Therapeutic gene delivery is an approach in biomedicine that aims to deliver functional genetic material, or "cargo," to specific cells in order to modulate gene expression patterns and correct disease phenotypes caused by genetic defects or abnormal expression. This genetic cargo can take many forms, such as healthy gene copies that replace a missing or defective gene, RNA molecules that can suppress the expression of an unwanted gene, or molecular tools that can edit the genome at specific locations. The concept of therapeutic gene delivery therefore stands in contrast to small-molecule or protein therapeutics that act on protein function downstream of the genome, and as such offers the potential to provide more fundamental and durable disease treatments.
Delivery of nucleic acids into cells has been one of the key challenges for the development of gene-based tools since the very early days. NAs are highly susceptible to degradation by ubiquitous nucleases, and their inherently negative charge can make it difficult to package and deliver them using small molecules or proteins. Viral vectors are an exception, as they have evolved to be highly efficient at transducing target cells, but have other limitations such as immunogenicity and genomic integration. Of the various non-viral systems that have been developed and evaluated over the years, LNPs have emerged as one of the most promising, with a good balance of performance and design flexibility. The major requirements of LNPs include packaging and protecting the NA payload from enzymatic degradation, providing stability during circulation in the bloodstream, and facilitating uptake by target tissues through surface chemistry and other means. Finally, LNPs must interact with cellular membranes to promote uptake, followed by an intracellular release of the NAs into the cytoplasm, where they can exert their desired effects.
As the effectiveness of a gene-based solution critically hinges on the ability of the delivery system to overcome a range of physiological and cellular barriers, a robust and high-performance delivery platform is necessary to realize the potential of gene-based solutions in practice. These delivery barriers include the inherent instability of NAs in biological fluids, limited penetration into tissues, non-specific uptake by off-target cells and, perhaps most importantly, cellular membranes and entrapment in endosomes. Therefore, advances in delivery technology, and in particular lipid nanoparticle systems are widely considered to be one of the key factors in the move to translate gene-based approaches from academic research to scalable real-world applications.
Current generation LNPs for gene delivery are far more sophisticated than just lipid droplets. Lipid nanoparticles are highly organized nanostructures and are self-assembled from several functional lipids, in precisely defined ratios. They typically have a diameter of 80–200 nm. LNP formulations consist of four essential lipids, each with a different, indispensable function. In a typical process, the lipids are dissolved in an ethanol–aqueous system and rapidly assembled by controlled mixing. The result is a complex, with a core of nucleic acid ionizable lipids and an enveloping lipid bilayer. PEGylated lipids form a soft brush-like corona on the exterior of the particle.
Fig.1 Cross-section diagram of a Lipid Nanoparticle showing mRNA payload (BOC Sciences Original).
Table 1. Key Components of Lipid Nanoparticles for Gene Delivery and Their Functions.
| Component Category | Primary Function | Common Examples |
| Ionizable cationic lipids | Core functional lipids of LNPs; become positively charged under acidic conditions to enable efficient nucleic acid encapsulation and promote endosomal escape | DLin-MC3-DMA, SM-102, ALC-0315 |
| Phospholipids | Form the structural backbone of the lipid bilayer, providing integrity and influencing membrane fluidity | DSPC, DOPE |
| Cholesterol | Intercalates within the lipid bilayer to stabilize particle structure, modulate membrane rigidity and fluidity, and support cellular uptake | Natural or modified cholesterol |
| PEGylated lipids | Localized on the particle surface; prevent aggregation via steric hindrance, reduce non-specific protein binding, and extend circulation time | DMG-PEG2000, DSG-PEG2000 |
Endocytosis is the main mechanism by which LNPs enter cells, which is an active, membrane-driven process. The precise route is determined by the physicochemical properties of the LNPs, including particle size, surface charge and density of PEGylated lipids on the surface of LNPs. Clathrin-mediated endocytosis is the most prevalent mechanism. It is triggered by an interaction between LNPs and a specific membrane-bound protein or receptor. This results in a membrane invagination, forming endosomal vesicles containing LNPs. Caveolin-mediated endocytosis and macropinocytosis are also possible depending on the formulation and cell type. The cellular uptake efficiency can be tailored by fine-tuning of the formulation. For example, a mildly positive surface charge will improve interaction with the negatively charged cell membrane. The type and amount of PEGylated lipids should also be optimized; high PEG density will increase stability in solution, but hinder membrane interaction and reduce uptake.
After cellular uptake, LNPs are enclosed in endosomes and their escape into the cytoplasm becomes the key rate-determining step for functional delivery. If not escaped from endosomes in time, these compartments will continue to mature and eventually fuse with lysosomes. This would result in degradation of the nucleic acid payload by lysosomal enzymes. Ionizable cationic lipids are essential for endosomal escape. In physiological conditions (neutral pH), ionizable lipids are largely uncharged and the resulting LNPs have a near-neutral charge. In the acidic endosome, as it matures, the amines of the ionizable lipids become protonated and positively charged. The positively charged ionizable lipids from LNPs then interact with the negatively charged phospholipids in the endosome membrane. Two mechanisms have been widely discussed: the "ion-pair" model assumes that electrostatic interaction between the ionizable lipids and endosomal phospholipids disrupts the membrane organization; the other "inverted hexagonal phase" model hypothesizes that the interaction between the ionizable lipids and endosomal membrane induces a rearrangement of the lipid from a lamellar phase to an inverted hexagonal phase with significantly reduced stability. Regardless of the exact mechanism, the interaction would lead to transient defects or pores in the endosomal membrane, through which the nucleic acids can be released into the cytosol. Endosomal escape is a necessary but not sufficient condition for the activity of the cargo. Once in the cytoplasm, mRNA is translated directly into target protein by ribosomes, while siRNA associates with the RNA-induced silencing complex to bind and degrade complementary messenger RNAs. When plasmid DNA is used for genome modification, another membrane (the nuclear envelope) must be traversed.
BOC Sciences combines formulation expertise with flexible customization to support optimized gene delivery strategies across development pipelines.
Whereas initial efforts in the field of LNPs focused on the optimization of formulation parameters, the development of new lipid chemistries has emerged as the main focus of the field. The early generations of cationic lipids were permanently positively charged and suffered from non-specific protein binding in the circulation as well as toxicity upon cellular uptake. As such, current efforts are now directed towards the design of next-generation lipids that are ionizable and can undergo chemical fine-tuning to meet desired charge properties as well as biodegradability requirements. This effort often includes the attachment of biodegradable linkers, such as ester groups, within the lipid structure. This results in lipids that are able to be rapidly broken down enzymatically into non-toxic by-products after completing their intracellular journey, allowing for significantly improved tolerability in the case of repeated dosing. In addition, further tuning of the lipid tail length and degree of saturation has been achieved, resulting in improved membrane fluidity, increased loading of nucleic acids, and improved release kinetics.
Table 2. Evolution of LNP Lipid Chemistry and Functional Improvements.
| Development Stage | Key Chemical Features | Technical Advantages | Primary Limitations |
| First generation | Permanently cationic head groups | High nucleic acid encapsulation; simple synthesis | Short circulation time; notable cytotoxicity; high non-specific interactions |
| Second generation | pH-sensitive ionizable head groups | Charged only under acidic conditions; reduced toxicity at physiological pH | Slower clearance; potential long-term accumulation |
| Third generation | Introduction of biodegradable bonds (e.g., ester linkages) | Rapid metabolic breakdown; significantly improved tolerability | More complex synthesis; requires precise control of degradation rates |
Targeted delivery to organs other than the liver is another significant achievement of the field. While LNPs have the natural proclivity to accumulate within the liver, strategies to enable more site-specific delivery to other organs have long been desired. This bottleneck was recently overcome with the introduction of Selective Organ Targeting (SORT) technology. SORT leverages the addition of a fifth component to the usual four-component LNP formulation that is termed a SORT molecule. The physicochemical properties of these molecules, such as having a known charge or degree of hydrophobicity, allows for alteration of the protein corona that the LNPs acquire in vivo, effectively redirecting the particles to a given organ (lung, spleen, or bone marrow, etc.). For instance, it has been shown that the addition of negatively charged SORT molecules to a formulation reprograms the delivery away from the liver to the spleen, while the inclusion of permanently cationic lipids instead can enable the delivery to pulmonary endothelial cells. In a similar manner, efforts to enable active targeting through antibody or ligand conjugation are being further developed. This allows LNPs to specifically bind to overexpressed receptors on certain tumor cells, further sharpening the accuracy of delivery.
Although significant progress has been made, the current low efficiency of endosomal escape is a major bottleneck. In fact, only a small percentage (generally estimated to be <2–3%) of internalized nucleic acids are released from endosomes and are able to carry out their desired function. This rate-limiting step can be overcome by creating large libraries of lipids for high-throughput screening to identify the structures with the best membrane fusion properties. Some efforts have also started to focus on developing cone-shaped lipids, whose shape is more conducive to perturbing endosomal membranes under the acidic endosomal environment. In addition, further tuning of the lipid tail length and degree of saturation has been achieved, resulting in improved membrane fluidity, increased loading of nucleic acids, and improved release kinetics. In addition to the intracellular challenges to LNP-mediated gene delivery, thermal instability is also a major hurdle to surmount. Through improved lyophilization methods and the discovery of novel cryoprotectants, LNPs may be more easily stored at room temperature for prolonged periods of time, eliminating the need for ultra-cold storage.
LNPs can be employed in a variety of ways to treat monogenic disorders. In protein replacement approaches, LNPs encapsulating mRNA, which encodes a functional protein, are administered. After endocytosis, typically by hepatocytes, the cell's translation machinery is used to produce the necessary proteins to restore the biological function that was lost due to a genetic mutation. LNPs have been employed to carry genome editing tools for more sophisticated applications. In a dual-LNP formulation, Cas9 mRNA and single-guide RNA (sgRNA) are co-delivered into the same cell and then assemble the CRISPR/Cas9 system in situ. This allows transient expression of the genome editor and can be an attractive approach: because the Cas9 protein is degraded shortly after genome editing is completed, there is less potential for off-target activity. These dual-LNP strategies have been shown to have strong proof-of-concept for conditions in which the production of aberrant proteins by the liver is the underlying pathology, including amyloidosis.
Applications of LNPs in cancer research and therapeutics are currently focused on immune modulation and tumor microenvironment reprogramming. LNPs are being developed for use in cancer vaccines. In this approach, LNPs carrying mRNA, which encodes tumor-associated neoantigens, are delivered to antigen-presenting cells. The target for these formulations is often dendritic cells. The neoantigen mRNA is translated and processed in the antigen-presenting cells and then activates cytotoxic T cells that can initiate an immune response against tumor cells. Compared with traditional vaccine approaches, LNP–mRNA vaccines have the advantages of a short design cycle and a multiantigen approach.
Table 3. LNP Cargo Strategies in Tumor-Oriented Gene Delivery.
| Cargo Type | Mechanism of Action | Intended Outcome |
| Tumor antigen mRNA | Immune activation | Trains the immune system to recognize and eliminate antigen-expressing tumor cells |
| Cytokine mRNA | Microenvironment modulation | Local expression of immune-stimulatory factors (e.g., IL-12) to convert immunologically "cold" tumors into "hot" tumors |
| siRNA / miRNA | Gene silencing | Downregulation of resistance-associated genes or key oncogenic drivers |
| Suicide gene DNA/mRNA | Direct cytotoxicity | Induces expression of enzymes that convert non-toxic prodrugs into cytotoxic compounds, leading to tumor cell apoptosis |
Gene delivery applications in cardiovascular disease have a high burden on formulation design because these treatments must be localized and tissue selective in their action. For example, lipid nanoparticles have been tested as potential agents for myocardial repair after injury. Delivery of modified mRNA, which encodes vascular endothelial growth factor (VEGF), stimulates angiogenesis in ischemic tissues and proliferation of cardiac progenitor cells. A key technical hurdle in these applications is preventing off-target uptake by other tissues.
Delivery to the brain for gene delivery applications is most often limited by the BBB. To address this challenge, surface functionalization of lipid nanoparticles with transferrin receptor–binding antibodies or targeted gangliosides has been pursued in recent research as a potential strategy for receptor-mediated transcytosis of LNPs across the BBB. Once in the brain, LNPs can carry genes that encode neurotrophic factors or ablate expression of mutant proteins that drive neurodegeneration, among other possibilities.
As a carrier, optimization of LNP formulation is still the most direct approach to achieve performance enhancement. In addition to lipid-type and -ratio design, active research interest has shifted from a conventional four-component system to highly complex and "smart" formulations. The key recent advances include the following:
First, the development of next-generation ionizable lipids is ongoing. The discovery of new lipid molecules with high delivery efficiency and low toxicity toward a specific target tissue is now a largely data-driven task. To expedite the process, lipid libraries are built in high-throughput manners and are combined with AI/ML-based models for in silico screening and prediction of new lead candidates. The resulting new lipids often carry unique head groups, linker chemistries, or hydrophobic tail structures to allow more refined control over physicochemical properties including pKa, biodegradability, and membrane fusion propensity.
Second, the roles of helper lipids have been further expanded. On one hand, some phospholipids like DOPE are now commonly used for their fusogenic ability, which is often related to their phase transition capability from lamellar to hexagonal phases and therefore an endosomal escape-promoting effect. On the other hand, variants of cholesterol are also being developed. Oxidized cholesterol and chemically modified cholesterol with additional functional groups have been explored to either offer reactive handles for covalent attachment of targeting ligands or allow more precise tuning of membrane fluidity and fusogenicity.
In addition, more fifth or even sixth functional components are being incorporated into LNPs. These may include hydrophobic small molecules with potentially synergistic activity or photosensitive components to enable on-demand and controlled release of the delivered cargo by light irradiation. Taken together, these developments have enabled the transformation of LNPs from simple nucleic acid encapsulation vehicles into highly complex and programmable gene delivery platforms.
Table 4. Innovation Directions for Functional Lipids in Next-Generation LNPs.
| Component Type | Innovation Objective | Development Directions and Examples |
| Ionizable cationic lipids | Improved targeting, reduced toxicity, enhanced endosomal escape | Development of organ-selective lipids (e.g., lung- or spleen-tropic lipids); incorporation of cleavable ester bonds for biodegradability; exploration of lipid libraries with diverse pKa ranges |
| Helper phospholipids | Dynamic responsiveness and functional synergy | Use of pH- or redox-sensitive phospholipids; selection of naturally membrane-disruptive lipids such as DOPE to support endosomal escape |
| Cholesterol and derivatives | Precise membrane modulation and functional integration | Use of chemically modified cholesterol (e.g., C24 bile acid derivatives) to tune fluidity; development of functionalized cholesterol as anchoring points for ligands |
| Surface-modifying molecules | Mitigation of PEG-related limitations; switchable stealth and targeting | Development of PEG alternatives (e.g., polysaccharides, polysorbates); use of cleavable PEG that detaches in vivo to expose hidden targeting motifs |
Cell- or organ-specific delivery is often required for efficacy improvement and off-target exposure reduction. In terms of targeting mechanism, the design and optimization of ligand-mediated active targeting, as well as "smart" responsive and physical targeting, have advanced significantly in recent years. For active targeting, first-generation ligands are commonly antibodies, peptides, and small molecules such as GalNAc. Nucleic acid aptamers are a new class of ligands that are gaining attention, with potential advantages including high affinity and selectivity toward a target molecule, and amenability to chemical stabilization and surface conjugation to LNPs. In parallel, more advanced active targeting strategies have been explored, where LNPs are engineered to be "smart" in the sense that they can sense surrounding environmental signals and change their behavior accordingly. For example, active targeting ligands are often shielded by neutral helper lipids or siRNA polymers during circulation, and they become exposed only under certain circumstances defined by, e.g., pH or enzymatic activity in a disease-associated microenvironment, enabling selective local activation. Physical targeting has also emerged as a potential direction. An external stimulus, including magnetic field when magnetic nanoparticles are added or focused ultrasound, can be applied to a desired area in close proximity to the target tissue/organ to further promote accumulation and trigger controlled release. Finally, specific organ targeting can also be achieved by tuning intrinsic physicochemical properties of LNPs. This ligand-free approach has attracted significant research interest for its structural simplicity and generality. Systematic lipid screening has enabled the production of LNPs with natural tropism toward certain organs such as the lung, spleen, bone marrow, or muscle, which can then directly and efficiently serve as the desired delivery platform for a wide range of applications.
Table 5. LNP Platform Products for Nucleic Acid Therapeutics.
| Product Category | Description | Price |
| Classic Ionizable Lipid LNP | LNPs composed of standard ionizable lipids, DSPC, cholesterol, and PEG-lipids, designed for fundamental delivery studies of mRNA, siRNA, and other nucleic acids, serving as a benchmark for evaluating basic LNP formulation performance. | Inquiry |
| Vaccine-Oriented Ionizable Lipid LNP | Optimized LNPs for tumor or infectious disease mRNA vaccines, enhancing nucleic acid delivery and immune activation, supporting antigen presentation, cytokine expression studies, and both in vitro and in vivo evaluation of vaccine candidates. | Inquiry |
| Biodegradable Lipid LNP | Formulated with cleavable ester-containing ionizable cationic lipids, these LNPs allow rapid metabolism after delivery, reducing long-term toxicity, and providing an ideal model for studying safety and tolerability in repeated dosing scenarios. | Inquiry |
| Targeted Ligand-Modified LNP | Covalently functionalized LNPs with ligands like GalNAc specifically bind hepatocytes or other target cell receptors, enabling active delivery of siRNA/mRNA and supporting studies of targeting efficiency and tissue-selective nucleic acid uptake. | Inquiry |
| Endosomal Escape-Enhanced LNP | LNPs with increased fusogenic lipid content or cone-shaped lipids optimize endosomal membrane disruption, enhancing cytoplasmic release of nucleic acids, and providing a reference system for studying endosomal escape mechanisms and delivery efficiency. | Inquiry |
The development of large-scale manufacturing technology has been a key enabler for lipid nanoparticles. The production of LNP must be scaled up from laboratory-scale microliter volumes to industrial-scale hundreds or thousands of liters in order to support commercial product supply. The main challenge associated with scale-up is how to tightly control critical nanoparticle properties, including particle size, size distribution, and encapsulation efficiency. Microfluidic technology has emerged as the industry standard. By rapidly mixing two fluid streams, a lipid-containing ethanol phase and an aqueous phase including nucleic acid and PEG-lipid, within microscale channels, microfluidic platforms enable highly reproducible self-assembly and encapsulation. Industrial-scale production has been achieved by developing continuous-flow microfluidic reactors with greatly improved efficiency, stability, scalability, and process control compared with batch-mixing platforms. In parallel, downstream processing has also been improved. For example, tangential flow filtration is now the commonly adopted method for buffer exchange, removal of residual solvents, and separation of unencapsulated nucleic acids, and it delivers higher efficiency and controllability than traditional dialysis methods. Sterile filtration and filling have also been optimized for LNP products to ensure stability and integrity.
Table 6. LNP Formulation, Design, and Evaluation Services.
| Service Name | Description | Price |
| LNP Formulation Service | Using automated microfluidics, hundreds of LNP formulations with novel ionizable lipids, helper phospholipids, cholesterol, and PEG-lipids are screened to optimize encapsulation, particle size, and in vitro transfection performance. | Inquiry |
| Targeted LNP Design Service | SORT technology introduces a fifth functional component to develop LNPs actively targeting liver, spleen, lung, or bone marrow, with in vivo biodistribution validation for organ-specific nucleic acid delivery studies. | Inquiry |
| LNP Synthesis Service | Custom design of ionizable cationic lipids with defined pKa, biodegradability, or membrane fusion properties, supported from chemical synthesis to preliminary biological evaluation based on structure-activity insights. | Inquiry |
| Continuous-Flow Microfluidic Process Development | Development and scale-up of continuous-flow LNP production from lab to commercial scale, ensuring consistent particle size, encapsulation efficiency, and other critical quality attributes. | Inquiry |
| Endosomal Escape Efficiency Quantification Service | Quantitative evaluation of LNP endosomal escape using pH-sensitive dyes and FRET techniques, providing key data to optimize formulations and enhance cytoplasmic nucleic acid release efficiency. | Inquiry |
| LNP Stability Study Service | For thermally sensitive systems like mRNA-LNPs, offering lyoprotectant selection, lyophilization optimization, and long-term stability studies to enable 2–8°C storage and reduce cold chain dependency. | Inquiry |
| Lipid Nanoparticles for Gene Delivery | Tailor lipid nanoparticles for intracellular delivery of DNA or RNA constructs, supporting gene expression, modification, or research applications. | Inquiry |
In summary, LNP manufacturing is moving toward fully automated, continuous, and digitally enabled production. By integrating real-time process analytical technologies for online monitoring of critical parameters such as particle size and pH, and coupling with feedback control systems, production conditions can be dynamically adjusted to ensure consistent product quality across different batches. This will serve as a solid industrial basis for large-scale, high-quality supply of gene delivery products worldwide.
