Compared with conventional drugs, nucleic acids have great advantages. The mechanism of action and high specificity of nucleic acids provide possible therapeutic approaches for viral infections, various cancers and inoperable genetic diseases with unmet clinical needs. In theory, gene therapy can achieve lasting or even curative effects. However, delivery of nucleic acids to active sites within cells is challenging due to their low stability in vivo and rapid extracellular host clearance. In addition, due to the negative charge, high molecular mass, and hydrophilicity of nucleic acids, their ability to penetrate cell membranes is poor. In order to solve the challenges related to nucleic acid drug molecules themselves, it is most important to develop delivery vector systems that can facilitate nucleic acid absorption into target cells. These delivery vectors themselves need to overcome the extracellular and intracellular barriers, tolerate nuclease activity in blood, enhance and assist cellular uptake of nucleic acid drugs, and facilitate endosomal escape of nucleic acid drugs after entering cells.
Gene delivery is usually divided into viral and non-viral delivery vectors. Viruses can be used to deliver genes of interest by insertion into the viral genome, followed by cellular infection and gene expression. Viruses with high DNA transfection efficiency, such as lentiviruses, retroviruses, adenoviruses and adeno-associated viruses, have long been used to treat diseases such as HIV, cancer and muscular dystrophy. Despite the high cell transfection rate of viral gene delivery systems, rapid clearance of existing antibodies, production of neutralizing antibodies against vectors, limited vector size (usually less than 7kb), and possible side effects leave wide space for the development of gene delivery technologies in biomedical research and clinical practice. In recent years, efforts have been made to design non-viral gene delivery systems based on lipids, polymers, peptides and inorganic compounds.
Lipid-based nanoparticles, polymer nanoparticles and inorganic nanoparticles are the three most common non-viral nanocarriers used for nucleic acid drug delivery (Figure 1).
1. Lipid-based Nanodelivery Systems
Lipid-based nano-delivery systems are the most widely used non-viral nucleic acid carriers, mainly because of their stable nanostructure in physiological fluids, and their fusion with negatively charged endosomal membranes enables efficient delivery of nucleic acids. Lipids are amphiphilic molecules containing a polar head group, a hydrophobic tail region, and a connector between the two domains. Lipid-based nano-delivery systems often contain other lipid components such as phospholipids, cholesterol, or polyethylene glycol components (Figure 2). The main differences between these nanoparticles depend on their lipid composition, synthesis parameters, and methods used for nucleic acid encapsulation. These delivery systems have been used to treat hereditary diseases (such as cystic fibrosis and propionic acid) and solid tumors.
2. Liposomes
Liposomes are composed of phospholipids with polar head groups and non-polar tails, as well as stabilizers such as cholesterol, which spontaneously self-assemble into vesicles due to their amphiphilic properties. Cationic lipids and amphoteric ions usually use electrostatic interaction between lipids and negatively charged nucleic acids to form cationic liposomes, thereby increasing encapsulation. Smaller liposomes are more likely to escape uptake by phagocytes (≤100 nm). However, the positive charge on the surface of nanoparticles may lead to nonspecific binding and immune stimulation of serum proteins, leading to toxicity. To this end, pegylated cationic liposomes were developed as substitutes. Liposomes can be prepared by three typical methods: membrane hydration, solvent injection and reversed-phase evaporation. These methods can achieve effective drug encapsulation, narrow particle size dispersion, and long-term stability.
3. Lipid nanoparticles (LNPs)
LNPs (50-100 nm) typically consist of ionizable and cationic lipids, cholesterol, phospholipids, and polyethylene glycol lipids, of which ionizable lipids play a major role in protecting nucleic acids from nuclease degradation. In addition, helper lipids, such as phospholipids and cholesterol, promote drug stability and membrane fusion, requiring approximately 30-40 mol% of helper lipids to effectively embed siRNA in LNPs. Polyethylene glycol lipids are formed by combining hydrophilic polyethylene glycol lipids polymers with hydrophobic lipid anchors to improve cyclic half-life and stability and prevent LNPs clearance. Low molecular weight polyethylene glycol lipids can reduce the adsorption of nonspecific proteins. In addition, polyethylene glycol lipid content determines particle size.
In contrast to liposomes, LNPs have micellar structure in the particle core. In addition, LNPs exhibit better kinetic stability and tougher morphology than liposomes. Ionizable LNPs have a near-neutral charge at physiological pH, but amine groups on ionizable lipids become protonated and positively charged at low pH, allowing assembly with negatively charged phosphate groups on nucleic acids. After complexation, the pH can be adjusted to a neutral or physiological pH for administration. After injection in vivo, ionizable LNPs can exude from the blood to target tissues. LNPs can then adsorb to the cell surface and enter the cell via endocytosis. The positively charged ionizable lipids help the endosomes escape and interact with the negative charges on the endosomal lipid membrane, causing the endosomes to become unstable and promoting nucleic acid release.
4. Polymer Nanoparticles
Polymer nanoparticles are prepared using natural polymers such as dextran, chitosan, cyclodextrin or synthetic polymers, which allows the manufacture of polymer nanoparticles with different compositions and structures. The most common forms of polymer nanoparticles are nanocapsules and nanospheres, which have multiple subclasses, such as polymers and dendrimers. Polymer nanoparticles are one of the most promising nano-delivery materials for nucleic acid drugs because of their simple synthesis, structural diversity, synthetic scalability, high transfection rate, gene immunogenicity and good biocompatibility.
5. Inorganic Nanoparticles
Inorganic nanoparticles have been studied for nucleic acid delivery and imaging, including gold nanoparticles, silica nanoparticles and iron oxide nanoparticles. They can be designed to a specific size, structure and geometry. Among inorganic nanoparticles, gold and iron oxide nanoparticles are generally considered as non-toxic nanomaterials.