Nano structured curcumin-silk fibroin drug delivery system by supercritical technology

Abstract

Chemotherapy is the most commonly used method in cancer therapy today, which always uses one or more chemotherapeutic drugs to prolong life or to reduce symptoms. Current chemotherapy drugs exhibit substantial adverse side-effects on normal tissues and organs of the human body if administered orally or by other traditional routes. Therefore, the major challenge in chemotherapy is the development of a drug delivery system (DDS) to deliver chemotherapeutic drugs safely and efficiently. Nanostructured DDS is a promising candidate in future therapy due to its high surface to volume ratio and better intracellular uptake efficiency. Current nanostructured DDSs are mainly dispensed orally; thus some of the existing problems can still not be addressed adequately, such as lack of tissue specificity as well as the complexity and expense of systems. Here, we supply an implantable, degradable, and controllable nanofibrous DDS, which not only can protect and deliver chemotherapeutic drugs to the specific site, but also can achieve controllable drug release accordingly, therefore minimizing the side-effect on normal cells. The purpose of this project was to develop novel nanostructured DDSs in supercritical CO₂ for cancer therapy. First, the natural compound curcumin (CM) was used as the drug model in this project due to its multi-functional bioactivities and low toxicity. In order to enhance the water solubility of CM, CM nanoparticles (NPs) with a spherical shape and mean particle size of ~325 nm were successfully prepared via solution-enhanced dispersion by supercritical CO₂ (SEDS) for the first time. The influence of process parameters on particle size was systematically investigated and the mechanism of CM NPs formation was also studied. The results indicate that both precipitation temperature and flow rate of the solution have a positive effect on particle size, while precipitation pressure shows a negative effect. The mechanism of NP formation was elucidated with the formation and growth of CM nuclei in the gaseous miscible phase evolved from initial droplets generated by the liquid-liquid phase split. Moreover, the water solubility and dissolution rate of CM NPs were higher than that of original CM powder. To characterize CM NPs and explore their application in biomedical fields, anti-bacterial, anti-oxidant, anti-cancer effect and cytotoxicity activities were evaluated. The results show that CM NPs exhibit a time-dependent intracellular uptake activity. CM NPs exhibit an improved inhibitory effect against Staphylococcus aureus and effective anti-oxidant activity. Meanwhile, CM NPs possess an enhanced anti-cancer effect due to the cell cycle arrest in the G2/M phase, accompanied by inducing apoptotic cells. Importantly, the cytotoxicity of CM NPs is obviously reduced. However, the water solubility and cell uptake efficiency of CM NPs is still limited. Secondly, to further increase the water solubility and cell uptake efficiency, CM was incorporated into silk fibroin (SF) carriers to generate CM-SF NPs via SEDS. The obtained CM-SF NPs (20 MPa pressure, 1:2 curcumin: silk fibroin ratio, 1% final concentration) showed irregular spherical shapes with a controllable particle size <100 nm and a narrow size distribution as well as high drug loading and encapsulation efficiencies. Meanwhile, the solubility of the CM was greatly increased, and CM was kept being released for 196 h. Moreover, the pressure during SEDS was such that it could induce the change in the secondary structure of the SF from α-helix to ß-sheet. Thus, more ß-sheet content in the SF caused a slower degradation rate and slow drug release. To explore the potential of CM-SF NPs in cancer therapy, in vitro intracellular uptake efficiency, anti-cancer effect, and cytotoxicity activities were evaluated. Higher water solubility and better time-dependent cellular uptake of CM-SF NPs was a strong indication of increased anti-cancer ability. MTS results show that the resulting CM-SF NPs possess enhanced dose- and time-dependent anti-cancer effects, which can be explained by the cell cycle arrest in the G2/M phase in association with inducing apoptotic cells. Importantly, CM-SF NPs exhibit low cytotoxicity on normal cell NCM460 but high toxic effect on cancer cell HCT116 at smart concentration (~10 g/mL). Finally, in order to achieve topical treatment of cancers with improved therapeutic efficiency and reduced side-effects, an implantable CM-SF nanofibrous matrix with controllable fibre diameter (<100 nm) was prepared via SEDS (20 MPa pressure, 8 mL/min flow rate, 35°C). Flow rate and concentration of the solution determined the formation of a nanofibrous structure. The drug release rate of CM-SF nanofibrous matrices can be modulated via ethanol vapor treatment (25-37oC, 6-12 h). Afterwards, the treated CM-SF nanofibrous matrices were used to evaluate the anti-cancer effect. The results show that treated CM-SF nanofibrous matrices have better intracellular uptake ability, which results in higher anti-cancer efficiency. The mechanism of inhibition on the growth of colon cancer cells can be explained by cell cycle arrest in the S phase in association with inducing apoptotic cells. Moreover, in vivo tumor inhibition results indicate that CM-SF nanofibrous matrices after ethanol vapor treatment also exhibit better tumor inhibition effect than other groups, which can be explained by the sustained drug release to the specific site. Therefore, it is concluded that the implantation of CM-SF nanofibrous matrices supplies a novel treatment of tumors in the future. In conclusion, these supercritical-based nanostructured DDSs (i.e. CM NPs, CM-SF NPs and CM-SF nanofibrous matrices) have great potential in the fields of cancer therapy, drug delivery and healthcare.