Enhancing Cisplatin Efficacy with Low Toxicity in Solid Breast Cancer Cells Using pH-Charge-Reversal Sericin-Based Nanocarriers: Development, Characterization, and In Vitro Biological Assessment

Abstract

Platinum-based chemotherapeutic agents are widely employed in cancer treatment because of their effectiveness in targeting DNA. However, this indiscriminate action often affects both cancerous and normal cells, leading to severe side effects and highlighting the need for innovative approaches in achieving precise drug delivery. Nanotechnology presents a promising avenue for addressing these challenges. Protein-based nanocarriers exhibit promising capabilities in the realm of cancer drug delivery with silk sericin nanoparticles standing out as a leading contender. This investigation focuses on creating a sericin-based nanocarrier (SNC) featuring surface charge reversal designed to effectively transport cisplatin (Cispt-SNC) into MCF-7 breast cancer cells. Utilizing AutoDock4.2, our molecular docking analyses identified key amino acids and revealed distinctive conformational clusters, providing insights into the drug-protein interaction landscape and highlighting the potential of sericin as a carrier for controlled drug release. The careful optimization and fabrication of sericin as the carrier material were achieved through flash nanoprecipitation, a straightforward and reproducible method that is devoid of intricate equipment. The physicochemical properties of SNCs and Cispt-SNCs, particularly concerning size, surface charge, and morphology, were evaluated using dynamic light scattering (DLS) and scanning electron microscopy (SEM). Chemical and conformational analyses of the nanocarriers were conducted using Fourier-transform infrared spectroscopy (FTIR) and circular dichroism (CD), and elemental composition analysis was performed through energy-dispersive X-ray spectroscopy (EDX). This approach aimed to achieve the smallest nanoparticle size for Cispt-SNCs (180 nm) and high drug encapsulation efficiency (84%) at an optimal sericin concentration of 0.1% (w/v), maintaining a negative net charge at a physiological pH (7.4). Cellular uptake and cytotoxicity were investigated in MCF-7 breast cancer cells. SNCs demonstrated stability and exhibited a pH-dependent drug release behavior, aligning with the mildly acidic tumor microenvironment (pH 6.0-7.0). Efficient cellular uptake of Cispt-SNC, along with DNA fragmentation and chromatin condensation, was found at pH 6, leading to cell apoptosis. These results collectively indicate the potential of SNCs for achieving controlled drug release in a tumor-specific context. Our in vitro studies reveal the cytotoxicity of both cisplatin and Cispt-SNCs on MCF-7 cells. Cisplatin significantly reduced cell viability at 10 μM concentration (IC₅₀), and the unique combination of sericin and cisplatin showcased enhanced cell viability compared to cisplatin alone, suggesting that controlled drug release is indicated by a gradient decrease in cell viability and highlighting SNCs as promising carriers. The study underscores the promise of protein-based nanocarriers in advancing targeted drug delivery for cancer therapy.

Figure 1

3D structure of cisplatin.

Figure 2

(A) The first grid box with center coordinates at x = 71.963, y = 90.989, and z = 102.494; dimensions of 80 × 76 × 62 Å; and a spacing of 0.869 Å. (B) The second grid box with a center at x = 114.997, y = 109.749, and z = 116.12, featuring dimensions of 60 × 52 × 92 Å with a 1 Å spacing. (C) The third grid box with center coordinates at x = 153.255, y = 129.716, and z = 134.526 nm; dimensions of 125 × 82 × 52 Å; and a 0.861 Å spacing.

Figure 3

Panel A illustrates the docking of sericin with cisplatin, revealing possible binding sites of cisplatin shown in boxes 1, 2, and 3, highlighted in yellow, purple, and orange, respectively. Notably, box 2 exhibits a higher likelihood of cisplatin binding compared to the other boxes. The most stable clusters in terms of energy and reproducibility, along with the most important amino acids involved in interactions, are shown in panels B, C, D, E, F, and G.

Figure 4

Size distribution analysis of (A) SNCs and (B) Cispt-SNCs. Zeta potential analysis of (C) SNCs and (D) Cispt-SNCs

Figure 5

SEM analysis of SNCs and Cispt-SNCs with the average size of 130 and 180 nm, respectively.

Figure 6

FTIR spectra of SNC, cisplatin, and Cispt-SNC.

Figure 7

(A) CD spectra of sericin and B-SNC. Sericin displayed a weak negative band at 218 nm, indicating a β-sheet structure. (B) The secondary structure was assessed using the K2D3 software.

Figure 8

EDX spectra of (A) SNCs (B) cisplatin (C) and Cispt-SNCs. EDX mapping images of the (D) element distribution of Cispt-SNCs, (E) cisplatin SEM-EDX, and (F) colorized elemental distribution (platinum is navy blue, nitrogen is yellow, chlorine is red, carbon is pale blue, and oxygen is purple).

Figure 9

(A) The in vitro release of cisplatin from SNCs at a pH value of 7.4. The release kinetics of cisplatin was analyzed using various mathematical models including first-order kinetics (B), Korsmeyer–Peppas (C), zero-order kinetics (D), Higuchi (F), and Hixson–Crowell (E).

Figure 10

MCF-7 cell viability after treatment with various concentrations of Cispt-SNCs and cisplatin. Results are mean ± SD, *p ≤ 0.05 compared to respective controls. Results are reported in five replicates.

Figure 11

Intracellular uptake of FITC-labeled Cispt-SNCs at pH 6 alongside DAPI staining in MCF-7 cells, displayed with a bar scale of 200 nm.