In situ synthesis of size-controlled melittin-polyhydroxyalkanoate nanospheres by microorganisms for plasmid delivery

A size-controlled polyhydroxyalkanoate (PHA) nanosphere, loaded with the amphiphilic cationic peptide melittin (Mel), was synthesized using E. coli to enhance the stability and delivery efficiency of Mel. It demonstrated that Mel efficiently mediated the delivery of plasmid DNA into cancer cells at concentrations below 1 μmol/L. To enhance Mel's expression, it was genetically fused with the PHA granule-associated protein (PhaP) through recombinant engineering. The fusion gene was inserted into the pET-28a(+) and transformed into an E. coli capable of synthesizing PHA nanospheres. This engineered strain not only produced Mel-PHA nanospheres but also regulated their size to approximately 100 nm under the control of PhaP, with a Mel loading concentration of 9.9 μmol/L. The purified Mel-PHA nanospheres could adsorb CRISPR/Cas12a plasmids. Optimal adsorption conditions were achieved by incubating the nanospheres and plasmids at 37 °C in PBS with a pH of 5.35 for 1 h, yielding a mass ratio of 2:1. Under simulated in vitro physiological conditions, the Mel-PHA@CRISPR exhibited stability in a culture medium containing fetal bovine serum for 24 h. Furthermore, the Mel-PHA@CRISPR were effectively internalized by HEK 293 A cells. This study successfully developed a self-assembled and size-controlled nanocarrier within E. coli for the delivery of CRISPR plasmids.