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A Review of In Vivo Delivery Strategies of Exogenous mRNA for Therapeutics and Vaccines

Messenger RNA (mRNA) technology is an emerging platform for the development of novel therapeutics and vaccines, especially for infectious diseases and cancer immunotherapy. However, mRNA delivery faces significant challenges in terms of stability, immunogenicity, and efficiency. Various strategies have been developed for mRNA delivery, such as naked mRNA injection, ex vivo loading of dendritic cells, cationic nano emulsion, cationic peptides, cationic polymers, and lipid nanoparticles.

An overview of delivery strategies and biomedical applications of mRNA. (Xiao Y, 2022) Fig.1 An overview of delivery strategies and biomedical applications of mRNA. (Xiao Y, 2022)

Naked mRNA Injection

Naked mRNA injection is the simplest and most direct strategy for mRNA delivery. It involves the direct injection of mRNA molecules into the target tissue, such as muscle, skin, or tumor, without any carrier or formulation. The injected mRNA molecules can be taken up by the surrounding cells through endocytosis or membrane fusion and then translated into the encoded proteins or antigens in the cytoplasm.

The main advantage of naked mRNA injection is its simplicity and low cost. It does not require any complex synthesis or purification steps, and it avoids the potential toxicity and immunogenicity of the carrier materials. Moreover, naked mRNA injection can achieve high and rapid expression of the encoded proteins or antigens, which can be beneficial for acute or emergency situations. However, naked mRNA injection also has several disadvantages, such as low efficiency, rapid degradation, and potential immune response. The naked mRNA molecules are vulnerable to nuclease activity in the extracellular and intracellular environments and thus have a short half-life and low stability. Moreover, the naked mRNA molecules have difficulty crossing the cell membrane and thus have a low transfection efficiency and a limited distribution. Furthermore, the naked mRNA molecules can activate the innate immune system, such as toll-like receptors and RIG-I-like receptors, which can induce interferon production and inflammation and thus affect the expression and function of the encoded proteins or antigens.

Ex Vivo Loading of Dendritic Cells

Ex vivo loading of dendritic cells with mRNA is another strategy for mRNA delivery. It involves the isolation of dendritic cells (DCs) from the blood or tissue of the patient or donor, the transfection of DCs with mRNA in vitro, and the reinfusion of the transfected DCs back into the patient or recipient. DCs are antigen-presenting cells that can activate T cells and induce adaptive immune responses. By loading DCs with mRNA encoding tumor antigens or viral antigens, the transfected DCs can present the antigens to the T cells and elicit specific and potent anti-tumor or anti-viral immunity.

The main advantage of ex vivo loading of dendritic cells with mRNA is its high specificity, potency, and safety. It can generate personalized and customized vaccines or immunotherapies based on the patient's or donor's own cells and antigens. Moreover, it can avoid the potential toxicity and immunogenicity of the carrier materials, as the mRNA molecules are delivered directly into the DCs without any formulation. Furthermore, it can achieve high and sustained expression of the encoded antigens, as the DCs can protect the mRNA molecules from degradation and provide a suitable environment for translation. However, ex vivo loading of dendritic cells with mRNA also has several disadvantages, such as complexity, cost, and variability. It requires a complex and labor-intensive process of isolating, culturing, transfecting, and reinfusing the DCs, which can increase the time, cost, and risk of contamination and infection. Moreover, it depends on the availability and quality of the DCs, which can vary from patient to patient or donor to donor and thus affect the efficiency and consistency of the transfection and the immunogenicity of the antigens.

Cationic Nano Emulsion

Cationic nano emulsion is a type of lipid-based formulation for mRNA delivery. It consists of a mixture of cationic lipids, neutral lipids, and water, forming nanosized droplets that can encapsulate mRNA molecules. The cationic lipids can interact with the negatively charged mRNA molecules and form complexes, which can protect the mRNA molecules from degradation and facilitate their uptake by the target cells.

The main advantages of cationic nano emulsion are its high stability, biocompatibility, and scalability. It can provide a stable and homogeneous formulation for mRNA delivery, which can be easily prepared and stored. Moreover, it can enhance the biocompatibility and biodistribution of the mRNA molecules, as the lipid components can mimic the natural cell membrane and avoid rapid clearance by the immune system. Furthermore, it can achieve high transfection efficiency and expression of the encoded proteins or antigens, as the cationic lipids can mediate the endosomal escape and cytoplasmic release of the mRNA molecules. However, cationic nano emulsion also has several disadvantages, such as potential toxicity and immunogenicity. The cationic lipids can cause cytotoxicity and inflammation, as they can disrupt the cell membrane and activate the innate immune receptors. Moreover, the cationic lipids can induce immunogenicity and interfere with the antigen presentation, as they can bind to the major histocompatibility complex (MHC) molecules and affect the recognition by the T cells.

Cationic Peptides

Cationic peptides are a type of synthetic or natural amino acid sequence that can bind and deliver mRNA molecules. They can form electrostatic interactions with the negatively charged mRNA molecules and form complexes, which can protect the mRNA molecules from degradation and facilitate their uptake by the target cells.

The main advantage of cationic peptides is their high transfection efficiency, biodegradability, and versatility. They can achieve high and rapid expression of the encoded proteins or antigens, as they can mediate the endosomal escape and cytoplasmic release of the mRNA molecules. Moreover, they can be biodegraded by the proteases in the cells and tissues, thus avoiding accumulation and toxicity. Furthermore, they can be modified and optimized to enhance their stability, specificity, and functionality. However, cationic peptides also have several disadvantages, such as potential toxicity and immunogenicity. The cationic peptides can cause cytotoxicity and inflammation, as they can disrupt the cell membrane and activate the innate immune receptors. Moreover, the cationic peptides can induce immunogenicity and interfere with the antigen presentation, as they can bind to the major histocompatibility complex (MHC) molecules and affect the recognition by the T cells.

Cationic Polymers

Cationic polymers are a type of synthetic or natural macromolecules that can bind and deliver mRNA molecules. They can form electrostatic interactions with the negatively charged mRNA molecules and form complexes, which can protect the mRNA molecules from degradation and facilitate their uptake by the target cells.

The main advantage of cationic polymers is their high transfection efficiency, biodegradability, and versatility. They can achieve high and sustained expression of the encoded proteins or antigens, as they can mediate the endosomal escape and cytoplasmic release of the mRNA molecules. Moreover, they can be biodegraded by the enzymes in the cells and tissues, thus avoiding accumulation and toxicity. Furthermore, they can be modified and optimized to enhance their stability, specificity, and functionality. However, cationic polymers also have several disadvantages, such as potential toxicity and immunogenicity. The cationic polymers can cause cytotoxicity and inflammation, as they can disrupt the cell membrane and activate the innate immune receptors. Moreover, the cationic polymers can induce immunogenicity and interfere with the antigen presentation, as they can bind to the major histocompatibility complex (MHC) molecules and affect the recognition by the T cells.

Lipid Nanoparticles

Lipid nanoparticles (LNPs) are a type of lipid-based formulation for mRNA delivery. They consist of a core of mRNA molecules surrounded by a lipid bilayer, which can protect the mRNA molecules from degradation and facilitate their uptake by the target cells. LNPs are the most widely used and clinically advanced strategy for mRNA delivery, especially for COVID-19 vaccines.

Schematic representation of components of LNPs. (Ramachandran S, 2022) Fig.2 Schematic representation of components of LNPs. (Ramachandran S, 2022)

The main advantage of LNPs is their high transfection efficiency, biocompatibility, and scalability. They can achieve high and sustained expression of the encoded proteins or antigens, as they can mediate the endosomal escape and cytoplasmic release of the mRNA molecules. Moreover, they can enhance the biocompatibility and biodistribution of the mRNA molecules, as the lipid components can mimic the natural cell membrane and avoid rapid clearance by the immune system. Furthermore, they can achieve high scalability and reproducibility, as they can be easily prepared and stored using microfluidic or extrusion methods. However, LNPs also have several disadvantages, such as potential toxicity and immunogenicity. The LNPs can cause cytotoxicity and inflammation, as they can disrupt the cell membrane and activate the innate immune receptors. Moreover, the LNPs can induce immunogenicity and interfere with the antigen presentation, as they can bind to the major histocompatibility complex (MHC) molecules and affect the recognition by the T cells. Therefore, the optimization of the lipid composition, size, charge, and surface modification of the LNPs is crucial for the safety and efficacy of mRNA delivery.

Conclusion

Taken together, each mRNA delivery strategy has its own strengths and weaknesses and different applications in biomedical fields. The choice of the optimal mRNA delivery strategy depends on several factors, such as the type and purpose of the mRNA, the target cells and tissues, the desired expression level and duration, the safety and efficacy profile, and the cost and feasibility of the production and administration. There is no one-size-fits-all solution for mRNA delivery, and the development of novel and improved mRNA delivery strategies is still a dynamic and challenging research area.

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