Preparation Of Synergetic Targeting Combined Chemotherapy Drug Her-HINP/PTX And Its Application In Breast Cancer Treatment

BackgroundBreast cancer is the second most common cause of cancer death and the most common cancer among women.It is estimated that there were 2,261,419 new cases globally in 2020,accounting for about 11.7%of all cases of all cancer types,even more than lung,prostate and colorectal cancers.Among 25%-30%breast cancer patients,tumor cells overexpress Human Epidermal Growth Factor Receptor type 2（HER2）,known as HER2 over expression,which has the characteristics of high invasiveness,rapid development,poor prognosis and easy metastasis and recurrence.Clinical studies have shown that targeted therapy can effectively inhibit the growth of HER2 overexpressed breast cancer and improve the cure rate.In 1998,the US Food and Drug Administration（FDA）approved trastuzumab（Herceptin?）as the first antibody-targeted therapy for breast cancer.In recent years,clinical trials of HER2 positive breast cancer have confirmed that initial trastuzumab combined with chemotherapy can increase the time to disease progression and overall survival compared to chemotherapy alone in the context of metastatic and adjuvant therapy^[1].However,the treatment of HER2 positive breast cancer still faces many difficulties in drug resistance and side effects^[2,3].Paclitaxel（PTX）,as the representative of Herceptin classical combination chemotherapy,is one of the most used traditional chemotherapy drugs today,but its water solubility is very limited（<0.03 mg/m L）.Traditional chemotherapeutic drugs are poor in water solubility and targeting,and have a large killing effect on normal cells,which leads to the occurrence of toxic side effects and adverse reactions further increased by conventional combined chemotherapeutic drugs.Therefore,how to improve the water solubility of traditional chemotherapeutic drugs,further improve the targeting of such chemotherapeutic drugs and reduce the toxic side effects and adverse reactions of conventional combined sequential drugs is still an urgent problem to be solved.In addition,studies have shown that synergistic targeting can further enhance the targeted release effect of anti-tumor drugs,while reducing the toxic and side effects of both drugs and improving the efficacy^[4,5].Objectives1.A new synergistic targeting combined chemotherapy drug Her-HINP/PTX with potential clinical application value was synthesized to reduce the drug resistance and toxic and side effects caused by single drug use and conventional drug combination,and improve the treatment effect.A series of characterization was carried out,its water solubility and stability were evaluated,and the drug loading rate and entrapment efficiency were calculated.2.To evaluate the in vitro response of Her-HINP/PTX to hyaluronidase:changes in morphology and particle size,drug release efficiency and fluorescence intensity after enzyme response.3.To study the biocompatibility,targeting and cytotoxicity of Her-HINP/PTX to tumor cells with different HER2 expression levels.4.To investigate the in vivo response of Her-HINP/PTX and evaluate its antitumor effect and biological safety in vivo.Methods1.Firstly,a synergistic targeting combined chemotherapeutic drug carrier HINP was constructed,and the hydrophobic chemotherapeutic drug PTX was loaded into the hydrophobic core of HINP by physical embedding.Finally,under the action of EDC/NHS,Her-HINP/PTX was covalently combined by dehydration condensation reaction by using-NH2 at the end of Herceptin and-COOH on the surface of HINP/PTX.The product of each step was determined by ¹H-NMR.The morphology,particle size,potential,absorption and emission spectra were characterized by TEM,DLS and FLS 980 fluorescence spectrometer.The final product Her-HINP/PTX was dispersed into ultrapure water,PBS and DMEM medium containing 10%heat inactivated fetal bovine serum,and stored at 37℃.The samples were digitally photographed at different times after dispersion,and the size of nanoparticles was measured by DLS to evaluate their water solubility and stability.The loading efficiency and drug loading content of PTX were determined by high performance liquid chromatography（HPLC）,and the coupling rate of monoclonal antibody Herceptin was determined by BCA protein quantitative method.2.An appropriate amount of synergistically targeting combined chemotherapy drug Her-HINP/PTX was dispersed in PBS and added with hyaluronidase（0.1 mg/m L）,incubated in an incubator at 37℃,100 rpm.The changes of morphology and size after hyaluronidase response in vitro were studied by TEM and DLS.The changes of fluorescence spectra and fluorescence intensity after enzyme response were recorded by FLS 980 steady-state transient fluorescence spectrometer and in-Vivo Master near-infrared imaging system for small animals In vivo.The release efficiency of enzyme responsive PTX was determined by dialysis.3.In order to study the targeting of Her-HINP/PTX,two breast cancer cells with different HER2 expression,SK-BR-3（HER2 high expression）,MDA-MB-231（HER2negative）and normal liver L02 cells were treated with fluorescent dye labeled Her-HINP and HINP,respectively.To further verify Herceptin’s targeting of HER2 and HA to CD44,we set up HA blocking group,Herceptin blocking group and HA+Herceptin double blocking group in SK-BR-3 breast cancer cells.After the nuclei and lysosomes were stained,the entry of different materials into the cells was observed by laser scanning confocal fluorescence microscope.In the study of cytotoxicity experiment,we studied two kinds of breast cancer cells with different HER2 expression,SK-BR-3（HER2 high expression）,MDA-MB-231（HER2 negative）and normal liver L02 cells.The experiment was divided into 6 groups.The Herceptin group,PTX group,HINP group,Her-HINP group,HINP/PTX group and Her-HINP/PTX group.The toxicity study was carried out at the drug concentration of 100、50、10、1、0.1、0.01、0.001μg/m L,Then CCK8 kit was used to monitor cell viability,and annexin V-FITC/PI apoptosis detection kit was used to further study the apoptosis of SK-BR-3 breast cancer cells with high HER2 expression.4.SK-BR-3 tumor bearing nude mice model was constructed.When the tumor grew to about 80 mm³,it was randomly divided into two groups:Her-HINP/PTX and HINP/PTX（n=3 in each group）,200μL were injected through caudal vein respectively（PTX=5 mg/kg）.At 6 h,12 h,24 h,36 h and 48 h after tail vein injection,NIR-Ⅱ small animal in vivo imaging system in vivo master was used for in vivo fluorescence signal monitoring under isoflurane anesthesia to observe the distribution and accumulation of two groups of chemotherapeutic drugs in tumor bearing nude mice.Similarly,SK-BR-3 tumor bearing nude mice model was used to evaluate the antitumor effect.The tumor bearing nude mice were randomly divided into the following 7 groups:Her-HINP/PTX group,HINP/PTX group,Herceptin group,Her-HINP group,PTX group,HINP group and normal saline group,with 5 rats in each group.The tumor size and body weight of tumor bearing mice were monitored respectively,and the mice were killed after 20 days of treatment.The main organs of mice in each treatment group were removed for HE staining histological analysis to evaluate the antitumor effect and biological safety in vivo of each treatment group.Results1.A new synergistic targeting combined chemotherapy drug Her-HINP/PTX was successfully synthesized,which has good water solubility and stability.When PTX:HINP（w/w）was 30%,it had high entrapment efficiency and drug loading rate,which were75.4±0.15%and 29.5±0.06%respectively.The antibody coupling rate of Herceptin was27.5%and the drug loading rate was 6.6%2.After incubation with hyaluronidase in vitro,the monodisperse Her-HINP/PTX became polydisperse,and the particle size changed significantly from 163.0±6.1 nm to53.2±7.5 nm and 228.6±14.1 nm.Under the stimulation of hyaluronidase,its size change in serum environment was similar to that in PBS.The size of monodisperse Her-HINP/PTX changed from 141.8±1.7 nm to 38.6±17.0 nm and 174.7±18.5 nm.It shows that the serum environment hardly affects the response of Her-HINP/PTX to hyaluronidase.In addition,TEM showed that Her-HINP/PTX changed from spherical to irregular under the action of hyaluronidase,which was consistent with the results of DLS.These results show that breaking the hyaluronic acid chain with hyaluronidase can effectively dissociate Her-HINP/PTX.The fluorescence intensity of IR-1048 was enhanced after Her-HINP/PTX was dissociated.The release of PTX was significantly higher than that in the experimental group without hyaluronidase.3.In vitro biocompatibility study,no obvious hemolytic reaction was observed at the concentration of 1600μg/m L,indicating that Her-HINP/PTX has good biocompatibility.In the study of cell targeting,SK-BR-3 cells with high HER2 expression can ingest a large amount of Her-HINP and can be double blocked by HA and Herceptin,while the uptake of HINP is low,indicating that the synergistic targeting combined chemotherapeutic drug Her-HINP/PTX has good CD44 and HER2 double targeting effect.The dual targeting effect of Her-HINP/PTX was further proved by the study of HER2 negative MDA-MB-231 cells and normal liver L02 cells.In the study of cytotoxicity,Her-HINP/PTX has good killing effect on HER2 positive tumor cells.In Her-HINP/PTX experimental group,the cell survival rate decreased significantly with the increase of its concentration.At the concentration of100μg/m L（Concentration of PTX）,only 23.96±2.59%of the cells remained alive.In the apoptosis experiment,the apoptosis rate of Her-HINP/PTX group also reached 69.45%,which further showed that Her-HINP/PTX had good antitumor effect in vitro.4.Her-HINP/PTX accumulated in the liver and tumor after tail vein injection.The fluorescence signal reached the peak at 24 hours after administration,and no obvious fluorescence signal was found in other tissues and organs.In the treatment of SK-BR-3tumor bearing nude mice,the tumor growth in Her-HINP/PTX treatment group was significantly inhibited,and no obvious toxic and side effects were observed through histological analysis.ConclusionsIn this study,a new synergistic targeting combined chemotherapy drug Her-HINP/PTX was successfully constructed,which has good water solubility,stability,biocompatibility and hyaluronidase response.The nano drug carrier constructed by HA encapsulated the hydrophobic chemotherapy drug PTX into the hydrophobic core of nanoparticles,which not only increased the water solubility and biocompatibility of PTX.Meanwhile,the release of enzyme responsive PTX also reduced the systemic toxicity and side effects of traditional chemotherapy drug PTX.The unique CD44 targeting ability and good biocompatibility of HA in the nano carrier make the nano drug delivery carrier have good research and application prospects.The near-infrared two region fluorescent molecule IR-1048 introduced into the system can be used to monitor the aggregation of drugs at the tumor site after being dissociated by hyaluronidase,so as to evaluate the therapeutic effect earlier.In addition,on the basis of the nano drug delivery carrier,we introduced the targeting molecule.The addition of Herceptin makes the nano drug have the double targeting effects of CD44 and HER2.The EPR passive targeting effect of nano materials and the active targeting effect on CD44 and HER2 make the chemotherapeutic drugs reach the tumor cells more accurately,realize targeted administration,increase the delivery efficiency of chemotherapeutic drugs at the tumor site,improve the treatment effect,so as to achieve the purpose of precision treatment.