The inhibitory effect of curcumin loaded poly (vinyl caprolactam) nanohydrogel on insulin fibrillation

Abstract

Amyloidosis refers to a group of diseases caused by the deposition of abnormal proteins in tissues. Herein, curcumin was loaded in a nanohydrogel made of poly (vinylcaprolactam) to improve its solubility and was employed to exert an inhibitory effect on insulin fibrillation, as a protein model. Poly (vinyl caprolactam), cross-linked with polyethylene glycol diacrylate, was synthesized by the reversible addition-fragmentation chain transfer method. The release profile of curcumin exhibited a first-order kinetic model, signifying that the release of curcumin was mainly dominated by diffusion processes. The study of curcumin release showed that 78% of the compound was released within 72 h. The results also revealed a significant decline in insulin fibrillation in the presence of curcumin-loaded poly (vinyl caprolactam). These observations confirmed that increasing the ratio of curcumin-loaded poly (vinyl caprolactam) to insulin concentration would increase the hydrogel's inhibitory effect (P-value < 0.05). Furthermore, transmission electron and fluorescence microscopies and Fourier-transform infrared spectroscopy made it possible to study the size and interaction of fibrils. Based on the results, this nanohydrogel combination could protect the structure of insulin and had a deterrent effect on fibril formation.

Keywords: Curcumin; Fibrillation; Insulin; Nano-hydrogel; PEGDA; PVCL; Poly (vinylcaprolactam); RAFT.

Fig. 1.

Curcumin release profile The profile of the cumulative release of curcumin from the Cur@PVCL system in terms of time (96 h) at pH= 7.5 (A). Matching various kinetic models with the cumulative release curve of Cur@PVCL (B).Cur, curcumin; PVCL, polyvinyl chloride.

Fig. 2.

The ThT binding test. Insulin fibrillation behavior in the presence of PVCL (A) and the presence of Cur@PVCL (B). The latter compound (B) indicated the insulin inhibitory effect. In these diagrams, the intensity has been plotted at 483 nm versus time at three different ratios of insulin to PVCL and insulin to Cur@PVCL. Cur, curcumin; PVCL, polyvinyl chloride.

Fig. 3.

The interaction of CR with the insulin fibrils Insulin fibrils were made in the presence of PVCL and Cur@PVCL and then, the interaction of CR with the fibrils was studied. The experiment was done using four different ratios of insulin to Cur@PVCL during 72 h of incubation. CR, Congo red; Cur, curcumin; PVCL, polyvinyl chloride.

Fig. 4.

DTT-induced insulin aggregation and studying the effects of applied compounds during the aggregation Comparison of the kinetics of DTT-induced insulin aggregation in the presence of PVCL and Cur@PVCL. Each sample contained 0.4 mg/mL insulin, 5 μl of 4 mM DTT, and different concentrations of PVCL and Cur@PVCL. The samples were analyzed at 43 °C for 20 min. The absorbance wavelength was fixed at 360 nm.DDT, dithiothreitol; Cur, curcumin; PVCL, polyvinyl chloride.

Fig. 5.

SDS-PAGE analysis of aggregated insulin in the presence of the studied compounds SDS-PAGE analyses of insulin aggregation in the presence of the hydrogels and curcumin alone. 15% non-reducing SDS-PAGE was applied for the test. Lane 1 indicates the obtained supernatant of insulin after inducing stress to the insulin sample in the presence of curcumin alone. Lanes 2, 3, and 4 stand for the obtained supernatants of insulin in the control state, in the presence of PVCL, and the presence of Cur@PVCL, respectively. SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; Cur, curcumin; PVCL, polyvinyl chloride.

Fig. 6.

The fluorescence microscopic images of insulin fibrils Under stress conditions, the fluorescence microscopic images of preformed insulin fibrils in the presence of PVCL and Cur@PVCL were taken. The test indicated the inhibitory effect of the Cur@PVCL hydrogel on insulin fibrillation. To prepare the fibrils for imaging, the samples at three different protein-compound ratios were incubated at 45 °C for 24 h. Then, 10 μl of each sample with 5 μl of ThT (100 μM) were uninformed on glass slides and were dried in a dark environment for 30 minCur, curcumin; PVCL, polyvinyl chloride.

Fig. 7.

Transmission electron microscopy of insulin fibrils Generated insulin fibrils in the presence of the maximum studied ratio of insulin to compounds (1:10) were selected for TEM analysis. The samples were prepared after a 24-h incubation under the stress condition. Cur, curcumin; PVCL, polyvinyl chloride.

Fig. 8.

SEC analyses of aggregated insulin specimens The SEC experiment has been shown for three different insulin samples after a 24-h incubation under stress conditions. The gel filtration was performed at a flow rate of 1 mL/min for 20 min. The absorbance wavelength was fixed at 210 nm. SEC, size exclusion chromatography; Cur, curcumin; PVCL, polyvinyl chloride.

Fig. 9.

Secondary structure analyses of the obtained insulin fibrils in different situations The secondary structures were evaluated by ATR-FTIR. Deconvolutions were done by the Origin Lab 2018b software. ATR-FTIR, attenuated total reflection-Fourier-transform infrared spectroscopy; Cur, curcumin; PVCL, polyvinyl chloride.