PEGylated pH-responsive peptide-mRNA nano self-assemblies enhance the pulmonary delivery efficiency and safety of aerosolized mRNA

Abstract

Inhalable messenger RNA (mRNA) has demonstrated great potential in therapy and vaccine development to confront various lung diseases. However, few gene vectors could overcome the airway mucus and intracellular barriers for successful pulmonary mRNA delivery. Apart from the low pulmonary gene delivery efficiency, nonnegligible toxicity is another common problem that impedes the clinical application of many non-viral vectors. PEGylated cationic peptide-based mRNA delivery vector is a prospective approach to enhance the pulmonary delivery efficacy and safety of aerosolized mRNA by oral inhalation administration. In this study, different lengths of hydrophilic PEG chains were covalently linked to an amphiphilic, water-soluble pH-responsive peptide, and the peptide/mRNA nano self-assemblies were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The in vitro mRNA binding and release, cellular uptake, transfection, and cytotoxicity were studied, and finally, a proper PEGylated peptide with enhanced pulmonary mRNA delivery efficiency and improved safety in mice was identified. These results showed that a proper N-terminus PEGylation strategy using 12-monomer linear monodisperse PEG could significantly improve the mRNA transfection efficiency and biocompatibility of the non-PEGylated cationic peptide carrier, while a longer PEG chain modification adversely decreased the cellular uptake and transfection on A549 and HepG2 cells, emphasizing the importance of a proper PEG chain length selection. Moreover, the optimized PEGylated peptide showed a significantly enhanced mRNA pulmonary delivery efficiency and ameliorated safety profiles over the non-PEGylated peptide and Lipofectamine TM 2000 in mice. Our results reveal that the PEGylated peptide could be a promising mRNA delivery vector candidate for inhaled mRNA vaccines and therapeutic applications for the prevention and treatment of different respiratory diseases in the future.

Development of polypeptide-based materials toward messenger RNA delivery

“…The targeting and accumulation within specific tissues or organs play a crucial role in achieving efficient mRNA transfection to target cells while minimizing potential side effects. 75,76 Targeting strategies for mRNA delivery can be broadly categorized into two main approaches: passive and active targeting. In the case of passive targeting, the enhanced permeability and retention (EPR) effect enables mRNA delivery systems to accumulate passively in tumour tissues.…”

“…Strategies such as surface PEGylation or decoration with polyanions can typically enhance passive targeting, thereby improving the serum stability and extending the blood circulation of gene vectors. 76 On the other hand, the active targeting strategy for mRNA delivery is to use targeting molecules, such as antibody, mannose, Arg-Gly-Asp peptide (RGD), 77 Cys-Lys-Lys-Lys (CKKK), 78 which specifically bind to receptors that are overexpressed on the surface of target cells, enhancing the uptake of the mRNA delivery system by those cells. For example, Dong et al demonstrated RGD modified mRNA nanocomplexes exhibited prolonged blood circulation and yielded much higher mRNA expression in tumours via intravenous administration compared to non-cRGD nanoformulation.…”

Opportunities and Challenges for Inhalable Nanomedicine Formulations in Respiratory Diseases: A Review

“…PEG modification is one of the more common modifications that can enhance the biosafety and bioavailability of nanoparticles, and is therefore favored by researchers. 102 …”

mRNA Cancer Vaccines: Construction and Boosting Strategies

“…Furthermore, modification of cationic peptides, such as polyethylene glycolization strategies, can achieve higher mRNA transfection efficiency. In one study, Xu et al modified the N-terminal end of cationic peptide LAH4-L1 with different chain lengths of PEG and found that 12 monomeric PEG-modified LAH4-L1 exhibited a significantly improved transfection efficiency …”

“…In one study, Xu et al modified the N-terminal end of cationic peptide LAH4-L1 with different chain lengths of PEG and found that 12 monomeric PEGmodified LAH4-L1 exhibited a significantly improved transfection efficiency. 127 2.3.4. Cell-Based Vectors.…”