| Objective To construct a p H/light dual-stimuli-responsive drug delivery system that combines different functions of antineoplastic drugs.The stability,safety and targeting of the drug delivery system were verified by in vivo and in vitro,and the response ability of the system to p H/ near infrared light and anticancer effect were evaluated,which provides a new strategy for combination therapy and targeted therapy of oral cancer.Methods 1.Construction and characterization of p H/light dual-stimuliresponsive drug delivery system.(1)The interferon(IFNα1β),photosensitizer Indocyanine green(ICG)and chemotherapeutic drug Doxorubicin(DOX)were mixed and triggered by water to obtain p H/light dual-stimuli-responsive IID.(2)The morphology of IID was observed by confocal microscope and transmission electron microscope,and the particle size and surface charge of IID were determined by Nano ZS90;(3)The synthesis and charge change of IID were detected by PAGE gel electrophoresis;(4)The encapsulation efficiency of ICG and DOX was determined by fluorescence spectrometer,and the encapsulation efficiency of IFN α 1 β was determined by enzyme-linked immunosorbent assay(ELISA).(5)The stability of IID in serum medium was detected by fluorescence spectrometer,and the photothermal effect of drug delivery system was detected by thermal imager.2.The response ability of drug delivery system IID to acidic environment.(1)The changes of particle size and surface charge of IID in different p H environments were detected by Nano ZS90.(2)The color changes and precipitation of IID in four different p H environments(p H=7.4,6.5,5.5,4.5)were observed,the release efficiency of DOX in p H environment was measured by fluorescence spectrometer.(3)The accumulation of IID and the release ability of DOX in acidic environment by PAGE gel electrophoresis and fluorescence spectrometer.3.Near infrared light triggers the IID to release drugs.(1)The controlled release effect of near infrared light on IID was observed by laser confocal microscope.(2)The release of DOX and ICG triggered by NIR were detected by PAGE gel electrophoresis.(3)The release efficiency of DOX and ICG triggered by NIR were detected by fluorescence spectrometer,the release efficiency of IFN α 1 β from IID triggered by NIR was detected by ELISA.4.Cytotoxicity of drug delivery system IID.(1)The IID uptake by Cal27 cell triggered by acidic environment and NIR was detected by laser confocal microscope and flow cytometry.(2)Cytotoxicity of IID was evaluated by CCK8 assay.5.The safety and anticancer effect of IID.(1)Establishment of subcutaneous heterotopic tumor model in nude mice: the cultured Cal 27 cells were subcutaneously injected into nude mice.When the tumor reached 0.5cm,the tumor tissue was taken and transplanted into the experimental nude mice to enlarge the number of tumor model mice.(2)The fluorescence signal of ICG was collected by IVIS Spectrum to evaluate the imaging ability of IID in tumor site.(3)the temperature change and thermal infrared image of tumor site were continuously collected by near infrared thermal imager to evaluate the photothermal treatment efficiency of IID.(4)The transplanted tumor model nude mice were randomly divided into 6 groups with 5 mice in each group.After 23 days of treatment,the experimental mice were anesthesia.The body weight and tumor size were measured every other day,and the change curves of body weight and tumor were drawn.Tumor tissue was taken and Ki67 index was observed to evaluate the anti-tumor ability of drug delivery system IID;main organs were taken for histological examination to evaluate the safety of the drug;(5)The level of IFN α 1 β,IL-1 β and IL12 in blood were measured by ELISA to evaluate the effect of IID on IFN α1β metabolism and immunity in nude mice.Results 1.Characterization of drug delivery system.(1)The IID solution was dark green,and dark green precipitation could be seen after centrifugation,and its properties were different from those of free IFN α 1 β,ICG and DOX.(2)Detection of particle size and charge of IID: the IID was a uniform round particle with a size of 380 nm under confocal microscope and transmission electron microscope.The diameter of IID measured by Nano ZS90 was 380±103 nm.(3)The results of PAGE gel electrophoresis showed that the negatively charged IFN α1β and ICG moved to the positive electrode,the positively charged DOX moved to the negative electrode,and the IID moved a strip to the positive electrode,suggesting negative charge,and the three were assembled successfully.(4)The entrapment efficiency of ICG in IID was 99.5±0.2%,and the entrapment efficiency of DOX was 22.0 ±0.4%.The encapsulation efficiency of IFN α1βwas 96.78 ±0.9%.(5)the fluorescence intensity of the characteristic peak of IID after assembly was significantly weaker than that of free DOX and ICG,and the fluorescence intensity of ICG and DOX in IID remained stable and consistent within 3 days.2.The response ability of drug delivery system IID to acidic p H.(1)The particle size of IID in acidic environment increased to 885 ±116nm at p H=6.5 compared with the particle size of IID in neutral environment p H7.4(380 ±103nm),the surface charge of drug delivery system changed from positive charge to negative charge.(2)Under acidic condition,IID aggregates and precipitates and releases DOX.(3)PAGE gel electrophoresis showed that the DOX released by IID could move to the negative electrode in acidic environment,and the fluorescence intensity of DOX and ICG in different p H environment showed that IID released DOX rapidly in acidic environment,while the fluorescence intensity of ICG had no obvious change,indicating that acidic environment caused IID to release DOX.3.Near-infrared light triggers the drug delivery system IID to release drugs.Under laser confocal microscope,it can be seen that IID in acid environment was triggered by NIR to release drugs rapidly,and with the increase of NIR trigger time,DOX was released more;gel electrophoresis showed that the drug delivery system released DOX moving to the negative and ICG moving to the positive after triggered by near-infrared light.Within 80 s,the release efficiency of DOX,ICG and IFN α1β were all 100%.4.Cytotoxicity of drug delivery system IID.The results of flow cytometry and laser confocal microscopy showed that acid environment and near infrared light trigger could promote the uptake of DOX and ICG in Cal 27 cell line.CCK8 showed that acid environment and near infrared light could promote drug killing of tumor cells.5.The safety and anticancer effect of drug delivery system IID.After receiving tail vein injection,the fluorescence signal of heterotopic tumor model nude mice was mainly concentrated in liver and kidney at 0.5h after ICG injection,and a small amount was located in tumor tissue.6h later,the signal in liver was very weak in ICG group,and there was no signal in tumor site.However,strong fluorescence signal could be seen in the tumor site 0.5h after injection of drug delivery system IID,and strong ICG fluorescence signal could still be seen in tumor tissue 24 hours later.The results of infrared thermography showed that in the drug delivery system IID group,the temperature of the tumor site increased rapidly to 42 ℃,and gradually increased with time,and then increased to 62 ℃to the platform stage after about 4min,while the temperature of the tumor site increased slowly in the ICG injection group,and finally reached the plateau stage at 42 ℃.The temperature of the tumor site in the normal saline group had no significant change in the light time.Both fluorescence imaging and photothermal imaging have proved that the drug delivery system IID has good targeting ability.Elisa results showed that the drug delivery system could significantly increase the concentration of IFN α1β in peripheral blood and promote the expression of inflammatory factor IL-1β,suggesting that IID played an immune effect and the immunity of nude mice was improved.Drug delivery system IID can significantly inhibit tumor growth and reduce the proliferation of tumor cells under the dual response of tumor acidic environment and near infrared light in vivo,as well as the multiple effects of adriamycin and interferon,while the body weight of nude mice has no significant change,indicating that IIID significantly reduces the systemic toxicity of chemotherapeutic drugs.Conclusions 1.Human interferon α1β for injection can be assembled as a carrier with photosensitizer ICG and chemotherapy drug DOX to form a drug delivery system with a size of about 380 nm and a negative charge on the surface,which can avoid rapid metabolism by the body in the process of circulation and show selective extravasation and retention in tumor tissue.2.The acidic condition of p H=6.5,which is similar to the tumor microenvironment,can trigger the change of the surface charge and size of the drug delivery system IID,which makes it accumulate and release the chemotherapeutic drug DOX in the tumor tissue,and realize the passive targeted delivery at the drug cell level.3.The IID accumulated in the tumor tissue,in response to near-infrared light,further triggers the drug delivery system IID to fully release DOX,ICG and INFα1β,further achieve accurate and efficient drug release,and achieve targeted therapeutic effect.4.Drug delivery system IID is a good near-infrared tumor imaging agent,which is conducive to real-time and clear monitoring of drug distribution in the body,it can efficiently enrich in the tumor site,and play a good photothermal treatment effect under the action of near-infrared light.5.PH/ optical double response drug delivery system IID can effectively increase the concentration of interferon in peripheral blood,immunotherapyphotothermal therapy-chemotherapy combined with multiple concurrent,significantly enhance the anti-tumor effect,and greatly reduce the side effects of drugs. |
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