Albumin Hyaluronic Acid Nanoparticles Containing Chemotherapy Drugs And Photothermal Agents For The Study Of Tumor Combination Therapy

Doxorubicin(DOX)is an anti-tumor antibiotic with a wide anti-tumor spectrum,which can be used for the treatment of breast cancer,liver cancer,bladder cancer and other cancers.However,due to the lack of targeting of DOX to tumor cells,the non-specific distribution of DOX into the blood circulation has caused many adverse effects,such as cardiotoxicity,myelosuppression,mucositis,and hair loss,limiting the clinical use of doxorubicin.Light therapy is a new method for tumor treatment,which has attracted wide attention due to its characteristics of precision treatment and non-invasive.In this study,doxorubicin was used as the model drug,Human Serum Albumin(HSA)and Hyaluronic acid(HA)were used as targeted vector materials,and photothermal agent IR780 was loaded simultaneously.Albumin hyaluronic acid nanoparticles(IR780/DOX-HSA-HA NPs)loaded with doxorubicin,a chemotherapy drug,and IR780,a photothermal agent,were obtained to improve DOX targeting and reduce toxic side effects,and to achieve the effect of combined chemotherapy and phototherapy,and to enhance the antitumor efficacy.In this study,Doxorubicin nanoparticles coated with albumin hyaluronic acid(DOX-HSAHA NPs)were first constructed,and the in vitro determination method of DOX was established by UV spectrophotometry,and the methodology was verified,including linear range,precision,specificity,recovery rate,etc.DOX-HSA-HA NPs were prepared by solvo-crosslinking method,and the formulation was optimized by single factor test and response surface method to obtain the best preparation conditions.The nanoparticles were characterized by particle size,potential,transmission electron microscopy,appearance,and in vitro release and hemolysis safety.The results showed that the nanoparticles had slowly release effect.The hemolysis results showed that the nanoparticles had no hemolysis risk and could be used for intravenous drug use.Subsequently,IR780 photothermal agent and IR780/DOX-HSA-HA NPs of chemotherapy drug DOX were prepared,and the nanoparticles were characterized by particle size,potential,transmission electron microscopy and appearance.The effect of different concentrations of HAase on particle size reduction was investigated by particle size shrinkage test.The results showed that in the presence of HAase,The size of the nanoparticles decreased continuously,indicating that the nanoparticles could be used for deep penetration of tumors.In vitro photothermal heating results show that IR780/DOX-HSA-HA NPs has an obvious heating effect,which can be used for photothermal treatment of tumors.Reactive Oxygen Specie(ROS)production in vitro results show that nanoparticles can produce a large number of ROS under irradiation.The results showed that IR780 encapsulated in nanoparticles could effectively alleviate the photoinstability of IR780 and improve the stability.Serum stability tests and the stability tests of nanoparticles dispersed in different media showed that nanoparticles had good serum stability.In different media,The particle size of nanoparticles did not change significantly and had good stability.The enzyme of nanoparticles and the drug release triggered by laser irradiation were investigated.The results showed that the drug release in nanoparticles was accelerated significantly in the presence of HAase and laser irradiation,and the drug release reached about 80% after 72 h.The drug release increased by about 30%,which proved that the nanoparticle has the ability of enzyme and laser triggering drug release when used in tumor therapy,which can enhance the effect of tumor therapy.Finally,it was verified through in vitro hemolysis test that IR780/DOX-HSA-HA NPs has no hemolysis risk,and it is a preparation that can be used for safe injection.Then,4T1 cells and HUVEC cells were used as cell models to investigate the antitumor activity of nanoparticles in vitro.The results of cell endocytosis fluorescence test and quantitative endocytosis test showed that after the addition of HA,the active targeting ability of nanoparticles was significantly enhanced,and the intake of HA nanoparticles by tumor cells was significantly increased.The results of cytotoxicity test showed that,The blank nanoparticles showed no obvious toxicity to cells,indicating that the nanoparticle materials were safe and non-toxic,and the nano drugs inhibited the survival rate of tumor cells in a concentration-dependent manner.The nanoparticles could significantly reduce the toxicity of free drugs and enhance the therapeutic effect.The production of ROS at the cell level was investigated.The results of fluorescence microscopy showed that the nanoparticles could produce more ROS under light,and thus had certain killing effect on tumor cells and played a role in photodynamic therapy.Meanwhile,the ability of nanoparticles to induce Immunogenic Cell Death(ICD)was investigated by immunofluorescence staining.By staining the ICD markers Calreticulin(CRT)and High Mobility Group protein B1(HMGB1),the staining results of the nanoparticle group were as expected.These results indicated that nanoparticles could induce immunogenic death of tumor cells.Finally,the antitumor activity of the nanoparticles in vivo was investigated using BALB/c mouse 4T1 tumor model.The results showed that the dual-loaded nanoparticle IR780/DOXHSA-HA NPs had a good anti-tumor effect,showing superior chemotherapy and phototherapy combined treatment effect,which was significantly superior to chemotherapy and phototherapy alone group,and had no effect on the body weight of mice after administration,indicating that the nanoparticle was a relatively safe and non-toxic drug.It can be used for intravenous administration.