Preparation Of 5-fluorouracil Nanoparticles Loaded On Red Carbon Dots And Its Imaging And Cellular Pharmacokinetics Study

5-Fluorouracil(5-FU)is one of the most commonly used anticancer drugs in clinical practice,which plays an important role in the treatment of cancer,but its clinical application is seriously affected by its irregular in vivo absorption,short biological half-life,poor selectivity and toxic side effects.In order to achieve targeting and visualization of drug delivery to cancer cells,improve the efficacy of 5-FU and reduce toxic side effects,this study designed a novel 5-FU nanoparticle with a prepared red fluorescent carbon dot with high fluorescence yield and good biocompatibility as a carrier and loaded with chemotherapeutic drug 5-FU to obtain a visual trace of liver cancer targeted slow-release 5-FU nanoparticle,and studied and analyzed the nanoparticle by ex vivo imaging.The kinetic behaviors and antitumor effects of the nanoparticles at the microscopic cellular level were studied by ex vivo imaging.The main research contens of this project are as follows:1.Firstly,N and B doped surface functionalized red fluorescent carbon dots(R-CDs)were prepared by hydrothermal method,and R-CDs were successfully loaded with chemotherapeutic drug 5-FU through hydrogen bonding to obtain CDs/5-FU nanoparticles,and then LPC/5-FU nanoparticles were prepared by linking PEG and target molecule lactobionic acid on the surface of R-CDs through amide reaction.They were characterized by laser particle size meter,transmission electron microscope(TEM),Fourier transform infrared spectrometer(FT-IR),ultraviolet spectrophotometer(UV),X-ray powder diffractometer(XRD),and fluorescence spectrophotometer.The results showed that the fluorescence spectra showed that the excitation wavelength of the prepared R-CDs was at 540 nm with excitation-dependent properties,and the light time and p H stability were good,and the quantum yield of the doped red carbon dots was calculated to be as high as 23.8%.Infrared spectra,XRD and TGA patterns proved the successful preparation of LPC/5-FU.2.A method for the determination of 5-FU content was established by high performance liquid chromatography(HPLC),and a methodological study was performed for the determination of drug loading and encapsulation rate of LPC/5-FU nanoparticles.The results showed that the method is characterized by high specificity,good precision and reproducibility,with a retention time of about 3 min,and can be used for the rapid determination of 5-FU.Good linearity was observed in the concentration range of 25μg/m L-200 μg/m L.The encapsulation rate of the prepared drug-laden nanoparticles was 76.4% and the drug loading was 37.6%.3.The in vitro release study and in vitro safety evaluation of LPC/5-FU nanoparticles showed that the free drug 5-FU was completely released within 4 h,and the release amount was 95.7%;the release amount of 5-FU in LPC/5-FU increased with the decrease of p H value of the release medium,and the release rate was the fastest and the release amount was the highest in PBS buffer solution at p H 5.5,reaching 87.7%.It reached 87.7%,indicating that the prepared drug-loaded nanoparticles had weakened intermolecular forces in the acidic buffer solution,which would lead to a more sustained and efficient release of the drug,and to a certain extent,the effect of slow release and prolonged drug efficacy was achieved.The in vitro safety of R-CDs and LPC/5-FU was evaluated by hemolysis assay,and the results showed that R-CDs and LPC/5-FU have good biocompatibility and good safety.LA molecules on the surface of LPC/5-FU nanoparticles need to specifically recognize the desialic acid glycoprotein receptor(ASGPR)to function,and there are fewer receptors on the surface of normal hepatocytes,so it is less toxic to 7702 cells.The inhibitory effects of free 5-FU and LPC/5-FU nanoparticles on two types of hepatocellular carcinoma cells(Hep G2 and SUN-739)were both concentration-dependent and time-dependent: with the prolongation of the action time,the 5-FU in LPC/5-FU nanoparticles was gradually released completely,and LPC/5-FU nanoparticles could achieve an inhibitory effect comparable to that of free 5-FU.4.The prepared red carbon dot R-CDs and LPC/5-FU nanoparticles were imaged ex vivo by inverted fluorescence microscopy and in vivo imager,and fluorescence microscopy successfully observed that R-CDs and LPC/5-FU nanoparticles emitted red fluorescence in the cytoplasm of Hep G2,and the cells took up R-CDs and LPC/5-FU nanoparticles in a time-dependent manner.The tail vein injection of LPC/5-FU nanoparticles into female Kunming mice was selected for in vivo imaging,and the distribution of R-CDs and LPC/5-FU nanoparticles in vivo could be observed.The experimental results showed that LPC/5-FU prepared with R-CDs as the carrier could achieve drug tracing and provide guidance for the study of the cellular kinetic behavior of this nanoparticle.5.Using hepatocellular carcinoma cells Hep G2 as the experimental object,we firstly established a HPLC method to determine the intracellular 5-FU content,and secondly studied the cellular uptake mechanism and cellular kinetic behavior.The LPC/5-FU entry pathway was studied at different times and with different uptake inhibitors.Results: The cellular uptake of LPC/5-FU was energy-dependent and mainly mediated by the involvement of clathrin in endocytosis.The intracellular 5-FU content was measured by high performance liquid chromatography and cell kinetic studies were performed according to the pharmacokinetic single-compartment model and statistical moments,which showed that the half-life of LPC/5-FU nanoparticles was prolonged,the intracellular retention time was longer and the cell kinetic behavior was improved compared with the control formulation of 5-FU and LC/5-FU,which may be the reason for the toxic effects of LPC/5-FU low toxicity and good in vitro anti-tumor effect.In vivo efficacy evaluation of 5-FU and LPC/5-FU nanoparticles were performed by establishing a tumor-bearing mouse model with Kunming female mice.In conclusion,N and B doped red fluorescent carbon dots R-CDs and LPC/5-FU fluorescent nanoparticles were successfully prepared in this study,and the fluorescent nanoparticles prepared with R-CDs proved to be excellent carrier materials for bioimaging and drug delivery,reducing the toxic side effects of 5-FU,improving the cellular pharmacokinetic behavior and enhancing the anti-tumor effects of drugs in vitro and in vivo.