In this study, a pH-induced self-assembly-based method has been developed to form silk fibroin nanoparticles (SFN-2) with a higher drug loading capacity (21.0 ± 2.1%) and cellular uptake than that of silk fibroin particles produced by a conventional desolvation method (SFN-1). Using the self-assembly method, rifampicin-encapsulated silk fibroin nanoparticles (R-SFN-2) were prepared with a size of 165 ± 38 nm at an optimum pH of 3.8. In silico analysis reveals that at acidic pH, the amino acid side chain charge neutralization of acidic residues, especially GLU64, promotes the formation of additional favorable interactions between the silk fibroin and the drug. The SFN-2 also possess a good aerosol property with a mass median aerodynamic diameter of 3.82 ± 0.71 μm and fine particle fraction of 64.0 ± 1.4%. These SFN-2 particles were selectively endocytosed by macrophages through clathrin- and caveolae-mediated endocytosis with a higher uptake efficiency (66.2 ± 2.1%) and were found to exhibit a sustained drug release in the presence of macrophage intracellular lysates. The cytokine and biomarker expression analyses revealed that SFN-2 could exhibit an immunomodulatory effect by polarizing the macrophages to an initial M1 phase and later M2 phase. Further, R-SFN-2 also exhibited an enhanced and sustained intracellular antibacterial activity against Mycobacterium smegmatis-infected macrophages than free rifampicin. Thus, the self-assembled silk fibroin particles with immunomodulatory action combined with a good aerosol and intracellular drug release property can be an attractive choice as a carrier for developing pulmonary drug delivery systems.
Keywords: antibacterial; drug loading; immunomodulatory; inhalable; intracellular; macrophages; self-assembly; silk fibroin.