Preparation And Properties Of Oxygen-carrying Dendritic Large-pore Mesoporous Silica Nanoparticles For Tumor Photothermal And Photodynamic Therapy

ObjectiveIn the recent years,the morbidity and mortality of malignant tumor has a increasing trend.The cancer can not be cured completely by traditional and single treatments such as chemotherapy,radiotherapy and surgery.At the same time,some severe side effects and postoperative reaction may occur.Thus,it becomes an extremely hard task to look for its new treatment.With the progress and development of medicine,people begin to use the combined strategies to treat cancer in order to improve the therapeutic efficiency of cancer.Photothermal therapy(PTT)is to gain the purpose of "burn" tumor tissue by using photosensitizer to convert absorb light energy into heat energy to increase the local temperature of tumor.It is a new treatment strategy and has some advantages such as no-ninvasive,safe and precise positioning.In order to improve the treatment effctiveness of photothermal and meanwhile,decrease the damage of normal tissue,it is very significant to consider which kind of phptosensitizer need be chosen.In this study,IR-780 iodide dye was selected to be as a photosensitizer to absorb light energy and convert it into heat under the near-infrared(NIR)light irradiation,meanwhile activate surrounding oxygen to generate cytotoxic reactive oxygen species(ROS).Since it not only has photothermal effect but also photodynamic effect,it can be used to treat tumor by both of PTT and photodynamic therapy(PDT).The effectiveness of tumor treatment can be improved obviously by combining these two methods.On the other hand,because of the rapid growth of tumor,their microenvironment is a hypoxia state,and PDT needs to consume oxygen.Therefore,it is very important to provide oxygen to tumor tissues for improving the effectiveness of PDT.In this paper,the dendritic large-pore mesoporous silica nanoparticles(DLMSN),one of the hot material in the filed of tumor diagnosis and therapy,was used as a carrier.It was used to carry IR780 and perfluorohexane(PFH)for delivering IR780 and oxygen to tumor tissues simultaneously to achieve better collaborative treatment effect of tumor.Here,PFH is a safe hydrophobic and oleophpbic oxygen carrier with high dissolved oxygen content.In order to improve its stability in aqueous solution and realize their tumor targeted delivery,cell membrane biomimetic nanocarrier technology was utilized to coat the white blood cell membrane(WBCm)on its surface in this study.Finally,a multifunctional nano-theraprutic system DLMSN-IR780/PFH@WBCm with PTT/PDT properties was obtained,and the effect of its combined therapy was preliminarily investigated in vitro.ContentsFirstly,the multifunctional DLMSN with WBCm coating and IR780 and PFH loading were successfully prepared by multistep synthesis in this study.As well as their structure and performance were characterized.Specific steps included the preparation of DLMSN,the modification of DLMSN inside and outside,loading IR780 and PFH in DLMSN,extraction of WBCm and coating WBCm on the surface of DLMSN.Secondly,the structure and properties of multifunctional DLMSN were characterized by transmission electron microscopy(TEM),dynamic laser light scattering(DLS),ultravi ol et-vi sibil e spectrophotometer(UV-vis)and energy dispersive X-ray spectrometer(EDS).Finally,the in vitro performance of this multifunctional DLMSN was preliminary investigated.The PTT/PDT properties and cytotoxicity of the multifunctional DLMSN were investigated in vitro though heating cure under NIR laser irradiation,the generated ROS level and MTT cytotoxicity assay using NIR laser,fluorescence spectrophotometer,laser confocal fluorescence microscope(LSCM)and enzyme markerMethodsIn this study,DLMSN was prepared by a dual templates method using hexadecyl trimethyl ammonium bromide(CTAB)as a main template and sodium salicylate(NaSal)as an auxiliary template.The sulfhydryl group(-SH)was modified on the surface of DLMSN by using Trimethoxysilylpropanethiol(MPTMS).Then the templates were removed,IR780 was loaded inside and fluorocarbon chain(-FC)was modified followed by PFH loading.Finally,WBCm was coated on the surface of the multifunctional DLMSN.In view of the difficulty of the separation of white blood cells from the blood of mice(the average count was 7.91 ×103/mm3)and it is difficult to extract enough WBCm to modify the multifunctional DLMSN.Thus,the mouse mononuclear macrophage Raw264.7 cell was used as the white blood cell model to be extracted the cell membrane and coating on the surface of the multifunctional DLMSN to obtain DLMSN-IR780/PFH@WBCm.Next,the structure and property of multifunction DLMSN in each step were characterized.The surface morphology,particle size(Dn)and dispersity of the DLMSN were observed by TEM.DLS was used to detect the hydraulic diameter(Dh),surface charge and poly dispersity index(PDI)of multifunction DLMSN.UV-vis spectrophometer checking was used to confirm whether IR780 was loading successfully.The element of DLMSN-IR780/PFH@WBCm was analyzed by energy dispersive X-ray spectrometer(EDS).Total protein analysis of WBCm was performed by coomassie brilliant blue staining.Finally,the performance of DLMSN-IR780/PFH@WBCm was evaluated in vitro.The heating curve of multifunction DLMSN was monitored by infrared thermal imager under NIR laser irradiation,and the generated ROS level of multifunction DLMSN was monitored by ROS fluorescence probe SOSG.The imtracellular ROS level generated by multifunction DLMSN was evaluated by LSCM.And the combine therapeutic efficiency of PTT/PDT by DLMSN-IR780/PFH@WBCm was evaluated by MTT assay.ResultsDLMSN-IR780/PFH@WBCm was successfully prepared from the result of TEM and DLS.The resultant multifunction DLMSN had uniform particle size and showed good dispersion.The average hydraulic diameter of DLMSN-IR780/PFH@WBCm was 129±3 nm and the Zeta potential was-21.03±1.2 mV.The results of UV-vis spectra of DLMSN confirmed that IR780 was successfully loaded into the DLMSN with a loading capacity of 1.8%.The loading capacity of PFH on DLMSN-IR780/PFH@WBCm was 10 μL/mg(1669%)by DLS.The results of total protein analysis with coomassie brilliant blue staining test showed that the characteristic protein WBCm on DLMSN-IR780/PFH@WBCm was consistent with that of Raw264.7 cell membrane protein,which showed that DLMSN-IR780/PFH@WBCm has the characteristic protein of Raw264.7 cell.Under NIR laser irradiation(808 nm,2 W/cm2),the solution temperature of DLMSN-IR780/PFH@WBCm solution(with IR780 content of 50 μg/mL)continuously rise and the temperature reached 51℃ at 10 min,meanwhile a large number of ROS were produced at the same time,indicating that DLMSN-IR780/PFH@WBCm has good photothermal and photodynamic performance;At the cellular level,after the mouse breast cancer 4T1 cell incubated with DLMSN-IR780/PFH@WBCm by NIR laser irradiation,it is obviously observed that there were a intense fluorescence signal in their cytoplasm by LSCM.It proves that there is a number of ROS in their cytoplasm.The results from MTT assay showed that DLMSN-IR780/PFH@WBCm could kill tumor cells better under NIR laser irradiation compared with the controls.ConclusionsIn this study,DLMSN was used as the carrier to carry IR780 and PFH,and then carry oxygen,followed by coating WBCm on the surface.The biomimetic DLMSN-IR780/PFH@WBCm nanosystem with high oxygen carrying,photothermal and photodynamic property was successfully constructed.The results in vitro experiments demonstrated that DLMSN-IR780/PFH@WBCm have both better photothermal and photodynamic effects which can effectively inhibit tumor cell growth under NIR laser irradiation through PTT/PDT combined therapy.