Development Of High Concentration Glutathione Intelligent Response Hybrid Gold Nanoparticles In Tumor Microenvironment For PAI/PPTT Of Tumors

Cancer is the general name of a group of genetic diseases.Because of the increasing morbidity and high mortality,the treatment of cancer has long been the focus of scientists.Traditional cancer treatment methods,such as chemotherapy,surgery and radiotherapy,have made great contributions,but they have limited therapeutic effects and great side effects,and cannot completely cure cancer.In the past few decades,nanotechnology engineering has developed rapidly in the biomedical field,among which plasma photothermal therapy（PPTT）is particularly concerned by scientists because of its advantages of addressing in time and space,high treatment efficiency and accurate treatment of tumor areas.Near infrared light（NIR）is often the first choice for PPTT,because the absorption/scattering of hemoglobin and water is relatively low in the biological transparent window（650-900 nm）,which makes NIR have high tissue penetration.Photoacoustic imaging（PAI）has deep tissue penetration and high spatial resolution,which shows great potential in tumor imaging diagnosis.The most important thing for efficient PPTT/PAI for tumor imaging and treatment is to find nano-materials with strong near infrared absorption and high photothermal conversion efficiency,which have both high biocompatibility and good pharmacokinetic characteristics.Gold nanoparticles（Au NPs）have attracted much attention of scientists because of their excellent optical properties.Au NPs not only has high photothermal conversion efficiency and good biocompatibility,but also has large specific surface area and is easy to modify functional molecules,which makes it widely used in non-invasive tumor treatment research.However,the problem is that generally speaking,small Au NPs are more suitable for intravenous administration,and reach the tumor area through blood circulation and enhanced permeation and retention effect（EPR）,because small particles stay longer and are less likely to be swallowed by reticuloendothelial system.However,only large spherical Au NPs（usually larger than 100 nm）have strong NIR absorption capacity necessary for tumor PPTT and PAI,but large particles are not conducive to blood circulation,and the drug dose reaching the tumor area is low and accumulated.In recent years,using the characteristics of physiological tumor microenvironment in tumor areas or tumor cells,Au NPs aggregates are formed in tumors,which can effectively avoid thrombus and reticuloendothelial system phagocytosis caused by particle aggregation in blood circulation and damage to normal tissues and cells.But up to now,most of the design schemes are complicated,costly and inefficient in photothermal conversion.Therefore,it is of great significance to design a simple and cheap tumor micro-ambient intelligence response Au NPs to achieve high-efficiency PAI and PPTT in vivo.In this paper,a new type of Au NPs with glutathione-stimulated aggregation in tumor microenvironment and excellent biocompatibility was prepared,which can be used for high-efficiency PAI/PPTT in tumor.The main research work is as follows:1.Glutathione sensitive polymer methoxy terminated cystamine polyethylene glycol（m PEG-CONH-ss-NH₂）was synthesized.The synthesis method is that the raw material methoxy-terminated carboxyl polyethylene glycol（m PEG-COOH,Mn=5000）purchased by zero-length cross-linking agent EDC/NHS forms an amide bond with the carboxyl group of cystamine dihydrochloric acid（Cys）,and the white flocculent product is obtained by purification such as dialysis and freeze-drying.The products were characterized by IR,XPS and ¹H NMR,which showed that the target products were synthesized successfully.2.Glutathione intelligent responsive hybrid gold nanoparticles（Au@PDA-ss-PEGm NPs）were prepared,and the Au IINPs,was selectively selected at 10 nm,20 nm,30 nm,40 nm,and the size of the nanoparticles was the most suitable for tumor therapy in vivo,taking into account the effect of photothermal therapy and the requirements of intravenous injection in vivo.For the polydopamine（PDA）coating layer,the most suitable thickness is selectively selected from 2 nm to 7 nm,which takes into account the sensitivity of intelligent response in vivo and the enhanced ability of photoacoustic imaging and photothermal therapy of tumor.The intelligent hybrid nanoparticles were characterized by transmission electron microscope（TEM）,ultraviolet-visible absorption spectroscopy（UV-vis）,infrared thermal imager,dynamic light scattering（DLS）and Zeta potential analyzer.3.The high concentration glutathione sensitive test of Au@PDA-ss-PEGm NPs was carried out in vitro,and Au@PDAPEGm NPs was used as the control sample for comparative experiment.Through the data analysis of TEM and UV-vis spectra,it is proved that Au@PDA-ss-PEGm NPs has significant intelligent response to high concentration of glutathione.Glutathione breaks the disulfide bond as a reducing agent,resulting in the shedding of the long-chain polyethylene glycol protective layer,and the particles exposed in phosphate buffer solution（PBS）form large aggregates because the surface electrostatic equilibrium is broken,resulting in coupled plasmon resonance（CPR）effect.The light absorption capacity in the near infrared region is greatly improved.UV-vis spectra show that the near-infrared light absorption capacity increases with the increase of glutathione concentration（0.001-100 m M）or treatment time（0-140min）.4.Experiments on photothermal efficiency and photoacoustic imaging effect of Au@PDA-ss-PEGm NPs were carried out in vitro,and comparative experiments were set up,which were characterized by infrared thermal imager and photoacoustic imager.The results show that Au@PDA-ss-PEGm NPs has excellent photothermal therapy and photoacoustic imaging ability,photothermal conversion efficiency is as high as 46.5%,and has good photothermal stability.With the increase of material concentration,the heating ability and photoacoustic signal intensity increased,and with the increase of glutathione concentration,the photoacoustic signal intensity also increased.5.The cytotoxicity test and confocal fluorescence imaging test of Au@PDA-ss-PEGm NPs were carried out in vitro,and a comparative experiment was set up,which was characterized by MTT method of cell light/dark toxicity test and AM/PI method of cell confocal fluorescence imaging.The results show that Au@PDA-ss-PEGm NPs has excellent biocompatibility and can kill Hela tumor cells irradiated by 808 nm laser（1.5W/cm²）.This conclusion is also confirmed by confocal fluorescence imaging.6.The photoacoustic imaging and photothermal treatment of tumors by Au@PDA-ss-PEGm NPs in vivo were tested,and a comparative experiment was set up.After intravenous injection of Au@PDA-ss-PEGm NPs into tumor-bearing mice,the photoacoustic signal intensity of tumor area was detected at different time points,and the best treatment time point（32 h）was found.Through the photothermal treatment of tumors under the guidance of photoacoustic imaging,the growth of surface tumors was successfully suppressed,and a large number of tumor cells were confirmed by tumor Hype staining sections.The results show that Au@PDA-ss-PEGm NPs can be rapidly metabolized out of the body in mice.