Messenger RNA (mRNA) is a promising biomolecule for various applications, such as vaccines, gene therapy, and gene editing. However, mRNA also faces several challenges and limitations that hinder its clinical translation. First, mRNA is highly susceptible to degradation by nucleases in the extracellular and intracellular environments, resulting in low stability and bioavailability. Second, mRNA can trigger innate immune responses and inflammation, leading to adverse effects and reduced efficacy. Third, mRNA has difficulty crossing the cell membrane due to its large size and negative charge, requiring efficient delivery systems to facilitate cellular uptake and endosomal escape. To overcome these challenges, various chemical modifications and supramolecular approaches have been developed to enhance mRNA properties and delivery. Chemical modifications involve covalently attaching functional groups to the nucleobases, sugar moieties, or phosphate backbone of mRNA, which can improve stability, reduce immunogenicity, and increase translational efficiency. Supramolecular approaches involve non-covalently assembling mRNA with other molecules or structures, which can protect mRNA from degradation, modulate immune responses, and mediate delivery.
Polymeric formulations are one of the most widely used supramolecular approaches for mRNA delivery. They involve the formation of polymeric complexes (polyplexes) or polymeric micelles with mRNA molecules, which can enhance the stability, protection, and delivery of mRNA.
Polyplexes are formed by the electrostatic interaction between positively charged polymers and negatively charged mRNA. The polyplexes can neutralize the charge of mRNA, reduce its size, and increase its stability against RNases. However, polyplexes also have some drawbacks, such as low transfection efficiency, high cytotoxicity, and difficulty in dissociation. Therefore, various polymers with different structures, properties, and functionalities have been developed and optimized to improve the performance of polyplexes. For example, polyethylenimine (PEI) is one of the most widely used polymers for mRNA delivery due to its high buffering capacity, which can facilitate endosomal escape. However, PEI also has high toxicity and immunogenicity, which limit its clinical application. To overcome these issues, PEI can be modified with different functional groups, such as PEG, targeting ligands, or pH-sensitive moieties, to enhance its biocompatibility, specificity, and responsiveness.
Polymeric micelles are another type of polymeric formulation for mRNA delivery. They are formed by the self-assembly of amphiphilic block copolymers, which have a hydrophobic core and a hydrophilic shell. The hydrophobic core can encapsulate mRNA molecules, while the hydrophilic shell can provide stability, stealth, and targeting abilities. Polymeric micelles have several advantages over polyplexes, such as lower toxicity, higher stability, and longer circulation time. Moreover, polymeric micelles can be easily tuned by changing the composition, structure, and ratio of the block copolymers. For example, poly(lactic-co-glycolic acid) (PLGA) is a biodegradable and biocompatible polymer that can form micelles with PEG or other polymers. PLGA micelles can protect mRNA from degradation and release it in a controlled manner. PLGA micelles can also be modified with different ligands, such as antibodies, peptides, or aptamers, to achieve active targeting of specific cells or tissues.
Polymeric formulations have been extensively studied and applied for mRNA delivery in various fields, such as cancer, disease models, and gene editing. For instance, polymeric micelles composed of poly(β-amino ester) and PEG were used to deliver mRNA encoding Cas9 and guide RNA for gene editing in vivo. The polymeric micelles showed high gene editing efficiency and low toxicity in mouse models of Duchenne muscular dystrophy and cystic fibrosis. Another example is the use of polyplexes composed of PEI and PEG to deliver mRNA encoding HIV gp120 for vaccination. The polyplexes induced specific antibodies against HIV infection after intranasal vaccination in mice.
| Polymeric Assemblies | Definition | Advantages | Disadvantages | Examples |
|---|---|---|---|---|
| Polyplexes | Polymeric complexes formed by electrostatic interaction between positively charged polymers and negatively charged mRNA | Neutralize the charge of mRNA, reduce its size, and increase its stability against RNases | Low transfection efficiency, high cytotoxicity, and difficulty in dissociation | PEI, PLL, poly(β-amino ester)s |
| Polymeric micelles | Polymeric formulations formed by self-assembly of amphiphilic block copolymers, which have a hydrophobic core and a hydrophilic shell | Provide stability, stealth, and targeting abilities, lower toxicity, higher stability, and longer circulation time | Require careful design and optimization of the block copolymers | PLGA, PEG, poly(phenylene ethynylene) |
Table 1. Summary of polymeric assemblies
RNA architectonics is a novel concept and method for mRNA engineering and delivery that involves the use of complementary RNA oligonucleotides to modify, protect, and assemble mRNA molecules. RNA architectonics can improve the stability, functionality, and delivery of mRNA by introducing various functional groups, such as cholesterol, PEG, or crosslinkers, to the mRNA via hybridization with RNA oligonucleotides. RNA architectonics can also form mRNA nanostructures, such as nanoparticles, nanorings, or nanoflowers, by self-assembly of multiple mRNA strands via RNA-RNA interactions.
RNA architectonics has several advantages over chemical modifications and polymeric formulations for mRNA delivery. First, RNA architectonics can preserve the translational activity of mRNA, as the RNA oligonucleotides can be easily removed by RNases in the cytoplasm. Second, RNA architectonics can enhance the stability of mRNA against RNases, as the RNA oligonucleotides can form secondary structures, such as hairpins or duplexes, to protect the mRNA. Third, RNA architectonics can control the release of mRNA, as the RNA oligonucleotides can be designed to respond to various stimuli, such as pH, temperature, or enzymes.
RNA architectonics has been applied for mRNA delivery in various fields, such as vaccines, immunomodulation, and nanostructures. For example, mRNA encoding HIV gp140 was hybridized with poly U oligonucleotides to form mRNA-poly U complexes, which showed enhanced stability, immunogenicity, and protection against HIV infection in mice. Another example is the use of mRNA encoding IL-12 and IFN-γ to form mRNA nanorings, which showed improved stability, functionality, and anti-tumor efficacy in mouse models of melanoma and colon cancer.
| RNA Architectonics | Definition | Advantages | Disadvantages | Examples |
|---|---|---|---|---|
| RNA oligonucleotides | Short RNA strands that can hybridize with mRNA to modify, protect, and assemble mRNA molecules | Preserve the translational activity, enhance the stability, modulate the immune responses, and control the release of mRNA | Require careful design and optimization of the sequence, structure, and function of the RNA oligonucleotides | Cholesterol, PEG, crosslinkers |
| RNA nanostructures | Nanoscale structures that can self-assemble from multiple mRNA strands via RNA-RNA interactions | Provide stability, protection, and delivery of mRNA, as well as functionality and diversity of the nanostructures | Require careful design and optimization of the shape, size, and function of the nanostructures | Nanoparticles, nanorings, nanoflowers |
Table 2. Summary of RNA architectonics
RNA architectonics and polymeric micelles are two promising supramolecular approaches for mRNA delivery that can be integrated to achieve synergistic effects, multifunctionality, and precise control. The integration of RNA architectonics and polymeric micelles can be achieved by covalent or non-covalent methods or by self-assembly.
Covalent methods involve the attachment of RNA oligonucleotides to the block copolymers, which can then form polymeric micelles with mRNA. This can enhance the stability, protection, and targeting of mRNA, as well as the responsiveness and functionality of the polymeric micelles. For example, a block copolymer composed of PEG and poly(β-amino ester) was conjugated with an RNA oligonucleotide containing a cholesterol moiety, which could hybridize with mRNA and form polymeric micelles. The polymeric micelles showed high mRNA loading, stability, and transfection efficiency, as well as pH-responsive release of mRNA.
Non-covalent methods involve the interaction of RNA oligonucleotides with the block copolymers, which can then form polymeric micelles with mRNA. This can improve the stability, protection, and delivery of mRNA, as well as the flexibility and versatility of the polymeric micelles. For example, a block copolymer composed of PEG and poly(lysine) was mixed with an RNA oligonucleotide containing a PEG moiety, which could interact with the poly(lysine) segment and form polymeric micelles. The polymeric micelles showed high mRNA loading, stability, and transfection efficiency, as well as reversible assembly and disassembly of mRNA.
Self-assembly methods involve the spontaneous formation of polymeric micelles with mRNA and RNA oligonucleotides, which can be driven by various forces, such as hydrophobic, electrostatic, or π-π interactions. This can enhance the stability, protection, and delivery of mRNA, as well as the simplicity and scalability of the polymeric micelles. For example, a block copolymer composed of PEG and poly(phenylene ethynylene) was self-assembled with mRNA and RNA oligonucleotides containing pyrene moieties, which could form π-π interactions with the poly(phenylene ethynylene) segment and form polymeric micelles. The polymeric micelles showed high mRNA loading, stability, and transfection efficiency, as well as light-responsive release of mRNA.
The integration of RNA architectonics and polymeric micelles has been applied for mRNA delivery in various fields, such as liver, lung, and skin. For example, polymeric micelles composed of PEG and poly(β-amino ester) conjugated with RNA oligonucleotides containing galactose moieties were used to deliver mRNA encoding luciferase to the liver. The polymeric micelles showed high liver specificity, stability, and transfection efficiency, as well as enhanced protein expression. Another example is the use of polymeric micelles composed of PEG and poly(lysine) mixed with RNA oligonucleotides containing mannose moieties to deliver mRNA encoding erythropoietin to the lung. The polymeric micelles showed high lung specificity, stability, and transfection efficiency, as well as increased hematocrit levels.
The RNA/polymer-based supramolecular approaches for mRNA delivery are a novel and promising field of research, which aim to improve the stability, functionality, and delivery of mRNA molecules by using RNA oligonucleotides and polymeric formulations. These approaches can overcome some of the drawbacks of conventional methods, such as chemical modifications and viral vectors, and provide flexibility, versatility, and responsiveness to various stimuli. However, these approaches also face some challenges and opportunities for future research and development, such as the optimization of the design, synthesis, and characterization of the RNA oligonucleotides and polymeric formulations, the evaluation of the biocompatibility, toxicity, and immunogenicity of the supramolecular complexes, and the improvement of the delivery efficiency, specificity, and functionality of the mRNA.
