RNA therapeutics, such as messenger RNA (mRNA) and small interfering RNA (siRNA), have emerged as a promising class of biologics that can modulate gene expression and protein synthesis in a specific and controlled manner. RNA therapeutics have the potential to treat a wide range of diseases, such as cancer, infectious diseases, genetic disorders, and autoimmune diseases, by targeting the underlying molecular mechanisms. However, RNA therapeutics also face significant challenges in terms of stability, delivery, and safety. RNA molecules are highly susceptible to degradation by nucleases in the biological environment and have poor membrane permeability and cellular uptake. Moreover, RNA therapeutics can induce unwanted immune responses and off-target effects, which may compromise their efficacy and safety.
To overcome these challenges, various types of nanocarriers have been developed to protect and deliver RNA therapeutics to the desired target cells and tissues. Among them, lipid nanoparticles (LNPs) are one of the most widely used and clinically advanced nanocarriers for RNA delivery. LNPs are self-assembled nanostructures composed of lipids, which can encapsulate RNA molecules within their hydrophobic core or associate them with their cationic surface. LNPs can shield RNA molecules from degradation, enhance their transmembrane transport and intracellular trafficking, and reduce their immunogenicity and toxicity. LNPs have demonstrated remarkable success in the development of mRNA-based vaccines for COVID-19 as well as siRNA-based therapeutics for various diseases.
LNPs are nanoparticles composed of lipids, which can encapsulate RNA molecules within their hydrophobic core or associate them with their cationic surface. LNPs can be classified into different types according to the composition and structure of the lipids, such as solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), and ionizable cationic lipid nanoparticles (iLNPs). The comparison and evaluation of different types of LNPs for RNA delivery can be based on several criteria, such as formulation, physicochemical properties, biological properties, and therapeutic performance. Based on the current literature, iLNPs are the most promising LNPs for RNA delivery, as they have demonstrated the highest efficacy and safety in preclinical and clinical studies. However, further optimization and investigation of iLNPs are still needed to address the remaining issues and challenges.
| Type | Components | Advantages | Disadvantages | Preparation Methods | Applications |
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| SLNs | A solid lipid core stabilized by a surfactant layer |
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| NLCs | A mixture of solid and liquid lipids |
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Similar methods as SLNs, but with different lipid ratios and temperatures |
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| iLNPs | Ionizable cationic lipids, which can bind to RNA molecules through electrostatic interactions |
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Table 1. Comparison of different types of LNPs for RNA delivery
LNPs face many challenges in terms of stability and delivery efficiency, which depend on various factors, such as the physicochemical properties of LNPs, physiological conditions, and biological barriers.
The physicochemical properties of LNPs, such as size, shape, charge, surface modification, etc., play a crucial role in determining the stability and delivery efficiency of LNPs. The size of LNPs affects their stability, circulation time, biodistribution, and cellular uptake. Generally, smaller LNPs have higher stability, longer circulation times, broader biodistribution, and higher cellular uptake than larger LNPs. However, the size of the LNPs should also match the size of the RNA molecules to ensure optimal encapsulation efficiency and release kinetics. The shape of LNPs affects their stability, diffusion, and interaction with cells. Spherical LNPs are more stable and diffuse faster than non-spherical LNPs, while non-spherical LNPs may have higher interaction and internalization with cells than spherical LNPs. The charge of LNPs affects their stability, aggregation, serum protein binding, and cellular uptake. Cationic LNPs have higher stability, lower aggregation, lower serum protein binding, and higher cellular uptake than anionic or neutral LNPs, but they may also induce more immunogenicity and toxicity. The surface modification of LNPs affects their stability, stealth, targeting, and endosomal escape. Polyethylene glycol (PEG) is a common surface modifier that can increase the stability and stealth of LNPs, prolong their circulation time, and reduce their immunogenicity and toxicity. However, PEG may also decrease the encapsulation efficiency and transfection efficiency of LNPs and induce anti-PEG antibodies. Targeting ligands, such as antibodies, peptides, or aptamers, can be conjugated to the surface of LNPs to enhance their specificity and affinity to the target cells or tissues. However, targeting ligands may also increase the complexity and cost of LNPs, and affect their stability and biodistribution. Endosomal escape agents, such as ionizable lipids, fusogenic peptides, or pH-sensitive polymers, can be incorporated into the LNPs to facilitate the release of RNA molecules from the endosomes to the cytosol. However, endosomal escape agents may also increase the toxicity and immunogenicity of LNPs and affect their stability and encapsulation efficiency.
The physiological conditions, such as pH, temperature, enzymes, serum proteins, etc., affect the stability and delivery efficiency of LNPs. The pH of the biological environment varies from acidic to neutral, depending on the location and function of the organs or tissues. LNPs should be stable at different pH values and be able to respond to pH changes to release the RNA molecules at the appropriate site. The temperature of the biological environment is usually around 37 °C but may fluctuate due to inflammation or fever. LNPs should be stable at different temperatures and be able to maintain their integrity and functionality. The enzymes of the biological environment, such as nucleases, lipases, proteases, etc., can degrade the RNA molecules, the lipids, or the surface modifiers of LNPs. LNPs should be resistant to enzymatic degradation and be able to protect the RNA molecules from the enzymatic attack. The serum proteins of the biological environment, such as albumin, immunoglobulins, and complement, can bind to the surface of LNPs, forming a protein corona that can affect the stability, biodistribution, cellular uptake, and immunogenicity of LNPs. LNPs should be able to avoid or regulate protein corona formation and be able to maintain their stealth and targeting properties.
The biological barriers, such as cell membrane, endosomal escape, lysosomal degradation, etc., affect the stability and delivery efficiency of LNPs. The cell membrane is a lipid bilayer that separates the cytoplasm from the extracellular environment and regulates the transport of molecules into and out of the cell. LNPs should be able to cross the cell membrane, either by passive diffusion, endocytosis, or membrane fusion, and be able to reach the target cells or tissues. Endosomal escape is a process that allows the LNPs and the RNA molecules to escape from the endosomes, which are membrane-bound vesicles that internalize the extracellular materials. LNPs should be able to escape from the endosomes, either by disrupting the endosomal membrane or by exploiting the endosomal sorting pathways, and be able to release the RNA molecules into the cytosol. Lysosomal degradation is a process that degrades the LNPs and the RNA molecules in the lysosomes, which are membrane-bound organelles that contain various hydrolytic enzymes. LNPs should be able to avoid or overcome lysosomal degradation, either by escaping from the lysosomes or by resisting the lysosomal enzymes, and be able to preserve the integrity and activity of the RNA molecules.
To enhance the stability and delivery efficiency of LNPs, various strategies have been developed, such as optimization of the formulation, combinations of different types of LNPs, co-delivery of different types of RNA molecules, and external stimuli. The optimization of the formulation involves the adjustment of the composition, ratio, and concentration of the lipids, the RNA molecules, and the surface modifiers, as well as the selection of the preparation methods, to achieve the optimal physicochemical and biological properties of LNPs. The combination of different types of LNPs involves the mixing or layering of different types of lipids, such as cationic, anionic, neutral, or ionizable lipids, to achieve the synergistic effects of LNPs, such as enhanced stability, transfection efficiency, and endosomal escape. The co-delivery of different types of RNA molecules involves the encapsulation or association of different types of RNA molecules, such as mRNA, siRNA, miRNA, or lncRNA, to achieve the multiple functions of LNPs, such as protein expression, gene silencing, gene regulation, or gene editing. The external stimuli involve the application of physical, chemical, or biological stimuli, such as light, heat, ultrasound, magnetic field, pH, enzymes, or antibodies, to trigger the release of RNA molecules from LNPs at the desired site and time.
There are some common LNP preparation techniques that are crucial for determining their physicochemical properties, biological properties, and therapeutic performance, such as high-pressure homogenization, microemulsion, solvent emulsification-evaporation, ethanol dilution, microfluidic mixing, and nanoprecipitation. The comparison and evaluation of different preparation techniquesare based on several criteria, such as simplicity, scalability, energy consumption, temperature, loading capacity, encapsulation efficiency, stability, transfection efficiency, and safety. According to the current literature, microfluidic mixing is the most promising preparation technique as it has demonstrated the highest performance and quality in terms of physicochemical properties, biological properties, and therapeutic performance. However, further optimization and investigation of microfluidic mixing are still needed to address the remaining issues and challenges.
| Technique | Advantages | Disadvantages | Examples |
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| High-pressure homogenization | Simple, scalable, and reproducible | Require high energy input and may cause the degradation of sensitive cargo | LNPs for mRNA delivery |
| Microemulsion | Allows precise control of particle size and composition | Require organic solvents and surfactants that may be toxic or immunogenic | LNPs for siRNA delivery |
| Solvent emulsification-evaporation | Suitable for hydrophobic cargo and high drug loading | Require organic solvents and multiple steps of emulsification and evaporation | SLNs for curcumin delivery |
| Ethanol dilution | Rapid, simple, and mild | May cause the aggregation or precipitation of LNPs | LNPs for pDNA delivery |
| Microfluidic mixing | Enables high-throughput screening and optimization of LNPs | Require specialized equipment and expertise | LNPs for mRNA and siRNA delivery |
| Nanoprecipitation | Solvent-free, one-step, and easy to scale up | Difficult to control particle size and stability | NLCs for doxorubicin delivery |
Table 2. Comparison of preparation techniques for LNPs
LNPs have demonstrated remarkable success in the delivery of various types of RNA molecules for a wide range of biomedical applications. For example, LNPs are the most widely used and clinically advanced nanocarriers for RNA vaccines, especially mRNA vaccines. On the one hand, LNPs can protect mRNA molecules from degradation, enhance their transfection efficiency, and facilitate their endosomal escape. On the other hand, LNPs also modulate the immune response by activating innate immune receptors, such as toll-like receptors (TLRs) and stimulators of interferon genes (STING). LNPs have been successfully applied for developing mRNA vaccines against various infectious diseases, such as influenza, Zika, rabies, and COVID-19.
However, LNPs also face many challenges in terms of safety and toxicity, which depend on various factors, such as the physicochemical properties of LNPs, physiological conditions, and biological barriers. The safety and toxicity issues of LNPs are mainly related to their immunogenicity, biodistribution, and elimination. Immunogenicity refers to the ability of LNPs to induce immune responses, which can be beneficial or detrimental, depending on the type and intensity of the response. Biodistribution refers to the distribution of LNPs in the body, which can affect their efficacy and safety. LNPs should be able to reach the target cells or tissues, but they may also accumulate in the non-target organs or tissues, causing adverse reactions or toxicity. Elimination refers to the clearance of LNPs from the body, which can affect their persistence and safety. LNPs should be able to be eliminated from the body, either by biodegradation, excretion, or metabolism, to avoid long-term accumulation or toxicity. However, LNPs may also be retained in the body, causing chronic inflammation or immunosuppression.
