Silk Fibroin-Coated Liposomes as Biomimetic Nanocarrier for Long-Term Release Delivery System in Cancer Therapy

Abstract

Despite much progress in cancer therapy, conventional chemotherapy can cause poor biodistribution and adverse side-effects on healthy cells. Currently, various strategies are being developed for an effective chemotherapy delivery system. Silk fibroin (SF) is a natural protein used in a wide range of biomedical applications including cancer therapy due to its biocompatibility, biodegradability, and unique mechanical properties. In this study, SF-coated liposomes (SF-LPs) were prepared as a biomimetic drug carrier. Physicochemical properties of SF-LPs were characterized by Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering, zeta potential measurement, and transmission electron microscopy (TEM). In vitro release of SF-LPs loaded with doxorubicin (DOX-SF-LPs) was evaluated over 21 days. Anticancer activity of DOX-SF-LPs was determined against MCF-7 and MDA-MB231 cells using the MTT assay. SF-LPs containing 1% SF exhibited favorable characteristics as a drug carrier. SF coating modified the kinetics of drug release and reduced the cytotoxic effect against L929 fibroblasts as compared to the uncoated liposomes containing cationic lipid. DOX-SF-LPs showed anticancer activity against breast cancer cells after 48 h or 72 h at 20 μM of DOX. This approach provides a potential platform of long-term release that combines biocompatible SF and phospholipids for cancer therapy, achieving efficient drug delivery and reducing side-effects.

Keywords: biomimetic nanocarrier; breast cancer; doxorubicin; drug delivery; liposome coating; liposomes; silk fibroin.

Figure 1

Schematic representation of liposomes containing soy phosphatidylcholine (soy PC), cholesterol (CH), and cationic stearylamine (SA) coated with SF via electrostatic interaction.

Figure 2

SDS-PAGE analysis of SF extracts at (A) 1 mg/mL and (B) 3 mg/mL, as determined by the measurement of absorbance at 280 nm. The estimated molecular weight of SF was 25.8 kDa.

Figure 3

FTIR spectra of SF and SF-LPs. (A) SF with and without treatment of methanol; (B) uncoated liposomes and SF-LPs using SF of 0.5–2.0%.

Figure 4

Physicochemical properties of liposomes with various amount of stearylamine (SA) in the liposomes containing soyPC, CH, and SA, including (A) particle size, (B) polydispersity index, and (C) zeta potential. Data are presented as the mean ± SD (n = 3). n.s. denotes not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 5

Representative TEM images of liposomes and SF-LPs with different SF concentrations. (A) Uncoated liposomes and SF-LPs when using (B) SF 0.5%, (C) SF 1.0%, and (D) SF 2.0%. In the presence of SF, a multilayer appearance was observed on the surface of liposomes.

Figure 6

Cell viability of L929 fibroblasts when treated with components of uncoated liposomes (LPs) and SF-LPs. The uncoated liposomes containing SA showed a reduction in cell viability by 20%, whereas SF-LPs could enhance cell proliferation. The lipid compositions were soy PC, CH, and SA at 10:1:3 (molar ratio), with total phospholipid concentrations of 0.25 mM and 5 mM; SF 1% (w/v) was used for coating liposomes. ** p < 0.01; * p < 0.05.

Figure 7

Comparison of in vitro release of DOX from uncoated liposomes and SF-LPs in PBS, pH 7.4 at 37 °C, over two periods. (A) Short-term release over the first 4 h (240 min) of drug release. (B) Long-term release over 21 days (500 h). The long-term release profiles were fitted with the Korsmeyer–Peppas kinetic model, M_t/M_∞ = K_kp × tⁿ, where M_∞ is the amount of drug at the equilibrium state, M_t is the amount of drug released over time t, K_kp is the constant incorporating structural and geometrical characteristics of the nanocarrier, and n is the diffusional exponent or drug release that determines drug release mechanism. The fitting curves demonstrate that the release behavior of DOX from uncoated liposomes and SF-LPs were governed by Fickian diffusion mechanism as a matrix system.

Figure 8

Cell viability of (A–C) MCF-7 and (D–F) MDA-MB-231 when exposed to DOX as a free drug and encapsulated in liposomes (DOX-LPs) or SF-coated liposomes (DOX-SF-LPs). SF-LPs were composed of soy PC:CH:SA (10:1:3) and 1% SF. The cells were treated with DOX in all forms at the final concentrations of 1, 10, and 20 μM for 24 h, 48 h, and 72 h. The percentage of cell viability was determined using the MTT assay. The cell viability was normalized within the control (PBS) of each incubation period as indicated by dashed lines (100% cell viability). Data are presented as the mean ± SEM, n = 3.