The Synthesis Of Vitamin-B12-conjugated Nanomedicines With Gastric Cancer Targeting,and Exploring The Mechanism Of Revering Drug Resistance

BackgroundGastric cancer(GC)is the second most common malignant tumor worldwide,and there are about 400000 new cases in China every year,accounting for 42%of the global total cases.To date,the incidence of the proximal gastric cancer increased since these 20 years.This severity of gastric cancer has become a major threat to national health in China.At present,the main treatments for the advanced gastric cancer were gastrectomy plus postoperative systemic chemotherapy.However,the prognosis of advanced gastric cancer remains poor.The major problems contributing to this circumstance were as followings:Firstly,single-modetraditional chemotherapy could induce dug tolerance ingastric cancereasily,while thepotential mechanism has not been fully elucidated.Consequently,the tumor drug resistanceinevitably leads to the low efficiency of chemotherapy.Secondly,intravenous drug delivery is one of the main methods for systemic chemotherapy.However,the absorbed drugs could not selectively bine to cancer cells,according tothe lacking of specific tumor-targeting ability.Furthermore,it is hard to achieve the enough concentration of drugs for inhibiting the growth of cancer cells.Therefore,it is necessary to developnovel medicineswith the tumor-targeting and reversal of drug resistance,while this new drug could exert an ffective,low toxic,comfortable,economical treatment for the gastric cancer patients,and finally improve the prognosis.In recent years,the use of effective drug delivery systems(DDSs)based on nanosized technologies has been proposed as a useful strategy for overcoming these issues.Such nanoparticles with high drug-load ability,could efficiently improve the capacities of drugs,enter cancer sites and realize intelligent drug delivery.However,these nanomedicines do not possess cancer-cell"targeting" capabilities.Emerging researches has focus on conjugating nanocarrier with ligands(e.g.,folate or antibodies)which could specifically bind to molecules in cancer cells,and finaly realize the presice treatment.Therefore,our study applied the VB12 as the targeted probe in gastric cancer based on its high uptake and transport pathway,and attempt to synthesize VB12-conjugatednanomicelles.Subsequently,the VB 12-conjugatednanomicelles were designed to encapsulate chemotherapeutic drugsin aqueous solution,and reverse drug resistance in gastric cancer.Finally,we explore the synthetic method and function mechanism of these VB12-conjugatednanomicelles.Part 1.Based on high drug loading efficiency,we develop sericin nanoparticles to highly encapsulate chemotherapy drugse,and explore both their function and mechanism for reversal of drug resistanceObject:To explore the synthesis method of amphiphilic sericin nanoparticles(NPs)and analyze their mechanism for reversal of drug resistance.Methods:The highly hydrophilic sericin and hydrophobic poly(γ-benzyl-L-glutamate)(PBLG)were synthesized together by the ring-opening polymerization and subsequently turned into amphiphilic nanomicelles,called sericin-PBLG.Then we synthesized sericin－PBLG-DOX by adding doxorubicin(DOX)to enhance the effect of chemotherapy.The physicochemical properties of sericin-PBLG-DOXmicelles were analyzed by the Ma Erwen Particle size Analyzer,transmission electron nicroscopy,near-infrared spectroscopy,nuclear magnetic hydrogen spectroscopy and drug release experiment.In addition,the bio-safety of sericin-PBLGmicelleswere valued by the hemolysis test,animal experiment and immunotoxicity.Finally,the laser confocal microscopy,transmission electron microscopy,cytotoxicity experiments,flow cytometry,western blotting and patient derived xenografts model mouses were used to detect the procession of uptake and release and the effective anti-tumor growth ability after treated with the sericin-PBLG-DOXmicelles.Results:1.Sericin-PBLG micelles have good biocompatibility both in vitro and in vivo.2.pH responsiveness ensures effective DOX release from sericin-PBLG-DOX towards nucleus.3.Sericin coating promotes DOX cellular uptake through the clathrin-mediated endocytosis pathway in the adriamycin-resistant cells,then located in lysosomes of tumor cells,ensures effective DOXrelease from sericin-PBLG-DOX towards nucleus.4.Compared with free adriamycin,the sericin binding adriamycin coulddramatically increase the drug sensitivities of MFC-7/ADR and HepG2/ADR cells,by activating caspase-3 apoptosis pathway and promote the anti-tumor growth effect both in vivo and in vitro.Conclusion:Sericin-PBLG-DOXmicelles could effectively and safely reverse drug resistance,which have potential value for clinical translation.Part 2.Based on the aggregation-induced emission effect,we develop the vitamin B12 fluorescent derivatives(VB12-AIE),and explore its intracellular imaging abilitiesObject:To explore the synthesis method of VB12.AIE and evaluate its intracellular imaging abilitiesMethods:We synthesize the VB12·AIE fluorescent derivatives,while the 1H NMR and the high performance liquid chromatography(HPLC)methods were applied to confirm the successful conjugation between the VB12 and AIE.In addition,the CCK-8 test was used to explore the safe concentration range of VB12,AIE.Finally,the intracellular uptake and the imaging abilities of VB12-AIE were evaluated by the fluorescence inverted microscope,laser confocal microscopy and flow cytometry.Results:1.The VB12-AIE fluorescent derivatives were successfully synthesized.2.The relative safe concentration of VB12-AIE ranged from lug/ml to 16ug/ml;3.After being absorbed by cells,VB12-AIEshowed red fluorescence under the excitation of 560nm wavelength.Conclusion:Our work reported a DPBP-NCS,an AIEgenlabeled vitamin B12 deratitibes,with deep-red emission.The VB12-AIE fluorescent derivatives have stable biological properties and good optical imaging effect.Therefore,this is a promising method to detect the novel uptake pathway of VB12,and it also could be used for imaging therapy of tumor.Part 3.Based on the tumor-targeting ability of VB12,we develop the VB12-combined sericin nanomicelles,and explore both their function and mechanism for reversal of drug resistanceObject:To explore the synthesis method of VB12-combined sericin nanomicelles,and analyze their mechanism for reversal of chemoresistance.Methods:VB12 was introduced on the surface of sericin-PBLG micelles to form VB12-sericin-PBLG micelles.Furthermore,paclitaxel(PTX)was also encapsulated to synthesize the VB12 targeted sericin nanoparticles(VB12-sericin-PBLG-PTX).The physicochemical properties of VB12-sericin-PBLG-PTX micelles were analyzed by the Ma Erwen Particle size Analyzer,transmission electron microscopy,near-infrared spectroscopy,nuclear magnetic hydrogen spectroscopy and drug release experiment.In addition,the safety ofVB12-seiricin-PBLG-PTX micelles were evaluated by the hemolysis test,animal experiment and immunotoxicity.Finally,the laser confocal microscopy,transmission electron microscopy,cytotoxicity experiments,flow cytometry,western blotting and patient derived xenografts model mouses were used to detect the procession of uptake and release and the effective anti-tumor growth ability after treated with the VB12-sericin-PBLG-PTX micelles.Results:1.VB12-sericin-PBLG-PTX micelles have good biocompatibility both in vitro and in vivo.3.VB12-sericin-PBLG-PTX micelles can transport more PTX into the PTX-resistant cells through the cobalamin transporter receptor(CD320-receptor)on the cell membrane.4.Compared with free PTX,the VB12-sericin-PBLG-PTXmicelles could change the mitochondrial membrane potentials(MTP),activate caspase-3 apoptosis pathway,then induce DNA damage of PTX-resistant cells,followed by inhibiting the anti-tumor growth effectively both in vivo and in vitro.Cornclusion:VB12-sericin-PBLG-PTXmicelles could effectively and safely reverse drug resistance of PTX-resistant cells through the CD320 receptor,which have potential value for clinical application.