Efficacy And Mechanism Of Chitosan-based Nanoparticles Carrying Docetaxel And Curcumin In The Treatment Of Lung Cancer

1.BackgroundLung cancer is the main cause of cancer-related death,and non-small cell lung cancer(NSCLC)is the most common type,with a 5-year survival rate of less than 20%.Although great progress has been made in the treatment methods including surgery,radiotherapy,chemotherapy and targeted therapy,the prognosis remains poor because most patients have reached advanced or extensive metastasis at the time of diagnosis.Among them,the limitation of drug selection restricts the treatment of lung cancer.In recent years,the research of lung cancer treatment has been further explored,in which nanomedicine research and immmunotherapy research have been widely concerned by researchers.The clinical conversion of some research results has also brought good news for patients.The serious side effects caused by traditional chemotherapy drugs can not only be significantly reduced through the delivery of nanocarriers,but also be enriched in the diseased tissues with the help of the passive targeting effect of nanocarriers,so as to play a better therapeutic role.Moreover,compared with traditional chemotherapy drugs,nanocarriers can precisely control the permeability and drug release efficiency of drugs in tissues by virtue of their own physical properties(including shape,size,charge,hydrophobicity and chemical composition).Tumor immunosuppressive microenvironment,as an increasingly concerned field,plays a key role in the process of drug resistance in tumor treatment.In order to solve this problem,more research work began to focus on the relationship between them.Therefore,the aim of this study is to explore the synergistic anti-tumor effect of new nanocarriers co-delivery of both traditional chemotherapy drug docetaxel and Chinese herbal medicine extract curcumin on lung cancer.2.Methods2.1 Synthesis and characterization of materialsSynthesis of nanocomposite micelles CMCS-BAPE(CB),mono-drug-loaded nanomicelles CMCS-BAPE-CUR(CB-C),and monodrug-loaded nanomicelles CMCS-BAPE-DTX(CB-D),Dual-drug-loaded nanomicelle CMCS-BAPE-DTX/CUR(CB-DC)and targeted dual-drug-loaded nanomicelle T7-CMCS-BAPE-DTX/CUR(CBT-DC)by chemical methods.Then,it was characterized by 1H NMR,FTIR,DLS and TEM.The release behavior of docetaxel(DTX)and curcumin(cur)in vitro was evaluated by HPLC and UV Vis.2.2 Uptake and antitumor effect of nano drug in vitro and in vivoThe uptake of nano carriers in lung cancer cells and xenografts in vivo was observed by confocal and flow cytometry;the killing effect of nano drugs on lung cancer cells in vitro was explored by CCK-8 and flow cytometry,and the anti-tumor effect of nano drugs was further verified by xenografts in vivo.2.3 Study on the immunomodulatory effect of nanodrugs in vivoC57bl/6J mice bearing CMT-64 lung cancer were constructed.After 24 hours,the subcutaneous transplanted tumor was obtained and divided into two parts.One part was fixed for immunohistochemistry(IHC),and then embedded,sliced,baked,dewaxed,hydrated,blocked,incubated first antibody,incubated second antibody,DAB staining,hematoxylin re staining and a series of operations were performed to detect the main immune cells in tumor tissue.In the other part,the content of immune cells in tumor tissue was detected by flow cytometry.The main immune cells(CD4+T cells,CD8+T cells,myeloid derived suppressor cells(MDSC)and regulatory T cells(Treg))in tumor tissue were labeled and detected followed by a series of operations,such as preparing single cell suspension,blocking,incubating surface protein antibody,permeating,incubating membrane protein antibody.3.Results3.1 Successful synthesis and characterization of nanomedicinesSuccessfully synthesized a targeted nanomedicine(CBT-DC)with a particle size of about 100-150nm.The 1H NMR results showed that the chemical shift of 3.90-4.20 ppm was attributed to the o-ch2-c=O structure of carboxymethyl chitosan,and the chemical shift of 6.89-7.76 ppm was attributed to the proton peaks on the benzene ring of pinacol benzoate and pinacol benzoate,indicating that the hydrophobic end was successfully coupled with carboxymethyl chitosan.There are a series of proton peaks of T7 peptide between the chemical shift of 4.3-1.1 ppm,which indicates the successful synthesis of CMCS-BAPE-T7.Compared with CMCS,CMCS-BAPE had stronger free primary amide bond at 3440-3300 cm-1,main characteristic peak of alkanes at 2900-2850 cm-1,vibration peak VC=C at 1650-1500 cm-1 and bending vibration peak ?=C-H at 1250-1000 cm-1,indicating that pinacol phenylborate and carboxymethyl chitosan were coupled by amide bond.The drug loading rates of DTX and CUR were 7.82%and 6.48%,respectively.3.2 Superior in vitro and in vivo uptake and antitumor effect of nanomedicinesCBT with cy5.5 had a higher uptake capacity than CB with cy5.5 by confocal microscopy and flow cytometry.CBT had a better uptake capacity than CB in subcutaneous xenografts in vivo.CCK-8 and flow cytometry showed that CBT-DC had better antitumor effect than free drug DTX,CUR,CB-D and CB-DC without targeting ability.In vivo subcutaneous tumor transplantation experiment,we injected the drug through the tail vein,CBT-DC can more significantly inhibit the growth of tumor,compared with the free single drug DTX and CUR,the nano drug CB-DC with dual drug also has better anti-tumor effect.3.3 Amelioration of immunosuppressive microenvironment of tumor by dual drug loaded nanomedicine CBT-DCIn immunocompetent C57bl/6j mice,post-injection of nanomedicines as planned.Firstly,the levels of IL-2 and IL-10 in blood were detected by ELISA.It was observed that there was no significant different in IL-2,while IL-10 which was positively correlated with M2 macrophages in CBT-DC group was significantly reduced which indicated that CBT-DC could effectively reduce its content and inhibit the unfavorable factors of tumor immunity.Then,IHC and FCM were performed to analyze the content of immune cells in tumor tissues,IHC results showed that NK cells were significantly increased in CNT-DC group,while Treg cells were decreased;FCM results were slightly different from IHC results,although CD4+and CD8+T cells and Treg cells had no significant changes,MDSC cells were significantly decreased,accumulating findings suggested that CBT-DC could reduce the content of immunosuppressive cells along with the increase of the content of NK cells.4.DiscussionWe confirmed that we have successfully obtained a new T7 targeted nanodrug(CBT-DC)through the synthesis,verification and characterization of the nanomaterials.The results of TEM and DLS showed that CBT-DC was a kind of regular spherical nanoparticles with a diameter of about 100-150 nm.The drug loading rates of DTX and cur were 7.82%and 6.48%respectively.In vivo and in vitro experiments have confirmed the superior tumor targeting function of nanocarrier,and verified the anti-tumor effect of CBT-DC is better than that of free single drug.Finally,we further explored the regulatory role of CBT-DC in tumor immunosuppressive microenvironment,and confirmed that CBT-DC can ameliorate tumor immune microenvironment by inhibiting the content of Treg and MDSC in immunosuppressive microenvironment,and increasing the infiltration of NK cells.