Rare Earth Nanocrystals Modified Hollow Mesoporous Manganese Oxide For Anti-Tumors Low Temperature Photothermal Therapy

Background:The current clinical oncologic methodologies,including surgery,radiotherapy,chemotherapy,immunotherapy and targeted therapy,do not achieve complete cure due to the drawbacks or limitations of the conventional strategies.In recent years,with the development and integration of materials science,medicine and biology,interdisciplinary research has provided new opportunities for tumor treatment.New treatment strategies based on nanotechnology have been widely concerned,such as photothermal therapy,photodynamic therapy,chemodynamic therapy,gene therapy and gas therapy,etc.The therapeutic effects of photothermal therapy on killing cancer cells and inhibiting solid tumors have been confirmed in applied basic research and clinical test,and this treatment does not cause complications and systemic toxicity.Photothermal therapy（PTT）is a promising anti-tumor treatment,which is based on the photothermal agents（PTAs）delivered to the tumor sites that inhibit cancer cells by converting light energy into heat energy after irradiation of light with specific wavelengths.The current research mainly focuses on high temperature PTT,which needs to increase the temperature on tumors over 50°C in order to achieve complete tumor elimination.However,such high temperature（temperature higher than 45°C）could not only ablate the tumors but also damage normal tissues around lesions through heat diffusion,as well as cause some other adverse biological effects.Therefore,the use of mild treatment temperature（below 45°C）to achieve efficient anti-tumor effect is of great significance for promoting PTT into the clinical treatment of tumors.Purpose:To achieve high-efficient mild PTT,this project intends to construct a low-temperature photothermal nanoplatform activated by tumor microenvironment:hollow porous H-MnO_x nanozymes were modified by the ultra-small-sized Na YF₄:Nd³⁺@Ca F₂ nanocrystals（Nd-NCs）on the surface and loaded hypoglycemic drug KL-11743 into the porous to construct H-MnO_x@Nd-NCs-KL-11743（MN-KL）.KL-11743（a glucose transporter GLUTs inhibitor）,which significantly reduce cellular glucose uptake,can block the energy source of tumor cells to inhibit the synthesis of heat shock proteins（HSPs）under heat stress.Ultra-small size Nd-NCs have both photothermal conversion performance and near-infrared second-region fluorescence imaging ability,which can achieve low-temperature photothermal effect and fluorescence imaging at the tumor site,and Nd-NCs can be metabolized out of the body due to ultra-small size.As a tumor microenvironment-responsive nanozyme,H-MnO_x not only can quench the luminescence of Nd-NCs in normal tissues to act as a’fluorescence switch’to facilitate the localization imaging of tumor sites,but also can produce a large amount of reactive oxygen species（ROS）and lipid peroxide（LPO）by catalytic reaction.Moreover,Mn²⁺/Mn³⁺/Mn⁴⁺released in response to the decomposition of H-MnO_x can generate·OH by Fenton-like reaction.The LPO and ROS can crosslink primary amines of proteins to destruct the structure and function of HSPs,which provides a second guarantee for the complete silence of HSPs in tumor cells.Ultra-small size Nd-NCs and ionic manganese element can also be excluded in vitro with metabolites,ensuring the biosafety of low-temperature photothermal therapy.Herein,we constructed a low-temperature photothermal nanoplatform that provide two-pronged strategy for the comprehensive removal of HSPs to realize mild PTT.Methods:（1）Preparation and characterization of MN-KL nanomaterials:Hollow porous MnO_x（H-MnO_x）was synthesized by using solid SiO₂nanospheres as templates.The rare earth nanocrystals Na YF₄:Nd³⁺@Ca F₂ were modified on the surface of H-MnO_x to obtain H-MnO_x@Nd-NCs（MN）with composite structure,and the final MN-KL composite nanomaterials were formed by MN loading small molecule inhibitor KL-11743.The MN-KL was fully characterized by TEM,HRTEM,XRD,XPS,BET,UV-visible-NIR absorption spectroscopy and Fourier transform infrared spectroscopy.（2）Performance tests of MN nanomaterials:The photothermal properties of MN were tested by infrared thermal imaging camera.The catalase-like activity（CAT）,Fenton-like reaction,oxidase-like activity（OXD）and glutathione oxidase-like activity（GSHO_x）of MN were tested by portable dissolved oxygen meter,methylene blue（MB）,1,3-diphenylisobenzofuran（DPBF）and 5,5’-dithiobis（2-nitrobenzoic acid）（DTNB）,respectively.At the same time,the tumor microenvironment responsiveness and NIR-II fluorescence imaging capability of MN were tested by UV-visible-NIR absorption spectrometer and NIR-II fluorescence imaging system.（3）Cell experiments based on MN-KL:The cell compatibility and toxicity of MN-KL were tested by MTT.The live and dead cells were stained by Calcein-AM/PI.The contents of ROS and LPO in the cells were detected by DCFH-DA and LPO detection kits.The changes of glucose level and ATP content in cells were detected by glucose detection kit and ATP detection kit.The expressions of HSP70 and GPX4 proteins were detected by Western blot（WB）.（4）Anti-tumor experiments in vivo based on MN-KL:CT26 cells were injected subcutaneously into female Balb/c mice to construct a mouse colon cancer xenograft model.After the tumor volume reached 100 mm³,MN-KL solution was injected in situ for low-temperature photothermal therapy.During the treatment,the tumor size and body weight of the mice were closely monitored,and the effect of low temperature photothermal therapy of MN-KL was evaluated after treatment.The biosafety of MN-KL was evaluated by biochemical analysis of blood and organs in mice.（5）Tumor localization imaging and in vivo metabolic analysis of MN:MN solution was injected into the tail vein of mice,and the fluorescence imaging of tumor site in mice was observed by near-infrared fluorescence imaging system.The metabolites of mice were collected by metabolic cages,and the element content in the metabolites was analyzed by ICP-MS to evaluate the catabolic ability of MN.Results:（1）The prepared MN nanomaterials presented hollow porous structure for loading the small molecule drug KL-11743.And the intrinsic existence of multivalent metal ions rendered MN with enzyme-mimic activities.（2）MN-KL can catalyze the generation of ROS and LPO in tumor cells,while simultaneously inhibiting glucose uptake and ATP synthesis of tumor cells.（3）MN-KL can down-regulate the expression of HSPs under heat stress by inhibiting the synthesis of HSP70 in tumor cells and consuming the existing HSP70,to achieve efficient low temperature photothermal therapy.（4）Low temperature photothermal therapy based on MN-KL can achieve effective ablation of tumors in mice,and no obvious side effects were observed.（5）MN nanomaterials can achieve specific NIR-II fluorescence imaging of mouse in tumor sites,and MN can be decomposed and eliminated from the body through feces and urine.Conclusions:H-MnO_x@Nd-NCs with abundant porous structures and enzyme-mimic activities were synthesized,followed by loading with hypoglycemic drug KL-11743,leading to the system named as MN-KL.By two-pronged strategy of GLUTs inhibitor and cascaded ROS storm,MN-KL achieves highly efficient mild PTT.In addition,MN-KL can also present specific NIR-II fluorescence imaging in tumor sites and the characteristics of catabolism due to the TME responsiveness,which ensures the biosafety in the process of low temperature photothermal therapy.