Currently, the development of nucleic acid drugs can be divided into two major directions: chemical modification and physical modification, both of which can individually or jointly enhance the stability of nucleic acid drugs both in vitro and in vivo, as well as the translation efficiency in specific tissues and cells. Various nucleic acid modification and delivery technologies have been developed, mainly including chemical modification, ligand coupling, and nano-delivery carriers.

Related Reading: "Progress in Research on In Vivo Delivery Strategies for Nucleic Acid Drugs (Part 1)"

Nanodelivery Carriers

1.Inorganic Nanocarriers

Inorganic nanocarriers refer to delivery systems primarily composed of inorganic materials. Commonly used inorganic nanomaterials include gold, silver, calcium phosphate, graphene oxide, quantum dots, and magnetic nanomaterials such as iron oxide. Inorganic nanomaterials have attracted significant attention due to their unique electrical, optical properties, biocompatibility, and low cytotoxicity. Gold nanomaterials possess flexible surfaces that allow nucleic acids to directly bind to gold nanoparticles. Some researchers have utilized the i-motif secondary structure to create a pH-sensitive siRNA-gold nanoparticle delivery system that can silence PLK1 (encoding enzymes necessary for stable chromosomes and mitosis), thereby inducing apoptosis in target cells. pH gradient changes can cause conformational changes in nucleic acids, inducing aggregation of siRNA-gold nanoparticles, promoting in vivo escape, and thus releasing siRNA. Stem cells have difficulty in absorbing foreign substances, making it difficult to use traditional non-viral vectors for drug delivery. Research has functionalized gold and silver nanoparticles with HIV-TAT peptides, enabling the carriers to effectively penetrate epidermal stem cells with low toxicity. Quantum dots are semiconductor-based monodisperse nanocrystals, and carbon quantum dots are one of the most typical applications. They typically have a size of less than 10 nm and possess characteristics such as low toxicity, high quantum yield, low photobleaching, good water solubility, easy surface modification, and chemical stability. Surface modifications such as PEI, ethylenediamine, spermine, and arginine have been widely applied to carbon quantum dots, and carbon quantum dots carrying cationic compounds can effectively transfect therapeutic plasmids into cells. Carbon quantum dots can be used in targeted cancer therapy, where folic acid-modified nitrogen-doped carbon quantum dots, combined with autophagy inhibitors, can achieve rapid (within 24 hours) inhibition of tumor cell growth with significant killing effects. Additionally, carbon quantum dots can serve as efficient fluorescent probes for nucleic acid labeling and real-time tracking of in vivo dynamic distribution. For example, liposomes encapsulating carbon quantum dots can be used for imaging tumor proliferation in blood vessels, and carbon quantum dots can be utilized to deliver siRNA for "visual" lung cancer treatment. Graphene, an allotrope of carbon, has become a novel nanomaterial due to its unique optical, thermal, and electrical properties. Graphene oxide exhibits π-π stacking non-covalent interactions, enhancing drug loading capacity and achieving controlled release. A stimulus-responsive nucleic acid delivery carrier composed of PEG, PEI, and graphene oxide enables controlled release by causing endosome rupture when graphene oxide nanoparticles absorb near-infrared radiation, leading to localized temperature elevation. Simultaneously, graphene oxide's high loading capacity allows for the assembly of graphene oxide, PEI, and sodium 4-styrenesulfonate into nanocomplexes for the simultaneous delivery of miRNA drugs and the anticancer drug doxorubicin.

2.Lipid Nanoparticle Delivery System

Lipid-based nanocarriers have become a research hotspot in the field of nucleic acid drug delivery. Lipids, being the primary component of cell membranes, readily fuse with the phospholipid bilayer of cell membranes, enhancing the cellular uptake of nanocarriers. Additionally, lipids are readily available, have good biocompatibility, and are biodegradable. Chemical modifications can also be applied to improve the targeting ability of these carriers. Currently, lipid nanocarriers under investigation include lipid complexes (lipoplex), lipid-polymer complexes (lipopolyplex), layer-by-layer nanoparticles (LbLNP), solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), and stable nucleic acid-lipid particles (SNALP).

(1)Lipid Complexes. One of the simplest delivery methods is to directly form lipid complexes by mixing nucleic acid drugs with cationic lipids, such as 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOTMA), through electrostatic attraction. The positively charged surface of lipid complexes facilitates their binding to negatively charged cell membranes and subsequent entry into cells. However, in vivo, they can interact with negatively charged biomacromolecules, leading to the displacement and leakage of negatively charged nucleic acid drugs, as well as the generation of charge-related physiological toxicity. Additionally, the stability of lipid complex formulations is not ideal, requiring immediate preparation before use.

(2)Lipid-Polymer Complexes. Lipid-polymer complexes are formed by electrostatically attracting positively charged polymer complexes, which are created by combining nucleic acid drugs with cationic polymers such as polyethylenimine (PEI), polylysine (PLL), polyarginine, polyamidoamine dendrimers (PAMAM dendrimer), and then encapsulating them with a lipid bilayer. The resulting nanoparticles have a surface that is largely neutral in charge. Compared to lipid complexes, lipid-polymer complexes can significantly improve delivery efficiency and stability. By modifying the surface of the complexes with hyaluronic acid or heparin, their immunostimulatory properties can be further reduced.

(3) Layer-by-Layer Nanoparticles. Layer-by-layer nanoparticles encapsulate nucleic acid drugs into multi-layered nanoparticles through sequential electrostatic interactions. The choice of a stable nano-core in the center and materials for each layer is crucial for the adsorption between adjacent layers. For instance, using negatively charged poly(lactic-co-glycolic acid) (PLGA) particles as the core, poly-L-arginine (poly-L-Arg) is first adsorbed onto the surface of the core to create a positively charged surface. This is then covered with negatively charged siRNA, followed by another layer of positively charged poly-L-arginine. Finally, a negatively charged hyaluronic acid layer coupled with CD20 antibodies is applied. The negatively charged outer layer of this multilayered nanoparticle avoids positive charge-related toxicity, and no significant toxicity was observed in animal experiments. However, the high material selection requirements and complex preparation process with poor reproducibility limit the application of layer-by-layer nanoparticles.

(4)Solid Lipid Nanoparticles (SLN) and Nanostructured Lipid Carriers (NLC). Solid lipid nanoparticles feature a solid cationic lipid core with a low melting point, where nucleic acid drugs are adsorbed onto the surface of the carrier matrix and stabilized by surfactants on the exterior. However, research has found that SLN may cause drug leakage due to the gradual growth of internal solid lipid crystals. Nanostructured lipid carriers, as an upgraded version, incorporate a small amount of liquid lipids into the matrix, reducing the degree of crystallization of the lipid core. This not only increases drug loading but also improves physical and chemical stability. Since the solid lipid core can also be used for drug loading, SLN and NLC can deliver lipophilic drugs. When Bcl-2 siRNA and paclitaxel were simultaneously encapsulated in SLN, in vitro results showed that both drugs could be released simultaneously.

(5)Stable Nucleic Acid Lipid Particles (SNALP). Stable Nucleic Acid Lipid Particles (SNALP) are lipid nanoparticles (LNP) containing ionizable cationic lipids, composed of positively charged/ionizable lipids, structural lipids, PEG lipids, and cholesterol. They can effectively prevent the degradation of nucleic acid drugs in endogenous environments and deliver drugs into cells. Due to their significant impact resulting from technological and commercial success, LNP in many literature specifically refers to SNALP. LNP typically have a particle size of 20 to 200 nm, which helps them overcome the shear force of body fluids (such as blood and lymph) and traverse tissue gaps. Research shows that LNP primarily enters cells through the formation of complexes with apolipoprotein E (ApoE) and subsequent endosome escape mediated by CLs/ILs after endocytosis mediated by low-density lipoprotein receptors. The in vivo distribution of LNP is closely related to the formulation and relevant receptors, and the study of LNP formulations is currently a hot and challenging research topic in RNA delivery.

Currently, three products using such delivery systems have been approved for market, including patisiran (trade name: Onpattro), an siRNA drug developed by Alnylam Pharmaceuticals for the treatment of hereditary transthyretin amyloidosis (hATTR), as well as the mRNA vaccines Comirnaty and Spikevax for preventing COVID-19 infection. The use of ionizable cationic lipids and the emergence of microfluidic mixing technology have resulted in drug-loaded particles with almost no surface charge. Ionizable cationic lipids are positively charged under acidic conditions but become nearly neutral in the environment of the body's circulatory system. During the preparation of lipid nanoparticles, solid lipid particles are formed by rapidly mixing an ethanol solution of lipids and an aqueous solution of nucleic acids under a pH of approximately 4. After dialysis to remove ethanol, the lipid particles are stored in a nearly neutral aqueous solution, resulting in a nearly neutral surface charge.

SNALP has achieved commercial success through a series of ingenious designs, but its problems are also apparent. Firstly, its delivery efficiency is limited, requiring high drug dosages to achieve therapeutic effects. However, as an injection that utilizes multiple lipid excipients, the toxicity of these excipients cannot be ignored. Additionally, PEG lipids have been reported to cause hypersensitivity reactions, and cationic lipids also have a certain pro-inflammatory effect. For example, Onpattro requires the use of antihistamines and hormone drugs before injection for control. With the emergence of the more efficient delivery system targeting the liver - N-acetylgalactosamine (GalNAc) conjugate delivery system, SNALP has gradually withdrawn from the siRNA delivery field. However, for mRNA and CRISPR gene editing drugs with poorer bioavailability and higher charge, the LNP delivery system is still the most effective way to address their delivery challenges.

Over the past half-century of accumulation, delivery technologies have gradually matured, enabling nucleic acid drugs to enter a period of rapid growth. Nucleic acid drugs have natural advantages in treating genetic diseases, providing valuable therapeutic opportunities for many patients with rare diseases. The development of nucleic acid drug delivery technology exhibits the characteristics of platformization. After the delivery technology matures, the speed of developing similar drugs based on the same technology platform will significantly accelerate. For example, leveraging the technical accumulation of lipid nanoparticles in the early stage, Moderna's mRNA COVID-19 vaccine took only 63 days from the completion of vaccine sequence selection and design to the first human administration, demonstrating unprecedented efficiency. It is foreseeable that the existing technology platform also holds great promise for future application in new therapeutic areas such as in vivo gene editing. At the same time, the delivery of nucleic acid drugs still faces numerous technical challenges. How to further improve delivery efficiency, reduce the toxicity of the delivery system, and achieve specific delivery to other tissues and organs outside the liver will be the research hotspots in this field. With further understanding of the in vivo mechanism of action of nucleic acids and related diseases, nucleic acid drug delivery systems are bound to usher in new breakthroughs and developments, contributing to providing safe and effective personalized treatment plans to benefit more patients.

References

[1] Chen Huan, Wei Lijun. Research Progress in Non-viral Delivery Systems for RNA Drugs [J]. Progress in Pharmaceutical Sciences, 2022, 46(11):

[2] Li Dan, Huang Yukun, Gao Xiaoling. Research Progress in RNA Drug Delivery [J]. Acta Pharmaceutica Sinica, 2023, 58(03):

[3] Wang Junfeng, Tan Manman, Wang Ying et al. Research Progress in the Modification and Delivery of Nucleic Acid Drugs [J]. Journal of Zhejiang University (Medical Sciences), 2023, 52(04):

About the Author

Xiaomichong, a pharmaceutical quality researcher, has been committed to pharmaceutical quality research and drug analysis method validation for a long time. Currently employed by a large domestic pharmaceutical research and development company, she is engaged in drug inspection and analysis as well as method validation.