Preparation And Pharmacokinetic Study Of CD44 Targeting Doxorubicin Nanoparticles

Objective:Doxorubicin（Dox）is a broad-spectrum anticancer drug.It has a good elimination effect on tumor cells at different stages,but also has strong toxicity to the heart and spinal cord,so it is used as a second-line drug.Chondroitin sulfate（CS）is a natural polysaccharide,which exists in mammalian extracellular matrix and cell surface.It has many excellent characteristics,such as anti-inflammatory,anti-oxidation,anti-tumor,and has good biocompatibility and anacoresis.The CD44 antigen is a type 1 transmembrane glycoprotein.It is relatively static in normal cell,but highly overexpressed in aggressive tumors.So it is often used as a drug target.Studies have shown that CS can be combined with CD44,so it can be targeted to the tumor.Experiments in this paper using CS modified Dox,gives Dox CD44 target,and then using the solvent evaporation method with PLGA form new CD44 targeted nanoparticles,make the drug not only has tumor cell’s target,and is more likely to reach the tumor cell,gets into the nucleus and effect curatively.Reduce the toxic and side effects of Dox while enhancing its efficacy.Methods:1.Synthesis of chondroitin sulfate-adriamycin（CS-Dox）by modifying Dox with purified and degraded CS.Then characterize it by Fourier transform infrared spectroscopy（FT-IR）,nuclear magnetic resonance spectroscopy（¹H-NMR）,thermogravimetric and differential thermal analysis（TGA/DSC）,transmission electron microscopy（TEM）and particle size distribution analyzer.2.Optimizing the preparation of Dox and CS-Dox’s PLGA nanoparticles,determining the optimal preparation of nanoparticles.Making the blank PLGA nanoparticles,Dox-PLGA nanoparticles and CS-Dox-PLGA nanoparticles by the optimal preparation and characterizing them by transmission electron microscopy（TEM）and particle size distribution analyzer.3.Using HT22 cells as normal cell,U251 cells as tumour cell,investigating the toxicity reduction of CS-Dox on normal cells;investigating the effects of Dox,CS-Dox and PLGA nanoparticles on the proliferation of U251 cells by CCK-8 method;investigating the way of U251 cell uptaking and transporting Dox,CS-Dox and their PLGA nanoparticles and the CD44 targets of CS-Dox and its PLGA nanoparticles on tumor cellsr under the laser scanning confocal microscope.4.Establishing a method for the determination of Dox concentration in rat plasma by fluorescence.Detecting Dox’s concentration in rat plasma by this method and carrying out pharmacokinetic analysis.Comparing Dox contents in mouse tissues by fluorescence spectrophotometry and analyzing them.Results:1.Dox was modified to synthesize chondroitin sulfate-adriamycin（CS-Dox）with purified and degraded CS.The results of infrared analysis（FT-IR）,¹H-NMR analysis,thermogravimetric and differential thermal analysis（TGA/DSC）showed that CS was combined with Dox backbone through an acyl bond.The synthesized CS-Dox micelles had spherical structure and regular morphology.The size of the hydrated particles was about120 nm,and the particle size was homogeneous and distributed evenly.The CMC of the synthesized micelles was about 0.0184 mg/mL.When CS-Dox concentration was greater than 0.0184 mg/mL,CS-Dox formed micelles in solution.2.Determined the optimal preparation of Dox nanoparticles,and prepared the blank nanoparticles,Dox-PLGA nanoparticles and CS-Dox-PLGA nanoparticles,their particle size was all around 150nm,PDI was small.They were of moderate size and regularly shaped in a round shape with uniform distribution and no adhesion.3.The toxicity of CS-Dox to normal cells was lower than that of Dox.Dox,CS-Dox and their nanoparticles had a good inhibitory effect on the proliferation of U251 cells,but the inhibitory effect of CS-Dox-PLGA on U251 cells is stronger than that of Dox and its nanoparticles.Compared with Dox,CS-Dox was more easily into the cell,CS-Dox-PLGA nanoparticles was more easily into the nucleus.After CS preincubation to U251,the ability of CS-Dox and its nanoparticles getting into cells was significantly weakened.4.Established the method for the determination of Dox in rat plasma by fluorescence.The method was simple,accurate,convenient and fast.Dox in plasma was detected by this method.When drugs entered the body,Dox levels in the blood decreased over time.The plasma area of AUC_0-24 and C_max of CS-Dox and its nanoparticles were higher than those of other groups.CS-Dox could be absorbed more and the curative effect was enhanced.When drugs got into the body,they were mainly concentrated in the liver and kidneys for metabolism.CS-Dox could reduce the distribution of Dox in the heartand increase the distribution of plasma.Conclusion:Dox modified with CS,which not only has the antitumor activity of Dox,but also has the target of CD44 molecular.That can make the drug specific to the tumor cells.The drug is prepared into PLGA nanoparticles,making it easier for the drug to enter the nucleus and play its antitumor effect.It also enhances the circulation time of the drug in the body and reduces the toxic and side effects of the drug on the heart.