Multi-responsive chitosan-based hydrogels for controlled release of vincristine

Abstract

As medical research progresses, the derivation and development of biological materials such as hydrogels have steadily gained more interest. The biocompatibility and non-toxicity of chitosan make chitosan hydrogels potential carriers for drug delivery. This work aims to develop two multi-reactive, safe, and highly swellable bio-hydrogels consisting of chitosan-graft-glycerol (CS-g-gly) and carboxymethyl chitosan-graft-glycerol (CMCS-g-gly), for sustained and controlled drug release, improved bioavailability along with entrapment in nanocarriers, which reduces side effects of vincristine sulphate. CS-g-gly and CMCS-g-gly are successfully prepared and fully characterized using analytical techniques. Under various conditions, the prepared hydrogels exhibit a high swelling ratio. Vincristine-loaded CS-g-gly (VCR/CS-g-gly), and CMCS-g-gly (VCR/CMCS-g-gly) show high encapsulation efficiency between 72.28-89.97%, and 56.97-71.91%, respectively. VCR/CS-g-gly show a sustained release behavior, and the maximum release of VCR from hydrogels reached 82% after 120 h of incubation. MCF-7 (breast cancer cell line) and MCF-10 (normal breast cell line) are evaluated for cell viability and apoptosis induction. The in-vitro anti-tumor efficacy is investigated using flow cytometry. The tetrazolium-based MTT assay of hydrogels shows no evidence of significant cytotoxicity in MCF-7 and MCF-10 cells. According to these findings, these hydrogels can effectively deliver drugs to MCF-7 and other breast cancer cells.

Fig. 1. Synthesis pathway of hydrogels.

a CS-g-gly, (b) CMCS-g-gly.

Fig. 2. Design strategy for CS-g-gly and CMCS-g-gly hydrogels.

a Gelling of CS and (b) gelling of CMCS-g-gly with NH4 + binder after 48 h.

Fig. 3. FT-IR spectra of the synthesized hydrogels.

a Chitosan, (b) Glycerol, (c) CS-g-gly, (d) CMCS, (e) CMCS-g-gly.

Fig. 4. ¹HNMR spectra of hydrogels.

a CS-g-gly and (b) CMCS-g-gly (500 MHz, D2O, 25 °C).

Fig. 5. SEM morphological characterization of Hydrogels.

a CS-g-gly, (b) VCR/CS-g-gly, (c) CMCS-g-gly, (d) VCR/CMCs-g-gly.

Fig. 6. Loaded and unloaded hydrogels obtained responses under the optimal conditions.

a hydrogel size, (b) Zeta potential, and (c) Polydispersity, (d) Schematic representation of zeta potential. Data represent means ± standard deviations (n = 3). For all charts, ***p < 0.001; **p < 0.01; *p < 0.05.

Fig. 7. Atomic force microscopy (AFM) images.

a CS-g-gly and (b) CMCS-g-gly hydrogels showing nanoparticles morphologies, TGA and DTG of (c) CS-g-gly and (d) CMCS-g-gly hydrogels.

Fig. 8. The effect of pH on swelling behavior, EWC and SR of hydrogels.

a CS-g-gly dry tablet in pH = 1.2, pH = 5 and pH = 7.4 after 24 h, (b) CMCS-g-gly dry tablet in pH = 5 and pH = 7.4 after 24 h, (c) CS-g-gly EWC (%), (d) CS-g-gly SR (%), (e) CMCS-g-gly EWC (%), (f) CMCS-g-gly SR (%), in different pH. Each experiment was performed and replicated three times, and the results were displayed using error bars to depict the variance.

Fig. 9. The effect of temperature on swelling behavior of hydrogels.

a CS-g-gly dry tablet, (b) CMCS-g-gly dry tablet, (c) CS-g-gly EWC (%), (d) CS-g-gly SR (%) (e) CMCS-g-gly EWC (%), (f) CMCS-g-gly SR (%), in different temperature after 2 and 5 h. Each experiment was performed and replicated three times, and the results were displayed using error bars to depict the variance.

Fig. 10. Encapsulation efficiency and drug release profile of prepared hydrogels.

Encapsulation efficiency of (a) CS-g-gly and (b) CMCS-g-gly. Drug release profile of (c) VCR/CS-g-gly and (d) VCR/CMCS-g-gly and free VCR in PBS (pH 7.4 and 5) after 120 h. Each value represents the mean ± SD of three independent experiments.

Fig. 11. Comparison of cytotoxic effect.

a, b Cs-g-gly, CMCS-g-gly, VCR/Cs-g-gly and VCR/CMCS-g-gly against MCF-7 cell lines and (c, d) Cs-g-gly, CMCS-g-gly, VCR/Cs-g-gly and VCR/CMCS-g-gly against MCF-10 cell lines, after 24 and 48 h incubation. Each experiment was performed and replicated three times, and the results were displayed using error bars to depict the variance.

Fig. 12. Annexin-V/-FITC/PI Flow cytometry analysis of MCF-7 breast cancer cells treated with the prepared hydrogels.

a CS-g-gly, (b) CMCS-g-gly, (c) CS-g-gly loaded with vincristine, and (d) CMCS-g-gly loaded for 48 h. The lower left (Q4) (Annexin−PI−) and the lower right (Q3) (Annexin+PI−) quadrants represent viable cells and early apoptotic cells. The upper right region (Q2) (annexin+PI+) and the upper left (Q1) (annexin− PI+) represent late apoptotic cells and necrotic cells.