The Application Of Charge-conversional Nano-composite In Combination Therapy For Cancer Treatment

In recent decades,the global incidence of malignant tumors has continued to rise.With the continuous development of medical technology,although the mortality rate of cancer has dropped significantly,cancer is still one of the most deadly disease that threaten the health of humans.At present,the main treatment methods for cancer are still surgery,chemotherapy,and radiotherapy,etc.Among them,chemotherapy is still the first choices for the treatment of terminal cancer.However,the main disadvantage of chemotherapy is the high toxicity towards normal tissues,which will cause various side effects such as bone marrow suppression,venous thrombosis,liver cell damage,and gastrointestinal toxic reactions.Moreover,multidrug resistance（MDR）will commonly occur in chemotherapy for cancer treatment,which greatly reduces the efficiency of treatment and is the main cause of tumor metastasis and recurrence.As developments in the cancer research,the scientists and clinician realized that combination of multiple modality therapy can improve the efficiency of tumor treatment,thereby overcoming the problem of MDR that caused by mono-chemotherapy.Furthermore,the emergence of new treatment methods such as photothermal therapy,photodynamic therapy,immunotherapy,etc.,has brought new hope for tumor treatment.On the other hand,the in vivo drug delivery process is also an important factor that affects the cancer treatment efficiency.The key points of the delivery process is as follows:1)effective delivery of nanomaterials to tumor tissues with prolonged blood circulation time;2)and effective accumulation in tumor sites;3)efficient drug release in tumor cells.Therefore,as a promising drug vehicle nanomaterials should display high biocompatibility,prolonged half life time in blood circulation,and effective accumulation in the tumor site.Moreover,stimuli-response in tumoral microenvironment should be achieved that the drug release can be controlled to increase the concentration of drug at the target site resulting in excellent anti-cancer efficiency.Therefore,it is necessary to choose a nanomaterial with special physical and chemical properties to solve the problem of in vivo delivery.In this paper,a series of charge-conversional drug delivery systems are designed and prepared to be applied to tumor combination therapy（such as photothermal-chemotherapy,photodynamic-chemotherapy）,which exhibited negative charged under normal physiological conditions and positive charge under acidic conditions at tumor site.The as prepared drug delivery systems with good bio-safety are expected to achieve effective delivery of therapeutic factors to tumor cells and overcome the MDR in tumor treatment which can provide a new strategy and the basic data for tumor combined therapy.The main contents of the experiments are listed as follows:（1）Firstly,gold nanorods（GNRs）were first synthesized by seed growth method.Then,the polypeptide polymer,PBGZL,was prepared by anionic ring-opening polymerization method by usingg-benzyl-L-glutamate N-carboxy-anhydride（BLG-NCA）,N-e-carbobenzyloxy-L-lysine N-carboxy-anhydride（ZLL-NCA）,and cystamine.DTT was used to reduce the disulfide bond in PBGZL to obtain SH-PBGZL.SH-PGLA-DOX was prepared via a pH-sensitive hydrazone bond.SH-PGLA was prepared by using HBr/CH₃COOH via a hydrolysis process.Finally,a series of pH-responsive zwitterionic polypeptide-conjugated gold nanorods were prepared through gold-thiol interactions,which were used for the combination of photothermal therapy and enhanced cancer therapy.Fourier infrared spectroscopy（FT-IR）,hydrogen nuclear magnetic spectroscopy（¹H-NMR）,ultraviolet-visible spectroscopy（UV-Vis）,transmission electron microscopy（TEM）and other techniques were used to investigate the chemical structure,particle size distribution,and microscopic morphology,and surface properties of the GNRs conjugates.The results of FT-IR,1H-NMR and UV-Vis showed that all the characteristic peaks corresponding to its structure.In addition,the nanorod-like morphology with 73 nm was seen through TEM characterization.The experimental results showed that the pH-responsive GNRs conjugates were successfully prepared.Various solutions simulating the tumor cell environment under different pH conditions were prepared to study the charge conversion property and in vitro drug release behavior.The experimental results showed that at acidic pH,the surface charge of the GNRs-PGLA/PGLA-DOX can convert from negative（-1.07 m V）to positive（16.96m V）,and achieve a high drug release of 71.36%at pH 5.0.Under 808 nm laser irradiation,it was shown that GNRs conjugates has high photothermal conversion efficiency.The confocal microscopy（CLSM）and MTT method were used to study cell uptake,biocompatibility,and cytotoxicity of the GNRs conjugates.The experimental results showed that GNRs-PGLA/PGLA-DOX has good biocompatibility to HUVEC and high toxicity to He La cells,and can be internalized by He La cells.Compared with simple chemotherapy and photothermal therapy,GNRs-PGLA/PGLA-DOX has a better therapeutic effect on He La under 808 nm laser irradiation.For in vivo anti-tumor experiment,an animal xenograft model of He La cells was established to study the anti-tumor efficiency.The tumor volume,tumor mass,body weight,and inhibitory effect were investigated.Compared with single chemical and the photothermal treatment group,under 808 nm laser irradiation the GNRs-PGLA/PGLA-DOX demonstrated excellent tumor inhibition effection combined cancer therapy.In summary,the as prepared GNRs conjugates in this experiment exhibited charge conversion property and pH-responsive drug release,which can be used in the combined chemo-photothermal therapy towards MDR in cancer treatment.（2）Secondly,the silica nanoparticles containing disulfide bond（SiO₂（S-S））were prepared by bis-(3-（triethoxysilyl）propyl-disulfide（BETSPD）as a raw material via a hydrolysis process.After modified with 3-aminopropyl triethoxy silane（APETS）,the modified silica nanoparticles（SiO₂（S-S）-DMA）were prepared by amidation reaction with 2,3-dimethylmaleic anhydride（DMA）.Finally,DOX and methylene blue（MB）were encapsulated in SiO₂（S-S）-DMA nanoparticles to obtain a pH-responsive photodynamic-chemotherapeutic composite nanomaterial SiO₂（S-S）-M-D-DMA.FT-IR and Raman scattering spectroscopy were used to confirm the existence of disulfide bonds.UV-Vis and fluorescence spectroscopy were used to make sure that DOX and MB have been successfully encapsulated.TEM-EDS photos showed that the size of spherical nanoparticles of SiO₂（S-S）-M-D-DMA were about 62 nm and also confirmed the presence of S element.All the above results indicate that the composite nano-material SiO₂（S-S）-M-D-DMA has been successfully prepared in this project.In order to study the charge conversion property and in vitro drug release behavior,various solutions were prepared to simulate the tumor cell environment under different pH conditions.The experimental results showed that in the acidic environment,the surface charge of SiO₂（S-S）-M-D-DMA can convert from negative charge（-13.93 m V）to positive charge（7.09 m V）indicating good charge conversion performance.As the concentration of glutathione inside the tumor is much higher than that in normal tissue,various solutions which simulated the tumor cell environment were prepared to study the drug release behavior of pH/reduction responsiveness.The results showed that SiO_2（）S-S-M-D-DMA achieved the highest drug release rate（75.69%）at pH 5.0 with 10 m M GSH,which proves that SiO₂（S-S）-M-D-DMA has reduction responsiveness and acid-sensitive drug release.In this study,DPBF was used to detect the production of singlet oxygen（¹O₂）of SiO₂（S-S）-M-D-DMA under different conditions under 660 nm laser irradiation.The experimental results showed that the degradation of DPBF in the pH 5.0 with 10 m M GSH group reached the maximum,that is,achieving the highest production efficiency of ¹O₂,which further proves that the prepared composite nanomaterials have excellent pH/reduction-responsive drug release.