A Multi-functionalized Nanocomposite Based On Gold Nanorods For Combating Multidrug Resistant Colorectal Cancer

Objective:The major hurdle to the successful chemotherapy of CRC is the emergence of multidrug resistance?MDR?,in which cancer cells exhibit a cross-resistant phenotype against multiple structurally and mechanistically unassociated drugs.Photothermal therapy?PPT?is a promising antitumor strategy for MDR tumors.Among various types of materials utilized in PTT,Gold nanorods?GNRs?,as a novel nano-material,has very high photothermal conversion efficiency in the near infrared region?NIR?.However,PPT might cause tumor metastasis and recurrence.In order to solve this problem,we prepared a novel multi-functional delivery system?DOX-loaded GNRs@mSiO₂@PHIS@TPGS?by using GNRs as cores to combat MDR colon cancer.Meanwhile,anti-tumor drug doxorubicin?DOX?was loaded into the nanocomposite,and the pH-sensitive poly-histidine?PHIS?was conjugated on the nanoparticle,which can induce pH stimulated drug release and endo/lysosome escape to increase drug efficacy.Moreover,d-?-tocopherol polyethylene glycol 1000succinate?TPGS?was assembled on the surface of the nanocomposite to realize drug intracellular retention by inhibiting P-glycoprotein and induce apoptosis.Compared with chemotherapy alone or photothermal therapy alone,the GNRs nanocomposite is a combination of photothermal-chemotherapy for drug-resistant colon cancer,which is expected to achieve better therapeutic effects and reduce systemic toxicity.Content:GNRs were first synthesized as cores,and coated with mesoporous silica enable the loading of antitumor drug DOX for chemo-photothermal therapy.To reverse DOX resistance,PHIS with pH responsivity was conjugated on GNRs@mSiO₂ to increase drug intracellular accumulation via efficient endo/lysosome escape;TPGS was then assembled on the surface of the nanocomposite to increase stability and realize drug intracellular retention by inhibition P-glycoprotein.The structure of the nanocomposite was confirmed by transmission electron microscopy?TEM?,UV-Vis-NIR spectra,fourier translation infrared?FT-IR?spectra and zeta potentials.The properties of the nanocomposite were evaluated,including the photothermal stability,the photothermal conversion efficiency,pH sensitive property and drug release profile.The intracellular delivery mechanisms and cytotoxicity were further investigated on MDR SW620/Ad300 cells.The in vivo experiments were finally performed to test the photothermal conversion property,antitumor efficacy and systemic toxicity.Methods:Preparation of the nanocomposite:First,GNRs were synthesized by using a seed-mediated growth method;second,GNRs were coated with mesoporous silica?GNRs@mSiO₂?using the sol-gel method,then introduced primary amino groups on their surface with an aminosilane APTES to conjugate with PHIS via an amide linkage.The resultant GNRs@mSiO₂@PHIS was loaded with DOX and further coated by TPGS through hydrophobic interaction.Characterization of the nanocomposite:The morphology,length-to-width ratio and dispersion state of GNRs coated with different surface layers were observed by using TEM.FT-IR spectra and zeta potentials were used to characterize the structures of the related products to comfirm the successful coating of mSiO₂,PHIS and TPGS on the surface of GNRs.UV-Vis-NIR absorption spectra was used to characterize the two surface plasmon resonance?SPR?bands consist of the weaker transverse SPR?TSPR?and the stronger longitudinal SPR?LSPR?.The photothermal conversion property of the nanocomposite was measured at different NIR irradiation time and at various nanocomposite concentrations with a power density of 2.5 W/cm² by using an NIR thermal camera.The pH-sensitivity of the nanocomposite was evaluated by the change of zeta potentials and drug release behavior at different pH value.To study the NIR-triggered drug release property,DOX release from the nanocomposite was performed in PBS at pH 5.0 under NIR laser irradiation at different time points.Evaluation of the nanocomposite in vitro:The MDR colorectal cancer cell line SW620/Ad300 were used to evaluate the cell viability after different treatments by MTT assay,then the DOX resistance-fold,the half inhibition concentration(IC₅₀)in each case and the reversal index?RI?were calculated.The ability of nanocomposite in reversing the drug resistant was further confirmed by the live-dead cell-staining assay.We first observed the intracellular accumulation of DOX in SW620/Ad300 cells after incubation with DOX-loaded GNRs@mSiO₂@PHIS@TPGS or free DOX for 1,2,and 4 h by confocal laser scanning microscopy?CLSM?,the endo/lysosome escape property was further evaluated.The celluar uptake and the drug efflux were also studied by flow cytometry.Evaluation of the nanocomposite in vivo:The in vivo heating efficacy of the nanocomposite in SW620/Ad300 tumor-bearing mice was assessed by the changes of tumor temperature under NIR laser irradiation.The in vivo antitumor efficacy of the nanocomposite was evaluated by pharmacodynamic experiment,including the changes in tumor volume and the images of excised tumors.Preliminary toxicity was evaluated by relative body weight changes and H&E staining of major organs in the nude mice?heart,liver,spleen,lung and kidney?..Results:The characteristics of the nanocomposite with an uniform and well-defined structure and a certain length-to-width ratio were confirmed by TEM,UV-Vis-NIR spectra,FT-IR spectra and zeta potentials.The TEM images showed the rod-shaped GNRs,mesoporous structure with a thickness of approximately 20.1±2.5 nm,the layer of mSiO₂@PHIS with a thickness of 21.7±1.9 nm andthe layer of mSiO₂@PHIS@TPGS with a thickness of 24.8±2.2 nm.The absorption of the nanocomposite in UV-Vis-NIR spectrum had no significant change after 2.5 W/cm²NIR laser irradiating for 1 h,indicating their good photothermal stability.DOX-loaded GNRs@mSiO₂@PHIS@TPGS suspensions exhibited a quick photothermal conversion under NIR exposure and reached above 43 ^oC after about 5min,indicating the good photothermal conversion property.As pH value decreased to6.0,the potential of nanocomposite converted to positivefrom negative.DOX was slowly released at pH 9.0,and the accumulated release drug was only 18.9%during 8days;whereas the accumulated release of drug was 42.6%at pH 6.5 in 8 days.When further dropped the pH to 5.0,66.1%of DOX was released,significantly higher than that at pH 6.5 or 9.0.DOX showed an obvious burst drug release at pH 5.0 after each NIR laser irradiation.To investigate the reversal of multidrug resistance in vitro by the DOX-loaded nanocomposite,the MDR colorectal cancer cell line SW620/Ad300 were used to evaluate the cell viability after different treatments.The resistance-fold of SW620/Ad300 to DOX was 175.5,which confirmed the SW620/Ad300 cells were DOX highly resistant.The intensity of NIR laser irradiation used has little effect on cell survival.The GNRs@mSiO₂@PHIS@TPGS nanocomposite itself showed a certain toxicity against SW620/Ad300 due to the anticancer activities of TPGS.The DOX-loaded GNRs@mSiO₂@PHIS@TPGS with NIR irradiation showed the lowest cell viability among all the treatment groups,and successfully reversed the multidrug resistance of SW620/Ad300 cells with the reversal index?RI?of 97.52,indicating the strong synergic effect of chemo-and photothermal therapy.The ability of nanocomposite in reversing the drug resistant was further confirmed by the live-dead cell-staining assay,which demonstrated that the combination of chemo-and photothermal therapy shows the strongest ability in reversing the MDR.In the in vitro cellular uptake tests,the amount of intracellular DOX increased with time in the cells treated with DOX-loaded GNRs@mSiO₂@PHIS@TPGS and some DOX was distributed in the nucleus at 4 h.On the contrary,the intracellular level of DOX in the cells treated with free DOX remained low for 4 h,and did not enter into the nucleus.At 4 h,the intracellular level of DOX in in the cells treated with the nanocomposite group is 2.3 folds of that in the cells treated with free DOX.In the cells treated with DOX-loaded GNRs@mSiO₂@PHIS@TPGS,the co-localization of DOX and endo/lysosomal compartments?yellow color?was obvious at the first two hours.At 4h,a small portion of DOX was observed in the endo/lysosomal compartments,while a large part of DOX were released into the cytoplasm and entered into the nucleus.The result demonstrated that the nanocomposite had efficient endo/lysosome escape property.After drug efflux for 2 h,the intracellular DOX level in the cells treated with DOX-loaded GNRs@mSiO₂@PHIS@TPGS did not show significantly changes;whereas that of the cells treated with free DOX largely decreased to 51.4%of the original drug level.The result indicated that the nanocomposite had the ability to prevent the drug effluxing from the MDR tumor cells,which probably due to the effect of TPGS.Overall,the results demonstrated that the DOX-loaded GNRs@mSiO₂@PHIS@TPGS nanocomposite could significantly increase the intracellular accumulation of DOX to overcome MDR,relying on its efficient intracellularly delivery and reduced drug efflux.To investigate the in vivo heating efficacy of the nanocomposite in SW620/Ad300tumor mice,tumor treated with PBS showed only a slight temperature increase within2 min under the laser irradiation?lower than 40.8°C?,while tumor treated with DOX-loaded GNRs@mSiO₂@PHIS@TPGS showed a rapid temperature increase as high as 54.5±0.6°C.In vivo antitumor efficacy tests demonstrated that the suppressive ability of tumor growth after different treatments were DOX-loaded GNRs@mSiO₂@PHIS@TPGSwithirradiation group>GNRs@mSiO₂@PHIS@TPGS with irradiation group>DOX-loaded GNRs@mSiO₂@PHIS@TPGS group>free DOX group>PBS group.The digital images of the tumors isolated at day 38 further confirm the above result.The preliminary toxicity study of the nanocomposite showed that the body weights of all the nude mice did not decrease significantly,indicating little systemic toxicity of the treatment.Furthermore,The H&E staining results of the main organs of the heart,liver,spleen,lung and kidney of nude mice showed no damages in the main organs of nude mice in all groups.Altogether,the in vivo experiments demonstrated that the nanocomposite could exert efficient chemo-and photothermal combination therapy against MDR colon cancer with little systemic toxicity.Conclusions:In this study,we successfully prepared the novel multi-functional drug delivery system DOX-loaded GNRs@mSiO₂@PHIS@TPGS nanocomposite,which has uniform dispersion,stable structure,good photothermal conversion efficiency,pH response and NIR laser-excited drug release performance.Compared with chemo-or photothermal therapy alone,the synergistic chemo-photothermal therapy using DOX-loaded GNRs@mSiO₂@PHIS@TPGS nanocomposite was demonstrated both in vitro and in vivo to exhibit higher therapeutic efficacywith little systemic toxicity.Therefore,the nanocomposite is expected to become a novel drug delivery nanosystem with a potential for synergistic photothermal therapy and chemotherapy to reverse drug-resistant cancer.