Glucose Transporter-mediated And Tumor Microenvironment Response To Novel Targeted Drug Delivery Systems

Cancer treatment remains an important program at present.Chemotherapy is dominant in the treatment of cancer.But,on the one hand,chemotherapeutic drugs have poor solubility and bioavailability,which result in the poor antitumor effects and severe adverse effects.On the other hand,the development of multidrug resistance results to the failure of chemotherapy.Tumor targeted drug delivery system(TDDS)has ability in improving the drug solubility and availability.And TDDS could enhance the drug distribution in tumor through enhanced permeability and retention(EPR)effect.It also could be further modified with targeting ligands,which could identify and interact with the tumor cell receptors,to improve the drug concentration in tumor cells.These approaches aim to strengthen treatment outcome and reduce adverse effect.However,at present,the TDDS remain having some drawbacks,such as poor tomor targeting ability,limited treatment outcome,and so on.The metabolic activity of tumor cells signicantly promoting compared with normal cells.The promoted glycolysis rates of tumor cells need intaking a large number of glucose to maintain balance.Thus,glucose transporter(GLUT)is overexpressed on the surface of tumor cells.The transporter-mediated tumor cellular uptake possesses faster transport rate and higher specificity compared with receptor-mediated cellular uptake.So glucose transporter has the potential to be employed as a target for tumor cell targeting.However,highly effective GLUT 1-mediated,tumor-targeting nanomedicines have not been widely developed.The intracellular and extracellular microenvironment on which tumor cells rely to survive is also significantly different from normal cells.The intracellular concentration of glutathione(GSH)in tumor cells is 4 times higher than that in the normal cells,and 5000 times higher than the extracellular concentration of GSH.This big discrepancy in the distribution of GSH could be developed to realize controllable drug release,which maximize the drug concentration in tumor cells,enhance treatment outcome of chemotherapy drugs and lower toxicity to normal tissues.The introduction of GSH-triggered disulfide cross-links to polymer nanocarriers can stabilize carriers against hydrolytic degradation and efficiently initiate drug release once the carriers are internalized in the target cells.However,these nanomedicines generally require sophisticated designs and complex fabrication procedures.The few clinically successful nanomedicines have shown that in addition to efficacy and safety,simplicity and cost play a decisive role in their translation into therapeutic products.Based on this,this study firstly dedigned a GLUT1 targeting and cellular microenvironment responsive TDDS for enhancing the anti-hepatocarcinoma efficacy of camptothecin.This system employed PAMAM dendrimer as the drug delivery carrier,the GLUT1 specific substrate D-glucose as the targeting ligand,and camptothecin as a model drug.The D-glucose was conjugated with PAMAM through a PEG linker,and the camptothecin was conjugated with PAMAM through disulfide bond.Finally,a Glucose-PEG-PAMAM-s-s-CPT(GPCC)conjugate was successfully synthesized.The conjugate was characterized by a 20 nm particle size,spherical structure,and faster drug release under high GSH concentration than normal physiological environment.The GPCC conjugate had higher cellular uptake for GLUT1-overexpressing HepG2 cells and HepG2 multilayer tomor sphere compared with control groups.While no significant cellular uptake of GPCC conjugate for GLUT1-lowexpressing L02 cells(p<0.05).The GPCC conjugate showed higher cytotoxicity and higher S phase arrest.The pharmacokinetic study indicated that the GPCC conjugate had long circulation effect(longer t1/2)and improved the bioavailability(higher AUC).The in vivo imaging experiment was conducted on a H22 hepatoma tumor-bearing mice model,and the results demonstrated that the GPCC conjugate could accumulated in tumor effectively,the GPCC conjugate has obvious tumor targeting ability.Pharmacodynamics results showed that the GPCC could effectively inhibited the growth of tumor,which demonstrated that the GPCC conjugate had better treatment outcome.And the tumor-bearing mice treated with the GPCC conjugate had minimal body weight loss,this result combined with the HE staining experimental result could demonstrate the lower toxicity of GPCC conjugate to normal tissues.The cause of multidrug resistance phenomenon of tumor cells is closely related to the increased expression of the efflux transporter p-gp.At present,there are several approaches to overcome MDR,such as Drug-loaded DDSs bypass the molecular domain responsible for the development of drug resistance;drug co-delivery systems to efficiently circumvent the MDR of cancer cells,including co-delivery of small molecular anticancer drug/macromolecular therapeutic gene,small molecular chemosensitizer/anticancer drug,and anticancer drug/suppressors of cellular resistance;effective mitochondrial targeted treatment strategies cancer mitochondria-directed drugs designed to trigger apoptosis(curbing the mitochondrial ROS production by delivery of antioxidants to the mitochondria,activating mitochondrial membrane permeability,targeting Bcl-2 proteins to inhibit or down-regulate anti-apoptotic action,delivering therapeutic genes for the expression of pro-apoptotic proteins including Bax,Bak)and so on.P-gp,as an ATP dependent transporter,consumes ATP when transporting substrate.Because mitochondria are the "energy factory" of cell,so it can be assumed that the MDR effect can be reversed to cut off the ATP supply for p-gp through mitochondria targeting.The mitochondria TDDS on overcoming MDR through mitochondria targeting to cut off the energy supply of P-gp is not reported.Moreover,mitochondrial targeting is usually not selective for tumor cells and normal cells,it can easily lead to serious side effects.Therefore,firstly achieving tumor cell targeting and further achieving mitochondrial targeting are the preferred options.The unique microenvironment of tumor tissue facilitates this 'cascading'targeting strategy.There is excessive expression of matrix metalloproteinases(MMP)in tumor extracellular microenvironment.It can also serve as an intelligent adjustment factor for TDDS to change the structure and various physical and chemical properties,such as surface charge,particle size etc,which can realize the special designed function,for example,multilevel targeting.Thus,we propose hypotheses from a new perspective,tumor cell and mitochondrial targeting could be achieved through the glucose ligands and MMP2,respectively,for overcoming the problem of MDR of tumor cells.Based on this,this study further designed a GLUT1 and MMP2 mediated mitochondria TDDS for overcong MDR of tumor cells.PAMAM dendrimer served as a drug delivery carrier,mitochondria targeting motif triphenylphosponium were mofified on the surface of PAMAM,glucosylated PEG were conjugated with PAMAM through MMP2 sensitive peptide(GPLGIAGQ),and the model drug paclitaxel was conjugated with PAMAM through GSH sensitive disulfide bond.Finally,the Glucose-PEG-peptide-Triphenylphosponium-PAMAM-Paclitaxel(GPp/TPP/PTX PAMAM)conjugate TDDS was constructed.The particle size of the GPp/TPP/PTX PAMAM conjugate was below 50 nm,and it reduced after treating with MMP2,indicating it was MMP2 sensitive.The in vitro drug release was evaluated under different concentration of GSH,and the drug release fron the GPp/TPP/PTX PAMAM conjugate was GSH sensitive.The result of cellular uptake conducted on the MCF-7/ADR cells and MCF-7/ADR multilayer tumor spheres,subcellular location,and cytotoxicity demonstrated that the GPp/TPP/PTX PAMAM conjugate could target to the GLUT1 over-expressing tumor cells and the mitochondria,it possessed stronger cytotoxicity,and lower MDR index.The MDR reversing mechanism of the GPp/TPP/PTX PAMAM conjugate could be explained by its ability in reducing the mitochondria membrane potential and expression of p-gp through the determination of mitochondria membrane potential,ATP content and p-gp content.The in vivo imaging and pharmacodynamics experiments were conducted on the MCF-7/ADR breast tumor-bearing nude mice,the results demonstrated the GPp/TPP/PTX PAMAM conjugate had better tumor targeting ability,better treatment outcome and lower toxicity.In summary,two GLUT1 targeting and tumor microenviorenment responsive TDDSs were designed to overcome the drawbacks of current anti-tumor drugs,they had the potential in improving the tumor targeting ability and pharmacodynamics,at the same time reducing system toxicity.They hold great promise in serving as TDDS.