Next-generation vaccines: Nanoparticle-mediated delivery of DNA and mRNA

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Next-generation vaccines: Nanoparticle-mediated delivery of DNA and mRNA

Next-generation vaccines: Nanoparticle-mediated delivery of DNA and mRNA.  Nucleic acid vaccines are an immune method designed to elicit an immune response similar to that of live attenuated vaccines.

In this method, DNA or messenger RNA (mRNA) sequences are delivered to the body to produce proteins that mimic disease antigens to stimulate the immune response. The advantages of nucleic acid vaccines include stimulation of cell-mediated and humoral immunity, easy design, rapid adaptation to changing pathogenic strains, and customizable multi-antigen vaccines.

However, help is needed to deliver fragile DNA/mRNA payloads, and nanoparticles (NPs) are one of the top candidates to mediate successful DNA/mRNA vaccine delivery.

Nanoparticle-mediated delivery is a non-viral delivery method that has many advantages to promote vaccine uncoating; they protect nucleic acid payloads from degradation and provide a versatile formulation strategy with a variety of biomaterial options to overcome cellular internalization Obstacles.Improve specific immune cell targeting through surface modification, and possibly use pH-sensitive materials to enhance endochromosome escape.

NPs improve the stability and effectiveness of vaccines, and can also make cocktail vaccines within one particle; NPs enable multiple nucleic acid vaccines to be co-delivered to the same target cell, so that the synergistic effect further enhances immunity. As shown in Figure 1, NPs can protect nucleic acids from being degraded by endonucleases and cleared by phagocytosis through the reticuloendothelial system.

Then the naked nucleic acids will face obstacles to enter the negatively charged cell membrane. NPs may be matched with specific cell receptors The surface ligand targets the cell and enters the cell through receptor-mediated endocytosis; inside the cell, the NP vaccine must escape the endosome to deliver the payload.

NPs are designed to respond to the acidic pH of endosomes, triggering endosome escape and intracellular payload release. Once in the cytoplasm, the DNA vaccine must further translocate to the nucleus for transcription.

Figure 1 Challenges of nucleic acid vaccines and solutions through NP-based transmission

Nucleic acids are delivered to cells because they are easily affected by endogenous nucleases. The dense negative charge of nucleic acids hinders cell internalization, and the presence of foreign nucleic acids in the cytoplasm triggers non-specific interferon reactions that are also obstacles to clinical transcription.

Therefore, the NP nucleic acid delivery system should effectively encapsulate negatively charged nucleic acids, protect endogenous enzymes, and promote cellular uptake and intracellular release. The combination of cationic polymer/lipid and negatively charged nucleic acid complexes can protect DNA/ mRNA is protected from endonuclease degradation and immune recognition.

Inorganic NPs with nucleic acid functionalization and other design features, such as the modularization of multivalent targeting ligands of nanoparticle components, can maintain a long-term thermal treatment dose and target Specific immune cells and lymph nodes (LNs) improve the effectiveness of the vaccine; cell penetrating peptides can be compounded with nucleic acids to improve delivery efficiency.

In addition, direct modification of DNA and mRNA molecules can be used to increase the effectiveness of the formulation.

This article introduces five types of newly developed nanoparticle platforms, clinical and preclinical trials.

1. Liposomes are tiny artificial vesicles with at least one lipid bilayer.

In liposome formulations of nucleic acids, self-assembly into spherical or amorphous structures is the most common, with lipids and nucleic acids dispersed throughout the bilayer. Neutral lipids can be used to achieve stability and transfection efficiency. The transfection efficiency depends on the geometry, the number of charged groups per molecule, the nature of the lipid anchor and the nature of the linkage.

As shown in the left panel of Figure 2, ionizable lipids are complexed with negatively charged mRNA at low pH, which promotes endocytosis and endocytosis; phospholipids provide structural integrity to the bilayer while supporting mRNA Escape from the endosome to the cell membrane, cholesterol helps stabilize LNPs and promote membrane fusion. The right panel of Figure 2 shows a low-temperature transmission electron microscope image of spherical LNPs with a multicellular structure.

Figure 2 Lipid nanoparticles are designed for cellular uptake and endosomal escape

2. Although liposomal nanoparticles are by far the most popular NP exchange reaction carrier, polymer nanoparticles (PNPs) are an excellent choice.

In recent years, polymers have been widely used for nucleic acid delivery, usually prepared from biocompatible and biodegradable polymers, in which the drug is dissolved, encapsulated, encapsulated, or attached to a nanoparticle matrix. The use of biodegradable polymer nanoparticles to control drug delivery has shown significant therapeutic potential.

They have a wide range of different chemical properties and physical properties, can protect the payload from degradation, enable the controlled release of gene chaperones, and are easy to modify the structure to adjust their physical and chemical properties, and show biocompatibility and biodegradability.

3. Natural polymers, such as chitosan and alginate, are polymers produced by biological cells.

Natural polymer nanoparticles are suitable for clinical applications due to their biocompatibility, biodegradability and low immunogenicity. Chitosan is a cationic polysaccharide and natural biopolymer. It has been used as an adjuvant and vaccine delivery system. Because it is non-toxic and biocompatible, it can penetrate the mucosal surface of epithelial cells and close intercellular connections. , With adhesive properties, can enhance the absorption of vinegar and drugs on the mucosal surface, and is one of the main ways for pathogens to enter the human body.

Polymer NPs show biocompatibility, stability and easy modification of chemical structure. Many vaccines need to be stored in extremely low-temperature refrigerators, which is a major drawback for diseases such as Ebola in developing countries, because low-temperature storage cannot be widely used. In addition, manganese is more biodegradable because of less material waste and sharp handling. In order to achieve these two goals, the Yang Group of Ebola virus vaccination proposed a novel method to coat polylactic acid-glycolic acid polynucleotide-lysine/polyglutamic acid/poly- with DNA vaccine.

Aminoglutaric acid (PLGA-PLL/PGA) nanoparticles are dissolved on the skin using PVA microneedle (MN) patch management (Figure 4). This formula relies on the properties of cationic PLGA-PLL nanoparticles combined with EboDNA vaccine (EboDNA) vaccine. The preparation can induce immune response in mice, and manganese is stable for at least 2 weeks at 37°C. The MN patch delivery system allows vaccinators to receive minimal training, and vaccine stability does not require refrigeration, all of which are very low-cost. These studies indicate that the focus in the field of MN-NP should shift to the transfection efficiency of microneedle in vivo preparations and the optimization of the MN-NP system.

Figure 3 A) Schematic diagram of the preparation process of PLGA-PLL/PGA-EboDNA. B) Schematic diagram of the production of the dissolved MN patch. C) Bright field image of MN patch, where PLGA-PLL-SRB (red) is wrapped in MNs.

4. Inorganic nanoparticles have extensively studied the delivery of nucleolytic acid.

Inorganic NPs are usually smaller in size than polymer/liposomal NPs, with a narrow size distribution and surface chemistry, which makes it easier for ligands to conjugate. Gold nanoparticles (AuNPs) are very stable inorganic nanoparticles. They exhibit a wide range of electromagnetic properties and are widely used in the research and development of their inherent optical properties and chemical modification of easy-to-draw surfaces.

Another type of inorganic NP, mesoporous mesoporous silicon nanomaterial particles (MSNs) are biodegradable and chemically stable nanostructured materials with unique macroporosity; this porosity allows a wide surface area to be used for NP surface chemistry Modularity and drug packaging, and allow many sites to carry nucleic acids efficiently. Song et al. reported the development of a single-cell microsphere DNA vaccine using rambutan-like single-cell microspheres as a gene carrier and adjuvant.

Rambutan-based MSNs were developed by copolymerization of resorcinol-formaldehyde (RF) resin and silica, showing unique pointed nanopatterns, and were further modified with cationic PEI (Figure 4), Ram–MSNs (A) and Ram-MSNs-PEI (b) transmission electron micrographs, the corresponding particle size distribution (c) and nitrogen adsorption isotherm (d) determined by DLS, have shown excellent pDNA delivery and effective protection of genes from nucleic acids Enzymatic degradation.

Figure 4 Rambutan-like mesoporous silica nanoparticles have good adsorption properties for pDNA

5. In addition to being used as vaccine drugs, peptides are also used to promote the delivery of nucleic acid vaccines.

Peptides used for nucleic acid delivery generate positive charges by excluding lysine and arginine residues, and combine with negatively charged nucleic acids to form nanocomposites. They can be used as nanocomposites, and the positive-negative ratio affects the formation of complexes. As a natural structure existing in all biological systems, peptide-based NPs are a natural method to provide nucleic acid vaccines.

Although the peptide-based NP system has recently had a harmful effect on the delivery of nucleic acid vaccines, it can be improved by developing an effective new complex library and expanding the library of materials that can produce peptide uncoating systems. Polylactic acid (PLA) NPs have been proven to encapsulate or adsorb various antigens and immunostimulant molecules, and can be absorbed by dendritic cells (DCs).

Since both the PLA-NP surface and the mRNA biological macromolecules are negatively charged, cationic CPPs have been used as cationic intermediates to load mRNA onto PLA-NPs (Figure 5). Negatively charged mRNA combines with cationic peptides (RALA, LAH4 or LAH4-L1) to form peptide/mRNA multimers. The complex is adsorbed on PLA-NPs to form PLA-NP/peptide/mRNA nanocomposite.

Figure 5 Carrierization of PLA-NPs mRNA with cationic peptide intermediates

DNA and mRNA vaccines have brought many benefits to modern vaccination systems, enabling them to quickly adapt to changing pathogen strains, as well as cheap and rapid artificial manufacturing capabilities. Due to the limitation of DNA/mRNA delivery with viral vectors, non-viral vectors use different materials such as lipids and polymers, inorganic molecules, peptides and their combinations to have various beneficial delivery characteristics. NPs provide groundbreaking opportunities to develop highly effective targeted therapies to improve the effectiveness of vaccines.

Each candidate NP vaccine needs to go through a series of clinical trials to evaluate its safety, immunogenicity and protection for humans. The following table describes the NP vaccine therapies currently undergoing different stages of clinical trials (Table 1). It is obvious that many are liposome-based, which represents a huge opportunity to improve and perfect the use of polymer, inorganic and peptide-based NPs. In clinical. In addition, only a few clinical trials involving NPs are based on nucleic acids, which indicates that the above-mentioned improvements in NP and nucleic acid design still have a great opportunity to fill the gap in NP-mediated nucleic acid delivery in clinical delivery.

Based on the successful experience in the clinical development process, further improving the design of DNA/mRNA payloads and NP formulations, and improving the understanding of the immune response induced by NP-mediated nucleic acid vaccines will produce faster immune vaccines, which may only require a few Can deal with infectious diseases and cancer in just one month.

Although the COVID-19 pandemic has caused huge damage and loss of life around the world, it has sounded the alarm for the world to design new technologies and concepts for vaccines. By solving the above challenges, we believe that the full potential of NP-mediated nucleic acid vaccines will be discovered in the future.

Next-generation vaccines: Nanoparticle-mediated delivery of DNA and mRNA

Next-generation vaccines: Nanoparticle-mediated delivery of DNA and mRNA

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