Insulin-cobalt core-shell nanoparticles for receptor-targeted bioimaging and diabetic wound healing

Abstract

Diabetic wounds represent a major issue in medical care and need advanced therapeutic and tissue imaging systems for better management. The utilization of nano-formulations involving proteins like insulin and metal ions plays significant roles in controlling wound outcomes by decreasing inflammation or reducing microbial load. This work reports the easy one-pot synthesis of extremely stable, biocompatible, and highly fluorescent insulin-cobalt core-shell nanoparticles (ICoNPs) with enhanced quantum yield for their highly specific receptor-targeted bioimaging and normal and diabetic wound healing in vitro (HEKa cell line). The particles were characterized using physicochemical properties, biocompatibility, and wound healing applications. FTIR bands at 670.35 cm^-1, 849.79, and 973.73 indicating the Co-O bending, CoO-OH bond, and Co-OH bending, respectively, confirm the protein-metal interactions, which is further supported by the Raman spectra. In silico studies indicate the presence of cobalt binding sites on the insulin chain B at 8 GLY, 9 SER, and 10 HIS positions. The particles exhibit a magnificent loading efficiency of 89.48 ± 0.049% and excellent release properties (86.54 ± 2.15% within 24 h). Further, based on fluorescent properties, the recovery process can be monitored under an appropriate setup, and the binding of ICoNPs to insulin receptors was confirmed by bioimaging. This work helps synthesize effective therapeutics with numerous wound-healing promoting and monitoring applications.

Fig. 1. Morphological characterization of ICoNPs (a) SEM micrographs indicating the nanoparticles to be around 10–15 nm at a scale of 100 nm. (b)TEM micrographs of ICoNPs show the nanoparticle size to be ∼13 nm at a scale of 50 nm (inset shows particle size at a scale of 5 nm) (c) EDS showing the presence of cobalt ions in the synthesized nanoparticles (inset shows the relative % of each atom present in the sample) (d) DLS spectra showing the hydrodynamic diameter of ICoNPs versus the % intensity of particles.

Fig. 2. Study for the structural variations in protein due to insulin–cobalt metal ion interactions to form ICoNPs. (a) Metal ion binding residues depict the interaction between chain B of insulin and cobalt ions. (b) The binding sites for cobalt ions at insulin protein with different amino acids within 3.55 Å distance along with the distance measured. (c) The binding score of metal ions with different amino acids in the protein chain (d) FTIR spectra of CoCl₂, insulin, and ICoNPs in the range 400–4000 cm⁻¹ indicate the formation of new bonds between Co–O (vibrations and stretching), CoO–OH bond, and Co–OH bonds which were otherwise not present. (e) Raman spectra of CoCl₂, insulin, and ICoNPs indicate the involvement of different functional groups of insulin in synthesizing ICoNPs.

Fig. 3. CD spectroscopy, release kinetics, and physical interaction studies of ICoNPs. (a) Circular dichroism spectroscopy studies showing the stability of insulin protein alone and after interaction with cobalt chloride (ICoNPs) (b) the plot shows the release kinetic studies to determine the % drug release from the ICoNPs (c) shows the drug release per hour in mg ml⁻¹ analysis of the specificity of interaction of insulin and CoCl₂ using (d) UV-visible absorption spectra of CoCl₂, insulin, and ICoNPs showing the peak of insulin alone and after interaction with cobalt (ICoNPs) at ∼272 nm (e) emission spectra of ICoNPs after excitation at 272 nm indicates the emission maxima at ∼300 nm. Inset shows the trailing of fluorescence from ∼420 nm onwards.

Fig. 4. MTT assay to determine the viability and growth rate of HEKa cells. The data shows the treatment of HEKa cells in comparison to the cells treated with varying concentrations (1.5, 7.5, 30, and 60 μM, respectively) of cobalt chloride, insulin, the mixture of insulin and cobalt chloride, and ICoNPs. The data were plotted as the mean value of three independent experiments.

Fig. 5. In vitro studies indicate nanoparticles' internalization into the cells and their role in bioimaging. (a and b) STEM analysis of cells after incubation with ICoNPs at different scales using a copper grid. (c) Interaction of ICoNPs with the cell membrane receptor leading to its movement inside the lipid bilayer (d) the presence of ICoNPs inside the cell membrane of HEKa cell when observed under a microscope indicating its internalization. The bioimaging of HEKa cells using varying concentrations of ICoNPs. (e) and (f) Shows HEKa cells without any treatment with ICoNPs in white and violet light, respectively. (g) and (h) Indicates the cells treated with 7.5 μM of ICoNPs; (i) and (j) show the cells after treatment with 30 μM of ICoNPs; (k) and (l) indicates the cells after treating them with 60 μM of ICoNPs at white and violet light respectively after a time duration of 3 hours which emits bright blue fluorescence.

Fig. 6. Promotion and monitoring of in vitro wound recovery of the diabetic and normal wound using ICoNPs. The nanoparticles induced better wound recovery in HEKa cells when compared with cobalt salt, insulin, or a mixture of both. The cells were incubated with a fixed concentration of all the solutions, that is, 30 μM. HEKa cells were taken as control (without any added formulations). (a) The figure shows diabetic wound healing using HEKa cells (a) 0 h, (b) 6 h, (c) 12 h, (d) 24 h; the cells treated with the salt solution of cobalt chloride after (e) 0 h, (f) 6 h, (g) 12 h, (h) 24 h; the HEKa cells after treatment with insulin protein (i) 0 h, (j) 6 h, (k) 12 h, (l) 24 h; the cells after treating them with a mixture of insulin and cobalt chloride are shown in figure (m) 0 h, (n) 6 h, (o) 12 h, (p) 24 h; cells after treatment with ICoNPs after a duration of (q) 0 h, (r) 6 h, (s) 12 h, (t) 24 h; respectively. (u) The plot shows the relative change in wound diameter in diabetic conditions after treatment with all the solutions respectively after specific time intervals. (b) The figure shows normal wound healing using HEKa cells (a) 0 h, (b) 6 h, (c) 12 h, (d) 24 h; the cells treated with the salt solution of cobalt chloride after (e) 0 h, (f) 6 h (g) 12 h (h) 24 h; the HEKa cells after treatment with insulin protein (i) 0 h (j) 6 h (k) 12 h (l) 24 h; the cells after treating them with a mixture of insulin and cobalt chloride are shown in figure (m) 0 h (n) 6 h (o) 12 h (p) 24 h; cells after treatment with ICoNPs after a duration of (q) 0 h (r) 6 h (s) 12 h (t) 24 h respectively. (u) The plot shows the relative change in wound diameter in normal conditions after treatment with all the solutions respectively after specific time intervals.