https://orcid.org/0000-0002-5906-0433
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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 LipofectamineTM 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.
Graphical abstract (TOC)
Schematic illustration of proper PEGylation strategy of pH-responsive LAH4-L1 peptide-mRNA nano self-assemblies enhances the pulmonary delivery efficiency of aerosolized mRNA.
Acknowledgments
We thank Cailing Yan, Shuping Zheng, and Minxia Wu of the Public Technology Service Center, Fujian Medical University, for the assistance in the IVIS bioluminescent imaging, flow cytometry, and transmission electron microscopy experiments and the assistance of the Laboratory Animal Center of Fujian Medical University for animal studies.
Authors’ contributions
Yingying Xu: conceptualization, methodology, analysis and interpretation of the data, the drafting of the paper, and revising it critically for intellectual content. Yijing Zheng: writing-original draft, investigation, validation, data analysis, software, and editing. Xuqiu Ding: methodology, investigation, data analysis, visualization. Chengyan Wang: animal study, investigation and data analysis. Bin Hua, Shilian Hong, Xiaoman Huang, and Jiali Lin: investigation, validation. Peng Zhang: resources, funding acquisition, writing-review. Wei Chen: project administration, funding acquisition, supervision, writing-review, and editing. All authors read and approved the final draft to be published.
Data availability statement
The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.
Disclosure statement
No potential conflict of interest was reported by the authors.
Ethical approval statement
The justification of intratracheally delivery of mRNA in mice is carefully evaluated and approved by the Laboratory Animal Welfare and Ethics Committee of Fujian Medical University, PR China. Although a A549 human lung epithelial cell model has been used in this study, a in vivo delivery safety and effectiveness evaluation of the vulnerable lung tissues is indispensable because a representative cell model could not fully disclosure the peptide/mRNA self-assemblies’ interaction with airway mucus and nuclease environment, as well as the potential toxicity toward different types of pulmonary epithelial cells and immune cells. A total of 144 BALB/c male mice with an average age of 8 to 12 weeks and body weight between 18 to 22 g were used. All mice were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. The mice were housed under a 12 h dark-light cycle at a constant temperature and with ad libitum feeding on water and standard laboratory chow. The experiment was carried out after one week of adaptation. Before intratracheal administration, the mice were anesthetized with intra-peritoneal injection of anesthetics (80 mg/kg ketamine and 4.5 mg/kg xylazine). A guiding cannula was gently intubated inside the trachea non-invasively to minimize the suffering of mice. The animal care and experimental protocols (the experimental design, justification for use of animals, animal treatment and disposal process) were all performed in accordance with protocols reviewed and approved by the Laboratory Animal Welfare and Ethics Committee of Fujian Medical University, PR China (the approved animal ethical number is IACUC FJMU 2022-Y-0683).
Supplementary information
A full description of detailed experimental methods, including the gel electrophoresis assay, particle size and morphology measurement, the MTT cytotoxicity assay, the mRNA cellular uptake and transfection, and in vivo mRNA delivery and biosafety studies, the fluorescent in vivo images of intratracheal administration of Cy5.5 fluorescent dye to mice (Figure S1) and pulmonary mRNA transfection study of PEG24LA/mRNA treated mice at 5:1 and 10:1 weight ratios (Figure S2 and S3) are provided in Supplementary Information (DOC).