Messenger RNA (mRNA) therapy and vaccination are emerging fields of biotechnology that aim to deliver mRNA molecules to specific cells in the body, where they can be translated into functional proteins for therapeutic or prophylactic purposes. Compared to conventional drugs and vaccines, mRNA-based approaches offer several advantages, such as high specificity, versatility, safety, and scalability. However, one of the major challenges of mRNA delivery is to protect the mRNA from degradation and facilitate its cellular uptake and endosomal escape. To overcome this challenge, lipid nanoparticles (LNPs) have been developed as efficient and biocompatible delivery systems for mRNA.
LNPs are self-assembled structures composed of a lipid bilayer enclosing an aqueous core, where the mRNA molecules are encapsulated. Among the various types of lipids that can be used to form LNPs, ionizable and cationic lipids play a crucial role in mediating mRNA delivery. These lipids can interact with the negatively charged mRNA and form complexes that are stable at physiological pH, but can release the mRNA upon encountering the acidic environment of the endosomes. Moreover, these lipids can also modulate the immune response to the delivered mRNA, either by enhancing or suppressing the activation of innate immune pathways.
One of the major challenges of mRNA therapy and vaccination is to deliver the mRNA molecules to the target cells in the body, where they can be translated into functional proteins. mRNA delivery faces multiple biological barriers, such as degradation by RNases, clearance by the immune system, and low permeability across the cell membrane. To overcome these barriers, various strategies have been developed to protect and transport mRNA, such as chemical modification, encapsulation in lipid nanoparticles (LNPs), and electroporation of antigen-presenting cells (APCs).
Chemical modification of mRNA involves replacing some of the nucleotides with modified ones, such as pseudouridine, 2'-O-methyl, or N1-methyl-pseudouridine, to increase the stability and reduce the immunogenicity of mRNA. These modifications can also enhance the translation efficiency and protein expression of mRNA by improving its folding and interaction with the ribosome. However, chemical modification alone is not sufficient to ensure effective mRNA delivery, and it may also introduce unwanted side effects, such as reduced activity, altered specificity, or increased toxicity.
Fig.1 Delivery and structural elements of mRNA therapeutics. 1
LNPs are self-assembled structures composed of a lipid bilayer enclosing an aqueous core, where the mRNA molecules are encapsulated. LNPs can protect mRNA from degradation and facilitate its cellular uptake and endosomal escape. LNPs are composed of four types of lipids—a helper lipid, a fusogenic lipid, cholesterol, and an ionizable or cationic lipid. The ionizable or cationic lipid is the key component that mediates the interaction between the LNP and the mRNA, as well as the LNP and the cell membrane. The ionizable lipid has a pKa value that allows it to be protonated at acidic pH and deprotonated at neutral pH. This property enables the LNP to form stable complexes with the negatively charged mRNA at physiological pH and to release the mRNA upon encountering the acidic environment of the endosomes. The cationic lipid has a permanent positive charge that can bind to the mRNA and the cell membrane, but it is more likely to cause cytotoxicity and immunogenicity than the ionizable lipid.
Electroporation of APCs is another strategy to deliver mRNA, especially for cancer immunotherapy. APCs are immune cells that can present antigens to T cells and activate them. Electroporation is a technique that uses electric pulses to create temporary pores in the cell membrane, allowing the mRNA to enter the cell. The mRNA can then be translated into antigens and displayed on the surface of the APCs, stimulating the T cell response against the tumor cells. Electroporation of APCs has the advantage of inducing a strong and specific immune response, but it also has some limitations, such as the need for ex vivo manipulation, the low efficiency and viability of the transfected cells, and the potential risk of autoimmunity.
The design and optimization of LNPs as mRNA delivery systems is a complex and multifactorial process that involves the selection and combination of different types of lipids, the determination of their optimal molar ratios, and the adjustment of various process parameters, such as the mixing speed, temperature, and pH. The goal of LNP design and optimization is to achieve high encapsulation efficiency, stability, transfection efficiency, and biocompatibility of the LNPs while minimizing their toxicity and immunogenicity.
One of the most critical components of LNPs is the ionizable or cationic lipid, which determines the interaction between the LNP and the mRNA as well as the LNP and the cell membrane. The ionizable lipid has a pKa value that allows it to be protonated at acidic pH and deprotonated at neutral pH. This property enables the LNP to form stable complexes with the negatively charged mRNA at physiological pH and to release the mRNA upon encountering the acidic environment of the endosomes. The cationic lipid has a permanent positive charge that can bind to the mRNA and the cell membrane, but it is more likely to cause cytotoxicity and immunogenicity than the ionizable lipid.
There are various types of ionizable and cationic lipids that have been developed and tested for mRNA delivery, such as DOTAP, DOTMA, DOPE, DLin-MC3-DMA, DLin-KC2-DMA, and DLin-6G. Each type of lipid has different physicochemical properties, such as hydrophobicity, head group size, charge density, and pKa value, that affect LNP formation, stability, and performance. Therefore, the choice of the ionizable or cationic lipid is a key factor in LNP design and optimization.
Another important component of LNPs is the PEG lipid, which is usually conjugated to a hydrophilic polymer, such as polyethylene glycol (PEG), to form a protective layer on the surface of the LNPs. The PEG lipid can increase the stability and circulation time of the LNPs, as well as reduce their aggregation and recognition by the immune system. However, the PEG lipid can also reduce the cellular uptake and endosomal escape of the LNPs, as well as induce an anti-PEG antibody response that can compromise the efficacy and safety of repeated administrations. Therefore, the optimal amount and type of PEG lipid should be carefully balanced in LNP design and optimization.
The other two components of LNPs are the helper lipid and the cholesterol, which are usually neutral or zwitterionic lipids that can enhance the stability and fluidity of the LNP membrane. The helper lipid can also facilitate the fusion of the LNP with the cell membrane and the endosomal membrane, thus promoting mRNA delivery. The cholesterol can also improve the interaction between the ionizable or cationic lipid and the mRNA, thus increasing the encapsulation efficiency. The optimal molar ratios of the helper lipid and the cholesterol depend on the type and amount of the ionizable or cationic lipid and the PEG lipid, as well as the size and charge of the mRNA.
The process parameters of LNP formation, such as the mixing speed, temperature, and pH, can also affect the LNP characteristics and performance. For example, higher mixing speeds can result in smaller and more uniform LNPs, but they can also cause shear stress and damage to the mRNA. Higher temperature can increase the fluidity and permeability of the LNP membrane, but it can also reduce the stability and shelf life of the LNPs. Higher pH can increase the ionization and charge of the ionizable lipid, but it can also reduce the encapsulation efficiency and stability of the LNPs.
To optimize the LNP design and process parameters, various experimental and computational methods have been developed and applied, such as factorial design, response surface methodology, artificial neural networks, and machine learning. These methods can help identify the optimal combinations of the LNP components and process parameters that can achieve the desired LNP characteristics and performance, as well as the interactions and effects of the different factors on LNP formation and function.
Fig.2 Representative LNP structure and ionizable lipids used in preclinical research and clinical trials. 2
The ionizable and cationic lipids used for mRNA delivery not only mediate the interaction between the LNP and the mRNA, as well as the LNP and the cell membrane, but also modulate the immune response to the delivered mRNA. Depending on the type and amount of the lipid, the LNP can either enhance or suppress the activation of innate immune pathways, which can have different implications for mRNA therapy and vaccination.
The innate immune system is the first line of defense against foreign invaders, such as viruses and bacteria. It consists of various types of immune cells, such as macrophages, dendritic cells, natural killer cells, and mast cells, as well as soluble factors, such as cytokines, chemokines, and complement proteins. The innate immune system recognizes the molecular patterns associated with pathogens or danger signals, such as double-stranded RNA, single-stranded RNA, CpG DNA, and lipopolysaccharide, through a set of receptors, such as toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs).
The activation of these receptors triggers a cascade of signaling events that lead to the production of pro-inflammatory and anti-viral mediators, such as interferons, interleukins, and tumor necrosis factor, as well as the maturation and migration of antigen-presenting cells (APCs), such as dendritic cells, to the lymph nodes, where they can activate the adaptive immune system. The adaptive immune system consists of T cells and B cells, which can recognize and eliminate specific antigens through the expression of antigen receptors, such as T cell receptors and antibodies. The adaptive immune system can also generate immunological memory, which can provide long-term protection against reinfection.
The ionizable and cationic lipids used for mRNA delivery can interact with the innate immune receptors and modulate their activation and signaling. For example, some ionizable lipids, such as DLin-MC3-DMA and DLin-KC2-DMA, can bind to TLR7 and TLR8, which are expressed in endosomes and recognize single-stranded RNA, and induce the production of type I interferons and pro-inflammatory cytokines. This can have a beneficial effect for mRNA vaccines, as it can provide an adjuvant effect that can enhance the antigen-specific immune response and the generation of immunological memory. However, this can also have a detrimental effect on mRNA therapy, as it can cause unwanted inflammation and toxicity, as well as reduce the protein expression and stability of the mRNA.
Other ionizable lipids, such as DLin-6G, can bind to TLR3, which is expressed in endosomes and recognizes double-stranded RNA, and induce the production of type III interferons and anti-inflammatory cytokines. This can have a beneficial effect for mRNA therapy, as it can reduce inflammation and toxicity as well as increase the protein expression and stability of the mRNA. However, this can also have a detrimental effect on mRNA vaccines, as it can suppress the antigen-specific immune response and the generation of immunological memory.
Fig.3 LNP-mRNA vaccines immune response process. 3
Therefore, the choice and optimization of the ionizable and cationic lipids for mRNA delivery should consider their immune activation and modulation effects and tailor them according to the desired outcome of mRNA therapy or vaccination. Moreover, the immune activation and modulation effects of the ionizable and cationic lipids may also depend on other factors, such as the type and amount of the mRNA, the target cell type and tissue, the route and dose of administration, and the co-delivery of other adjuvants or immunomodulators. Therefore, further studies are needed to elucidate the mechanisms and factors that influence immune activation and modulation by ionizable and cationic lipids and to develop strategies to optimize the safe and efficacious use of mRNA therapeutics.
In summary, ionizable and cationic lipids play a crucial role in mediating the interaction between the LNP and the mRNA, as well as the LNP and the cell membrane, and modulating the immune response to the delivered mRNA. Depending on the type and amount of the lipid, the LNP can either enhance or suppress the activation of innate immune pathways, which can have different implications for mRNA therapy and vaccination. Therefore, the choice and optimization of the ionizable and cationic lipids for mRNA delivery should consider their immune activation and modulation effects and tailor them according to the desired outcome of mRNA therapy or vaccination.
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