Peptide Functionalization Of Luminescent Gold Nanoparticles And The Related Tumor Imaging And Therapy

Ultra-small luminescent gold nanoparticles（AuNPs）have received great attention by many scientific researchers for their uses in optical detection,biosensors,cell imaging and disease diagnosis due to their tunable optical properties,functionalized surface modification,good biocompatibility and effective renal clearance.In view of the current low fluorescence quantum yield and tumor targeting efficiency of luminescent AuNPs,as well as the challenges faced in biomedical application research,in this paper,we prepared GSH-functionalized near-infrared（NIR）emitting AuNPs with different configurations by using a typical thiolate peptide,glutathione（GSH）containing anchoring groups of-SH,-COOH and-NH₂,as surface ligand and controlling their surface chemistry,to further explore the differences in the physical,chemical properties and cellular interaction of NIR-emitting AuNPs with different surface ligand configurations.Meanwhile,the effects of surface ligand configuration on the metabolic pathways,biodistribution and tumor targeting efficiency of luminescent AuNPs in vivo were also investigated.In addition,we designed a cyclopeptide（CP）that spontaneously assembled into nanofibers with controllable lengths,which could be utilized for in situ assembly of NIR-emitting AuNPs into ordered and well-controlled one-dimensional（1D）nanostructures（AuNPs@CP）with rapid lysosome escape,achieving the controllable self-assembly of luminescent AuNPs.The AuNPs@CP was used as nanovectors to load plasmid DNA（p DNA）with overexpression of p53 gene,whose transfection capacity in vitro and in vivo was also studied.In consideration of the tumor growth inhibition of p53 protein,the antitumor effects of the AuNPs@CP-p DNA in MDA-MB-231 tumor-bearing nude mice were evaluated after i.t.injection for consecutive times.The detailed results are summarized as follow:Since the protonation or deprotonation could affect the binding strengths of the anchoring groups of-NH₂ and-COOH in GSH with gold,the luminescent AuNPs were synthesized under different p H values to achieve the controllable modification of surface ligand around AuNPs.The protonation of the weak anchoring groups of-NH₂ and-COOH at low p H value（e.g.,p H2.0）would facilitate the formation of N-Auanchoring sites,while the deprotonation at high p H value（e.g.,p H 10.0）would enhance the anchoring sites of COO-Auand more exposed surface-NH₂.The various ultrasmall luminescent AuNPs（11B-AuNPs,11A-AuNPs,07B-AuNPs and07A-AuNPs）were prepared by adjusting the molar ratio of GSH to HAuCl₄（1.1:1 and 0.7:1）at p H～10.0 or p H～2.0.Their physical and chemical properties,as well as their interactions with cells were investigated.It reveals that more exposed-NH₂ on the surface of AuNPs could increase the endocytosis efficiency and promote their interaction with cells/cell membranes.Further,weak anchoring groups（such as-COOH,-NH₂）played an important role in regulating the thiolate ligands to synthesize luminescent AuNPs with different anchoring sites.Furthermore,by using the luminescent AuNPs with different anchoring sites,the effects of different surface ligand binding forms on the metabolic pathways,biodistribution and tumor targeting efficiency of AuNPs in vivo were evaluated.It discovers that the more exposed surface-COOH would promote their rapid body clearance through the kidneys,and the AuNPs with more exposed-NH₂ were mainly eliminated from kidney but also partly from liver.In addition,the AuNPs with more exposed surface-NH₂ showed prolonged blood circulation and retention,which enhanced the in vivo transportation of the AuNPs to the tumor site,resulting in 4.4 and2.3-fold targeting efficiency of 11B-AuNPs and 07B-AuNPs than that of 11A-AuNPs and 07A-AuNPs,respectively.Above results are helpful to further understand the effect of weak binding sites（COO-Auor N-Au）for luminescent AuNPs on their biological behavior,and provide guidance for the design of nanoprobes or drug delivery systems with high tumor targeting efficiency in clinical applications.In order to improve the fluorescence quantum efficiency of AuNPs and broaden their applications in biomedical field,we designed a cyclopeptide that can spontaneously assemble into nanofibers,which could be utilized for in situ assembly of NIR-emitting AuNPs into AuNPs@CP with controllable length,uniform morphology and rapid lysosome escape.The AuNPs@CP exhibited NIR emission with a maximum at 810 nm,and the quantum yield（QY）was measured to be 5.9%in water using fluorescein as reference,which was～6 times higher than those of the previously reported NIR-emitting AuNPs（～1%）.As the self-assembling cyclopeptide is rich in arginine,the guanidine group on the arginine could interact with the negatively charged phosphate group on the lysosomal membrane,which destroys the stability of the membrane,leading to the rapid lysosomal escape of AuNPs@CP.In addition,the rapid lysosomal escape ability confers AuNPs@CP as excellent nanovectors for in vitro gene transfection,which shows high gene transfection efficiencies in construction of cell line with～7.5-fold overexpression of p53 protein.In order to expand the application researches of 1D luminescence AuNPs@CP in vivo,we evaluated its biological safety.It found that AuNPs@CP-p DNA would spread into the blood from the tumor site with the extension of time after injection.Finally,it would be metabolized by the kidney and liver,and thus excreted in the urine and feces with the clearance half-life(t_1/2α=2.41±0.66 h).At the same time,we also discussed the gene delivery efficiency and anti-tumor therapy of AuNPs@CP-p DNA in vivo.The results showed the tumor growth of mice i.t.injected with AuNPs@CP-p DNA exhibited the significant decrease as measured from the tumor volumes,and the tumor weight from sacrificed mice treated with AuNPs@CP-p DNA were reduced by 64.9%.Finally,the main organs showed no obvious histological changes after i.t.injection of AuNPs@CP-pDNA as compared to the control group（i.t.injected with saline）.Above results indicated that the 1D AuNPs@CP as new biological nanovectors with low toxicity,high load efficiency and delivery ability,which show a great application prospect in gene delivery,drug visual monitoring,anti-tumor therapy.