| Objective:(1) To establish an assay method of paclitaxel and separate Low densitylipoprotein (LDL) from fresh human plasma.(2) Synthesis of polyethylene glycol graftedchitosan coupled decanoic acid.(3) Select the method for the preparation of PTX loadedmicelles, then optimize the process conditions and modify the drug loaded micelles usingLDL Then characterized and investigate the in vitro characteristics of them.(4) Evaluatethe in vivo acute toxicity, anti-tumor activity and the in vivo targeting characteristics of thePTX-LDL-ACD micelles compared with the PTX-INJ.(5) In addition, to investigate the invitro cell toxicity，intracellular uptake and Cell apoptosis of PTX-LDL-ADC micellescompared with the PTX-ADC and PTX.Methods:(1) The determination of paclitaxel was established by High Performance LiquidChromatography (HPLC). The KBr density gradient centrifugation was used to isolateLDL from human plasma and the protein of LDL was determined then.(2) The Chitosanderivatives were characterized by IR,1H NMR, wide angle X-ray diffraction (WAXD) anddifferential scanning calorimetry (DSC). In addition, the solubility in different solvent andthe solubilization capacity of insoluble drugs were determined too.(3) The best methodwas used to prepare the drug loaded micelles and optimized. EDC·HCl and NHS was usedto help LDL to connect micelles. The dialysis bag methods were used to analyze therelease rate determination of two kinds of drug loaded micelles.(4) The LD50ofPTX-ACD-LDL was synthesized by acute toxicity test of mice. The animal tumor modelswere established and the in vivo growth curve and the inhibition rate of tumor in nude micewere confirmed then. The histological observation was conducted after HE staining.Thetargeting Cy7-LDL-ACD was establishmented and its character was studies as well.(5)Thecell toxicity of micelles was detected by MTT method. And its intracellular uptake andsubcellular distribution in A549cells were studied by copolymer rubber microscopy andfluorescence microscopi. Results:(1) The standard curve of PTX was established and its regression equation was y=33.784x+6.073, R2=0.99996, PTX has good linearity among the range of0.5～200μg·mL-1. Low density lipoprotein (LDL) was isolated from fresh human plasma bysequential density gradient ultracentrifugation with a size distribution of (28.7±5.1) nm andPDI was0.208. The morphology of LDL was round spherical observated by Transmissionelectron microscope observation. And the LDL protein (not concentrated) concentrationwas0.808mg·mL-1measured by Bradford method.(2) The molecular weight of theamphiphilic block copolymer arginine-chitosan-decanoic acid (ACD) is about8.3kD; andthe critical micelle concentration of ACD was3.299×10-2mg·mL1tested by fluorescencespectrometry. The solubility test showed that ACD could easily dissolve in the range of pH1～12and self-assemble to form a micelle solution with light blue opalescence in water.(3)The drug loaded micelle was preparated by optimizated ultrasonic method and thebest preparation process was as follows: the concentration of ACD was3mg·mL-1andthe drug loading ratio was3∶10with the ultrasound number of200times. Theinfrared and gel electrophoresis experimentes showed thatLDL successfully connected to drug loaded micelles and its release was followedthe Higuchi equation,and the skeleton type release model characteristics.ACD micelleshave a mean size of166.3nm, polydisperse index of0.2and a potential of+21.75～+29.80mV determined by malvern zetasizer.(4) PTX-ACD and PTX-ACD-LDL group’s LD50and95%confidence limits of mouse intravenous injection were65.36(40.92~94.70)mg·kg-1and70.81(44.33~102.59) mg·kg-1respectively. The results showed that, the LD50of PTX-LDL-ACD group of ICR mice by intravenous injection was2.4times of thePTX-INJ group，and the LD50of PTX-ACD group was2.2times of the PTX-INJ group.The order of tumor inhibition: PTX-LDL-ACD> PTX-ACD> PTX-INJ, in whichPTX-LDL-ACD group’s inhibition rate reached63.42%.After intravenously through tailveins1h, two micelles systemic dispersion in the body, and fluorescence is moreconcentrated in the tumor site within6h; within12h, micelles concentrated in liver andtumor of nude mice observed clearly; within72h, strong fluorescence still was observed innude mice tumor of ACD-LDL group.(5) The cytotoxicity of micelle PTX-LDL-ADC>PTX-ADC; and the intracellular uptake amount of cells to absorb PTX-LDL-ADC>PTX-ADC; The and the impact on the cell apoptosis PTX-LDL-ADC>PTX-ADC.Conclusion: The HPLC method for the determination of content of paclitaxel was established and the high purity LDL was separated successfully in our experiment. Thedrug loaded PTX-ACD micelles was prepared and the LDL was successfully connected tothe micelle. PTX micelles can successfully increased drug solubility, and sustained releaseof drugs. Acute toxicity experiments showed that the PTX-ACD and the PTX-LDL-ACDhas lower toxicity than the PTX-INJ. In addition, the tumor inhibition experiments showedthat the inhibitory effect of LDL modified micelles were much better. The Cy7-labeledLDL-ACD showed a more obvious targeting in vivo by imaging offluorescence.Experimental results, compared with Taxol and loaded micelles withoutmodified the LDL, chitosan nanoparticles modified with the low-density lipoprotein (LDL)can activly combine with low density lipoprotein receptor (LDLR) of cancer cells,increased cytotoxicity significantly. |
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