| Multidrug resistance(MDR)is among the major obstacles for cancer treatment.Nano-carriers provide an effective method to overcome MDR for their unique properties.Polyamidoamine(PAMAM)dendrimers have capacities for endo-lysosomal escaping and nuclear trafficking.Thus the PAMAM dendrimers can deliver DNA-toxic drugs into the nuclei.However,the intrinsic toxicity of PAMAM dendrimers due to their highly positive charge impedes their applications.Pluronic is a kind of triblock copolymers with essential anti-MDR properties.PAMAM dendrimers are modified with pluronic to improve the safty and curative effect.Pluronic F127(PF127)-modified PAMAM dendrimer conjugates were designed.1H-NMR,FTIR and ninhydrin assay proved the successful synthesis of PAMAM-PF127.The decreased zeta potential of PAMAM-PF127 reduced the hemolytic toxicity and cytotoxicity compared with PAMAM.Then doxorubicin(DOX)was encapsulated by PAMAM-PF127.The results showed that the prepared DOX/PAMAM-PF127 could increase cellular accumulation,achieve nuclear trafficking,and finally improve cell apoptosis of MCF-7/ADR cells.Furthermore,pluronic F68(PF68)with excellent biocompatibility was used to modify PAMAM.Whether or not the conjugation number of PF68 can influence the property of PAMAM-PF68 needs to be clarified.Three kinds of PAMAM-PF68 conjugates were prepared with different conjugation numbers of PF68.PAMAM-n1 PF68,PAMAM-n2 PF68 and PAMAM-n3 PF68 are used to represent the final products(n represents the conjugation number of PF68,which is 31,54 and 79 respectively).The drug-loading capacity,size and cytotoxicity were clarified.Increased cytotoxicity against MCF-7/ADR cells was observed for the DOX-loaded PAMAM-PF68 conjugates with high number of PF68 modification.However,over modification of PF68 could induce low drug loading efficiency,increased size and decreased uniformity.DOX/PAMAM-n2 PF68 was chosen to further elucidate the mechanisms of overcoming MDR and the anti-MCF-7/ADR tumor activity in vivo.The results showed that the cellular uptake of DOX/PAMAM-n2 PF68 was via caveolae-mediated endocytosis.Then the complexes could escape from the lysosomes via the proton sponge mechanism of PAMAM.Furthermore,the conjugates regulated the function of the mitochondria,causing the increase of reactive oxygen species(ROS),the loss of mitochondrial membrane potential to promote cell apoptosis,and the decrease of ATP to inhibit the activity of drug efflux pumps,such as P-gp.Finally,DOX/PAMAM-n2 PF68 could delivery DOX into the nuclei.The regulation of MDR-related gene expression,such as the increased expression of Topo Ⅱα and the decreased expression of Bcl-2,could improve cell apoptosis of MCF-7/ADR cells.Besides,improved penetration and inhibition efficiency of MCF-7/ADR tumor spheroids was achieved by DOX/PAMAM-n2 PF68 compared with free DOX.The in vivo anti-tumor assay showed that the complexes could induce effective accumulation and enhance chemotherapeutic efficiency in MCF-7/ADR tumors with low cardiotoxicity in vivo.Based on the above study,PAMAM G2-PF68(PPF68)was modified to graphene oxide(GO)via diselenide bond(prone to be oxidized or deoxidized)to achieve spatial and temporal control of drug release.The controllable ROS production can be induced by near-infrared(NIR)light to induce the breakage of diselenide bond.Then the PPF68 will be divorced from GO to cause the fast release of drug.The characterization by 1H-NMR and FTIR proved the successful synthesis of GO-PPF68.The results of zetasizer and AFM treated by H2O2 showed that the GO-PPF68 was ROS-sensitive.The photosensitizer indocyanine green(ICG)and chemotherapeutic agent DOX were co-delivered to form ICG/DOX/GO-PPF68 to achieve controllable synergistic treatment of chemotherapy and photothermal therapy.The in vitro release behavior of DOX from ICG/DOX/GO-PPF68 was pH-sensitive,temperature-dependent and ROS-sensitive.Furthermore,the anti-MCF-7/ADR tumor activity of ICG/DOX/GO-PPF68 was investigated in vitro and in vivo.ICG/DOX/GO-PPF68 showed higher cytotoxicity against MCF-7/ADR cells under NIR laser irradiation.The mechanism study showed that the cellular uptake of DOX/PAMAM-n2 PF68 was via caveolae-mediated endocytosis.ICG/DOX/GO-PPF68 could increase DOX accumulation in MCF-7/ADR cells under NIR laser irradiation.Afterwards,the lysosome escape was achieved with the help of GO-PPF68 under NIR laser irradiation.Then the fast increase of ROS level led to the rapid release of vast DOX.Finally,DOX was delivered into the nuclei.At 48 h,the MCF-7/ADR cells treated by ICG/DOX/GO-PPF68 with NIR laser irradiation showed decreased expression of ABCB1 gene,GST-pi gene,HSF1 gene,and P-gp,which would benefit the MDR reversal.ICG/DOX/GO-PPF68 could achieve better penetration in MCF-7/ADR tumor spheroids under NIR laser irradiation.The results of in vivo study showed that ICG/GO-PPF68 could effectively accumulate in the tumor site with stronger pthotothermal effect.The MCF-7/ADR tumors were significantly inhibited with reduced tumor volume after the treatment with ICG/DOX/GO-PPF68 under NIR laser irradiation.And the cardiotoxicity of DOX could be significantly reduced.The results demonstrate that the novel nano-carriers can effectively overcome MDR.The study will provide theoretical and experimental evidence for the treatment of drug-resistant tumors. |
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