Lipid nanoparticles (LNPs) are nanoscale delivery systems made from lipids designed to encapsulate and transport therapeutic molecules, such as RNA (CRISPR-RNA, siRNA, or mRNA), DNA, or small molecules, into specific cells or tissues. LNPs are non-viral vectors that allowed the delivery of vaccines such as the SARS-CoV-2 or COVID-19 vaccine during the recent pandemic. LNPs are widely used in medicine and biotechnology because they improve the stability, bioavailability, and targeted delivery of drugs and genetic materials. Formulating conjugated oligonucleotides, including siRNA or tcDNA-based oligonucleotides, into lipid nanoparticles (LNPs) enhances therapeutics' delivery more effectively while ensuring stability.
| 
| 
|
LNPs enable drug delivery by offering a safer and more efficient way to deliver therapeutic drugs, especially for nucleic acid-based treatments. Their versatility and adaptability make them a cornerstone of modern pharmaceutical and biomedical research. The formulation of lipid nanoparticles (LNPs) is a precise process. Correctly formulated LNPs offer stability, functionality, and the ability to encapsulate therapeutic molecules effectively.
Components of Lipid Nanoparticles are:
Ionizable Lipids have a neutral charge at physiological pH but become positively charged in acidic environments, facilitating cargo release into the target cells.
Phospholipids stabilize the lipid bilayer and help maintain the structural integrity of the nanoparticle.
Cholesterol enhances stability and fluidity, contributing to the nanoparticle's robustness.
PEGylated Lipids are lipids modified with polyethylene glycol (PEG), which improves circulation time in the bloodstream by reducing immune recognition.
LNPs can encapsulate hydrophilic (water-soluble) and hydrophobic (water-insoluble) molecules. Surface functionalization of LNPs with specific ligands enables targeted delivery. Since LNPs are biodegradable, drugs encapsulated into LNPs have reduced long-term toxicity.
Typical applications are:
Cancer Therapy: LNPs allow the targeted delivery of chemotherapeutic drugs to tumor cells.
Diagnostics: LNPs can carry imaging agents for disease detection.
Gene Therapy: LNPs enable the delivery of siRNA, mRNA, or CRISPR components for treating genetic disorders.
Vaccines: Modern mRNA vaccines utilize LNPs, such as vaccines for COVID-19 (Pfizer-BioNTech and Moderna).
Table 1: Key Components of LNPs.
| Component | Function | Structure |
| Ionizable Lipids | Essential for encapsulating nucleic acids (e.g., mRNA, siRNA). Becomes positively charged in acidic environments (like endosomes) to facilitate cargo release. Example: DOTMA | 
|
| Phospholipids | Contribute to the structural integrity of the lipid bilayer. Example: DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine). | 
|
| Cholesterol | Improves stability and fluidity. |  |
| PEGylated Lipids | Provide steric stabilization and extend circulation time by reducing immune clearance. Example: DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene glycol). | 
Amino-PEG-DSPE |
| Therapeutic Cargo | Nucleic acids (e.g., mRNA, siRNA, DNA) or small molecule drugs. | 
|
Tabe 2: Formulation Methods.
| Method | Description | Advantages & Disadvantages |
| Microfluidic Mixing | A widely used technique for LNP production. Lipids dissolved in ethanol are mixed with the aqueous phase containing the therapeutic cargo, for example, mRNA in buffer. Rapid mixing induces self-assembly of lipids into nanoparticles. | Scalable and reproducible. Allows fine control over particle size (~50–150 nm). |
| Thin Film Hydration | Lipids dissolved in organic solvent are evaporated to form a thin lipid film. The film is rehydrated with an aqueous solution containing the cargo. The mixture is sonicated or extruded to form nanoparticles. | Simple setup. Less reproducible and harder to scale. |
| Ethanol Injection | Lipids in ethanol are rapidly injected into the aqueous phase containing the cargo under controlled stirring. Nanoparticles form spontaneously at the interface. | Simple process. Less control over particle size |
Table 3: Parameter for Optimization
| Parameter | Optimization | Coditions & Analysis |
| Lipid Ratios | Adjusting the ratios of ionizable lipids, cholesterol, phospholipids, and PEGylated lipids affects stability, encapsulation efficiency, and cell uptake. | pH Conditions: The aqueous phase often has a low pH to promote effective nucleic acid encapsulation. Particle Size: Controlled through lipid-to-cargo ratios, flow rates (in microfluidics), and extrusion pore sizes. |
| Quality Control Metrics | Particle Size and Distribution Encapsulation Efficiency Stability Zeta Potential | Measured by dynamic light scattering (DLS). Assessed using UV-Vis spectroscopy or fluorescence. Monitored over time at storage conditions. Indicates surface charge and stability. |
| Typical LNP Composition Example mRNA Delivery | Ionizable Lipid: 50% Cholesterol: 30% Phospholipid: 10% PEG-lipid: 10% | The formulation process is crucial to ensure the LNPs are optimized for efficient delivery, reduced toxicity, and effective therapeutic outcomes. |
| Analysis and Characterization | LNP purity and composition | Liquid chromatography (LC). |
| LNP formulation stability | Differential Scanning Calorimetry. |
| mRNA | LC and liquid chromatography tandem mass spectrometry (LC-MS). |
| Size | Dynamic Light Scattering |
| Charge | Zeta Potential Analyzer |
| Stability | Transmission and scanning electron microscope |
| Morphology | Atomic force microscope |
| Encapsulation Efficiency (%) | Plate reader Cryogenic electron microscopy |
References
Amici A, Pozzi D, Marchini C, Caracciolo G. The Transformative Potential of Lipid Nanoparticle-Protein Corona for Next-Generation Vaccines and Therapeutics. Mol Pharm. 2023 Nov 6;20(11):5247-5253. [PMC]
Felgner PL, Gadek TR, Holm M, et al. Lipofection: a highly efficient, lipid‐mediated DNA‐transfection procedure. Proc Natl Acad Sci U S A. 1987;84:7413‐7417. [PMC] [PubMed] Synthesis of cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).
Jayaraman M, Ansell SM, Mui BL, Tam YK, Chen J, Du X, Butler D, Eltepu L, Matsuda S, Narayanannair JK, Rajeev KG, Hafez IM, Akinc A, Maier MA, Tracy MA, Cullis PR, Madden TD, Manoharan M, Hope MJ. Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo. Angew Chem Int Ed Engl. 2012 Aug 20;51(34):8529-33. [PMC]
John, R.; Monpara, J.; Swaminathan, S.; Kalhapure, R. Chemistry and Art of Developing Lipid Nanoparticles for Biologics Delivery: Focus on Development and Scale-Up. Pharmaceutics 2024, 16, 131. [mdpi]
Jung HN, Lee SY, Lee S, Youn H, Im HJ. Lipid nanoparticles for delivery of RNA therapeutics: Current status and the role of in vivo imaging. Theranostics. 2022 Oct 24;12(17):7509-7531. [PMC]
Hou, X., Zaks, T., Langer, R., Dong, Y.; Lipid nanoparticles for mRNA delivery. Nat Rev Mater 6, 1078–1094 (2021). [nature]
Ma Y, Li S, Lin X, Chen Y. A perspective of lipid nanoparticles for RNA delivery. Exploration (Beijing). 2024 Apr 15;4(6):20230147. [PMC]
Mehta M, Bui TA, Yang X, Aksoy Y, Goldys EM, Deng W. Lipid-Based Nanoparticles for Drug/Gene Delivery: An Overview of the Production Techniques and Difficulties Encountered in Their Industrial Development. ACS Mater Au. 2023 Aug 21;3(6):600-619. [PMC]
Samaridou E, Heyes J, Lutwyche P. Lipid nanoparticles for nucleic acid delivery: current perspectives. Adv Drug Deliv Rev. 2020;154‐155:37‐63. [PubMed]
Schlich M, Palomba R, Costabile G, Mizrahy S, Pannuzzo M, Peer D, Decuzzi P. Cytosolic delivery of nucleic acids: The case of ionizable lipid nanoparticles. Bioeng Transl Med. 2021 Mar 20;6(2):e10213. [PMC]
Wang R, Xiao R, Zeng Z, Xu L, Wang J. Application of poly(ethylene glycol)-distearoylphosphatidylethanolamine (PEG-DSPE) block copolymers and their derivatives as nanomaterials in drug delivery. Int J Nanomedicine. 2012;7:4185-98. [PMC]
Zalipsky S, Brandeis E, Newman MS, Woodle MC. Long circulating, cationic liposomes containing amino-PEG-phosphatidylethanolamine. FEBS Lett. 1994;353(1):71–74. doi: 10.1016/0014-5793(94)01013-7. [PubMed]
---...---
Bio-Synthesis Inc. is pleased to offer a large variety of oligonucleotides and peptides for a number of research applications, including LNPs, COVID 19 peptides, analysis and vaccine development!
---...---