Genome editing techniques are used to modify DNA sequences for many purposes, including basic genetic research, crop engineering, and the study of disease. Genome editing tools are often large, complex biomolecules that can't readily pass through cell membranes. Efficient and safe delivery methods are thus required. Lipid nanoparticles (LNPs) have been an important solution in this case.
LNPs are spherical vesicles made up of self-assembling, specialized lipids, and are typically between 50 and 100 nm in diameter. Nucleic acids or ribonucleoprotein complexes can be encapsulated by the LNPs for genome editing. In a similar role, LNPs have been used for the delivery of genome editing components.
Compared to viral vectors and other non-viral delivery systems, LNPs offer multiple benefits:
High Delivery Efficiency
LNPs protect genome editing components from degradation by nucleases in the bloodstream. Surface modifications, such as PEGylation, extend circulation time, increasing the likelihood of reaching target cells. Delivery efficiency in liver cells can exceed 70%.
Favorable Safety Profile
Unlike viral vectors, LNPs do not integrate into the host genome, eliminating insertional mutation risks. Their lipid components are metabolizable, and they generally trigger minimal inflammatory responses. Multiple dosing is feasible, supporting scenarios that require repeated genome editing.
Scalable Manufacturing
The manufacturing processes for LNPs have been validated in mRNA vaccine production, allowing rapid scale-up. Production costs decrease with scale, making LNPs suitable for broad applications.
Flexible Payload CapacityA single LNP can simultaneously carry multiple nucleic acids, supporting multi-gene editing. Pre-assembled ribonucleoprotein complexes can also be delivered to accelerate the onset of editing activity.
Fig.1 Comparison of LNP and AAV safety profiles in genomic editing (BOC Sciences Original).
Table 1. Key Advantages of LNPs in Genome Editing.
| Advantage | Performance | Comparison with Viral Vectors |
| Delivery Efficiency | Liver-targeted delivery 70–90% | Similar to lentivirus but without integration risk |
| Safety | No genome integration, metabolizable | AAV integration risk is low but present |
| Immunogenicity | Low inflammation, suitable for repeated dosing | Viral vectors often trigger immune responses, limiting repeat dosing |
| Payload Capacity | 4–10 kb mRNA plus multiple guide RNAs | AAV packaging limited to ~4.7 kb |
| Production Speed | Process development to production in 2–4 weeks | Viral vector prep requires 6–8 weeks |
| Cost Control | < $10 per dose | Viral vectors typically > $100 per dose |
LNPs encapsulate negatively charged nucleic acids through electrostatic interactions. A typical formulation contains four key lipid types:
Table 2. Typical LNP Components and Functions.
| Lipid Type | Representative Molecules | Mass Fraction | Core Function |
| Ionizable Lipids | DLin-MC3-DMA, SM-102 | 35–50% | Nucleic acid binding, endosomal escape |
| Helper Phospholipids | DSPC, DOPE | 10–20% | Stabilize bilayer, promote membrane fusion |
| Structural Lipids | Cholesterol | 35–45% | Fill gaps, enhance particle stability |
| Surface-modifying Lipids | PEG2000-DMG | 1.5–3% | Prolong circulation, reduce aggregation |
LNPs are formed by rapidly mixing lipid and nucleic acid solutions, with the nucleic acids encapsulated in the internal aqueous phase. Particle size can be precisely controlled by adjusting flow rates and lipid ratios.
LNPs are usually taken up by cells through endocytosis. The apoE proteins present on the particle surface can bind to LDL receptors present on the cell surface to mediate clathrin-mediated uptake. The endocytosis is more efficient for liver cells because of their high LDL receptor expression.
After cellular uptake, LNPs get entrapped in early endosomes. Endosomes then mature and acidify (pH 7.4 → 5.5) to facilitate endosomal escape of the nucleic acid. Uptake usually happens within 30–120 min. The efficiency of cellular uptake is largely determined by particle size and surface charge; spherical particles with size 50–100 nm and mild cationic charge (1.5–3.5) are found to be optimal.
The key event of delivery is the endosomal escape of the payload. Ionizable lipids get protonated under acidic endosomal pH, which increases the positive charge on the particle. This results in the destabilization of the endosomal membrane and the release of the payload into the cytoplasm. The escape is also thought to occur by the "proton sponge effect," in which osmotic swelling upon protonation of the ionizable lipids results in endosomal rupture.
This event is usually achieved within 1–4 h of cellular uptake. The released mRNA is directly available for translation in the cytoplasm by ribosomes to form functional proteins that further assemble to form the desired protein complex. Alternatively, delivery as a pre-formed ribonucleoprotein complex can lead to direct entry of RNA into the nucleus bypassing translation. This usually takes place within 30 min of release. The escape efficiency of LNPs can be increased by adjusting lipid pKa (6.2–6.8), addition of helper phospholipids to enhance membrane perturbation, and altering PEG density.
Tissue-specific delivery can be done by:
Active targeting: Modification of the particle surface with targeting ligands such as GalNAc for liver hepatocytes, folate for tumor cells, etc.
Passive targeting: The natural architecture of tissues can be exploited, such as fenestrated liver sinusoidal endothelium allows natural accumulation of the particles.
Editing efficiency is influenced by a wide range of factors. Guide RNA design is crucial for target specificity, while codon optimization can be performed for efficient translation. Nuclear localization signals also influence the nuclear import of the particle.
Table 3. Strategies to Enhance LNP Genome Editing Efficiency.
| Optimization | Method | Efficiency Improvement |
| sgRNA design | Select high-specificity sequences, avoid off-targets | 3–5× specificity increase |
| Protein optimization | Dual nuclear localization signals, codon optimization | 2–3× editing efficiency increase |
| Particle formulation | Adjust N/P ratio 3–6, lipid phase transition<37°C | 50–80% increase in delivery efficiency |
| Endosomal escape | Use ionizable lipids with pKa 6.2–6.8 | Escape rate increased from 20% → 40% |
| Targeting modification | Add 0.5–2% ligand-conjugated lipid | 5–10× increase in specific uptake |
Off-target editing can be monitored using genome-wide detection techniques. High-fidelity protein variants can further minimize unintended edits. For transient applications, mRNA is preferred due to rapid degradation within 72 hours, reducing off-target risks. In plants, LNP-based delivery has successfully edited wheat and maize protoplasts, opening new avenues for crop improvement.
BOC Sciences delivers customizable lipid nanoparticle solutions designed to enhance stability, delivery efficiency, and functional performance.
The effectiveness of LNPs is strongly determined by their lipid composition, particularly the ionizable lipids. Although established ionizable lipids have been used effectively, the need for improved alternatives that can be applied across a wider tissue and cell type range has driven efforts to design libraries of new ionizable lipids with new chemical backbones. Libraries of candidate lipids have been designed and created via rational design and combinatorial chemistry.
These lipids are optimized to meet multiple objectives, including enhanced stability in the blood circulation, improved endosomal escape in a broad range of cell types, and minimized intrinsic immunogenicity. For instance, ionizable lipids with biodegradable bonds (such as ester linkages) can be rapidly degraded after their delivery task is done, and thus could improve safety. Formulations developed for non-liver tissues such as lung, spleen, or eye have also exhibited unique advantages likely linked to differential interaction with these tissue microenvironments.
Table 4. Lipid Nanoparticle Products for Genome Editing Delivery.
| Product | Description | Price |
| Ionizable Lipid–Based Genome Editing LNP | Ionizable lipid–based LNP enabling efficient nucleic acid encapsulation and endosomal escape for genome editing research. | Inquiry |
| PEGylated Long-Circulating LNP | PEG-modified LNP designed to extend circulation time and improve stability for gene editing delivery. | Inquiry |
| mRNA-Loaded Editing LNP | LNP optimized for mRNA-based editing payloads, supporting transient expression and controlled activity. | Inquiry |
| Multi-Component Co-encapsulation LNP | LNP supporting simultaneous delivery of multiple nucleic acid or protein components for complex editing systems. | Inquiry |
| GalNAc-Modified Targeting LNP | GalNAc-functionalized LNP enhancing selective delivery efficiency in hepatocyte-focused research models. | Inquiry |
| Size-Tuned Microfluidic LNP | Microfluidic-produced LNP with precise size control for formulation optimization and mechanistic studies. | Inquiry |
| pH-Responsive Endosomal Escape LNP | pH-responsive LNP designed to enhance endosomal membrane disruption and intracellular payload release. | Inquiry |
Despite preferential accumulation of systemically administered LNPs in the liver (often via apolipoprotein-mediated pathways), tissue-selective targeting still requires surface functionalization of LNPs with targeting ligands that are covalently conjugated or physically inserted to the surface. One successful and well-explored approach utilizes N-acetylgalactosamine (GalNAc) ligands for hepatocyte-specific targeting.
GalNAc binds to asialoglycoprotein receptors that are selectively overexpressed in liver cells, and thus, GalNAc-based LNPs are taken up almost exclusively by hepatocytes, which has been harnessed for systemic delivery of small nucleic acid therapeutics and is being adapted for genome editing nucleoprotein complexes.
A number of other targeting ligands are being developed for other tissues, often based on antibody fragments, peptides, or aptamers that target specific surface markers or receptors. These function as "postal codes" that allow LNPs to navigate complex in vivo environments and enter target cells, which can include tumor cells, immune cells, pulmonary endothelial cells, or neurons. Also emerging are strategies for "stealth" targeting, in which LNPs have conditionally activated targeting, i.e., they remain non-targeting and inert until they reach certain microenvironments, like acidic tumor pH or in the presence of certain enzymes, which may help improve targeting specificity and limit off-target systemic exposure.
As mentioned previously, many of the key physicochemical properties of LNPs that are known to affect their in vivo fate and function, including their biodistribution, cellular uptake, and eventual genome editing efficiency are determined by their particle size and surface charge (zeta potential).
Particle Size Optimization: Particle size of LNPs is known to be a key determinant in their in vivo distribution and cellular uptake mechanisms. For example, smaller LNPs in the size range of 30–50 nm may have better penetrance across dense tissue barriers or entry into certain cell types, while larger LNPs in the size range of 80–150 nm may be more rapidly captured by the mononuclear phagocyte system and thus have a higher liver and spleen accumulation. Microfluidic-based production methods can be tuned to have a tighter control over particle size, which can allow fine-tuning and optimization of particle size for desired tissue targeting. For example, an effective pulmonary delivery vehicle may require an LNPs of a particular size range to achieve deposition in the alveolar space.
Charge Optimization: Surface charge affects not only particle stability in the circulation, interactions with cell membranes, and even its protein corona formation. Mildly positive, neutral or even slightly negative LNPs are more stable in the circulation. However, a moderate level of positive charge may be desirable for initial interactions with negatively charged cell membranes to facilitate cellular uptake. However, the key is to tune the particle surface properties to be sufficiently interactive to allow cellular uptake without high levels of nonspecific binding, aggregation, and clearance. Modern LNP designs incorporate "smart" charge switching by carefully tuning the ratio of ionizable lipids and PEGylated lipids, which allows particles to be overall neutral while circulating in blood but become positively charged in the lower pH environment of endosomes.
Table 5. Lipid Nanoparticle Solutions for Gene and Nucleic Acid Delivery.
| Service Category | Description | Price |
| Lipid Nanoparticle Formulation | Design and optimize lipid nanoparticle compositions to achieve efficient delivery, stability, and controlled release for various therapeutic and research applications. | Inquiry |
| Lipid Nanoparticle Manufacturing | Scalable production of lipid nanoparticles with consistent quality, ensuring reproducibility and suitability for preclinical studies or industrial applications. | Inquiry |
| Lipid Nanoparticle Encapsulation | Efficiently load nucleic acids, proteins, or small molecules into lipid nanoparticles while preserving bioactivity and ensuring precise release in target cells. | Inquiry |
| Lipid Nanoparticles Synthesis | Chemical synthesis and assembly of lipids to produce nanoparticles with defined size, composition, and properties for research and therapeutic use. | 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 |
| Lipid Nanoparticle Stability | Assess and optimize lipid nanoparticle stability under storage and physiological conditions to ensure long-term integrity and consistent performance. | Inquiry |
| Lipid Nanoparticle Characterization | Analyze lipid nanoparticles' size, charge, morphology, and encapsulation efficiency to confirm quality, reproducibility, and functionality for research or therapeutic use. | Inquiry |
The efficiency of LNP-mediated genome editing ultimately depends on overcoming intracellular barriers, with endosomal escape recognized as the rate-limiting step.
Enhancing Endosomal Escape: Next-generation ionizable lipids are designed to rapidly and effectively disrupt endosomal membranes under acidic conditions. Structure-activity relationship studies examine how lipid molecular features, head group size, linker type, and tail unsaturation, affect membrane perturbation, guiding rational lipid design.
Facilitating Nuclear Delivery: For editing post-mitotic or differentiated cells, efficient transport of genome editing complexes from the cytoplasm into the nucleus is critical. Strategies include co-encapsulation of nuclear import-enhancing peptides or chemical conjugation to promote nuclear entry. Alternatively, delivery can exploit transient nuclear membrane breakdown during cell division, which requires precise control of component release kinetics.
Coordinated Multi-Component Delivery: Precise genome modifications often require co-delivery of multiple components, such as editing proteins, guide RNAs, and DNA repair templates. LNPs can co-encapsulate these components, but formulations must be optimized to ensure proper assembly after release, avoiding degradation or dilution that would compromise editing efficiency.
One major limitation of current LNPs is their intrinsic liver tropism. While advantageous for targeting liver-associated conditions, this property restricts applications in other organs such as the heart, muscles, nervous system, and lungs. Expanding therapeutic reach depends on developing novel delivery systems.
Future breakthroughs will rely on creating advanced ionizable lipid libraries through high-throughput screening and rational design, aimed at escaping liver capture and targeting specific tissues such as pulmonary endothelial cells, cardiomyocytes, or select neurons. Some studies have identified LNP formulations with natural tropism toward lungs or spleen. In parallel, active targeting strategies are becoming more sophisticated, moving beyond traditional liver-targeting ligands to include molecules that recognize specific cell surface markers—for example, peptides or antibody fragments that interact with blood-brain barrier transporters to enable central nervous system delivery.
Safety requires precision. Off-target activity is when the editing machinery alters genomic sites other than the intended target and can lead to unwanted mutations and cellular dysfunction. Avoiding this potential outcome requires several layers of protection. First, optimize the guide sequence with improved computational algorithms for maximum specificity.
Second, the delivery of high-fidelity editing proteins is key. Engineered high-fidelity variants that bind less strongly to imperfectly matched sequences can often be obtained with high target efficiency but greatly reduced off-target activity. Third, transient delivery methods—such as using RNA instead of DNA—prevent the editing machinery from lingering inside of cells. In the future, new editors that avoid double-strand cleavage of DNA, such as base editors or prime editors, may further decrease off-target activity by design.
However, many therapeutic applications require specific repair of genes (e.g., homology-directed insertion of the correct sequence), rather than disruption of a gene. It has been difficult to reliably achieve high-efficiency repair in vivo, especially in non-dividing cells, because the cell's default is to use an error-prone repair pathway to repair double-strand breaks.
Approaches to increase efficiency have included optimizing the design of the donor DNA template and co-delivery to ensure that it is delivered to the nucleus at the same time as the editing components. In addition, small molecules or protein cofactors could be co-delivered to help promote precise repair pathways or temporarily inhibit error-prone repair pathways. Newer editing technologies, such as prime editing, allow precise modification of the sequence without inducing a double-strand break.
Genome editing has also introduced a host of ethical and societal issues that need to be taken into account, which are amplified by the possibility of using highly efficient LNP delivery. There is broad consensus that germline editing is unacceptable given that the changes are heritable and the long-term implications are difficult to predict. Somatic genome editing should be limited to therapeutic or preventative purposes and be subject to appropriate ethical oversight. Other issues include ensuring informed consent, long-term follow-up on safety and possible off-target side effects, and equitable access to these novel therapies.
Future developments of LNP-mediated genome editing are likely to take several of the following directions:
Smart and modular LNPs: Development of "intelligent" nanoparticles that are sensitive to specific biological cues (e.g., disease-associated enzymes, pH) for spatiotemporal control over the release of the editing payload.
Iterative improvement of editing tools: Combination of LNP platforms with next-generation editors that have higher precision, efficiency, and safety to target a broader range of conditions.
Manufacturing and accessibility: Optimization, automation, and scale-up of LNP manufacturing for broader patient access by bringing down the costs.
Biological evaluation of long-term effects: Comprehensive preclinical assessment of prolonged efficacy, immune responses, and organ-level effects of in vivo genome editing.
Table 6. Key Challenges of LNP-Mediated Genome Editing and Strategies to Address Them.
| Core Challenge | Impact | Key Strategies & Future Directions |
| Tissue Targeting Limitations | Systemically administered LNPs predominantly accumulate in the liver, limiting delivery to other tissues (brain, lungs, muscles) | High-throughput screening of novel ionizable lipids for tissue-specific tropism; development of active targeting ligands (peptides, aptamers) for cell-specific recognition; exploration of local administration routes (e.g., intrathecal or intravitreal injections) |
| Off-Target Effects | Editing tools may act at unintended genomic sites, causing potential harmful mutations | Use high-fidelity protein variants; optimize guide sequences via computational algorithms; employ transient delivery methods to shorten active window; transition to editors that do not require double-strand breaks |
| Low In Vivo Precision | Homology-directed repair efficiency is low in somatic, especially non-dividing, cells | Optimize donor DNA template design and co-delivery; regulate cellular repair pathways with auxiliary factors; prioritize editing systems that do not rely on double-strand breaks |
| Safety and Ethical Considerations | Long-term safety unknown; germline editing risks; equitable access concerns | Adhere to strict ethical boundaries for somatic editing; establish long-term monitoring systems; standardize and scale production to reduce costs and promote fairness |
By progressively addressing these scientific and technological challenges within a robust ethical framework, LNP-mediated genome editing has the potential to transform medical practice, offering curative possibilities for millions of patients with genetic disorders, cancer, and other intractable diseases.
