| Objectives:Oral squamous cell carcinoma(OSCC)is a common malignant tumor of the head and neck.Strategies of the treatment for the early stage(stage I and II)of OSCC are mainly surgery and radiotherapy.However,for the most patients are often in the middle or late stage(stage III to IVB)at the time of presentation,the local recurrence rate is still as high as 30%and the 5-year survival rate is less than 40%,which requires combined chemotherapy.However,owing to chemotherapeutic drugs lack tumor specificity in clinic,they often form a large amount of ineffective accumulation in healthy tissues,which causing serious toxic-side effects to the body.The nano-drug delivery system can improve tumor targeting,significantly prolong the circulation time in vivo,and reduce the side effects of chemotherapy drugs.Carbon monoxide(CO),as a gas signal molecule,can selectively induce apoptosis of tumor cells,is an important member of gas tumor therapy.Unfortunately,the free diffusion of CO across the membrane is difficult to ensure effective therapeutic concentrations,moreover,the existing CO prodrugs,such as CORM,release unstably and faster,which limits their clinical application.In this investigation,we used metal carbonyl compound C6Fe2O6S2(hereinafter referred to as Fe-CO)that is a CO-prodrug with high ROS responding as the loaded drug to release the gas.Adriamycin(DOX)was connected with DSPE-PEG through Hyd functional group to realize that DOX could respond to release in the low pH microenvironment of tumor.The nano drug carrier system(NPs)was constructed by self-assembly,and cRGD(cyclic RGD)was used to modify the nano drug carrier system to achieve tumor targeting effect.A large number of experiments have confirmed that this new nano-drug delivery system has superior drug loading ability,excellent responsive drug release ability in tumor microenvironment,and significant tumor targeting ability,and combinate tumor chemotherapy and gas therapy as tumor-targeting therapy for OSCC,which providing a new scheme for the treatment of OSCC.Methods:1.We used the metal carbonyl compound C6Fe2O6S2(referred to as Fe-CO),as the encapsulated drug,is a precursor drug with high ROS responding to release CO.After the chemical grafting reaction,DOX was connected to DSPE-PEG by Hyd functional group with low pH-responsive ability,and the polymer DSPE-PEG-cRGD with tumor-targeting effect was prepared.The polymer was self-assembled by nanoprecipitation method which prepared three groups of nano-drug delivery systems CO/mDOX-NPs,CO/hDOX-NPs and CO/hDOX/RGDNPs.The particle size of the nano-drug delivery system was detected by dynamic light scattering(DLS)and TEM,and the drug loading capacity was detected by an ultramicrospectrophotometer.2.We selected human squamous cell carcinoma SCC25 and CAL27 cells as the research objects.The CO-P fluorescent probe was used to detect the intracellular CO release ability of CO/mDOX-NPs,CO/hDOX-NPs and CO/hDOX/RGD-NPs,co-localization with lysosomes was used to evaluate the endosome escape of nano-drug delivery system.3.CCK8 and Calcein-AM/PI flow cytometry apoptosis detection were used to evaluate the inhibitory effect of the new drug-loading systems CO/mDOX-NPs、CO/hDOX-NPs、CO/hDOX/RGD-NPs for tongue squamous cell carcinoma cells.The Wound Healing test and Transwell migration test were used to detect the inhibitory migration of tongue squamous cell carcinoma cells.4.DCFH-DAI fluorescent probe was used to detect the change of intracellular ROS(reactive oxygen species)level,and ELISA kits were used to detect the changes of cytokines TNF-α and IL-1β in the medium,to cogitate the related factors.5.SCC7 was implanted subcutaneously into C57 female mice to establish the animal model of tongue squamous cell carcinoma.After injecting three groups of drug-loaded nanosystems CO/mDOX-NPs、CO/hDOX-NPs、CO/hDOX/RGD-NPs through the caudal vein,the tumor growth was observed,and the changes in tumor volume and mouse weight were recorded regularly.After the observation period,the mice were killed and tumor tissues were collected;At the same time,we used the small animal in vivo three-dimensional optical imaging analysis system to carry out fluorescence imaging on the tumor tissue,main organs(heart,liver,spleen,lung,and kidney)and some muscle tissues around the tumor collected from tumorbearing mice and mice killed,and then detect the distribution of NPs in the three groups.Results:1.The results of the NMR analysis suggested that DSPE-PEG 2000-cRGD was successfully synthesized;HPLC results showed that the synthesized DSPE-PEG 2000-hyd-dox had a good pH-responsive ability when hydrazone bond(Hyd)was broken under low pH conditions and could release DOX stably.The results of particle size analyzed by DLS and TEM showed that CO/mDOX-NPs、CO/hDOX-NPs、CO/hDOX/RGD-NPs were about 110nm、110nm and 140nm,respectively,indicating that the prepared nano-drugs were small and uniformly dispersed.Then the absorbance value at 326 nm was measured by ultramicro spectrophotometer,and the results were fitted with the standard curve of Fe-CO.The CO drug loading rates of the nano drug loading system were calculated to be 15.89%,18.55%and 19.31%,respectively,which indicated that the nano drug prepared by us had high Fe-CO drug loading effect.2.CO-P fluorescence results showed that compared with free Fe-CO,NPs could stably release CO in cells.The results showed that the fluorescence intensity of CO probe in CO/hDOX/RGD-NPs group was the most significant and had a stable time intensity effect.The results of lysosomal co localization showed that CO/hDOX/RGD-NPs group was more likely to be absorbed by tongue squamous cell carcinoma cells in unit time,and endosome escape occurred successfully.3.CCK8 results showed that NPs could significantly inhibit the growth of tongue squamous carcinoma cells,and the inhibitory results showed a dose-dependent effect.Meanwhile,we found that the inhibitory effect of three groups of NPs on tongue squamous cell carcinoma was higher than that of free Fe-CO and DOX drugs,especially the CO/hDOX/RGDNPs group showed the minimum IC50(half inhibitory concentration)value.The results of Wound Healing test and Transwell migration test showed that NPs could inhibit the migration of tongue squamous cancer cells,which was significantly better than that of free drug group,and the migration rate and the number of migrating cells of the nano drug delivery system CO/hDOX/RGD-NPs were lowest.4.The results of Calcein-AM/PI double staining and flow cytometry showed that CO/hDOX/RGD-NPs had the highest proportion of apoptosis compared with the two groups of nano drug delivery systems,which again proved that the new nano drug delivery system CO/hDOX/RGD-NPs had excellent ability to induce TSCC cell apoptosis.5.The ROS fluorescence results showed that NPs could increase the ROS level in OSCC,especially CO/hDOX/RGD-NPs group could most significantly increase the intracellular ROS level,which was consistent with the results of cell growth inhibition and apoptosis.ELISA results showed that the use of free DOX alone could stimulate the express of TNF-α,and IL-1βin tongue squamous cell carcinoma cells.In contrast,the prepared nano drug-loading systems CO/mDOX-NPs,CO/hDOX-NPs,CO/hDOX/RGD-NPs could reduce the expression levels of TNF-α and IL-1β.6.In vivo antitumor results of NPs showed that all three groups of drug-loaded nanosystem systems could inhibit tumor growth,especially CO/hDOX/RGD-NPs NPs could actively target tumor tissues with excellent tumor enrichment ability and remarkable antitumor efficacy;The results of intravital three-dimensional optical imaging in small animals showed that the drugloaded nanosystem CO/hDOX/RGD-NPs possessed excellent active targeting ability,able to achieve efficient enrichment within tumor tissues,and had low toxic side effects.Conclusions:In this investigation,DSPE-PEG-Hyd-DOX polymer with pH-responsive release of DOX and DSPE-PEG-cRGD polymer with tumor-targeting ability were prepared by chemical grafting reaction.Using CO prodrug C6Fe2O6S2(referred to as Fe-CO)with ROS-responsive release ability,three groups of NPs were prepared by self-assembly method.Therefore,we developed a new nano-drug delivery system that can achieve tumor targeting,pH and ROS dual response,and deliver CO prodrug/DOX to achieve combined treatment,which enhances the therapeutic effect of OSCC.The nano drug delivery system CO/hDOX/RGD-NPs could achieve the active targeting delivery to tongue squamous carcinoma cells by cRGD-specific recognition of integrin ανβ3,which was more easily untaken by tongue squamous carcinoma cells.The results of in vitro experiments showed that CO/hDOX/RGD-NPs could rapidly and stably release CO and DOX in tongue squamous carcinoma cells,stimulate the increase of intracellular ROS level,and also significantly inhibit the proliferation and migration of OSCC cells,promote the apoptosis of OSCC cells,and showed good anti-inflammatory effect at low concentrations;the vivo anticancer results demonstrated that CO/hDOX/RGD-NPs exhibited a remarkable ability to inhibit tumor growth,and significantly reduced ineffective enrichment in vivo Therefore,we believe that the CO/hDOX/RGD-NPs nano-drug delivery system has low toxicity,high tumor specificity and remarkable therapeutic effect on OSCC,which provides a reliable new method for the treatment of OSCC. |
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