Oral Docetaxel And Bevacizumab Co-delivery Nanoparticles Improve Intestinal Absorption And Enhance Anticancer Activity

BackgroundCancer is not only harmful to human health,but also affects people's psychology to overcome the disease.Moreover,lung cancer has become the highest mortality of cancer patients in our modern life.Non small cell lung cancer(NSCLC)accounts for more than two-thirds of all types of lung cancer.In the past,we mainly used surgery,radiotherapy,and then chemotherapy to cure NSCLC.But these practices in the past will cause injury to patients to a large extent.With the progress of modern medicine and science and technology,studies have shown that combined chemotherapy can also be used to treat NSCLC.Currently,the National Cancer Comprehensive Network(NCCN Guide(?))oncology clinical practice guidelines recommend the combination of Bevacizumab(BVZ)and Docetaxel(DTX)in the treatment of non-small cell lung cancer(https://www.nccn.org/professionals/physician?gls/default.aspx).Nano drug delivery(NDD)technology has made great progress.It is widely used in chemotherapy drug delivery to reduce toxicity and improve therapeutic effect.Based on the concept of combined chemotherapy,the Nano drug co-delivery system(NDCDS)refers to the loading of two or more anti-cancer drugs with different physical and chemical properties and different pharmacological mechanisms into the same nano drug delivery system in.Compared with free drug combined chemotherapy,it has the advantages of effectively breaking through the biological barrier,high targeting,precise drug release,etc.,and can better produce synergistic effects of drugs.Nanoparticle oral delivery has good convenience and safety,and can protect some protein macromolecular drugs from secretion or enzyme damage.Therefore,nano oral drug delivery is an academic topic that researchers pay attention to in recent years.Due to the shortcomings of the intravascular drug delivery system,such as poor patient compliance,with the development of modern nanotechnology,oral nano drug delivery systems have emerged.It has the advantages of maintaining plasma drug concentration,low side effects,and good patient compliance.Therefore,it has been recognized by researchers and developed rapidly.AimIn this study,based on the concept of combined chemotherapy,we designed and prepared oral co delivery nanoparticles of DTX and BVZ.Carboxymethyl chitosan(CMC)and poly(lactic acid glycolic acid)(PLGA)were used as carriers for DTX(CPNP_DTX),and methoxypolyethylene glycol poly(?-amino ester)(m PEG-PAE)was used as carriers for BVZ(PPNP_BVZ).Then,the two types of nanoparticles are physically mixed in mass ratio to form mixed co delivery nanoparticles,called CPNP_DTX&PPNP_BVZ.In this study,small molecule chemotherapeutic drugs and monoclonal antibody drugs combined with delivery nanoparticles act on non-small cell lung cancer cell line A549.The physicochemical characterization,drug absorption and anticancer activity were studied.Methods1.Preparation and physicochemical characterization of nanoparticlesCPNP,CPNP_DTX and CPNP_Rh123 were prepared by emulsion solvent evaporation method.The surface morphology and distribution of nanoparticles were observed by transmission electron microscope.The surface morphology and distribution of CPNP,CPNP_DTX,PPNP and PPNP_BVZ were observed by transmission electron microscope.The dispersion of fluorescent traceable CPNP_Rh123 and PPNP_BVZ-FITC nanoparticles after physical mixing was observed by laser confocal microscopy.The size and surface charge of nanoparticles were measured by Zeta PALS high resolution potentiometry and particle size analyzer.To verify the pharmaceutical properties of the drug,we used Fourier transform infrared spectroscopy(FT-IR)and X-ray diffraction analysis(X-RD).The encapsulation rate(EE%),drug loading rate(DL%)and in vitro drug release of the nanoparticles were calculated by UV-Vis spectrophotometer,and the above methods were used to analyze whether the drug was successfully encapsulated in the nanoparticles.2.Study on plasma concentration of drugs in vivoThe plasma concentrations of free drugs and nano drugs were determined by microplate method in mice before peak,when peak and after peak respectively.3.Promote drug absorptionHuman colorectal adenocarcinoma Caco-2 cells were used to simulate human intestinal epithelial cells.The CCK-8 kit was used to evaluate the toxicity of nanomaterials to Caco-2 cells,and to detect the toxicity of drugs to Caco-2 cells.Normal saline,Rh123+BVZ-FITC and CPNP_Rh123+PPNP_BVZ-FITC were orally administered to mice.After acting for a certain period of time,the intestinal tissue was taken out,and the absorption of the three groups of samples in the intestine was observed with a laser confocal scanning microscope.Caco-2 cells were pretreated with inhibitors of different absorption modes,and then treated with CPNP_Rh123 and PPNP_BVZ-FITC respectively.The possible absorption modes of two kinds of nanoparticles were explored by comparing the absorption effects of different treatment groups.4.Enhance anticancer activityIn order to evaluate the anticancer effect of nanoparticles,A549 cells were selected.Use the CCK-8 kit to evaluate the safety of nanomaterials to A549 cells,detect the toxicity of drugs to A549 cells,and calculate the median inhibitory concentration(IC₅₀)and combination index(CI).Subsequently,we selected the ratio of DTX(10?g/m L)to BVZ(250?g/m L)based on the cytotoxicity results,and mixed CPNP_DTX and PPNP_BVZto form CPNP_DTX&PPNP_BVZ according to the drug loading rate(DL%).A549 cells were treated with CPNP_Rh123+PPNP_BVZ-FITC for 1 h,2 h,4 h and 6 h,respectively.Subsequently,the uptake of nanoparticles by A549 cells at different time was observed by confocal laser scanning microscope.Result1.Physical and chemical characterization of nanoparticlesThe results of TEM,particle size analysis and potential measurement show that the size of the prepared nanoparticles is less than 200 nm,the size of the nanoparticles is uniform,the dispersion is good,and the nanoparticles are negatively charged.Infrared spectroscopy and X-ray diffraction analysis showed that the drug was successfully loaded into nanoparticles.Through particle size analysis,the average particle sizes of CPNP,CPNP_DTX,PPNP,PPNP_BVZ,CPNP_DTX&PPNP_BVZ were169.10±8.29 nm,192.0±9.48 nm,98.92±0.14 nm,82.07±0.19 nm,132.60±0.058 nm,respectively.The two kinds of nanoparticles were detected by UV-Vis spectrophotometer and calculated to show higher encapsulation rate and drug loading rate.The encapsulation rate of CPNP_DTX was 86.11±5.07%,the drug loading rate was26.34±1.55%,PPNP_BVZ The encapsulation rate is 85.50±0.33%,and the drug loading rate is 0.54±0.01%.In vitro drug release experiments showed that nanoparticles had better controlled drug release characteristics2.Study on plasma concentration of drugs in vivoIn the study of drug plasma concentration,whether the plasma drug concentration is before,at,or after the peak,compared with the free drug group Free DTX and Free BVZ-FITC,after single oral administration of nano drugs CPNP_DTX and PPNP_BVZ-_FITC,the plasma drug concentration of DTX and BVZ-FITC increased.The plasma drug concentration in CPNP_DTX was increased by 53.78%compared with the free drug,and the PPNP_BVZ-FITC was increased by 67.73%compared with the free drug.3.Promote drug absorptionIn vitro cytotoxicity test showed that both CPNP and PPNP nanomaterials showed no obvious toxicity to Caco-2 cells,and could be used as drug delivery carriers.However,the survival rate of Caco-2 cells was still over 80%.At the same time,the intestinal absorption experiment in mice showed that the nanoparticles had good intestinal absorption effect.4.Enhance anticancer activityThe study of drug toxicity showed that CPNP and PPNP nanomaterials were basically non-toxic to A549 cells.Nano drug has significant toxic effect on A549 cells.Among them,when CPNP_DTX&PPNP_BVZ combined delivery,it has obvious anti-cancer activity,and through further exploration,it is concluded that CPNP_DTX and PPNP_BVZhave synergistic effect.The results of drug uptake by A549 cells showed that drug loaded nanoparticles could accumulate in A549 cells for a long time.As time goes on,more and more nanoparticles are absorbed by cells.ConclusionIn this study,we have successfully prepared binary blended nanoparticles for drug co-delivery nanoparticles(BBN-DCD),which can be used for oral delivery of DTX and BVZ.BBN-DCD NP consists of two kinds of drug loaded nanoparticles:CMC-PLGA-DTX NP(or CPNP_DTX)and m PEG-PAE-BVZ NP(or PPNP_BVZ).The nano co-delivery system can overcome the intestinal barrier,improve the intestinal absorption of small molecule chemotherapy drug DTX and large molecule monoclonal antibody drug BVZ,and significantly enhance the anti-cancer effect.Therefore,our study reveals the potential of binary mixed small molecule chemotherapy combined with macromolecular antibody drugs,and nanoparticles can be used as oral drug delivery system.