Construction And Anti-breast Cancer Evaluation Of Gambogic Acid-chlorin E6 Targeted Nanoparticles Based On Self-assembly

Objective:In order to solve the shortcomings of active constituent of traditional Chinese medicines gambogic acid（GA）such as poor solubility and low bioavailability,as well as the limitations of the tumor environment on PDT treatment,this study is based on the design principle of self-assembly,prepared the carrier-free self-assembled nanoparticles GA-Ce6-FA NPs containing the active ingredient GA of traditional Chinese medicine anti-tumor,including GA with multifunctional active ingredient of traditional Chinese medicine,Chlorin e6（Ce6）with PDT function,and Targeting ability of folic acid（FA）.The construction and preparation of GA-Ce6-FA NPs not only realizes the co-delivery of chemotherapeutics and photosensitizers,but also can deplete the GSH in the tumor through GA to further enhance the PDT therapeutic effect and has significant anti-tumor efficacy in vivo and in vitro.The preparation of the nanoparticles provides a new idea for co-loading and synergistic treatment of active ingredients of traditional Chinese medicine and PDT drugs.Methods:（1）Use the trace GSH detection kit to determine the ability of consume intracellular GSH by GA;Use the CCK8 method to investigate the synergistic effect of GA and Ce6 in MCF-7 cells;Use the self-assembly method to prepare GA-Ce6 NPs and GA-Ce6-FA NPs,and investigate the influence of drug molar ratio,stirring time,drug concentration and other factors on the formation of nanoparticles;Establish drug content detection methods through ultraviolet and HPLC to determine the drug loading and encapsulation efficiency of nanoparticles.（2）Use TEM,UV,fluorescence,etc.to characterize the formulation;Through the influence of SDS and Na Cl to prove nanoparticles formation mechanism;Through the particle size,PDI,etc.to investigate the stability of nanoparticles in different media;Through different p H to investigated the in vitro release of the nanoparticles.（3）Through CLSM and flow cytometry,investigated the cellular uptake capacity of the nanoparticles,proved the FA receptor target of the nanoparticles orientation;Evaluate the cytotoxicity of nanoparticles on cell lines expressing different FA receptors;Use Annexin V-FITC/PI cell apoptosis experiment to evaluate the apoptotic ability of nanoparticles.（4）The DPBF and DCFH-DA fluorescent probes were used to prove the ability of Ce6 to produce ROS;The trace GSH detection kit is used to prove the ability of nanoparticles to consume GSH.（5）Evaluate the blood safety of the nanoparticles through hemolysis and coagulation experiments;establish the MCF-7 cell subcutaneous tumor model,observe the distribution of the nanoparticles in vivo through the small animal imaging system,and further evaluate the pharmacodynamics of anti-breast cancer by the nanoparticles in vivo;Evaluate the safety of nano preparations by HE staining.（6）Observe the fluorescence imaging of GSH and ROS in the tumor by Thiol Tracker Violet and DCFH-DA fluorescent probes staining and prove the anti-tumor ability mechanism of the nanoparticles.Results:（1）GA could decrease the relative GSH content in cells with concentration dependence at the cell level;When the molar ratio of GA and Ce6 were 1:1 and 2:1,the CI value were 0.92±0.04 and 0.73±0.07,respectively,with synergistic effect;GA-Ce6 NPs were prepared under the following conditions:GA:Ce6 molar ratio with1:1,10 min of stirring time,0.6 mg/m L of Ce6,3 mg/m L of GA,the particle size was145.4±8.91 nm,the PDI was 0.121±0.008,the Zeta potential was-24.2±4.1 m V,the encapsulation efficiency of GA and Ce6 were 89.3±4.96%and 95.8±3.18%,respectively,and the loading efficiency were 51.71±3.75%and 48.3±3.86%,respectively.After adding FA,when Ce6:GA:FA were 1:1:0.2,the best preparation process of GA-Ce6-FA NPs was obtained.The particle size was 135.4±8.79 nm,the PDI was 0.093±0.014,and the Zeta potential was-25.2±3.6 m V;The encapsulation efficiency of GA,Ce6,and FA were 93.25±1.78%,95.45±4.96%,and 98.73±5.27%,respectively,and the loading efficiency were 48.5±2.53%,47.79±2.94%,and 3.71±0.19%,respectively.（2）GA-Ce6-FA NPs had an obvious Tyndall effect,nearly spherical particles,and the nanoparticles absorption peak intensity of ultraviolet and fluorescence was lower than that of the original drug;Proved the nanoparticles was formed by hydrophobic interaction and electrostatic adsorption between molecules;The stability test results showed that the particle size,PDI and Zeta potential of GA-Ce6-FA NPs have no significant changes within 9 days;In vitro release results showed that GA-Ce6-FA NPs in the medium of p H 5.0,the cumulative release rates of GA and Ce6were 56.91±4.6%and 72.61±4.6%,respectively,which showed the ability of p H-responsive release.（3）The uptake of nanoparticles in MCF-7 cells was time-dependent,and the uptake rate of GA-Ce6-FA NPs was best;After excess FA incubation,The cellular uptake of GA-Ce6-FA NPs was reduced,indicating that GA-Ce6-FA NPs are targeted to cell sites through FA receptors for endocytosis;The results of cytotoxicity experiments showed that in MCF-7 cells,after laser irradiation,the IC₅₀ value of GA-Ce6 NPs was 2.817±0.265μM,and the IC₅₀ value of GA-Ce6-FA NPs was 2.074±0.19μM.Compared with the Ce6,GA and GA-Ce6 NPs group,after laser irradiation GA-Ce6-FA NPs had the strongest ability to induce apoptosis of MCF-7cells,with an apoptotic rate of 64.51%.（4）Compared with GA and Ce6,nanoparticles showed a stronger GSH consumption ability,and GA-Ce6-FA NPs had a stronger ability to generate ROS at the cellular level,which proved that GA could enhance the effect of PDT by consuming GSH,which has the endogenous synergistic anti-tumor ability.（5）Compared with the control group,the nanoparticles had a good blood safety;The results of in vivo distribution showed that GA-Ce6-FA NPs could enhance the accumulation of the drug at the tumor site;The results of the efficacy evaluation of anti-breast cancer in vivo,it showed that the GA-Ce6-FA NPs group had the strongest tumor inhibition rate（88.2%）after laser irradiation,and it had a significant inhibitory effect on tumors;The results of each tissue section proved the biological safety of the nanoparticles.（6）The results of the determination of GSH and ROS in tumor tissues further showed that GA could reduce GSH and facilitate the generation of ROS after the administration of nanoparticles,thereby enhancing the PDT effect of Ce6.Conclusions:A carrier-free targeted nanoparticles GA-Ce6-FA NPs containing the active ingredient of traditional Chinese medicine GA and the photosensitizer Ce6 was prepared first time.The uniform particle size,the high encapsulation efficiency and loading efficiency,the good stability,and the p H-responsive release ability.At the cellular level,GA and Ce6 not only had a synergistic effect,but also could actively target through FA receptors to increase the uptake of GA-Ce6-FA NPs.And in the GA-Ce6-FA NPs,GA could consume excess GSH in tumor cells,thereby enhanced the effect of ROS produced by Ce6 to kill tumor cells,further improving the ability of PDT treatment,and improving the nanometer by the endogenous synergy of GA and Ce6 the ability of the formulation to inhibit cell proliferation.GA-Ce6-FA NPs had a good blood safety.After in vivo administration,GA-Ce6-FA NPs could be passively and actively targeted to the tumor site,further increased the accumulation of drugs in the tumor,and exerted a synergistic anti-breast cancer ability,achieved the endogenous synergistic antitumor effect of the combined treatment of active ingredients of traditional Chinese medicine and PDT.