Codelivery of resveratrol melatonin utilizing pH responsive sericin based nanocarriers inhibits the proliferation of breast cancer cell line at the different pH

Abstract

Protein-based nanocarriers have demonstrated good potential for cancer drug delivery. Silk sericin nano-particle is arguably one of the best in this field. In this study, we developed a surface charge reversal sericin-based nanocarrier to co-deliver resveratrol and melatonin (MR-SNC) to MCF-7 breast cancer cells as combination therapy. MR-SNC was fabricated with various sericin concentrations via flash-nanoprecipitation as a simple and reproducible method without complicated equipment. The nanoparticles were subsequently characterized for their size, charge, morphology and shape by dynamic light scattering (DLS) and scanning electron microscope (SEM). Nanocarriers chemical and conformational analysis were done by fourier transform infrared spectroscopy (FT-IR) and circular dichroism (CD) respectively. In vitro drug release was determined at different pH values (7.45, 6.5 and 6). The cellular uptake and cytotoxicity were studies using breast cancer MCF-7 cells. MR-SNC fabricated with the lowest sericin concentration (0.1%), showed a desirable 127 nm size, with a net negative charge at physiological pH. Sericin structure was preserved entirely in the form of nano-particles. Among the three pH values we applied, the maximum in vitro drug release was at pH 6, 6.5, and 7.4, respectively. This pH dependency showed the charge reversal property of our smart nanocarrier via changing the surface charge from negative to positive in mildly acidic pH, destructing the electrostatic interactions between sericin surface amino acids. Cell viability studies demonstrated the significant toxicity of MR-SNC in MCF-7 cells at all pH values after 48 h, suggesting a synergistic effect of combination therapy with the two antioxidants. The efficient cellular uptake of MR-SNC, DNA fragmentation and chromatin condensation was found at pH 6. Nutshell, our result indicated proficient release of the entrapped drug combination from MR-SNC in an acidic environment leading to cell apoptosis. This work introduces a smart pH-responsive nano-platform for anti-breast cancer drug delivery.

Figure 1

Structures of resveratrol (A) and Melatonin (B).

Figure 2

SEM analysis of (A) 0.2% sericin nano-capsule with the average size of 265 nm; (B) 0.3% sericin nano-capsule with the average size of 557 nm; (C) 0.5% sericin nano-capsule with the average size of 662 nm, and (D) 1% sericin nano-capsule with the average size of 918 nm.

Figure 3

SEM analysis of 0.1% sericin nano-capsule with the average size of 127.9 nm.

Figure 4

Physicochemical characterization of NDCDS (RES + MEL-SCN).

Figure 5

The FT-IR spectra of melatonin (MEL), Resveratrol (RES), SNC, and MR-SNC.

Figure 6

CD spectra of sericin, B-SNC, MR-SNC, MEL, MEL + RES and RES (A); sericin spectrum demonstrated a weak negative band at 218 nm, which was assigned to β-sheet. The K2D3 software output of sericin secondary structure (B).

Figure 7

The in vitro release of MEL and RES from MR-SNC under different pH values (7.4, 6.5, 6), up to 55 h.

Figure 8

(A) MCF-7 cells viability after treatment with various concentrations of B-SNC, RES + MEL, and MR-SNC at different pHs. Results are mean ± SD for 3 different experiments, *P ≤ 0.05 compared to the respective controls. (B) Means Comparission of MCF-7 cells viability at various pHs in three groups. Results are mean ± SD for 3 different experiments, *P ≤ 0.05 compared to the respective controls.

Figure 9

Intracellular uptake of FITC labeled MR-SNC (500 µg/mL, pH 6) and DAPI staining of MCF-7 cells. Bar scale, 200 nm.

Figure 10

The charge reversal process may disrupt the electrostatic interactions between the sericin amino acids side chains (carboxyl and amino) in SNC at cancer environment PH, releasing MEL and RES.