Construction Of Tumor Targeted Camptothecin Nanodelivery System For The Synergistic Antitumor Therapy Of Drug-Resistant Three Negative Breast Cancer

Background:Camptothecin,extracted from Camptotheca acuminata Decne.,has antitumor effect through acting on the topoisomerase I to inhibit DNA synthesis in the tumor cells.The intact lactone ring of camptothecin is an essential group to inhibit topoisomerase I.Camptothecin kills tumor cells in the form of cell apoptosis,and is prone to multidrug resistance,thus leading to the chemotherapy failure.Therefore,it is very important to deliver camptothecin in the form of lactone ring into the tumor cells and overcome the drug resistance of tumor to camptothecin.Ferroptosis is a programmed cell death form different from apoptosis and plays an important role in tumor treatment.The essence of ferroptosis is that the excessive free iron can catalyze the hydrogen peroxide in the cell to produce a large amount of ROS,to oxidize the lipid on the cell membrane,and to form a large amount of lipid peroxide.Therefore,the delivery of iron ions into the tumor cells is expected to kill them through ferroptosis pathway,thus overcoming the drug resistance.Dopamine can coordinate with metal ions,which can undergo oxidative self-polymerization under the alkaline condition to form polydopamine（PDA）nanoparticle with strong cell adhesion capability.Polydopamine nanoparticles have excellent photothermal conversion ability and p H responsive release behavior.The network structure of polydopamine can effectively load the drugs.In addition,the amino group in dopamine is conducive to structural modification.Connecting folic acid to dopamine can enhance the active tumor targeted ability of polydopamine nanoparticles.As it is difficult to use antibodies and immunotherapy for the triple negative breast cancer,chemotherapy has become the main method.However,traditional chemotherapy is prone to drug resistance,and collaborative treatment has become a promising method to treat the triple negative breast cancer and overcome the drug resistance.Objective:Dopamine and folic acid modified dopamine were used as starting materials to prepare the tumor active targeted CPT/Fe@PDA-FA nanoparticles loaded with camptothecin and iron through oxidative autopolymerization.CPT/Fe@PDA-FA were delivered and targeted to the tumor site to achieve efficient entry into the tumor cells and release camptothecin and iron ions under the stimulation of intracellular p H.Combined with the photothermal properties of nanoparticles,CPT/Fe@PDA-FA could achieve combination therapy of cell apoptosis,ferroptosis and photothermal treatment to kill tumor cells and conquer drug resistance in multiple ways.Methods:1.Using dopamine and folic acid as starting materials,the folic acid modified dopamine（DA-FA）was synthesized by amidation reaction.The structure was characterized and confirmed by ¹H NMR and IR spectra.2.Through oxidative autopolymerization,the coordination of phenolic hydroxyl group in dopamine with iron ion,as well as theπ-πstacking of polydopamine grid structure and camptothecin plane structure,the tumor active targeted nanoparticles with efficient loading of camptothecin and iron were obtained by using dopamine and different proportion of DA-FA.The particle size,polydispersity index,zeta potential,CPT loading,iron loading,stability,morphology and elemental analysis of the nanoparticles were evaluated by dynamic laser particle size analyzer,high performance liquid chromatography,ultraviolet spectrophotometer,scanning electron microscope and transmission electron microscope.3.After CPT/Fe@PDA,CPT/Fe@PDA-FA5,CPT/Fe@PDA-FA10 and CPT/Fe@PDA-FA20 were incubated with the cells,the uptake of nanoparticles was observed by confocal laser scanning microscope（CLSM）to screen the best tumor targeted nanoparticle.The photothermal conversion properties of CPT/Fe@PDA-FA10nanoparticles were investigated by infrared thermography.The p H responsive drug release characteristics,in vitro drug release kinetics and hemolysis of CPT/Fe@PDA-FA10nanoparticles were investigated.4.MTT,cell clone formation and live/dead cell staining experiments were used to investigate the inhibitory activity of CPT/Fe@PDA-FA10 nanoparticles on the proliferation of drug-resistant triple negative breast cancer cells in vitro.5.MTT,ROS and LPO staining,GSH and MDA content determination,biological transmission electron microscope and western blot experiments were used to observe the cell proliferation activity,detect the contents of ROS,LPO,GSH and MDA,observe the morphology of mitochondria and observe the expression of ferroptosis related proteins（GPX4 and SLC7A11）,to investigate the activity of CPT/Fe@PDA-FA10 in inhibiting drug-resistant triple negative breast cancer cells through ferroptosis in vitro.6.Apoptosis staining,flow cytometry and western blot were used to investigate the activity of CPT/Fe@PDA-FA10 in inhibiting drug-resistant triple negative breast cancer cells through apoptosis in vitro.7.MTT competitive growth inhibition experiment and CLSM competitive uptake experiment were used to investigate the tumor active targeted effect and cell uptake mechanism of CPT/Fe@PDA-FA10.8.By injecting 4T1-Luc/CPT drug-resistant cells into the breast in situ,the nude mouse model of in situ drug-resistant triple negative breast cancer was constructed.The photothermal transformation performance of CPT/Fe@PDA-FA10 in nude mice with in situ drug-resistant triple negative breast cancer was observed by infrared thermography.9.The volume of in situ drug-resistant triple negative breast cancer was measured by vernier caliper,and the fluorescence intensity of tumor in nude mice was photographed by small animal imaging system in vivo to investigate the inhibitory effect of CPT/Fe@PDA-FA10 on the growth of in situ drug-resistant triple negative breast cancer in nude mice.10.Through H&E staining,TUNEL staining,ROS staining,LPO staining,Ki67staining,western blot experiment,MDA and GSH content detection and Caspase 3,Bcl-2,GPX4 and SLC7A11 immunofluorescence staining of in situ drug-resistant triple negative breast cancer tissue,the mechanism of CPT/Fe@PDA-FA10 against in situ drug-resistant triple negative breast cancer was investigated.11.The toxicity of CPT/Fe@PDA-FA10 to nude mice was investigated by weighing the nude mice bearing tumor,detecting the activities of alanine aminotransferase（ALT）and aspartate aminotransferase（AST）,as well as the contents of serum urea nitrogen（BUN）and creatinine（CREA）in the serum of nude mice,and H&E staining of heart,liver,spleen,lung and kidney tissues of mice.Results:1.The folic acid modified dopamine target material was synthesized and confirmed as the target compound by ¹H NMR and IR spectra.2.The average particle size of CPT/Fe@PDA,CPT/Fe@PDA-FA5,CPT/Fe@PDA-FA10 and CPT/Fe@PDA-FA20 was between 200-400 nm,and the polydispersity index（PDI）was less than 0.2.The iron loading was about 10%,and the encapsulation efficiency was about 50%.The loading of camptothecin was about 12%,and the encapsulation efficiency was about 38%.CPT/Fe@PDA,CPT/Fe@PDA-FA5,CPT/Fe@PDA-FA10 and CPT/Fe@PDA-FA20 remained stable at 4℃for 28 days.3.The CLSM results showed that the 10%target modified nanoparticle CPT/Fe@PDA-FA10 had the best uptake effect.The results of transmission electron microscope and scanning electron microscope showed that CPT/Fe@PDA-FA10 were spherical,regular in shape and good in homogeneity,and contained C,N,O and Fe elements.The observation results of infrared thermal imager showed that the nanoparticles had photothermal conversion properties that were dependent on irradiation time,laser power and nanoparticles concentration.Drug release experiments showed that CPT/Fe@PDA-FA10 had an acid sensitive drug release ability,and the drug release behaviors conformed to biexponential and biphase dynamics equation.The hemolysis test results showed that CPT/Fe@PDA-FA10 did not produce hemolysis.4.The IC₅₀ of the constructed 4T1-Luc/CPT drug-resistant cells was 21483 nmol/L,and the resistance index（RI）was 144,which met the criteria for successfully constructing drug-resistant cells.The results of MTT experiment,clone formation experiment and live/dead cell staining experiment showed that CPT/Fe@PDA-FA10+Laser could play a significant antitumor role through apoptosis,ferroptosis and photothermal therapy,and show good antitumor effect on drug-resistant tumor cells.5.The addition of ferroptosis inhibitor Fer-1 or DFO could inhibit the antitumor effect of CPT/Fe@PDA-FA10.The content of ROS,LPO and MDA in the CPT/Fe@PDA-FA10group increased significantly,while the content of GSH decreased significantly.The color of mitochondria deepened and the density of bilayer membrane increased.The expression of SLC7A11 protein and GPX4 protein observed by western blot test was significantly decreased.The results showed that CPT/Fe@PDA-FA10 could play an antitumor role through ferroptosis pathway.6.The proportion of flow cytometry apoptosis staining in the CPT/Fe@PDA-FA10+Laser group was the highest.The expression of Caspase 3 protein observed by western blot experiment was significantly increased,and the expression of Bcl-2 protein was significantly decreased.The results showed that CPT/Fe@PDA-FA10 could play an antitumor role in the way of apoptosis.7.The addition of competitive inhibitor FA did not change the inhibitory activity and cell uptake of non-targeted CPT/Fe@PDA on 4T1-Luc/CPT drug-resistant cells,but significantly reduced the antitumor effect and uptake of CPT/Fe@PDA-FA10 on 4T1-Luc/CPT drug-resistant cells.The above experiments showed that CPT/Fe@PDA-FA10 had the characteristics of active tumor targeted.Chlorpromazine,an inhibitor of the reticulin pathway,and genistein,an inhibitor of the caveolae pathway,were incubated with the cells in advance to significantly reduced the amount of CPT/Fe@PDA-FA10 absorbed by cells,indicating that nanoparticles were mainly absorbed by 4T1-Luc/CPT drug-resistant cells through the reticulin pathway and caveolae pathway.8.The results of small animal imaging system in vivo showed that the nude mouse model of in situ drug-resistant triple negative breast cancer was successfully constructed.After laser irradiation,the temperature of CPT/Fe@PDA-FA10 group increased significantly,indicating that CPT/Fe@PDA-FA10 had good photothermal conversion performance in vivo.The change trend of tumor volume and fluorescence intensity showed that CPT/Fe@PDA-FA10 showed significant inhibitory effect on the growth of in situ drug-resistant triple negative breast cancer through apoptosis,ferroptosis and photothermal treatment.9.The H&E staining results of breast cancer tissue showed that the number of cancer cells and the number of heteromorphic nuclei in the tumor tissue of the CPT/Fe@PDA-FA10+Laser group were reduced.The content of ROS,LPO and MDA in tumor tissue of CPT/Fe@PDA-FA10+Laser group increased significantly.The expression of GPX4 and SLC7A11 was significantly decreased by immunofluorescence staining and western blot.The above results together showed that CPT/Fe@PDA-FA10+Laser group could effectively inhibit in situ drug-resistant triple negative breast cancer through ferroptosis pathway.10.The number of TUNEL and Caspase 3 positive cells in tumor tissue of CPT/Fe@PDA-FA10+Laser group increased significantly,while the number of Bcl-2positive cells decreased significantly.The expression of apoptosis related protein Caspase 3in breast cancer tissue of the CPT/Fe@PDA-FA10+Laser group was significantly increased and the expression of Bcl-2 was significantly reduced observed by western blot.The above results together showed that CPT/Fe@PDA-FA10+Laser group could effectively inhibit in situ drug-resistant triple negative breast cancer through apoptosis.11.There was no significant change in the body mass of nude mice in CPT/Fe@PDA-FA10+Laser group,indicating that CPT/Fe@PDA-FA10+Laser had no toxicity.The activities of ALT and AST as well as the contents of BUN and CREA in the serum of nude mice in each treatment group were within the normal range,indicating that each treatment group had no damage to the liver and kidney functions of nude mice bearing tumor.No obvious morphological changes were observed in the H&E staining of the heart,liver,spleen,lung and kidney tissues of the tumor-bearing nude mice in each treatment group,indicating that no damage was caused to the heart,liver,spleen,lung and kidney tissues of the tumor-bearing nude mice in each treatment group.Conclusions:The folic acid modified dopamine was synthesized as carrier to construct camptothecin-based apoptosis/ferroptosis/photothermal triple-combination therapeutic system.The optimized nanoparticle CPT/Fe@PDA-FA10 could efficiently load camptothecin and iron,which had active tumor targeted ability,acid sensitive drug release property,excellent photothermal conversion performance and antitumor efficacy in vitro and in vivo.The antitumor studies have shown that CPT/Fe@PDA-FA10 could effectively aggregate in the tumor tissue and be actively absorbed into tumor cells.Under laser irradiation,CPT/Fe@PDA-FA10 could significantly kill drug-resistant triple negative breast cancer cells,and exhibit no obvious side effects on the major tissues and organs.The strategy provided a new idea for the construction of a three combined system based on camptothecin from traditional Chinese medicine to treat drug-resistant triple negative breast cancer,and provided experimental support for the formulations research and clinical application of the active ingredient camptothecin in traditional Chinese medicine.