Nonviral targeted mRNA delivery: principles, progresses, and challenges

Abstract

Messenger RNA (mRNA) therapeutics have garnered considerable attention due to their remarkable efficacy in the treatment of various diseases. The COVID-19 mRNA vaccine and RSV mRNA vaccine have been approved on the market. Due to the inherent nuclease-instability and negative charge of mRNA, delivery systems are developed to protect the mRNA from degradation and facilitate its crossing cell membrane to express functional proteins or peptides in the cytoplasm. However, the deficiency in transfection efficiency and targeted biological distribution are still the major challenges for the mRNA delivery systems. In this review, we first described the physiological barriers in the process of mRNA delivery and then discussed the design approach and recent advances in mRNA delivery systems with an emphasis on their tissue/cell-targeted abilities. Finally, we pointed out the existing challenges and future directions with deep insights into the design of efficient mRNA delivery systems. We believe that a high-precision targeted delivery system can greatly improve the therapeutic effects and bio-safety of mRNA therapeutics and accelerate their clinical transformations. This review may provide a new direction for the design of mRNA delivery systems and serve as a useful guide for researchers who are looking for a suitable mRNA delivery system.

Keywords: delivery obstacles; mRNA therapeutics; nonviral delivery; targeted delivery systems.

FIGURE 1

Schematic illustration of mRNA therapeutics. (A) The central dogma. mRNA therapeutics work through the expression of functional proteins or peptides in cytoplasm. (B) For disease prophylaxis or immunotherapy, mRNA encoding an antigen is internalized by somatic cells (e.g., muscle cells) or antigen‐presenting cells (APCs) after intramuscular injection. Then, antigens expressed in the cytoplasm are degraded by proteasomes, and then effector cells (e.g., T cells and B cells) are activated to detect and eradicate pathogens directly. The therapeutic antibodies can also be produced by mRNA for passive immunity. (C) mRNA has been used for protein replacement therapy, encoding transmembrane, intracellular, and secreted proteins. (D) mRNA can be applied to encode the Cas9 protein for gene editing in vivo. (E) mRNA encoding reprogramming factors can reprogram cells into induced pluripotent stem cells in vitro, which can differentiate into desired functional cells for tissue regeneration.

FIGURE 2

The in vivo obstacles encountered in the mRNA delivery process. (A) The phosphodiester bonds in the structure of mRNA are highly susceptible to degradation by RNA enzymes in the physiological environment. (B) mRNA‐containing formulations, such as LNPs, will rapidly get covered with various circulatory proteins and form the protein corona after intravenous administration. It causes the nanoparticles to be easily captured by MPS (e.g., macrophages in the liver) and cleared out of the body. (C) Furthermore, a portion of LNPs can enter target cells. The encapsulating mRNA can be released into the cytoplasm via (a) membrane fusion and (b) proton sponge effect‐mediated lysosomal escape.

FIGURE 3

The schematic illustration of mRNA‐loading mechanisms. (A) The stable nanoparticles can be formed with negatively charged mRNA and positively charged polymers or lipids. (B) The mRNA can coordinate with metal ions and self‐assembly form spherical nanoparticles while retaining the integrity and biological function of RNA. (C) Furthermore, a novel class of nucleobase‐lipids termed DXBAs enables them to bind to oligonucleotides via the H‐bonding (principle of complementary base pairing) and p‐p stacking with reduced toxicity in vitro and in vivo.

FIGURE 4

The common structures of the delivery systems. (A) Lipid nanoparticles (LNPs) are typically prepared by microfluidics with traditional (cationic or ionizable) lipids or functional lipids, cholesterol, and helper lipids. (B) The polymer‐based vectors, including polyethyleneimine (PEI), ionizable amphiphilic Janus dendrimer (IAJD), and chitosan, can encapsulate mRNA to form stable nanoparticles via simple mixing.

FIGURE 5

The various applications of targeted mRNA delivery systems. (A) Lung‐targeted applications. Exosomes are decorated with various integrins and tetraspanins via the nebulization route. LNPs bind specific antibodies (e.g., anti PECAM) via the intravenous route. (B) Liver‐targeted applications. LNPs bind the targeting ligand (e.g., ATRA, GalNAc) via the intravenous route. (C) Brain‐targeted applications. LNPs bind the targeting ligand (e.g., glucose, RVG) via retro‐orbital injection; binding of glucose to GLUT1 and RVG to nAChR allows LNPs to be endocytosed by endothelial cells. (D) Spleen‐targeted applications. MC3‐based LNPs modified by PS are delivered to the spleen by binding to macrophages via the intravenous route. (E) Kidney‐targeted applications. Polyplexes decorated with cyclam bind to CRCX4 receptors for delivery to injured tubule cells via the intravenous route. Biorender was used for this figure.

FIGURE 6

The applications of cellular targeted delivery. (A) Dendritic cells. Glucosylated nanovaccines targeting Glut‐1 on DCs deliver both conventional antigens and tumor‐specific neoantigens, triggering DCs maturation and robust adaptive immune responses. (B) T cells. The synthesis of LNPs engineered to transport mRNA to T cells was achieved by substituting cholesterol with hydroxycholesterol within their structure and design. (C) Macrophages. The dual targeting of both tumor cells and tumor‐associated macrophages in breast cancer therapy developed by utilizing folate receptors highly expressed on these cells. (D) Tumor cells. Lipid–peptide nanocomplexes to deliver mRNA to the murine B16‐F10 melanoma tumor.