Silk fibroin-based nanoparticles drug delivery system by supercritical CO₂technology

Abstract

Current cytotoxic chemotherapy in the field of biomedical engineering, especially cancer therapy has been limited by poor efficiency and high toxicity due to immediate drug release, non-specific tissue distribution, degradation by chemical and enzymatic hydrolysis, and low cellular uptake efficiency. The purposes of this study were to develop a silk fibroin-based nanoparticles drug delivery system by supercritical CO₂ technology to decrease the adverse side effects of the drugs and enhance drug performance. To achieve the objectives of the research, in the first part, silk fibroin (SF) nanoparticles were fabricated via solution-enhanced dispersion by supercritical CO₂ (SEDS) for the first time successfully. The influence of process parameters on particle size and SF nanoparticles formation mechanism were investigated. The results indicated that precipitation temperature, concentration and flow rate of SF solution have a positive effect, while precipitation pressure has a negative effect. The nanoparticle formation mechanism was elucidated with the formation and growth of silk fibroin nuclei in the gaseous miscible phase evolved from initial droplets generated by the liquid-liquid phase split. Secondly, to characterize silk fibroin (SF) nanoparticles and explore their application in drug delivery systems, ethanol was used to treat drug loaded SF nanoparticles for inducing water insolubility. The SF nanoparticles demonstrated excellent biocompatibility, time-dependent and concentration-dependent cellular uptake properties, and thus can be used as drug carrier. However, the drug leakage is a critical issue in the process of ethanol treatment. Thirdly, in order to overcome the disadvantages of SF nanoparticles and develop a novel SF/PPP nanoparticles drug carrier with the advantages of natural SF and synthetic PLLA-PEG-PLLA (PPP) polymer, a modified SEDS process was designed to prepare the SF/PPP composite nanoparticles. The results of biological evaluation showed that the silk fibroin can improve the property of PPP as drug carrier. SF/PPP nanoparticles possessed much better biocompatibility and can accelerate cell adhesion and internalization compared with PPP nanoparticles. Furthermore, in order to study the application of SF/PPP nanoparticles in cancer therapy, anti-cancer drug, paclitaxel (PTX) was loaded into SF/PPP nanoparticles by the SEDS process. The resulting PTX loaded SF/PPP (PTX-SF/PPP) nanoparticles do not need induction of water solubility so that the drug loss of PTX loaded silk fibroin nanoparticles in post-treatment can be avoided. PTX-SF/PPP nanoparticle could enhance the solubility of PTX and exhibited controlled drug release property. Especially the drug release rate in PBS solution could be accelerated with decrease of the pH value from 7.4 to 6.0. This property can benefit the tumor-specific therapy due to the unique tumor environment. MTS assay demonstrated in vitro anti-tumor activity of the PTX-SF/PPP nanoparticles for MCF-7 and HePG-2 cells after one week was slightly higher than that of free PTX. In terms of the characteristics of nanoparticles, PTX-SF/PPP nanoparticles have potential application in the field of tumor therapy. Finally, in order to enhance the targeting efficiency of the SF/PPP nanoparticles to the tumor site, a tumor-specific ligand, folic acid (FA), was grafted onto the surface of the SF/PPP nanoparticles successfully by conjugation of the amino group on the silk fibroin with the carboxylic group of folic acid activated by EDC/NHS. Flow cytometric analysis showed that the FA-SF/PPP possessed a higher cellular uptake by MCF-7 tumor cell than SF/PPP nanoparticles. However, MTS assay indicated that in vitro anti-tumor activity had no significant difference between PTX-loaded FA-SF/PPP and PTX-loaded SF/PPP nanoparticles. Due to the tumor-targeted delivery, high drug load and controlled drug release property, the PTX-FA-SF/PPP nanoparticles can be utilized as an effective drug delivery system for tumor targeted therapy. In conclusion, silk fibroin-based nanoparticles drug delivery systems including SF, SF/PPP, and FA-SF/PPP nanoparticles have been developed by supercritical CO₂ technology successfully. These nanostructured drug delivery systems have potential application in the field of biomedical engineering, especially cancer therapy.