An Antitumor Strategy Of Mild Photothermal Therapy Based On Au-pd Dual Precious Metal Nanozymes

BackgroundAt present,China is facing with the severe reality that the cancer morbidity and mortality rate are continuously rising,the number of cancer patients is increasing,and the survival rate of cancer patients is low in five years.Evidently,there have a long way to go in the fight against cancer for us.Traditional treatment methods which include surgery,radiotherapy,chemotherapy and systemic adjuvant therapy have been moderately effective,nevertheless there are still limitations.The development of new drugs and technologies for the treatment of cancer has become a consensus among researchers worldwide.Photothermal therapy（PTT）used as a non-invasive tumour ablation method enables the effective removal of the primary tumour by killing the cancer cells.Alternatively,excessive temperatures are likely to cause thermal damage to surrounding normal tissues and can lead to harmful biological effects.Although the use of mild photothermal therapy（m PTT,42°C-45°C）activates the expression of heat shock proteins（HSPs）in tumour cells,it can lead to heat resistance and diminishes the therapeutic effect.Therefore,inhibition of the expression of HSPs is the key to achieving efficient m PTT.What’s more,reactive oxygen species（ROS）are reactive chemicals,mainly consisting of superoxide anions（·O₂^-）,hydroxyl radicals（·OH）and singlet oxygen（¹O₂）,which play an important role in cell signalling and homeostasis in the body.What’s more,the reactive oxygen species（ROS）produced by the nanoenzyme could cross-link with the primary ammonia structure of proteins,thereby directly disrupting the structure and function of HSPs.As a nanomaterial with mimetic enzymatic activity,the unique multifunctional catalytic activity of nanoenzymes enables them to catalyse the production of cytotoxic molecules specifically in the tumour microenvironment（TME）and are widely used in tumour therapy.In this regard,peroxidase-like（POD）activity catalyzes the generation of highly cytotoxic hydroxyl radicals（·OH）from H₂O₂overexpressed in TME;and peroxidase-like（CAT）activity converts H₂O₂to O₂,which not only effectively alleviates the lack of oxygen in tumour tissues,but also activates oxidase-like properties（OXD）catalytic activity;moreover,OXD activity catalyzes the generation of·O₂^-from O₂,and the generated·O₂^-is partially converted to ¹O₂by reacting with H⁺under acidic TME conditions.Therefore,regulation of intracellular ROS stress levels by nanoenzymes is expected to improve the efficacy of cryo-photothermal therapy by reducing the thermotolerance of cancer cells.PurposeDue to the low-cost,high stability,ease of preservation and remarkable physicochemical properties of nanoenzymes,as well as their extensive and efficient catalytic activity,they are used in a wide range of applications.Thus,this study focused on the design of a precious metal-based gold-palladium（Au@Pd,AP）nanoenzyme with triple enzymatic activity of catalase,oxidase-like and peroxidase-like enzymes,and it can generate large amounts of ROS in TME using its acidic environment and overexpressed H₂O₂through this three catalytic enzymatic activities to fully exploit the chemical effect of disrupting the function of HSPs and damaging tumour cells.In consideration of the excellent photothermal conversion efficiency of two precious metals,gold-palladium,this topic combines AP nanoenzymes with photothermal therapy for ROS-mediated enhancement and m PTT for better anti-tumour effects.Methods（1）Preparation and characterisation of gold-palladium nanoenzymes with core-shell structure:Gold nanorods were synthesised by using the template method of seed growth,and gold nanorods were etched with hydrochloric acid to leave room for palladium growth after encapsulation of mesoporous silica（m Si O₂）,then the gold-palladium nanoenzymes with mesoporous silica encapsulation were synthesised under the reduction of ascorbic acid,finally,Au@Pd nanoenzymes with core-shell structure were obtained by etching the mesoporous silica shell with sodium hydroxide.X-ray powder difractometer（XRD）,transmission electron microscope（TEM）and elemental analysis were used to determine the crystalline phase,structure,morphology and elemental composition and distribution of the products.And malvern particle size analysis of nanomaterials for size,dispersion and charged properties.UV-Vis absorption spectroscopy assessed the ability of the material to absorb laser light at different wavelengths.（2）Simulated enzymatic activity and photothermal effects of Au@Pd nanoenzymes:A portable dissolved oxygen meter was used to detect changes in oxygen production following the addition of H₂O₂and Au@Pd nanoenzymes,thereby verifying their catalase-like（CAT）activity.1,3-Diphenylisobenzofuran（DPBF）,3,3’5,5’-tetramethylbenzidine（TMB）and o-phenylenediamine（OPD）were used as substrate probes to detect oxidase-like（OXD）and peroxidase-like（POD）activities respectively.The photothermal conversion efficiency of Au@Pd nanoenzymes was calculated by using an infrared thermal imaging camera to record real-time temperature changes under laser irradiation at a wavelength of 1064 nm,and the experiment was repeated to demonstrate the good photothermal stability of Au@Pd nanoenzymes.In this work,Au@Pd nanoenzymes was able to catalyze the production of three reactive oxygen species（·OH,·O₂^-,¹O₂）in a simulated tumor microenvironment as measured by ESR（Electron spin-resonance spectroscopy）.（3）Cell experiments in vitro:2’,7’-Dichlorofluorescein diacetate（DCFH-DA）was employed as a probe to detect intracellular ROS levels using laser confocal microscopy.Meanwhile,The three intracellular levels of reactive oxygen species were detected with DHE,HPF,and SOSG probes,respectively.Also,MTT and live-dead double-staining kit（Calcein-AM/PI）were used to detect cytocompatibility,viability and death;protein blotting analyzed to detect intracellular HSP70 expression levels.（4）Tumor suppressor experiments in vivo:Tumour models were constructed by subcutaneous injection of 4T1 cells into the mammary fat pads of female Balb/c mice aged 6-8 weeks.After intratumoral injection of Au@Pd nanoenzymes and mild PTT,to measure the tumour volumes and mouse body weights and assess treatment outcomesby ROS/HSP70 tissue section staining.Then,tissue sections of major organs were stained with hematoxylin and eosin（H&E）,moreover,blood biochemical analyses were performed to assess the bioin vivo compatibility of Au@Pd nanoenzymes.Results（1）The Au@Pd nanoenzymes had a gold-palladium bimetal with an overall core-shell structure.The length to diameter ratio of the gold nanorods could be controlled by restricting the etching time of the gold nanorods,while the growth space of the metallic palladium could be suitably limited.（2）Metallic palladium,a widely used catalyst in industrial production,imparted Au@Pd nanoenzymes mimetic enzymatic activity in the TME environment,greatly enriching the properties of simple gold nanorods.With its simulated triple enzyme（CAT-like,OXD-like and POD-like）activity,Au@Pd nanoenzymes was capable of generating large amounts of ROS in TME,also exerting an enhancing effect on CDT.（3）Cell experiments in vitro confirmed that Au@Pd nanoenzymes was quite biocompatible,with essentially no damage to mouse L929 cells,while it had significant concentration-dependent toxicity to mouse breast cancer 4T1 cells.Subsequently,a significant increase in ROS in 4T1 cells in the presence of Au@Pd nanoenzymes was observed under laser confocal microscopy using DCFH-DA,DHE,HPF and SOSG probes,respectively,and the addition of near-infrared（NIR）light conditions further increased the ROS content.At the same time,the results of MTT and live-dead cell staining experiments also corresponded to the above results,indicating that a synergistic anti-tumour effect was produced under the strategy of Au@Pd nanoenzymes combined with photothermal,which laid the foundation for the subsequent in vivo tumour suppression experiments.Protein blot analysis revealed that under the activation of NIR,the production of large amounts of ROS by Au@Pd nanoenzymes in the TME could deplete the HSP70 expressed by heat stress,resulting in a significant reduction of tumour cell thermal resistance and enhancing the therapeutic effect of m PTT.（4）The strategy of Au@Pd nanoenzymes combined with photothermal therapy achieved inhibition of in situ tumour growth through ROS-mediated m PTT.ConclusionThe Au@Pd nanoenzymes nano-therapeutic system was successfully constructed with 1064 nm laser excitation in the NIR II region in this study.Apparently,the Au@Pd nanoenzymes nano-enzyme synthesized based on precious metal materials has multi-class enzymatic biocatalytic activity in TME,which can achieve the accumulation of ROS in tumour cells,and inhibit the expression of HSP70 under heat stress to augment the mild photothermal therapeutic effect,and showed better anti-tumour effects in both in vivo and ex vivo experiments.