The Preparation Of Cell-penetrating Peptide Functionalized Luminescent Gold Nanoparticles And Their Applications In Tumor Imaging And Therapy

Due to the ultrasmall size,tunable optical properties,easy surface modification,good stability and biocompatibility,luminescent gold nanoparticles(AuNPs)have attracted great attention in biomarker analysis,cellular mechanism research,disease diagnostics and so on.The regulation and functionalization of their surface ligands can not only change their optical properties,improve their stability and biocompatibility,but also endow them specific functions,which is significant to improve their application value and broaden their application fields.The investigations of accurately controlling the interaction between functional nanoparticles and living cells,as well as their metabolism and disease targeting properties in vivo,play a significant role in the development of nanomedicine and the diagnostics of diseases.Therefore,we synthesized cell-penetrating peptide functionalized endogenous dual-emitting AuNPs by regulating the surface ligand density of ultrasmall luminescent AuNPs,achieved the real-time visual imaging of living intracellular pH and exploration of their endocytosis mechanism.Then,we systematically studied the difference and mechanism of interaction between AuNPs with different surface ligand density and tumor cells,as well as the effect of surface ligand density of AuNPs on the production of singlet oxygen and its mechanism for their application in photodynamic therapy.Finally,AuNPs with different surface ligand density were applied to the tumor targeting imaging in vivo,and the effects of surface ligand density on the targeting of tumors and metabolism in vivo were investigated.The detailed research contents and results are summarized as follows:The ultrasmall endogenous dual-emitting AuNPs with emissions both at 810 nm and 615 nm were synthesized by regulating the added ratio of ligands in the reaction solution.The real-time visual imaging of intracellular pH and the localization of organelles with their pH could be realized by using the ratio signals of dual emissions of ultrasmall AuNPs.We also found that the introduction of slight number of cell-penetrating peptide on the surface of AuNPs could improve their cellular interaction dynamics and internalization through direct cellular membrane penetration but endocytosis.In this chapter,the exploration of the endocytosis pathway of dual-emitting CR-AuNPs revealed that cellular internalization of AuNPs involved multiple pathways,which further deepens the understanding of the mechanism of interaction between ultrasmall AuNPs and living cells.The research in this chapter performs a new method for the design of functionalized endogenous dual-emitting AuNPs and their application in sensing and imaging at the cellular level,which also provides a new idea for monitoring cellular state and function.By comparing the membrane binding and endocytosis of ultrasmall AuNPs with different surface ligand densities,surface ligand density-regulated the interaction process between ultrasmall AuNPs and cells was revealed.It was found that gold nanoparticles with low ligand density showed fast cellular interaction and strong membrane-binding but low cellular internalization proportion,while AuNPs with high surface coverage displayed a weak cellular interaction and membrane-binding but more cellular uptake.In addition,we also found that slight modification of cell-penetrating peptide could be an efficient means to tune cellular interaction dynamics and internalized amount of AuNPs through direct membrane penetration.At the same time,the change of ligand density of AuNPs would also induce the change of their emissions,especially for the pH-responsive dual emissions.This series of optical changes make the AuNPs to be a very useful optical probe for subcellular imaging and tracking.These findings greatly enrich the understanding of the interaction mechanism between ultrasmall luminescent AuNPs and cells,and provide a feasible strategy for the design of surface-regulated functionalized nanoparticles and their interaction with cells.We also systematically compared the generated efficiency of singlet oxygen of ultrasmall AuNPs with different ligand density,explored their generation mechanism and their cytotoxicity for tumor cells in a wide concentration range for their applications in photodynamic therapy to tumor cells.By using the ABDA as singlet oxygen indicator,it was proved that the ultrasmall luminescent AuNPs with long luminescent lifetime could generate singlet oxygen under the illumination,and their generation mechanism of singlet oxygen was closely related to the content of Au(I).The cytotoxicity of the tumor cells confirmed that the AuNPs with different ligand densities could be used for photodynamic therapy at lower concentration conditions(0.5-1 nM)without FBS in culture environment,and also disclosed that the AuNPs modified with the cell-permeable peptide had stronger photodynamic therapy effect on the tumor cells.Furthermore,it is confirmed that the cell-permeable peptide can greatly increase the cytotoxicity of the 05CR-AuNPs with low ligand density in a high concentration range,even under the condition without illumination or with the FBS in culture environment.In addition,the ultrasmall luminescent AuNPs were substantially nontoxic to normal cells,demonstrating their good biological safety and tumor-specific targeting capacity,which is benefit to their applications in therapy of tumors.The study in this chapter not only provides the deeper understanding of the biological safety of the ultrasmall luminescent AuNPs,but also widens the application of the AuNPs in the biomedicine.By comparing the difference of the in vitro and in vivo tumor targeting of the utrasmall luminescent AuNPs with different ligand density,we found that these ultrasmall luminescent AuNPs not only showed good biocompatibility,and the various metabolism and clearance routes,but also displayed high-efficient in vitro and in vivo tumor targeting.By changing the surface ligand density of the ultrasmall luminescent AuNPs,it is possible to regulate a variety of in vivo metabolism-related parameters including plasma concentration,metabolic clearance rate,and clearance route of AuNPs,and their in vivo distribution to improve the tumor targeting.In addition,by a study of the cell-level and animal-level of ultrasmall luminescent AuNPs modified with a small number of cell-permeable peptides,CR-AuNPs containing a small number of cell-permeable peptide exhibited higher tumor cell targeting,but were easily captured by the RES organ and reduced their in vivo tumor targeting.On the contrary,GSAuNPs showed the higher in vivo tumor targeting,especially 05GS-AuNPs with the low ligand density,their tumor targeting efficiency could reach to 5.01%,which is higher than that of the reported ultrasmall luminescent GS-AuNPs.The research in this chapter not only expands the application of the ultrasmall nanoparticles in the disease targeting,but also provides a new way for the preparation of the nanomedicine with biosafety.