Preparation Of Serum Albumin Biomimetic PLGA Nanoparticles And Its Application In Cancer Chemotherapy And Photothermal Therapy

Polylactic acid-co-glycolic acid(PLGA)is a kind of degradable high molecular polymer materials widely used in the preparation of nanocaliths.It has the advantages of non-toxicity,non-irritability,degradability and good biocompatibility,and can protect drugs with low solubility and poor stability in biological environments.However,PLGA nanoparticles are easy to be recognized by macrophages and phagocytosis in human body.Wrapping albumin in the outermost layer of PLGA nanoparticles can effectively improve this situation,thus extending the in vivo circulation time and improving pharmacokinetics.Albumin modification can also increase tumor targeting.Cabataxel(CTX),as the second generation of taxane antitumor drugs,has poor water solubility,lack of targeting to tumor tissues,and has certain toxic and side effects.Therefore,in this study,the bionic PLGA of Human Serum Albumin(HSA)was adopted as the nano-carrier loaded chemotherapy drug CTX to improve its shortcomings and enhance its antitumor activity.A large number of studies have shown that the combination of multiple therapies to treat tumors is significantly more effective than one therapy alone.Photothermal therapy is mild and has few side effects,which can effectively enhance the anti-tumor effect when combined with chemotherapy.Therefore,the near-infrared dye Indocyanine Green(ICG)approved by FDA and CTX are jointly loaded on human serum albumin bionic PLGA nanoparticles in this study,in order to achieve the purpose of photothermal chemotherapy combined therapy and enhance the effect of tumor treatment.In this study,the bionic albumin PLGA nanoparticles(CTX-PLGA/HSA NPs,CPHNs)loaded with cabataxel were prepared.A method for the determination of carpacel content based on high performance liquid chromatography was established,and the method was verified.The specificity,precision and recovery rate were all in line with the requirements for the determination of carpacel content.At the same time,the determination methods of CPHNs encapsulation rate and drug loading were determined.CPHNs were prepared by ultrasound-emulsion method.The single factor of CTX dosage,PLGA dosage,HSA solution concentration,type of oil-phase solvent,oil-water phase volume ratio,ultrasonic power,ultrasonic time and stirring time were investigated using particle size,polydispersion coefficient,encapsulation rate and drug loading as evaluation indexes.The three factors that had the greatest influence on the particle size and encapsulation rate of nanoparticles were selected and combined with Box-Behnken Design response surface optimization method to optimize the prescription and determine the optimal prescription of CPHNs.The average particle size of CPHNs was109.90±0.79 nm(PDI was 0.145±0.010),and the Zeta potential was-11.14±2.49 mV.The CPHNs were spheroid and uniform in size,with obvious HSA modification layer.The encapsulation rate of CPHNs was 60.97±1.58%,and the drug loading was 2.40±0.36%.Then,albumin bionic PLGA nanoparticles(ICG-CTX-PLGA/HSA NPs,ICPHNs)loaded with both ICG and CTX were prepared.ICG and 3%PVA solution were added into the aqueous phase as emulsifiers.The other preparation methods were the same as CPHNs.The UV spectrum analysis showed that ICG and ICPHNs had the same absorption peak,which proved the successful preparation of ICPHNs.Compared with CPHNs measured by laser particle size analyzer,the size of ICPHNs was slightly larger,about 119.72±0.79 nm(PDI was 0.174±0.025),Zeta potential was-7.12±2.89,and uniform size spherical nanoparticles were observed by transmission electron microscopy,with obvious HSA modification layer.After the addition of ICG,there was no significant change in the encapsulation rate of CTX,which was about61.23%,and that of ICG was 72.56%.The preliminary stability study showed that the particle size of ICPHNs in ultra-pure water,normal saline and PBS did not change significantly within5 days,and the stability of icph Ns in fetal bovine serum remained good within 72h.ICPHNs also greatly improve their fluorescence stability compared with free ICGs.Cyclic photothermal heating experiments show that ICPHNs can improve the photothermal stability of ICG.In vitro photothermal heating experiments show that the nanoparticles with ICG concentration of 15μg/mL can be heated to more than 50℃ under the irradiation of 808nm near-infrared laser with1.5W/cm~2.In vitro release experiments showed that ICPHNs had a certain sustained release effect,and the release effect of nanoparticles with HSA modified layer was different from that of nanoparticles without modified layer.Light-triggered drug release experiments have proved that light can heat up the nanoparticles and crack them,so that chemotherapeutic drugs can be released faster at specific sites to achieve therapeutic effects.In vitro detection of Reactive Oxygen Species(ROS)shows that ICPHNs produce more ROS than free ICGs and have better anti-tumor effects.Hemolysis test showed that the percentage of hemolysis of ICPHNs samples was less than 5%,indicating that the preparation was safe and non-toxic and suitable for intravenous administration.In order to demonstrate the antitumor activity of ICPHNs in vitro,4T1 cells were used as the model for in vitro antitumor activity experiments.After the cells were incubated with ICPHNs by laser irradiation,DCFH-DA fluorescent probe was used to detect the production of reactive oxygen species in cells.The results showed that a large number of reactive oxygen species could be produced by laser irradiation after ICPHNs was incubated with cells,so as to kill tumor cells.Cytotoxicity of the blank vector PLGA/HSA NPs was tested.The results showed that the blank vector had no obvious cytotoxicity.Cytotoxicity tests were performed on free ICG,IPHNs+Laser,ICPHNs and ICPHNs+Laser.The results showed that the cell survival rate of combined photothermal chemotherapy was significantly lower than that of photothermal therapy and chemotherapy alone,which proved that ICPHNs could achieve the purpose of combined therapy and enhance the therapeutic effect.The cell uptake experiment showed that the modification of nanoparticles by HSA and laser irradiation could increase the uptake of nanoparticles by cells,thus improving the anti-tumor effect.Immunofluorescence staining showed that CRT fluorescence was enhanced and HMGB1 fluorescence was weakened after ICPHNs treatment,which proved that ICPHNs could cause stronger ICDs than free ICGs and enhance the killing effect on tumor cells.Finally,a mouse subcutaneous 4T1 tumor model was established to study the antitumor activity in vivo.It was found in the experiment that the body weight of mice in the ICPHNs+Laser combined treatment group did not change significantly,while the tumor volume and weight decreased significantly,which proved that ICPHNs had no obvious toxicity and could achieve the purpose of combined treatment of tumor with good antitumor activity in vivo.