Nucleic acids are essential biomolecules that store and transmit genetic information in living organisms. However, their therapeutic applications are limited by their low stability, poor cellular uptake, and immunogenicity. To overcome these challenges, various strategies have been developed to deliver nucleic acids into cells, such as viral vectors, liposomes, polymers, and nanoparticles. Among them, PTD/nucleic acid complexes are a promising class of hybrid molecules that combine a cell-penetrating peptide (PTD) with a nucleic acid (DNA, mRNA, or oligonucleotide) to achieve efficient cellular delivery and biological activity of the nucleic acid. PTDs are short peptides that can translocate across cell membranes and carry cargoes into cells. By conjugating PTDs to nucleic acids, PTD/nucleic acid complexes can enhance the stability, specificity, and functionality of nucleic acids and enable their applications in various fields, such as gene therapy, drug delivery, and biosensing. However, PTD/nucleic acid complexes also face some challenges and limitations, such as low stability, poor specificity, and an immune response. Therefore, it is important to explore and optimize the strategies for generating PTD/nucleic acid complexes, which can affect their structure, properties, and performance. The strategies for generating PTD/nucleic acid complexes can be classified into three main categories: covalent conjugation, non-covalent interaction, and self-assembly. Each category has its own advantages and disadvantages, and different methods have been developed and applied within each category.
Covalent conjugation involves the formation of a covalent bond between the PTD and the nucleic acid, either directly or through a linker molecule. The covalent bond can be either cleavable or non-cleavable, depending on the type of reaction and the linker used. Covalent conjugation can provide high stability, precise stoichiometry, and controlled release of the nucleic acid, but it can also introduce potential toxicity, immunogenicity, and loss of nucleic acid function.
The most common reactions used for covalent conjugation are amine-carboxyl, thiol-maleimide, thiol-thiol, hydrazide-aldehyde, and gold-thiol, which can generate amide, thioether, disulfide, hydrazine, gold-thiol, and triazole rings as covalent linkages, respectively. The choice of the reaction and the linker depends on the availability and accessibility of the functional groups on the PTD and the nucleic acid, the reaction conditions, and the desired properties of the complex. For example, the amine-carboxyl reaction can be performed under mild conditions and can use various linkers, such as polyethylene glycol (PEG), to increase the solubility and biocompatibility of the complex. However, this reaction can also cause side reactions and heterogeneity in the product. The thiol-maleimide reaction can be more specific and efficient, but it requires the introduction of thiol and maleimide groups on the PTD and the nucleic acid, respectively, which can be challenging and costly. The thiol-thiol reaction can be simpler and faster, but it can also result in the aggregation and oxidation of the complex. The hydrazide-aldehyde reaction can be reversible and pH-sensitive, which can allow the release of the nucleic acid in acidic environments such as endosomes or lysosomes. However, this reaction can also be slow and incomplete and require the modification of the nucleic acid with aldehyde groups, which can affect its structure and function. The gold-thiol reaction can be used to attach PTDs to gold nanoparticles (AuNPs) that can carry nucleic acids on their surface, which can enhance the stability, delivery, and activity of the nucleic acid. However, this reaction can also cause aggregation and instability of the AuNPs and require the optimization of the size, shape, and surface charge of the AuNPs.
Non-covalent interaction is another way to make PTD/nucleic acid complexes. Unlike covalent conjugation, which uses strong bonds to link the PTD and the nucleic acid, non-covalent interaction uses weak forces to attract them. These forces can be different, such as electrostatic, hydrophobic, or π-π interactions. They depend on the charge, polarity, and shape of the PTD and the nucleic acid. Non-covalent interaction has some benefits, such as low toxicity, high versatility, and reversible binding of the nucleic acid. But it also has some drawbacks, such as low stability, variable stoichiometry, and competition with serum proteins.
Electrostatic interaction is the most common force in PTD/nucleic acid complexes. It happens when groups with opposite charges attract each other. For example, PTDs that have positive charges, such as polyarginine, polylysine, or TAT, can bind to nucleic acids that have negative charges, such as DNA, RNA, or oligonucleotides. The strength of the electrostatic interaction can change with the pH, the ionic strength, or the presence of other charged molecules in the solution.
Hydrophobic interaction is another force in PTD/nucleic acid complexes. It happens when groups that do not like water avoid contact with it. For example, PTDs that have non-polar groups, such as penetratin, transportan, or pVEC, can bind to nucleic acids by inserting their non-polar groups into the grooves or the minor face of the nucleic acid. The strength of the hydrophobic interaction can increase with the temperature, the polarity, or the presence of other hydrophobic molecules in the solution.
A third non-covalent force involved in PTD/nucleic acid complexes is π-π interaction, which is caused by the overlap of the electron clouds of aromatic rings. For example, aromatic PTDs, such as tryptophan-rich peptides, can bind to nucleic acids by stacking their aromatic rings with the bases of the nucleic acid. The strength of the π-π interaction depends on the orientation, the distance, and the polarity of the aromatic rings. π-π interaction can be influenced by changing the solvent, the conformation, or the presence of other aromatic molecules in the solution.
Self-assembly is a different way to make PTD/nucleic acid complexes. In this strategy, the PTD and the nucleic acid do not need to be linked or attracted, but they can form nanostructures, such as micelles, liposomes, or nanoparticles, by themselves or by external stimuli. Nanostructures can act as shields for the nucleic acid and help it enter and leave the cells to work better. Self-assembly has some good points, such as high efficiency, biocompatibility, and responsiveness. But it also has some bad points, such as complex synthesis, low loading capacity, and aggregation.
Self-assembly can happen because of different factors, such as the molecular structure, the environmental conditions, or the external stimuli. For example, some PTDs have two parts-one likes water and one does not. These PTDs can form micelles or vesicles in water and put nucleic acids inside their water-hating cores or bilayers. The size and shape of the micelles or vesicles can change with the concentration, the temperature, or the pH of the solution. Some PTDs can also make nanoparticles with nucleic acids by themselves or by external stimuli, such as light, heat, or magnetic fields. These nanoparticles can have different shapes, such as spheres, rods, or stars, and different properties, such as fluorescence, magnetism, or catalysis. The making and working of the nanoparticles can change with the type, the ratio, and the sequence of the PTD and the nucleic acid.
One example of PTD/nucleic acid complexes generated by covalent conjugation is a TAT-DNAzyme complex, which was designed to target and cleave the mRNA of the oncogene Bcl-2 in cancer cells. The TAT peptide was covalently linked to the DNAzyme by a thiol-maleimide reaction, and the complex was able to enter the cells and induce apoptosis by reducing Bcl-2 expression. Another example is a PEG-TAT-siRNA complex, which was designed to deliver and silence the gene of the HIV-1 integrase in infected cells. The PEG-TAT peptide was covalently linked to the siRNA by an amine-carboxyl reaction, and the complex was able to protect the siRNA from degradation and enhance its cellular uptake and gene silencing efficiency.
Both polyarginine-aptamer complexes and transportan-oligonucleotide complexes are generated by non-covalent interactions. The polyarginine peptide was non-covalently associated with the aptamer by electrostatic interaction, and the complex was able to enhance the stability and specificity of the aptamer and inhibit the thrombin activity. And the transportan peptide was non-covalently associated with the oligonucleotide by hydrophobic interaction, and the complex was able to produce a fluorescence signal upon hybridization with the target microRNA miR-21 in biological samples.
In addition, a complex of peptide amphiphile and DNA, generated by the self-assembly process for gene delivery, can form nanofibers that carry plasmid DNA into cells, make micelles that trap the plasmid DNA in their water-repelling core, as well as release the plasmid DNA when the pH changes and transfer the genes to the cells effectively.
Different methods of creating PTD/nucleic acid complexes have their own advantages and disadvantages, as well as challenges and opportunities. For instance, by covalently linking the PTD and the nucleic acid, the complex can be stable and precise, but it may also be toxic and damage the nucleic acid function. To improve this method, we can use linkers that can release the nucleic acid at the target site and coatings that can shield the complex from the immune system. In the same way, the complex generated by non-covalent interactions can be flexible and reversible, but it may also be unstable and inconsistent. A possible improvement for this method is to optimize the charge, polarity, and shape of the PTD and the nucleic acid, as well as coatings that can prevent the complex from being degraded or interfered with. Furthermore, self-assembly can make the complex efficient and biocompatible, but it can also complicate synthesis and cause aggregation. One way to enhance this strategy is to adjust the size, shape, and surface charge of the nanostructures and to use nanoparticles that can carry multiple nucleic acids or other molecules on their surface.
| Method | Type of bond | Stability | Toxicity | Loading capacity | Release mechanism | Example |
|---|---|---|---|---|---|---|
| Covalent conjugation | Covalent bond, either direct or through a linker, cleavable or non-cleavable | High | Potential | Precise | Controlled | TAT-DNAzyme complex |
| Non-covalent interaction | Non-covalent force, such as electrostatic, hydrophobic, or π-π interaction | Low | Low | Variable | Reversible | Polyarginine-aptamer complex |
| Self-assembly | Self-organization or external stimuli, forming nanostructures such as micelles, liposomes, or nanoparticles | Moderate | Moderate | Low | Responsive | Peptide amphiphile-DNA complex |
Table 1. A Comparison of Different Strategies for Generating PTD/Nucleic Acid Complexes
