A Novel Au-Se Bond Gold Nanoplatform And Its Application In Pyroptosis Events

Pyroptosis is a new form of programmed cell death（PCD）,which is closely related to the activation of inflammation-related Caspase proteins stimulated by a series of microbial infections or endogenous danger signals.Pyroptosis is widely involved in the occurrence and development of various diseases,including cardiovascular diseases,central nervous system diseases,and tumors.In-depth study of cell pyroptosis will help to understand its role in the occurrence and development of related diseases,and provide new ideas for clinical prevention and treatment.With the deepening of the research on pyroptosis,the relationship between pyroptosis and the occurrence and development of tumors has attracted extensive attention.However,the limitation of the understanding of the pyroptosis signaling pathway has greatly restricted the establishment of pyroptosis-related tumor therapy methods.The specific detection of pyroptosis-related molecules is of great significance to fully understand the pyroptosis pathway and effectively utilize its signal transduction to achieve tumor therapy.Calcium is the most abundant mineral in the human body and is involved in a variety of life processes such as muscle contraction,enzyme activation,immune responses,and neuronal activity.At the same time,as the second messenger of intracellular signal transmission,Ca²⁺also plays an important role in maintaining cell shape,controlling cell membrane permeability and cell excitability,and regulating cell cycle.Usually,intracellular Ca²⁺concentration is in a state of dynamic equilibrium.Once there is a huge fluctuation,such as"calcium overload",cell function will be seriously disturbed and cell death will be caused.Recent studies have found that intracellular calcium overload may be involved in the activation of the pyroptosis-related NLRP3inflammasome,but its mechanism remains unclear.Therefore,there is an urgent need for a simple and effective accurate detection tool to realize the study of the process related to pyroptosis.Gold nanomaterials have been widely used in biosensing and therapy due to their high specific surface area,stable chemical properties,good biocompatibility,and good optoelectronic properties.Au-S bonds can be formed between compounds containing sulfhydryl groups（-SH）and gold nanomaterials.Therefore,Au-S covalent bonding is a common method for combining biomolecules with gold nanomaterials.Traditional thiol-functionalized gold nanomaterials are widely used in fluorescence,photoacoustic,Raman,electrochemical sensing and other fields.However,under complex physiological environment,the loading of thiolated recognition units on the surface of gold nanomaterials is low,and it is easily replaced by a high concentration of biological thiols in cells,which leads to the distortion of detection results.Therefore,how to fundamentally solve the interference of high concentrations of thiols on Au-S bonds in vivo is of great significance for the application of ngold-based nanomaterials in the biological field.Selenium is an essential trace element in living organisms.Its chemical valence is variable,its bonding methods are diverse,and it is involved in many physiological and pathological processes.It has similar chemical properties to sulfur.In a large number of previous studies on selenium,we found that Au-Se bonds have higher stability than Au-S bonds.Based on this,in this paper,a revolutionary new technology is proposed by taking advantage of the higher stability of Au-Se bonds—reconstructing traditional Au-S nanoplatforms with Au-Se nanoplatforms.Compared with the traditional Au-S platform,the Au-Se platform has higher selectivity and stability,and can effectively avoid the distortion of detection results caused by high concentrations of biothiols in cells.In this work,we used the high-sensitivity probe constructed by this nanoplatform to explore the signaling pathway in the process of pyroptosis,and further study the relationship between calcium overload and pyroptosis.In addition,the platform has also been successfully used for gold-based precise photothermal therapy of tumors.This research is mainly divided into the following parts:（1）Thiol-functionalized gold nanomaterials are widely used in biomedical fields.However,in complex physiological environments,the recognition units loaded on the surface of gold nanomaterials through Au-S bonds are easily replaced by high concentrations of biothiols in cells,resulting in false positive detection results.To fundamentally address the intracellular instability of Au-S bonds,we designed a new selenol-modified Au-Se nanoplatform（Au-Se NPF）by exploiting the higher stability of Au-Se bonds.Compared with conventional Au-S NPFs,Au-Se NPFs exhibited superior anti-interference ability in the presence of millimolar glutathione（GSH）with higher selectivity and stability.The platform was successfully applied to high-fidelity imaging of intracellular Caspase-9,showing better selectivity and sensitivity during biological detection.Therefore,the platform construction method is simple and feasible,providing a revolutionary new way for the design of gold nanosensing probes and their applications in chemical sensing and clinical detection in the future.（2）Pyroptosis is a new type of cell death method,which is closely related to the inhibition of tumor cell development.However,the pyroptotic pathway（PP）induced by different stimuli remains unclear.Therefore,in order to study the PP of tumor cells,we modified three fluorophore-labeled peptide chains capable of specifically detecting Caspases-1/3/4 proteins on the surface of gold particles（Au NPs）through Au-Se bonds,and designed a multicolor fluorescent nanoprobe（Cas-NP）achieves highly selective and sensitive detection of the activation of Caspases-1/3/4proteins during pyroptosis.In addition,we realized the selenization modification of Caspase-4peptide chain（Cyanine-5-LEVD-Se H）for the first time,overcoming the complex and expensive problems of the synthesis of selenized peptide chain in previous commercial synthesis.Using this probe,we successfully observed the activation of Caspase-1/3/4 proteins during tumor cell pyroptosis induced by adenosine triphosphate（ATP）or lipopolysaccharide（LPS）,as well as the interaction between the three proteins.This work provides an effective tool for studying the complex molecular mechanism of pyroptosis,and provides a new idea for the development of effective therapeutic pyroptosis inhibitors.（3）The latest study found that Ca²⁺can promote the activation of NLRP3 inflammasome,and NLRP3 inflammasome is thought to be closely related to the activation of pyroptosis-related protein Caspase-1.However,how to effectively deliver Ca²⁺into tumor cells and to study whether calcium overload can trigger Caspase-1 protein-mediated pyroptosis still faces great challenges.In this chapter,we prepared acid-responsive hybrid nanoparticles（Ca CO₃-Au-PLGA）.Ca CO₃-Au-PLGA exists stably at normal physiological p H,but in the acidic lysosomal environment of tumors,it rapidly disintegrates,releasing Ca²⁺,triggering calcium overload,and simultaneously releasing gold nanoprobes responsive to Caspase-1 protein.Through confocal imaging,it can be found that calcium overload in tumor cells triggers the activation of Caspase-1 protein,which confirms that the cell death process triggered by calcium overload is a pyroptosis process related to Caspase-1 protein.This work provides an effective tool for the targeted release of calcium ions in tumor cells and the study of calcium ion-related pyroptosis.（4）Thiol-functionalized gold nanomaterials are also widely used in photothermal therapy（PTT）of tumors.However,during prolonged blood circulation,biothiols would interfere with Au-S bond-modified gold nanomaterials,thereby reducing the PTT effect.Therefore,we try to overcome this problem by utilizing the Au-Se nanoplatform established in the previous chapter.In this chapter,we designed an acid-induced aggregation of selenol-functionalized zwitterionic Au NPs（Au-Se-C₄-N₆）to enhance nanomaterial delivery efficiency and retention in the tumor region.Compared with thiol-functionalized zwitterionic Au NPs（Au-S-C₄-N₆）,Au-Se-C₄-N₆ exhibited better stability and resistance to biological thiol interference ability during blood circulation in vivo.It can achieve effective dispersion and aggregation in the normal area and tumor area,and improve the PTT effect.In addition,the results of in vivo studies found that Au-Se-C₄-N₆ had a more pronounced anticancer effect（5-fold reduction in tumor volume）compared with Au-S-C₄-N₆.This result demonstrates that selenol-modified acid-induced aggregation zwitterionic gold nanoparticles is an effective strategy to improve the stability and PTT effect.It is also demonstrated that the Au-Se nanoplatform can be used as an effective platform to improve the delivery efficiency of nanomaterials,thereby improving the clinical therapeutic effect of tumors.