| Tumors are among the most devastating human diseases. Chemotherapy has been one of the three major treatment methodologies for malignant tumors, while the significant side effect and the low stability of the therapeutic agents decrease the therapeutic efficiency, decline the compliance of the patients, and limit the utilization of the chemotherapy.Docetaxel (DTX) is a semisynthetic taxoid derived from the European yew tree, Taxus baccata, and it is a mitotic spindle poison that accelerates the microtubule assembly from tubulin and blocks the depolymerization of the microtubule. It has been demonstrated extraordinary anticancer effects both in vitro and in vivo against a variety of tumors including lung, ovaries, breast, leukemia, malignant melanoma, etc. Although docetaxel produced profound effects on several cancer cells in vitro, the clinical test didn't show satisfactory effect in patients with tumor, mainly due to that docetaxel lacks tissue and cell specificity. As a result, a drug delivery system targeted to tumor, especially tumor neovasculature, represents an attractive prospect for future therapy.In the present study, DTX was taken as the model drug to be wrapped up into nanostructured lipid carriers (NLC) by modern nano-drug delivery technique. Three novel docetaxel-loaded nanostructured lipid carriers were prepared in this study. The first system was the passive docetaxel-loaded nanostructured lipid carriers (DTX-NLC); the second one was the poly (ethylene glycol) (PEG) modified docetaxel-loaded nanostructured lipid carriers (nNLC); the third one was antibody modified docetaxel-loaded nanostructured lipid carriers (tNLC). The three biodegradable NLC were evaluated in terms of pharmaceutical characteristics and bio-characteristics, respectively. The main methods and results were as follows:1. Determination of entrapment efficiency and drug contentSolution-ultracentrifugation method was applied to separate free DTX and the DTX-loaded NLC. The drug concentration was then determined by HPLC method. 2. Preliminary Studies on passive docetaxel-loaded nanostructured lipid carriers (DTX-NLC)DTX-NLC was prepared by the modified film ultrasonication-dispersion method. Then lyophilization was carried out to improve the stability of the DTX-NLC. The freeze-dried DTX-NLC was characterized by particle size distribution, zeta potential and entrapment efficiency. In vitro cytotoxicity of DTX-NLC was evaluated by MTT assay against three human cancer cell lines and one murine malignant melanoma (B16). The apoptosis was first detected by assessment of nuclear morphology staining with Hoechst 33342 and AnnexinV-FITC kit was used to measure the percentage of apoptosis induced by DuopafeiR or DTX-NLC. Cell cycle analysis was assessed by flow cytometry. In vivo antitumor efficacy was evaluated in Kunming mice bearing B16. The morphology of DTX-NLC was approximately spherical, the particle size and zeta potential of the freeze-dried DTX-NLC were 203.67±4.15 nm and-31.17±2.20 mV, respectively. The entrapment efficiency and drug loading of freeze-dried DTX-NLC were 88.9±1.02%and 4.25±0.35%, respectively. The in vitro release results showed that the drug release from DTX-NLC was slower than from DuopafeiR and followed the Weibull equation. Compared with DuopafeiR, DTX-NLC revealed more cytotoxicity against A549 cells by inducing more apoptosis and more G2/M arrest. The inhibition rates of DuopafeiR, DTX-NLC (10mg/kg) and DTX-NLC (20mg/kg) were 20.42%,51.53%and 90.36%, respectively, indicating that DTX-NLC could more effectively inhibit tumor growth. The results of the body weight variations of mice also showed that even if the dose of DTX-NLC was increased, DTX-NLC also have lower toxicity during the therapeutic procedure. These results suggested that DTX-NLC might be a promising drug delivery system for cancer therapy. To our knowledge, this was the first report about DTX-NLC for murine malignant melanoma treatment.3. Preliminary Studies on PEG modified docetaxel-loaded nanostructured lipid carriers (nNLC) and antibody modified docetaxel-loaded nanostructured lipid carriers (tNLC).Vascular endothelial growth factor receptors (VEGFRs) are overexpressed on the surface of a variety of tumor cells and on tumor neovasculature in situ, which are potential targets for tumor-and vascular-targeting therapy. This study aimed to develop a VEGFR-mediated drug delivery system to target chemotherapeutic agents to VEGFR2-overexpressed tumor cells and tumor neovasculature endothelial cells in vitro and in vivo. PEG modified docetaxel-loaded nanostructured lipid carriers (nNLC) and docetaxel-loaded targeted nanostructured lipid carriers (tNLC) were designed and prepared with DSPE-PEG-NH2 as a linker. The cellular cytotoxicity, cellular uptake, in vivo therapeutic effect and biodistribution of nNLC and tNLC were investigated. The mean particle size of tNLC was 168.70±2.07 nm (polydispersivity index 0.195±0.009) with zeta potential-28.89±1.3 mV, entrapment efficiency 98.43±0.51%, the drug loading 5.55±0.06%(w/w) and an ligand average coupling efficiency of 3.34±2.63%. As control, nNLC showed the mean particle size 160.23±2.57 nm (polydispersivity index 0.212±0.01), zeta potential -29.34±1.73 mV, entrapment efficiency 93.24±0.93%and the drug loading 5.95±0.07%(w/w). The mean size of FITC-tNLC was 162.23±3.45 nm (polydispersivity index 0.198±0.01). Cytotoxicity of tNLC against two human cancer cell lines and B16 was superior to DuopafeiR and nNLC. The tNLC also showed better tolerant and antitumor efficacy in murine model bearing B16 compared with DuopafeiR or nNLC. The studies on cellular uptake and biodistribution indicated that the better antitumor efficacy of tNLC was attributed to both the increased accumulation of drug in tumor. Compared with DuopafeiR, NLC distinctly changed the distribution of DTX in vivo and increased the concentration of drug in tumor greatly. The overall targeting efficiency (Te) of tNLC in tumor was improved from 4.04%(DuopafeiR) to 6.41%(1.59 times), the relative taking efficiency (re) and the maximum concentrations (Ce) in tumor were 2.63 and 1.35 respectively; The Te of nNLC in tumor was improved from 4.04% (DuopafeiR) to 4.54%(1.12 times), re and Ce in tumor were 1.92 and 1.16, respectively. The tumor targeting effect of tNLC was higher than those of nNLC. The relative tumor accumulation of nNLC and tNLC to DuopafeiR at 6h after intravenous administration was 294%and 533%, respectively. These findings suggested that tNLC designed to bind specifically to VEGFR-2 could be used to deliver DTX to the tumor endothelium and tumor and may inhibit tumor growth.In this study, water insoluble DTX was successfully incorporated into NLC for the aim to realize the target anticancer chemotherapy. DTX-NLC had high entrapment efficiency and stability by lyophilization. The DTX-NLC showed higher efficacy and lower toxicity in a murine malignant melanoma model when compared with DuopafeiR. PEG modified NLC display dramatically increased blood circulation in comparison to DuopafeiR. In addition, the anti-VEGFR2 labeled NLC showed specific tumor and tumor endothelium targeting efficiency. Therefore, the NLC presented in this paper could significantly enhace therapeutic efficacy and decrease the toxicity. Among them, the anti-VEGFR2 labeled NLC might be a promising targeting formulation for cancer treatment. |
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