Precise Gradient Separation Of Water-Soluble Near-Infrared-Ⅱ Luminescent Gold Nanoclusters For Imaging Research

Atomically precise gold nanoclusters（AuNCs,<2 nm）are a family of ultra-small nanoparticles,composing of a few to several hundred gold atoms and a layer ligand molecule with a defined structure and composition.Due to the strong quantum size effects,AuNCs exhibit unique physicochemical and optical properties,which have received extensive attention in the fields of biological imaging,biosensing,disease diagnosis and personalized nanomedicine,and so on.Compared with the polydispersity and heterogeneity of traditional gold nanoparticles（AuNPs）,the defined structure and composition of AuNCs is an ideal molecular model for studying the surface chemistry and the relationships between the structure and properties of nanoparticles.In recent years,a series of important achievements have been achieved in the synthesis and structural elucidation of atomically precise AuNCs,but most of these are hydrophobic and difficult to be applied to organisms.However,strategies for the synthesis of water-soluble AuNCs are inadequate,and most of the obtained AuNCs are polydisperse with multiple-core size components,making it impossible to accurately study the mechanism of nano-bio interaction and metabolism of AuNCs in vivo,which has been considered as a serious roadblock in clinical translation.Therefore,significant efforts are urgently required to establish simple,efficient,and universal separation approaches for the preparation of well-defined water-soluble AuNCs with monodispersion.To achieve this goal,we developed an Mn²⁺-guided gradient separation method to accurately control the size variation of AuNPs in the sub-nanometer region and systematically studied the separation mechanism.Utilizing this separation strategy,a large scale of water-soluble atomically precise AuNCs was synthesized and applied to investigate the transport and toxicity of AuNPs with the sub-nanometer differences（approximately 0.5 nm）in vivo.Finally,a novel approach was designed for the synthesis of bi-thiolate-ligand protected Au₂₅NCs with unique absorption in the second near-infrared region（NIR-II）.Au₂₅NCs protected by bi-thiolate ligand exhibited excellent physiochemical and optical properties and showed unique advantages in metastatic lymph node images.The detailed research contents are summarized as follows:The highly water-soluble MPS-AuNPs with polydisperse size distributions were synthesized by the typical size-focusing method.When Mn²⁺was introduced into the pre-synthesized MPS-AuNPs,the size distribution of AuNPs step-wisely decreased as the concentration of Mn²⁺increased.The atomically precise Au₂₅MPS₁₈ was easily obtained when the Mn²⁺concentration increased to 0.5 mmol/L,even at low-speed centrifugal（4000 g,5 min）.We found that[Mn（OH）]⁺was the effective species for inducing gradient separation of large-sized AuNPs in the solution.Under alkaline conditions,the introduced Mn²⁺is transformed to form[Mn（OH）]⁺,and then preferentially combined with large-sized AuNPs in the solution to form nanoaggregates through synergistic coordination（-SO-Mn）and hydrogen bonding（-SO₃···HO-Mn）,thereby realizing gradient centrifugation of AuNPs of different sizes.This separation strategy can be extended to the purification of Au₂₅SG₁₈ in glutathione（GSH）-protected AuNPs solution,showing excellent universality and extensibility,and providing a new method for the separation and purification of water-soluble AuNCs at atomically precise levels.Based on the above research,trace amounts of Mn²⁺were introduced during the synthesis of polydisperse MPS-AuNPs to precisely control the gradient decreasing of AuNPs size in solution at the nanoscale,even to the atomically precise level.We found that the Mn²⁺-mediated in situ synthesis co-separation and purification strategy can synthesize a large number of water-soluble atomically precise Au₂₅MPS₁₈,and increasing the aging temperature can shorten the reaction time to less than 1 h.Further investigation shows that the mechanism of Mn²⁺-guided in situ synthesis was consistent with the principle of post-added Mn²⁺,and the strong separation ability was derived from the metal manganese（Mn⁰@[Mn（OH）]⁺）that formed during the Na BH₄ reduction process.In addition,the Mn²⁺-guided in situ synthesis strategy could easily and precisely control the size difference of AuNPs in solution at the sub-nanometer scale（approximately 0.5 nm）,which could be used to investigate the size-dependent sub-organ distribution,clearance,and toxicity in the sub-nanometer regime.We found that even sub-nanometer size differences of AuNPs could also cause significant differences in their distribution,metabolism,and toxicity in the liver and spleen.This discovery performs a new idea for the synthesis of water-soluble atomically precise AuNCs,but also promotes the research on the transport and toxicology of metal nanoclusters in vivo at the sub-nanometer region or atomic level,providing the possibility for the future industrial production and clinical translation of AuNCs.In order to further enhance the physicochemical properties of water-soluble atomically precise AuNCs,the atomically precise bi-thiolate-protected Au₂₅NCs with NIR-II absorption and emitting were synthesized by introducing negatively charged thiolate ligands collocating with positively charged thiolate ligands in steps during the synthesis process.The obtained bi-thiolate-protected Au₂₅NCs showed a unique optical absorption at 795 nm and 1055 nm,and the photothermal conversion efficiency can reach 44.1%under 1064 nm laser irradiation.Furthermore,we discovered that the unique absorption at 1055 nm derives from the distortion of the Au₂₅NCs structure due to the charge anisotropy and the intramolecular hydrogen bond of the ligands on the surface.Compared with traditional mono-thiolate-protected Au₂₅MPS₁₈,the bi-thiolate-protected Au₂₅NCs exhibited zwitterion-like properties,which can effectively avoid the non-specific interaction between proteins,cells and tissues,and reduce the accumulation in tissues and organs.In addition,the bi-thiolate-protected Au₂₅NCs showed a high resolution in lymph node imaging and lit up metastatic lymph nodes within a few minutes,achieving a rapid diagnosis of metastatic lymph nodes,and also providing a 6–8 h imaging window for clinical lymph node dissection.This synthesis strategy not only provides a new idea for the design and synthesis of novel AuNCs but also provides the possibility for simultaneous conduct fluorescence imaging with AuNCs excited at 1064 nm,further improving the penetration depth and resolution of optical imaging,as well as the diagnosis of disease areas,providing a guarantee for surgical navigation and photothermal therapy.