduce nonspecific interactions. Research indicates that the control of ionizable lipids' pKa at or below 7 is a key parameter for both nucleic acid encapsulation and intracellular activities. Cholesterol is used to tune membrane fluidity and rigidity for structure integrity and membrane fusion. Helper phospholipids, such as DSPC, form the lipid bilayer. PEG-modified lipids are exposed on the particle surface, which creates a hydration layer for extended circulation and prevention of rapid clearance. By rationally tuning the composition and physicochemical properties of the four components, uniform nanoparticles with the diameter typically ranging from 50 to 100 nm can be generated. Such particle size is favorable for tissue penetration and cellular uptake.
The siRNA is a double-stranded RNA (dsRNA) with a length of about 21–23 nucleotides. siRNA in the cytoplasm searches for the complementary sequence in the messenger RNA (mRNA) and by RNA interference mechanism initiates the degradation of target mRNA. It silences the target gene expression and can be used for specific knockdown of the protein of interest. Theoretically, siRNA can be used against any gene. siRNA as a free entity has a lot of biological limitations. The high concentrations of serum ribonucleases cleave unprotected siRNA very quickly. The highly negative charged siRNA has difficulty passing through the negatively charged membrane of the cell. When it enters the cell, it can end up in endosomes from where it cannot escape. As a result, the free siRNA has a very short half-life and no functional bioavailability. LNPs were created to protect the siRNA physically by encapsulation, use the properties of nanoparticles to facilitate the uptake of particles into cells, and have functional groups on the surface of LNPs to facilitate escape from the endosome.
Endocytosis is the primary route of cellular uptake for lipid nanoparticles. Nanoparticles adsorb serum proteins upon reaching the tissue of interest, which is called protein corona. The protein corona is mainly composed of apolipoprotein E (ApoE), which is important for liver-targeted delivery, as it mediates specific binding to LDLR expressed on the surface of hepatocytes and internalization of nanoparticles via receptor-mediated endocytosis. Nanoparticle accumulation in tumor associated tissues is mainly due to the enhanced permeability and retention (EPR) effect. Aberrant vascular structures within tumors can allow nanoparticles to extravasate and passively accumulate. Binding of nanoparticles to cell surface receptors can also be achieved by decoration of targeting ligands (e.g., RGD peptide, folate group) to enhance uptake into tumor cells. Following receptor mediated endocytosis, nanoparticles are enclosed in early endosomes with a mildly acidic pH (~6.0) and may mature into late endosomes, with a lower pH (~5.0), which eventually fuse with lysosomes.
Interaction of LNPs with cell membranes dictates delivery efficiency. The surface charge of ionizable lipids is close to neutral at physiological pH and thus it avoids the nonspecific protein binding and minimizes off-target interactions. Binding of a ligand to a cell surface receptor triggers invagination of the local membrane. Clathrin-coated pits form at the cell surface and eventually pinch off to form an endocytic vesicle. Membrane fusion is governed by lipid composition. Lipids with unsaturated acyl chains increase fluidity and therefore enhance lipid mixing with the cell membrane. Cholesterol modulates membrane rigidity and bending elasticity which helps in maintaining nanoparticle stability during membrane deformation. However, long PEG chains may sterically hinder the close membrane approach of LNPs. Therefore, the molecular weight of PEG used in most lipid formulations is typically limited to 2000 Da. Escape from endosomes is considered as the rate-limiting step for siRNA delivery, as majority of the internalized nanoparticles are not able to escape and get degraded. LNPs utilize the chemistry to circumvent this challenge. In late endosomes, the endosomal pH triggers the ionization of ionizable lipids resulting in acquisition of positive charges by cationic lipids. Positively charged lipids electrostatically interact with the negatively charged endosomal phospholipids (e.g., phosphatidylserine) via ion pairing. The transient and reversible interaction of these ion pairs within the bilayer weakens the membrane, leading to membrane fusion or disruption and consequent release of the siRNA into the cytoplasm. This is sometimes referred to as a modified "proton sponge"–like effect. Protonation of the ionizable lipids is coupled with chloride ion influx into the endosome and water entry leading to an osmotic imbalance, resulting in endosomal swelling and rupture. Experimental evidence showed that ionizable lipids with pKa~6.5, can mediate endosomal escape efficiencies of >80%. Unsaturated acyl chain degree also influences endosomal escape efficiency. Lipids containing two double bonds such as linoleoyl chains perform better than saturated acyl chain or more unsaturated acyl chain lipids. It is likely related to favorable membrane fusion property and phase transition.
Surface properties and particle size predominantly influence the biodistribution of lipid nanoparticles. A neutral or slightly negative surface charge avoid detection by the MPS and prolong circulation time. ApoE-mediated recognition by LDLR is the key determinant for liver-selective delivery which explain the preferential accumulation of most LNP formulations in the liver. An optimal particle size of 80 nm is the best for hepatocyte uptake, since they are able to pass the fenestrated endothelium and at the same time do not lose efficient cellular internalization. EPR effect along with active targeting mechanism mostly contribute to tumor targeting. Nanoparticles with smaller diameters (~50 nm) have been reported to have deeper penetration, whereas the particles with larger diameters (>100 nm) accumulate closer to the periphery of the tumor. Targeting ligands on the surface of LNPs can also significantly increase tumor specific uptake. Targeting RGD peptide to the tumor specific integrin receptor αvβ3 that overexpressed in the tumor associated neovasculature have shown to improve distribution and uptake of LNPs. By optimizing the lipid composition and surface functionalization, LNPs can be tailored to target other organs and tissue.
Fig.1 Diagram of LNP-siRNA intracellular delivery and gene silencing mechanism.
BOC Sciences delivers scalable lipid nanoparticle solutions with tailored compositions to support diverse siRNA delivery strategies and research objectives.
The primary advantage of LNPs for siRNA delivery is that they greatly improve the efficiency of siRNA across biological barriers and to its intracellular site of action. Naked siRNA is highly negatively charged and thus it is difficult to cross the negatively charged cell membrane; in addition, naked siRNA is rapidly cleared and degraded in circulation. Lipid nanoparticles can encapsulate and protect the siRNA, and their nanoscale size and surface characteristics enable them to efficiently enter cells through physiological processes such as endocytosis. In addition, LNPs can achieve targeting in a following two fashions: Passive targeting: This involves the enhanced permeability and retention (EPR) effect for nanoparticles to passively accumulate at a pathological tissue or organs (such as solid tumors). Active targeting: Surface functionalization of nanoparticles with targeting moieties (ligands), for example, antibodies, peptides or small molecules, to allow LNP to bind to specific biomarkers on the surface of the target diseased cell and achieve high-precision targeting at cellular or tissue level, greatly reducing off-targeting effect on healthy tissues.
As nucleic acid macromolecules, siRNA encounters two major issues in a physiological setting: being degraded by RNases (ribonucleases) in serum and being rapidly filtered by the kidney. Lipid nanoparticles physically encapsulate siRNA and shield it from nuclease attack, significantly prolonging its half-life from minutes to hours. The small size (larger than the renal filtration cutoff) and PEGylated surface also enable LNPs to be less recognized and cleared by the MPS, further prolonging their circulation time. In aggregate, these factors increase the effective dose of siRNA reaching the target tissue and systemically improve its bioavailability.
Earlier generations of nucleic acid delivery systems, such as certain cationic polymers or liposomes, could cause strong cytotoxicity or a robust innate immune response (such as interferon responses) due to the excessive positive charges, which precludes many applications. The development of lipid nanoparticles, especially ionizable cationic lipids that are overall neutral at physiological pH, greatly minimizes nonspecific membrane disruption and cytotoxicity. In addition, by optimizing lipid composition, and using highly purified, chemically modified siRNA (such as 2'-O-methyl modifications) to minimize activation of immune recognition pathways such as Toll-like receptors, both the safety window and tolerability of the therapy can be improved without compromising its therapeutic efficacy.
The lipid nanoparticle platform is highly modular and flexible. The four major components can be readily modified or even replaced to systematically tune the physicochemical properties and biological properties of the nanoparticles. This flexibility means that a single platform can be optimized for delivering siRNA targeting different disease markers, ranging from liver diseases and solid tumors to central nervous system disorders, by tailoring the formulation for specific applications.
Table 1. Customization of Lipid Nanoparticle Components and Their Functions.
| Adjustable Component | Purpose of Adjustment | Potential Impact on Nanoparticle Properties |
| Ionizable cationic lipids | Optimize endosomal escape, reduce toxicity | Modify pKa, membrane fusion ability, metabolic pathways |
| Helper lipids (e.g., DSPC, DOPE) | Adjust membrane fluidity and stability | Influence phase transition temperature, endosomal escape mechanisms (e.g., promote hexagonal phase formation) |
| Cholesterol | Modulate membrane rigidity and in vivo stability | Enhance structural integrity, affect interactions with lipoproteins |
| PEGylated lipids | Control circulation time and targeting | Long-chain PEG increases circulation time; short-chain or detachable PEG enhances cellular uptake; targeting ligands for active targeting |
| siRNA Sequence and Modifications | Target specific disease markers, enhance stability | Determines molecular targets; chemical modifications reduce immunogenicity and enhance nuclease stability |
Since cancer is typically a disease resulting from a number of genetic lesions that dysregulate normal cellular functions, e.g. activation of an oncogene or inactivation of a tumor suppressor gene, siRNA based therapy is well suited to target many pathogenic genes simultaneously. Conjugation of siRNA to a lipid nanoparticle formulation would allow delivery of siRNA into tumor cells to silence oncogenes that are required for tumor growth and survival, tumor invasion, or drug resistance. For example, siRNA targeting known cancer-driver mutations, such as KRAS mutations (common in pancreatic and lung cancers) or the BCR-ABL fusion gene (chronic myelogenous leukemia), could be loaded into the nanoparticles and delivered. To more actively target tumor tissue and improve efficacy and/or reduce systemic toxicity, tumor-specific ligands, such as folate or transferrin receptor antibody fragments, could be attached to the nanoparticle surface to facilitate active tumor targeting.
Viruses, unlike bacteria, are much more dependent on host cell replication machinery and other components, and this also leaves a smaller range of targets for traditional small molecule drugs. Therefore, siRNA can be targeted directly to conserved portions of the viral genome itself or viral messenger RNAs that are required for viral replication, and by this mechanism act earlier in the viral life cycle. For example, siRNA that target the nucleoprotein gene of the influenza virus or other highly conserved sequence regions of the HIV genome could be designed and synthesized. Lipid nanoparticles could then be used to package and deliver these siRNAs to virus-infected cells (e.g., respiratory epithelial cells or immune cells) where they bind to the viral RNA and cleave it, preventing replication and spread of the virus. This would provide a rapid platform approach for the generation and evaluation of siRNA therapeutics against emerging and pandemically-relevant viral infections.
A common cause of genetic disease is a single gene defect, resulting in the accumulation of toxic proteins or a lack of a functional protein. An siRNA-based therapy can be employed to treat such conditions, either by silencing of the mutant allele (allele-specific silencing) or by knockdown of a toxic protein. Lipid nanoparticles can aid in the delivery of such a therapy. A classic example of this is in the treatment of transthyretin amyloidosis, where the siRNA is packaged into a lipid nanoparticle that is preferentially taken up by the liver and then silences the expression of the transthyretin protein to reduce amyloid deposits that cause nerve and heart damage. Similar approaches are being developed for other conditions that are expressed by the liver, such as hypercholesterolemia (targeting the PCSK9 gene) or α1-antitrypsin deficiency.
In a number of chronic inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease, various inflammatory cytokines and inflammatory signaling pathways are activated in a dysregulated fashion. siRNA can be designed and used to specifically reduce the expression of key inflammatory mediators (such as TNF-α, IL-1β, IL-6) or of their upstream signaling proteins. Lipid nanoparticles can be used to deliver the therapeutic siRNA into activated immune cells (macrophages, T cells) or into inflamed tissues (synovial membranes, intestinal mucosa). By designing the lipid nanoparticle to specifically target the site of inflammation (e.g., using EPR effect, or by modifying ligands that can bind to surface molecules of inflammatory cells), one could achieve site-specific suppression of inflammation rather than a systemic immunosuppression, and thus represent a novel therapeutic approach for these diseases.
Table 2. Product Categories of Lipid Nanoparticles for siRNA Delivery.
| Product Name | Description | Price |
| Ionizable Lipid LNP | This lipid nanoparticle is designed based on ionizable lipids with tunable pKa values, enhancing siRNA endosomal escape and promoting intracellular release. | Inquiry |
| PEGylated LNP | PEGylation extends the circulation time of lipid nanoparticles, reduces nonspecific interactions with the immune system, enhances stability, and prevents premature clearance. | Inquiry |
| Tumor-targeted LNP | Designed for tumor therapy, this lipid nanoparticle is surface-modified with targeting ligands such as RGD peptides or folate to specifically recognize tumor cells, enhancing targeted delivery. | Inquiry |
| CNS-targeted LNP | Designed to cross the blood-brain barrier, this lipid nanoparticle carries therapeutic molecules like siRNA for precise delivery to treat neurological diseases such as Alzheimer's and Huntington's disease. | Inquiry |
| Inhalable LNP | Designed for pulmonary diseases, this inhalable lipid nanoparticle has an optimized lipid formulation that allows it to remain in the lungs and promotes siRNA uptake by alveolar epithelial cells. | Inquiry |
| Muscle-targeted LNP | Modified lipids enhance the targeting of muscle tissues, making this LNP suitable for treating muscle-related diseases such as Duchenne muscular dystrophy. | Inquiry |
| Long-acting LNP | A lipid nanoparticle formulation for ophthalmic diseases, designed as a long-acting, sustained-release product delivered via intravitreal injection, providing prolonged therapeutic effects in the eye. | Inquiry |
The most important recent progress in the formulation of lipid nanoparticles has been the rational design and application of new ionizable cationic lipids. Traditional lipid nanoparticles often have certain bottlenecks in efficacy and safety. The newly developed generation of lipids are rationally optimized in chemical structure to achieve a balance between efficient endosomal escape and biocompatibility. For instance, a series of new lipids have been designed by introducing biodegradable ester linkages or specific branched structures. These structures allow for faster and more complete metabolic clearance of the lipid once it has completed the task of delivering siRNA. This greatly reduces the risk of long-term toxicity. In addition, the selection and combination of helper lipids have been further optimized. In addition to the traditional phosphatidylcholine derivatives, phospholipids such as distearoylphosphatidylethanolamine (DSPE) have been used to increase membrane fusion ability or promote non-bilayer phase formation. This further improves the efficiency of endosomal escape. By systematically optimizing the formulation design, the next generation of lipid nanoparticles can achieve stronger gene silencing effects at lower doses and expand delivery beyond the liver.
Engineering the surface of lipid nanoparticles to achieve site-specific delivery beyond the liver has been an area of focus for innovation. New strategies for surface modification are mainly concentrated in two aspects: prolonging circulation and tissue accumulation, and enabling cell recognition. One important innovation is the development of detachable or responsive PEG coatings. While traditional PEG layers can prolong circulation time, they can also inhibit cellular uptake. New designs of PEG-lipids allow the PEG layer to be broken or peeled off in response to specific conditions such as the acidic pH or high concentration of certain enzymes in the tumor microenvironment. This can expose the nanoparticle core or the underlying targeting ligands. This can help promote active cellular uptake. Another innovation is the development and use of new targeting ligands. In addition to traditional antibody fragments and peptides, small molecule ligands (such as PSMA ligands for targeting prostate cancer cells) and nucleic acid aptamers are becoming increasingly popular due to their small molecular size, low immunogenicity, and ease of synthesis and modification. These ligands can be precisely anchored to the surface of the nanoparticle. They can act as an "intelligent navigation system" that can guide the carrier through complex biological environments and specifically bind to receptors on diseased cells. In this way, precise delivery can be achieved from the level of tissue to that of cells.
siRNA-based lipid nanoparticle therapies have rapidly developed from concept demonstration to the development of broad-spectrum therapeutic applications. In terms of genetic diseases, following the successful demonstration in transthyretin amyloidosis, similar approaches are actively being developed to target other diseases of liver origin. siRNA therapies targeting the angiopoietin-like protein 3 gene to lower LDL-C for the treatment of familial hypercholesterolemia are being developed, and targeting of mutant alpha-1 antitrypsin genes is being developed for the treatment of alpha-1 antitrypsin deficiency. In cancer treatment, in addition to exploring the application of lipid nanoparticles for gene knockout of a single gene such as KRAS, combination synergistic and multi-target therapy is also emerging as a research direction. For example, lipid nanoparticles that co-load siRNAs that silence the programmed cell death ligand 1 (PD-L1) gene with immune checkpoint-related genes are being designed to reshape the tumor immune microenvironment. Combination of siRNA-loaded lipid nanoparticles with chemotherapy drugs, immune stimulators, or photothermal therapy to achieve enhanced anti-tumor effects is also being developed.
Table 3. Examples of Progress in siRNA Therapies for Extrahepatic Delivery Using Lipid Nanoparticles.
| Target Tissue/Disease | Main Technological Strategy | Example Potential Targets |
| Central Nervous System Diseases | Receptor-mediated transport across the blood-brain barrier; intrathecal or intracranial injection formulations | Huntington's disease-related mHTT gene, Alzheimer's disease-related BACE1 gene |
| Pulmonary Diseases | Inhalable formulations; optimized lipid composition for lung retention and alveolar epithelial cell uptake | Cystic fibrosis transmembrane conductance regulator (CFTR) gene, Respiratory syncytial virus (RSV) gene |
| Muscular Diseases | Development of muscle-targeting ionizable lipids; modification with muscle cell-targeting peptides | Duchenne muscular dystrophy-related anti-myostatin gene exon skipping |
| Ocular Diseases | Development of long-acting, sustained-release intravitreal formulations | Vascular endothelial growth factor (VEGF) gene, Age-related macular degeneration-related targets |
The design process of lipid nanoparticles is based on a clear definition of the therapeutic target (target cell type, delivery route, expected pharmacokinetics, etc.). The synthesis core is to mix four different lipid components with siRNA in an aqueous solution to achieve self-assembly. The most widely used method is microfluidic mixing, where lipids dissolved in ethanol are mixed with siRNA dissolved in acidic buffer at pre-controlled flow rates in micro-size channels. During the mixing process, the lipids in the ethanol phase diffuse into the aqueous phase, and at the same time, due to the pH change, the ionizable lipids are protonated and combined with the negatively charged siRNA. The lipid molecules then spontaneously self-assemble around the siRNA-lipid complex to form structurally ordered nanoparticles. This process is rapid, easily controlled, and highly reproducible, making it suitable for both small-scale laboratory development and large-scale industrial production.
Size, surface charge, and surface modifications are all important physicochemical properties of lipid nanoparticles that ultimately determine their fate in vivo and should be well optimized. Particle size: It is generally controlled within 80-100 nm. Particles with a larger size are rapidly cleared by the mononuclear phagocyte system, while particles with a smaller size pass through the blood vessels too quickly, resulting in reduced accumulation in the target tissue and increased renal clearance. At the same time, the polydispersity index should be as low as possible to ensure batch-to-batch reproducibility and predictability in vivo. Surface charge (Zeta potential): Nanoparticles with a near-neutral or slightly negative charge reduce nonspecific interactions with blood components and prolong circulation time. This parameter can be finely tuned by adjusting the lipid composition and degree of PEGylation. Surface properties: These are mainly tuned by the type and ratio of PEGylated lipids. The length and density of PEG chains will directly affect the "stealth" ability of nanoparticles and the dynamics of interaction with target cells. Optimization of surface properties requires a balance between long circulation time and effective uptake by cells.
Table 4. Optimization Goals and Impact of Key Physicochemical Parameters for Lipid Nanoparticles.
| Parameter | Optimization Goal Range | Main Impact on Delivery Efficiency |
| Particle Size | 80-100 nm (for intravenous delivery) | Affects circulation time, tissue penetration, uptake pathways, and clearance mechanisms |
| Polydispersity Index | < 0.2 | Affects batch uniformity, in vivo distribution consistency, and product quality |
| Zeta Potential | -10 mV to +5 mV (at physiological pH) | Affects plasma protein adsorption, immune recognition, circulation stability, and cellular interactions |
| Encapsulation Efficiency | > 90% | Determines effective payload, affecting therapeutic efficacy and potential side effects |
Scaling from laboratory to meet commercial demands is a critical step in the technology's translation. Continuous flow production, based on microfluidic principles, has become the industry standard. This technology transfers the mixing process from static beakers to precisely controlled continuous flow systems, enabling accurate control of mixing efficiency, temperature, and residence time, ensuring high consistency of product quality from milliliter to hundred-liter scale production.
Table 5. Lipid Nanoparticle Services for siRNA Delivery at BOC Sciences.
| Service Category | Description | Price |
| LNP Formulation Service | Provides end-to-end formulation development services, including lipid component design, multi-formulation library creation, high-throughput preparation, and in vitro/in vivo evaluations. | Inquiry |
| LNP Surface Modification Service | Offers comprehensive services for selecting ligands, designing conjugation strategies, and coupling ligands to LNP surfaces to enable active targeting of specific tissues or cells. | Inquiry |
| siRNA-LNP Process Development | Delivers process development and optimization services for siRNA-LNP, including scale-up from laboratory to production using microfluidic-based technologies, ensuring consistency and robustness. | Inquiry |
| LNP Formulation Analysis Service | Provides detailed physicochemical characterization of LNPs (e.g., particle size, PDI, zeta potential), siRNA encapsulation rates, and stability studies with biological activity tests. | Inquiry |
| LNP Stability Solutions | Offers services for evaluating LNP stability, including screening cryoprotectants, optimizing lyophilization curves, and developing processes to address storage challenges for siRNA-LNP systems. | Inquiry |
| Lipid Nanoparticles for siRNA Delivery | Encapsulate and deliver siRNA molecules using lipid nanoparticles to achieve targeted gene silencing with improved uptake and reduced off-target effects. | Inquiry |
As a kind of complex colloidal system, in addition to containing biomacromolecules (siRNA), lipid nanoparticles also have a series of stability problems in long-term storage, which are also challenges in production and industrialization. Physicochemical instability may manifest as siRNA leakage, aggregation or fusion of nanoparticles, lipid hydrolysis or oxidation, and chemical degradation of siRNA. To solve these problems, a combination of multiple strategies is usually needed. The first and most basic one is formulation optimization, that is, to select appropriate lipids, optimize the cholesterol ratio, and control the degree of PEGylation to improve the structural rigidity of nanoparticles. Lyophilization is currently the most widely used method to achieve long-term storage of the final product, where the nanoparticle dispersion is dehydrated under low temperature (usually -80 ℃) to form a solid cake, and then stored at low temperature to prolong the shelf life of the product. Lyophilization process also needs to carefully select appropriate cryoprotectants (e.g. sucrose, trehalose) to avoid structural damage during dehydration. After rehydration, the nanoparticles should quickly restore the original size distribution and biological activity. In addition, low-temperature storage and transportation conditions for the product are also very important to ensure stability.
