Preparation And Evaluation Of Multistage Antitumor Drug System Based On Carbon Dots Loaded Liposomes

Nanoparticles have proven to be effective drug carriers in diagnosis and therapy of cancer.But they faced a contradictory issue that NP with large size appear weak tumor penetration,meanwhile small size resulted in poor tumor retention.Herein,we fabricated doxorubicin conjugated carbon dots(DOX-CDs)and indocyanine green loaded liposomes(LPs)named DOX-CDs-ICG-LPs using a modified reverse phase evaporation process,and with high incorporation in the aqueous core.The DOX-CDs-ICG-LPs showed higher monodispersity,preferable fluorescence stability than free ICG or DOX.With laser irradiation,the DOX-CDs-ICG-LPs appeared higher temperature response,effective DOX release.Additionally,the fluorescence of DOX and ICG in DOX-CDs-ICG-LPs was also seen during in vitro subcellular localization.Compared to chemotherapy alone or photothermal therapy alone,the combined treatment of DOX-CDs-ICG-LPs with laser irradiation induced synergistical apoptosis and death of HepG2 cells.Antitumor activity in vivo suggested that DOX-CDs-ICG-LPs could achieve higher anti-tumor activity against H22 tumor cells and inhibit H22 tumor growth in vivo compared with DOX-CDs and ICG-LPs.It is worth noting that no systemic toxicity was observed after repeated administration of DOX-CDs-ICG-LPs with laser irradiation.Therefore,well-defined DOX-CDs-ICG-LPs have great potential in targeting cancer imaging and chemo-therothermal therapy.Using citric acid as carbon source and ethylenediamine as passivation agent,a carbon dots with higher QY was synthesized in one step by hydrothermal method.Further,the nuclear targeting sequence NLS was bonded to the surface,and the antitumor drug doxorubicin was loaded onto the CDs with a bridge of pH-sensitive quinone bonds.The structure of CDs was confirmed by FT-IR and NMR.XPS,XRD and TEM techniques were used to analyze and characterize the composition,microstructure and surface morphology of CDs.The results showed that CDs were well spherical dispersed with particle size about 3.8 nm.The CDs solution showed bright blue fluorescence under ultraviolet light irradiation.The excitation and emission wavelengths were 350 and 450 nm,respectively.It showed that DOX and NLS were successfully bonded to the surface of CDs.The in vitro release in different pH media was investigated and controlled release was achieved at pH 5.5.Choosing Hep G2 cells as the model,the cytotoxicity of CDs,CDs-NLS and DOX-CDs was examined by MTT assay.The results showed that CDs and CDs-NLS had no obvious cytotoxicity and good biocompatibility.DOX-CDs showed lower cytotoxicity at low concentrations,while high concentrations showed consistency with DOX.The liposome nanocomposite carrier of DOX-CDs-ICG-LPs was prepared by reverse evaporation method.The prescription and dosage of EPG were investigated.The morphology and surface charge of the nanocomposite carrier were evaluated by transmission electron microscope and Malvern particle size analyzer.The optical properties were determined by UV-visible and fluorescence spectra.The drug loading and entrapment efficiency of DOXCDs and ICG were measured by dextran microcolumn centrifugation,and the stability of DOX-CDs-ICG-LPs under high temperature and light conditions was further investigated.Photothermal conversion properties of DOX-CDs-ICG-LPs were evaluated using 808nm NIR lasers.The obtained DOX-CDs-ICG-LPs nanocomposite carriers had good dispersibility and surface potential.After ICG was loaded by liposomes,both the maximum absorption wavelength and the maximum emission wavelength showed a red shift,and the fluorescence intensity was also greatly enhanced.DOX-CDs-ICG-LPs significantly improved the stability of ICG under light and heat conditions.Under continuous NIR laser irradiation(808 nm),the photothermal properties of ICG were positively correlated with the concentration and irradiation power.Human Hepatocellular Carcinoma HepG2 cells were used to study the effects of different concentrations of DOX,DOX-CDs and DOX-CDs-ICG-LPs on tumor cells.Cytotoxicity and in vitro cell imaging was performed by MTT colorimetric method and laser confocal microscopy(CLSM),respectively.Cell uptake behavior was investigated by flow cytometry.Quantitative evaluation of cell apoptosis caused by DOX-CDs-ICG-LPs was performed using AO/EB double staining and Annexin V-FITC/Pl double staining.Cytotoxicity results showed that after laser irradiation,free ICG and ICG-LPs showed concentration and irradiation time-dependent cytotoxicity within a certain concentration range.The DOX and ICG showed synergistic cytotoxicity after 808 nm laser irradiation in DOXCDs-ICG-LPs group.Cell imaging experiments showed that DOX-CDs could enter the nucleus and achieve nuclear imaging.The NIR laser irradiation of DOX-CDs-ICG-LPs increased the concentration of CDs and DOX in cytoplasm and nucleus,and the cell uptake of DOX-CDs increased significantly.Compared with DOX,DOX-CDs nanocomplexes could induce apoptosis and reduce cell necrosis.The DOX-CDs-ICG-LPs nanocomplex carrier group showed higher apoptosis rate and necrosis rate,and could effectively bind chemotherapy and photothermal of DOX and ICG.Using H22 tumor-bearing mice as a model,the distribution of DOX-CDs-ICG-LPs in mice was investigated by in vivo imaging techniques using the fluorescence properties of CDs and ICG.The anti-tumor effect of DOX-CDs-ICG-LPs in vivo was evaluated,and toxicity to each major organ was performed by H&E staining.Immunohistochemistry was used to study the effect of nanocomplex on the proliferation and apoptosis of tumor cells.The results showed that the DOX-CDs-ICG-LPs nanocomposite carriers were concentrated in tumors and were few distributed in other organs.After laser irradiation,the DOX-CDs in the tumor site was stronger and more evenly distributed,indicating a great long circulation and tumor targeting.The nanocomposite could inhibit the growth of H22 tumors with an average tumor inhibition rate of 85.1%,which had little ill effect on the weight of mice.DOX-CDs-ICG-LPs could reduce cardiotoxicity induced by doxorubicin.In addition,they significantly inhibited the proliferation of H22 tumor cells and induced tumor cell apoptosis.