Abstract
Co-delivery of different species of protein-encoding polynucleotides, e.g., messenger RNA (mRNA) and plasmid DNA (pDNA), using the same nanocarrier is an interesting topic that remains scarcely researched in the field of nucleic acid delivery. The current study hence aims to explore the possibility of the simultaneous delivery of mRNA (mCherry) and pDNA (pAmCyan) using a single nanocarrier. The latter is based on gelatin type A, a biocompatible, and biodegradable biopolymer of broad pharmaceutical application. A core-shell nanostructure is designed with a thermally stabilized gelatin–pDNA coacervate in its center. Thermal stabilization enhances the core’s colloidal stability and pDNA shielding effect against nucleases as confirmed by nanoparticle tracking analysis and gel electrophoresis, respectively. The stabilized, pDNA-loaded core is coated with the cationic peptide protamine sulfate to enable additional surface-loading with mRNA. The dual-loaded core-shell system transfects murine dendritic cell line DC2.4 with both fluorescent reporter mRNA and pDNA simultaneously, showing a transfection efficiency of 61.4 ± 21.6% for mRNA and 37.6 ± 19.45% for pDNA, 48 h post-treatment, whereas established commercial, experimental, and clinical transfection reagents fail. Hence, the unique co-transfectional capacity and the negligible cytotoxicity of the reported system may hold prospects for vaccination among other downstream applications.
Highlights
Nucleic acid-based therapies are currently moving with vast strides towards increasingly broader clinical application
We varied the gelatin to a plasmid DNA (pDNA) ratio first (Figure 2) and five gelatin to pDNA mass ratios were investigated (100:1, 70:1, 50:1, 30:1, 20:1 and 1:1 w/w)
We found that using a gelatin to pDNA mass ratio of 30:1 and mixing temperature of 37 ◦ C resulted in the smallest particle size and PDI, with a negative zeta potential
Summary
Nucleic acid-based therapies are currently moving with vast strides towards increasingly broader clinical application. Investigating nucleic acids (NA) as vaccination tools has been for years one of the most advanced fields of research for nucleic acid-based therapies. Many ongoing clinical trials investigate mRNA-based vaccines for rabies, influenza H7N9, influenza H10N8, cytomegalovirus, human metapneumovirus, parainfluenza virus 3, respiratory syncytial virus, and Zika, among others [3]. Until recently, no NA-based vaccines have been approved for human use. This situation has been rapidly changing following the outbreak of SARS-CoV-2 in 2019. In December 2020, the mRNA vaccines of Pfizer/BioNTech (BNT162b2) [5] and Moderna (mRNA-1273) [6] were the first vaccines to receive approval for emergency use in humans against SARS-CoV-2
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