Study On Antitumor Preparation Loading 10-Hydroxycamptothecin Polymer Prodrug For Chemotherapy Combined With Phototherrnal Therap

In this paper we prepared a pH-responsive tumor targeting drug delivery system in combination with photothermal therapy and chemotherapy on the base of tumor microenvironment.Gold nanoparticle of the system is the core,drug is an intermediary,and PEG is outer layer.the system uses EPR effect of tumor tissue passively targeting to tumor.The drug carried is 10-HCPT-Hyd-PEG which is a conjugate of 10-HCPT and PEG with a linker containing a pH sensitive hydrazone bond and a sulfur atom.10-HCPT-Hyd-PEG is connectted to the gold nanoparticles through its sulfur atom which built a ligand covalent bond with gold nanoparticles to form 10-HCPT-Hyd-PEG@AuNPs.Due to the acidic microenvironment of tumor tissue and lysosomes in cells,the linker between HCPT and PEG which contains a hydrazone bond ruptures and drug release after endocytosis.At the same time,under the near-infrared laser irridation,surface plasma turbulence effect of gold nanoparticles can convert light energy into heat which increases local temperature and further to kill tumor cells.Normal tissues without near infrared laser irradiation is not damaged.Spherical gold nanoparticles were prepared by reduction method with gold chloride acid as raw material,sodium citrate as stabilizer and reducing agent.Size of gold nanoparticles is about 30 nm.Using UV absorption spectrum,fluorescence spectrum,HRTEM,DLS technology for characterization of gold nanoparticles.Results showed that blank gold nanoparticle surface is weakly electronegative,and appearanced wine red with good stability.Particle size and potential did not change significantly after one month on room temperature.Maximum UV absorption wavelength is 520 nm.A conjugate 10-HCPT-Hyd-PEG was synthesized.A linker containing a sulfur atom and a pH responsive hydrazone bond was used which ruptures under low pH condition and release drug.A gold-sulphur bond connectted 10-HCPT-Hyd-PEG to the surface of gold nanoparticles.By which,an antineoplastic drug delivery system of 10-HCPT-Hyd-PEG@AuNPs was prepared with chemotherapy and phototherapy property.UV absorption spectra and fluorescence spectra analysis,HRTEM,DLS,XPS technology were used to characterize the conjugate.HPLC method was used for determining drug loading,drug release in different pH condition in vitro and irradiation stability after laser irradiation.Agarose gel electrophoresis method was used to test the effect of 10-HCPT-Hyd-PEG on activity of DNA topoisomerase I.It was found that under neutral condition,10-HCPT-Hyd-PEG is stable,but in acidic solution(<pH 6.0),hydrazone bond ruptured and drug release quickly.10-HCPT-Hyd-PEG@AuNPs can meet the requirements for drug-loading with good stability and heating speed is not affected after 808 nm near infrared laser irradiation.Agarose gel electrophoresis results showed that 10-HCPT-Hyd-PEG has no inhibitory activity on DNA topoisomerase I and it is an inactive prodrug of 10-HCPT.Using nano fluorescent microscope,laser confocal microscopy and Flow cytometry test to investigate cellular uptake;using MTT method to detect the cell toxicity in MCF-7,HepG2 and SW480 cells;using DNA ladder method to detect tumor cell apoptosis in MCF-7 cells;using western blotting method to detect the Bcl-2 protein expression after near infrared laser irradiation.Results show that the cellular uptake of 10-HCPT-Hyd-PEG was fast and to maintain a high concentration inside the cell for a long time.No exocytosis was quickly observed,which is helpful to lasting efficacy.Nano-fluorescence microscope observed that gold nanoparticles can quickly enter cell through cell endocytosis.After near infrared laser irradiation,the cell toxicity,apoptosis rate of 10-HCPT-Hyd-PEG@AuNPs group were significantly improved in three tumor cell lines,and the Bcl-2 protein expression was significantly lower than 10-HCPT-Hyd-PEG group and 10-HCPT group.Axillary tumorburdened animal model of nude mice was set up using MCF-7 cells and in vivo pharmacokinetics,tissue distribution and targeting research were studied.The results showed that circlation time of 10-HCPT-Hyd-PEG and 10-HCPT-Hyd-PEG@AuNPs in tumorburdened nude mice is significantly higher than that of free 10-HCPT solution.The order of 10-HCPT amount in tumor,liver and lung is:10-HCPT-Hyd-PEG@AuNPs>10-HCPT-HydPEG>10-HCPT soluttion.Results show that 10-HCPT-Hyd-PEG@AuNPs has better targeting property to tumor,liver and lung.The order of drug distribution in the heart and kidney is:10-HCPT-Hyd-PEG@AuNPs<10-HCPT-Hyd-PEG<10-HCPT.Which indicates that 10-HCPT-Hyd-PEG@AuNPs preparation show lower toxicity to heart and kidney.Determine tumor growth after given 10-HCPT-Hyd-PEG,10-HCPT-Hyd-PEG@AuNPs,10HCPT,blank AuNPs and saline control for 14 days.Result is that tumor inhibition effect of each group is:10-HCPT-Hyd-PEG@AuNPs>10-HCPT-Hyd-PEG>10-HCPT>AuNPs.Anti-tumor effect of 10-HCPT-Hyd-PEG@AuNPs group after the near infrared laser is greatly increased compared with 10-HCPT solution group.The results show that the antitumor effect of photothermal therapy combined with chemotherapy has been markedly enhanced compared with pure chemotherapy.Evaluate the toxicity of different drug delivery systems by analysis of experimental animals’ organ pathological slices after given different preparations,weight loss of experimental animals and blood phase.Results show that the the order of toxicity is AuNPs<10-HCPT-Hyd-PEG@AuNPs<10-HCPT-Hyd-PEG<10-HCPT.Preliminary toxicity research shows that:10-HCPT-Hyd-PEG@AuNPs preparation has good biological compatibility,lower toxicity,and do not influence weight of animals.It has no blood toxicity.