In Vivo Aggregation of Clearable Bimetallic Nanoparticles with Interlocked Surface Motifs for Cancer Therapeutics Amplification.

Although renal-clearable luminescent metal nanoparticles (NPs) have been widely developed, their application to efficient cancer therapy is still limited due to low reactive oxygen species (ROS) production. Here, a novel system of clearable mercaptosuccinic acid (MSA) coated Au-Ag bimetallic NPs is designed to enhance ROS production. The results show that the strong COO-Ag coordination bonds between the carboxylic acid groups of MSA and Ag atoms on the Au-Ag bimetallic NPs could construct high-rigidity interlocked surface motifs to restrict the intrananoparticle motions for enhanced ROS generation. Moreover, bimetallic NPs exhibit pH-responsive self-assembly capability under the acidic environment inside lysosomes of cancer cells at both in vitro and in vivo, restricting the internanoparticle motions to further boost ROS production. The well-designed bimetallic NPs show high tumor targeting efficiency, fast elimination from the body through rapid liver biotransformation, and extensive destruction to cancer cells, resulting in good security and prominent therapeutic performance.

Intrarenal pH-Responsive Self-Assembly of Luminescent Gold Nanoparticles for Diagnosis of Early Kidney Injury.

Metabolic acidosis-induced kidney injury (MAKI) is asymptomatic and lack of clinical biomarkers in early stage, but rapidly progresses to severe renal fibrosis and ultimately results in end-stage kidney failure. Therefore, developing rapid and noninvasive strategies direct responsive to renal tubular acidic microenvironment rather than delayed biomarkers are essential for timely renoprotective interventions. Herein, we develop pH-responsive luminescent gold nanoparticles (p-AuNPs) in the second near-infrared emission co-coated with 2,3-dimethylaleic anhydride conjugated ß-mercaptoethylamine and cationic 2-diethylaminoethanethiol hydrochloride, which showed sensitive pH-induced charge reversal and intrarenal self-assembly for highly sensitive and long-time (~24 h) imaging of different stages of MAKI. By integrating advantages of pH-induced intrarenal self-assembly and enhanced interactions between pH-triggered positively charged p-AuNPs and renal tubular cells, the early- and late-stage MAKI could be differentiated rapidly within 10 min post-injection (p.i.) with contrast index (CI) of 3.5 and 4.3, respectively. The corresponding maximum CI could reach 5.1 and 9.2 at 12 h p.i., respectively. Furthermore, p-AuNPs were demonstrated to effectively real-time monitor progressive recovery of kidney injury in MAKI mice after therapy, and also exhibit outstanding capabilities for drug screening. This pH-responsive strategy showed great promise for feedback on kidney dysfunction progression, opening new possibilities for early-stage diagnosis of pH-related diseases.

Noncovalent Interaction Guided Precise Photoluminescence Regulation of Gold Nanoclusters in Both Isolate Species and Aggregate States.

Designing luminophores bright in both isolate species and aggregate states is of great importance in many emerging cutting-edge applications. However, the conventional luminophores either emit in isolate species but quench in aggregate state or emit in aggregate state but darken in isolate species. Here we demonstrate that the precise regulation of noncovalent interactions can realize luminophores bright in both isolate species and aggregate states. It is firstly discovered that the intra-cluster interaction enhances the emission of atomically precise Au25(pMBA)18 (pMBA=4-mercaptobenzoic acid), a nanoscale luminophore, while the inter-cluster interaction quenches the emission. The emission enhancing strategies are then well-designed by both introducing exogenous substances to block inter-cluster interaction and surface manipulation of Au25(pMBA)18 at the molecular level to enhance intra-cluster interaction, opening new possibilities to controllably enhance the luminophore's photoluminescence in both isolate species and aggregate states in different phases including aqueous solution, solid state and organic solvents.

Transcytosis-Based Renal Tubular Reabsorption of Luminescent Gold Nanoparticles for Enhanced Tumor Imaging.

Transcytosis-based tubular reabsorption of endogenous proteins is a well-known energy-saving pathway that prevents nutrient loss. However, utilization of this well-known reabsorption pathway for the delivery of exogenous nanodrugs remains a challenge. In this study, using the surface mimic strategy of a specific PEPT1/2-targeted Gly-Sar peptide as a ligand, renal-clearable luminescent gold nanoparticles (P-AuNPs) were developed as protein mimics to investigate the transcytosis-based tubular reabsorption of exogenous substances. By regulating the influential factors (H+ content in tubular lumens and PEPT1/2 transporter counts in tubular cells) of Gly-Sar-mediated transcytosis, the specific and efficient interaction between P-AuNPs and renal tubular cells was demonstrated both in vitro and in vivo. Efficient transcellular transportation significantly guided the reabsorption of P-AuNPs back into the bloodstream, which enhanced the blood concentration and bioavailability of nanoparticles, contributing to high-contrast tumor imaging.

Glutathione-Activated Emission of Ultrasmall Gold Nanoparticles in the Second Near-Infrared Window for Imaging of Early Kidney Injury.

Biomarker-activatable luminescent probes with high sensitivity and specificity show great promise in advanced bioimaging applications. However, the lack of stable biomarkers at an early stage is currently a major obstacle for sensitive early disease imaging. Herein, we develop a facile in vivo ligand exchange strategy to achieve renal-clearable activatable luminescent gold nanoparticles (AuNPs), which are independent of biomarkers for sensitive and long-time imaging of early kidney injury. Significantly activated emission in the second near-infrared region (∼1026 nm) is realized from the ligand exchange of triphenylphosphine-3,3',3″-trisulfonic acid (TPPTS)-coated AuNPs (∼1.4 nm, TPPTS-AuNPs) with quantitative amounts of glutathione (GSH). The abundant GSH in cells, particularly in liver sinusoids, is then demonstrated successfully to activate the emission of TPPTS-AuNPs with an extremely low background for both cell imaging and in vivo visualization of visceral organs (e.g., liver and kidneys). In addition, the in vivo GSH-exchanged TPPTS-AuNPs show enhanced interactions with acidic renal tubular epithelial cells, resulting in sensitive (contrast index, ∼3.9) and long-time (>6.5 h) noninvasive monitoring of acidosis-induced early kidney injury. This facile ligand exchange strategy opens new possibilities for designing activatable luminescent probes independent of biomarkers for earlier disease diagnosis and treatment.

Redox cascade reaction for kinetic resolution of racemic α-methylbenzylamine and biosynthesis of α-phenylethanol.

Chiral α-methylbenzylamine and α-phenylethanol are important building blocks for the industrial production of optically active drugs, bioactive compounds. Methods for the simultaneous synthesis of chiral α-methylbenzylamine and α-phenylethanol remain rare. Herein, a biocatalytic redox cascade reaction composed of ω-transaminase, aldo-keto reductase, and glutamate dehydrogenase for chiral α-methylbenzylamine and α-phenylethanol synthesis from racemic α-methylbenzylamine was constructed. A novel ω-transaminase and two different chiral aldo-keto reductases were demonstrated in the cascade reaction. The cosubstrate and redox equivalents were regenerated simultaneously by glutamate dehydrogenase. Using the approach, (R)-α-phenylethanol, (S)-α-phenylethanol, and (R)-α-methylbenzylamine were prepared with excellent stereoselectivity (ee > 99.7%). Furthermore, semi-preparative-scale biotransformation of racemic α-methylbenzylamine was conducted. The production of (R)-α-phenylethanol reached 26.05 mM at 24 h, and the production of (S)-α-phenylethanol reached 25.44 mM at 32 h. Taken together, a novel idea was proposed for the efficient and green synthesis of chiral α-methylbenzylamine and α-phenylethanol, which had great potential for industrial application. KEY POINTS: • Excellent stereoselectivity chiral α-methylbenzylamine and α-phenylethanol were synthesized. • A novel ω-transaminase demonstrated the catalysis toward (S)-α-methylbenzylamine. • Two novel aldo-keto reductases demonstrated the conversion toward acetophenone.

Luminescent Gold Nanoparticles with Controllable Hydrophobic Interactions.

The construction of luminescent gold nanoparticles (AuNPs) with highly redshifted emission in the second near-infrared window (NIR-II) and good biocompatibility is still challenging. Herein, using an amphiphilic block copolymer (ABC) template with controllable hydrophobic interactions in the diverse forms of unimers and micelles, we report a facile strategy for redshifting the emission and enhancing the biological interactions of luminescent AuNPs. While the uniform clusters of NIR-II AuNPs are formed in situ inside the hydrophobic cores of ABC micelles with strong interparticle hydrophobic interactions and enhanced emission at 1080 nm with a high quantum yield (QY) of 1.6%, the rigid NIR-II AuNPs are generated with strong intraparticle hydrophobic interactions as ABC unimers on the surface, leading to a redshifted emission of 1280 nm with a QY of 0.25% and enhancing the affinities toward injured intestinal mucosa in colitis imaging. These findings open new possibilities for the design of highly redshifted luminescent AuNPs with enhanced biological interactions.

Manganese(II)-Guided Separation in the Sub-Nanometer Regime for Precise Identification of In Vivo Size Dependence.

A precise understanding of nano-bio interactions in the sub-nanometer regime is necessary for advancements in nanomedicine. However, this is currently hindered by the control of the nanoparticle size in the sub-nanometer regime. Herein, we report a facile in situ Mn2+ -guided centrifugation strategy for the synthesis of large-scale ultrasmall gold nanoparticles (AuNPs) with a precisely controlled size gradient at the sub-nanometer regime. With the discovery that [Mn(OH)]+ , especially metallic manganese (Mn0 @[Mn(OH)]+ ) nanoparticles, could selectively interact with larger AuNPs through synergistic coordination and hydrogen bonding to form aggregates, we also realized the fast (<1 h) synthesis of water-soluble atomically precise Au25 with high yields (>56 %). We further demonstrated that sub-nanometer size differences (approximately 0.5 nm) significantly alter non-specific phagocytosis of AuNPs in the reticuloendothelial system macrophages, elimination rate, and nanotoxicology.

Luminescent Gold Nanoparticles with Discrete DNA Valences for Precisely Controlled Transport at the Subcellular Level.

Ultrasmall luminescent gold nanoparticles (AuNPs) with excellent capabilities to cross biological barriers offer great promise in designing intelligent model nanomedicines for investigating structure-property relationships at the subcellular level. However, the strict surface controllability of ultrasmall AuNPs is challenging because of their small size. Herein, we report a facile in situ method for precisely controlling DNA aptamer valences on the surface of luminescent AuNPs with emission in the second near-infrared window using a phosphorothioate-modified DNA aptamer, AS1411, as a template. The discrete DNA aptamer number of AS1411-functionalized AuNPs (AS1411-AuNPs, ≈1.8 nm) with emission at 1030 nm was controlled in one aptamer (V1), two aptamers (V2), and four aptamers (V4). It was then discovered that not only the tumor-targeting efficiencies but also the subcellular transport of AS1411-AuNPs were precisely dependent on valences. A slight increase in valence from V1 to V2 increased tumor-targeting efficiencies and resulted in higher nucleus accumulation, whereas a further increase in valence (e.g., V4) significantly increased tumor-targeting efficiencies and led to higher cytomembrane accumulation. These results provide a basis for the strict surface control of nanomedicines in the precise regulation of in vivo transport at the subcellular level and their translation into clinical practice in the future.

Rapid Biotransformation of Luminescent Bimetallic Nanoparticles in Hepatic Sinusoids.

Liver sequestration, mainly resulting from the phagocytosis of mononuclear phagocyte system (MPS) cells, is a long-standing barrier in nanoparticle delivery, which severely decreases the disease-targeting ability, leads to nanotoxicity, and inhibits clinical translation. To avoid long-term liver sequestration, we elaborately designed luminescent gold-silver bimetallic nanoparticles that could be rapidly transformed by the hepatic sinusoidal microenvironment rich in glutathione and oxygen, significantly different from monometallic gold nanoparticles that were rapidly sequestrated by Kupffer cells due to the much slower biotransformation. We found that the rapid sinusoidal biotransformation induced by the synergistic reactions of glutathione and oxygen with the reactive silver atoms could help bimetallic nanoparticles to avoid MPS phagocytosis, promote fast release from the liver, prolong blood circulation, enhance renal clearance, and increase disease targeting. With the fast biotransformation in sinusoids, liver sequestration could be turned into a beneficial storage mechanism for nanomedicines to maximize targeting.

Noninvasive photoacoustic computed tomography/ultrasound imaging to identify high-risk atherosclerotic plaques.

PURPOSE: Noninvasive detection of high-risk plaques is still challenging. In this study, we aimed to noninvasively assess αvß3-integrin expression using a customed photoacoustic (PA) computed tomography (PACT)/ultrasound (US) system in atherosclerotic lesions of varying degrees of severity and to explore its potential value for detecting high-risk plaques. METHODS: We constructed αvß3-integrin-targeted ultrasmall gold nanorods (AuNRs) with cyclo Arg-Gly-Asp (cRGD) and tested their properties. Employing C57BL/6 J (wild-type, WT) mice and apolipoprotein E gene knockout (ApoE-/-) mice fed either a chow diet or a high-fat/high-cholesterol diet (HFHCD), we established varying degrees of lesion severity. In vivo PACT/US imaging was performed to assess αvß3-integrin expression in the 4 groups by cRGD-AuNRs. Further histopathologic examination was conducted to evaluate the plaque vulnerability indicators. RESULTS: The data showed that cRGD-AuNRs exhibited excellent photothermal conversion capacity, stability, targeting ability, and biocompatibility. The immunohistochemical results indicated that αvß3-integrin was upregulated with increasing aggravation of the lesions. In vivo PACT/US imaging showed good consistency with αvß3-integrin expression. Notably, ApoE-/- mice fed a HFHCD showed an abrupt PA intensity increase compared with the other groups. The histopathologic examination verified that the atherosclerotic plaques of ApoE-/- mice fed the HFHCD developed unstable phenotypes. Correlation analysis showed that PA intensity was mainly related to inflammation and angiogenesis among all of the indicators. CONCLUSION: Our data indicated that αvß3-integrin is an effective indicator of plaque instability, and noninvasive PACT/US molecular imaging assessment of αvß3-integrin holds promise in detecting high-risk plaques.

Discovery and therapeutic implications of bioactive dihydroxylated phenolic acids in patients with severe heart disease and conditions associated with inflammation and hypoxia.

Our initial studies detected elevated levels of 3,4-dihydroxyphenyllactic acid (DHPLA) in urine samples of patients with severe heart disease when compared with healthy subjects. Given the reported anti-inflammatory properties of DHPLA and related dihydroxylated phenolic acids (DPAs), we embarked on an exploratory multi-centre investigation in patients with no urinary tract infections to establish the possible pathophysiological significance and therapeutic implications of these findings. Chinese and Caucasian patients being treated for severe heart disease or those conditions associated with inflammation (WBC ≥ 10 ×109/L or hsCRP ≥ 3.0 mg/L) and/or hypoxia (PaO2 ≤ 75 mmHg) were enrolled; their urine samples were analyzed by HPLC, HPLC-MS, GC-MS and biotransformation assays. DHPLA was detected in urine samples of patients, but undetectable in healthy volunteers. Dynamic monitoring of inpatients undergoing treatment showed their DHPLA levels declined in proportion to their clinical improvement. In DHPLA-positive patients' fecal samples, Proteus vulgaris and P. mirabilis were more abundant than healthy volunteers. In culture, these gut bacteria were capable of reversible interconversion between DOPA and DHPLA. Furthermore, porcine and rodent organs were able to metabolize DOPA to DHPLA and related phenolic acids. The elevated levels of DHPLA in these patients suggest bioactive DPAs are generated de novo as part of a human's defense mechanism against disease. Because DHPLA isolated from Radix Salvia miltiorrhizae has a multitude of pharmacological activities, these data underpin the scientific basis of this medicinal plant's ethnopharmacological applications as well as highlighting the therapeutic potential of endogenous, natural or synthetic DPAs and their derivatives in humans.

Highly Controllable Nanoassemblies of Luminescent Gold Nanoparticles with Abnormal Disassembly-Induced Emission Enhancement for In Vivo Imaging Applications.

Assembly-induced emission enhancement has been widely observed for luminophors but it causes emission decrease after disassembly. The construction of unique nanoassemblies with disassembly-induced emission enhancement (DIEE) is demanded. Herein, by taking advantages of alterable siloxane bridge crosslinking states, we report a facile strategy to fabricate water-soluble nanoassemblies of luminescent gold nanoparticles (AuNPs) with abnormal DIEE. The fabricated AuNP nanoassemblies with dominant interparticle crosslinking showed a redshifted emission at ≈1070 nm with quantum yields (QYs) of 1.8 %. After disassembly, the nanoassemblies with increased intraparticle crosslinking exhibited a unique DIEE (>6 folds, QYs, 12 %) due to the enhanced ligand-to-metal charge transition. Besides, the disassembly facilitated renal clearance of AuNPs to reduce nonspecific retention in the body, opening new possibilities for designing highly emissive renal-clearable nanostructures toward more bioapplications.

Weak Anchoring Sites of Thiolate-Protected Luminescent Gold Nanoparticles.

Surface functionalization of gold nanoparticles (AuNPs) with thiolate ligands is a successful strategy for controlling their stability, nanotoxicity, circulation, and interaction with biological environments as leading nanomedicines. However, the effects of the weak anchoring groups of NH2 and COOH have been long-term ignored because of the well-recognized strong anchoring site of S-Au. Herein, the authors achieve controllable weak anchoring sites of the luminescent AuNPs using a typical thiolate peptide such as glutathione with anchoring groups of SH, COOH, and NH2 . Additionally, they establish that not only the strong anchoring site of S-Au, but also the weak anchoring sites from N-Au and COO-Au are critical to the behavior of AuNPs at both in vitro and in vivo levels. These results open up new possibilities for the fundamental understanding of the significance of the weak anchoring sites in the future surface functionalization of nanomedicines toward advanced theranostics.

Concentration-Dependent Subcellular Distribution of Ultrasmall Near-Infrared-Emitting Gold Nanoparticles.

The ability to accurately control the subcellular distribution of nanomedicines provides unique advantages on understanding of cellular biology and disease theranostics. The nanomedicine concentration is a key factor to affect the theranostic efficiency and systematic toxicity. Herein, we unravel a concentration-dependent subcellular distribution of near-infrared-emitting gold nanoparticles (AuNPs) co-coated with glutathione and a cell-penetrating peptide CR8 (CR-AuNPs), which shows a strong membrane-binding at high concentration but more endocytosis for mitochondria targeting at the low concentration region. Attributing to high content of AuI and microsecond luminescent lifetimes, these AuNPs can catalyze dissolved oxygen to generate singlet oxygen (1 O2 ) efficiently. Combining with the concentration-dependent subcellular distribution, the luminescent AuNPs show photocytotoxicity in the relative low concentration region. These findings facilitate the fundamental understanding of the biological behaviors and potential cytotoxicity of ultrasmall luminescent AuNPs toward future theranostics.

Enhanced Ultrasound Contrast of Renal-Clearable Luminescent Gold Nanoparticles.

Renal-clearable nanoparticles are typically fast eliminated through the free glomerular filtration, which show weak interaction with the renal compartments and negligible ultrasound signals, raising challenges in direct imaging of kidney diseases. Here, we report the ultrasmall renal-clearable luminescent gold nanoparticles (AuNPs) with both pH-induced charge reversal and aggregation properties, and discover that enhanced ultrasound contrast could be facilely acquired through the increased tubular reabsorption and in situ aggregation of AuNPs in renal tubule cells in injured kidneys. The tuning elimination pathway of the renal-clearable luminescent AuNPs is further demonstrated to provide a synergistical fluorescence and ultrasound imaging strategy for diagnosing early kidney injury with precise anatomical information.

Strict DNA Valence Control in Ultrasmall Thiolate-Protected Near-Infrared-Emitting Gold Nanoparticles.

Realizing robust DNA functionalization with strict valence control in the sub-2-nm thiolate-protected luminescent gold nanoparticles (AuNPs) is highly demanded but remains unsolved due to their unique Au(0) core and Au(I)-S shell structures. Herein, we report a facile strategy using phosphorothioates (ps)-modified DNA (psDNA) as a template for in situ growth of near-infrared (NIR)-emitting AuNPs with precisely controlled DNA valence. In addition, the particle size could be finely tuned in ultrasmall ranges from 1.3 to 2.6 nm with regulation of the ps length of psDNA. The ultrasmall NIR-emitting AuNPs bearing strict DNA valence are also demonstrated to be as powerful building block for well-organized one-dimensional assembly and optical probe for targeted cellular imaging. Such a facile strategy in decoration of luminescent AuNPs with strict DNA valence provides a new pathway for development of surface-functionalizable ultrasmall metal nanoplatforms toward various downstream applications.

Bidirectional Regulation of Singlet Oxygen Generation from Luminescent Gold Nanoparticles through Surface Manipulation.

Singlet oxygen (1 O2 ) generation has been observed from ultrasmall luminescent gold nanoparticles (AuNPs), but regulation of 1 O2 generation ability from the nanosized noble metals has remained challenging. Herein, the 1 O2 generation ability of ultrasmall AuNPs (d ≈ 1.8 nm) is reported to be highly correlated to the surface factors including the amount of Au(I) species and surface charge. By taking the advantages of facile in situ PEGylation, it is discovered that a high amount of Au(I) species and surface charge results in strong ability in generation of 1 O2 , whereas a relative low amount of Au(I) species and surface charge leads to weak ability in 1 O2 production. A feasible general strategy is then developed to controllably regulate the 1 O2 generation efficiency of the AuNPs through facile ligand exchange with positively-charged or negatively-charged thiolated ligands. The AuNPs as nanophotosensitizer for 1 O2 generation in the cellular level is also demonstrated to be highly controllable through surface ligand exchange with synergistical effects of 1 O2 generation ability and subcellular distribution to lysosome or mitochondria. The strategy in the bidirectional regulation of 1 O2 generation from ultrasmall AuNPs provides guidance for future design of nanosized metal nanomedicine toward specific disease diagnosis and treatment.

Amphiphilic Block Copolymer-Guided in Situ Fabrication of Stable and Highly Controlled Luminescent Copper Nanoassemblies.

Assembling instable ultrasmall nanoparticles (NPs) into uniform nanoarchitectures with excellent stability and controllability in aqueous solution is still challenging. Herein, taking the advantage of controllable size and shape of amphiphilic triblock copolymer template, we report a facile and robust strategy for in situ fabrication of highly luminescent Cu nanoassemblies with uniform morphology and remarkable stability. The dominant number of encapsulated CuNPs in an assembly can be controlled through regulating hydrophobic core size by varying block segments of the template. The cross-linking by a multidentate thiol ligand largely enhances the emission and stability of the Cu nanoassemblies in physiological environment. By virtue of their intriguing features, the Cu nanoassemblies can be applied to possible biomedical applications. These findings establish our approach as a facile and feasible method for preparing stable and well-controlled ultrasmall metal NP-based assemblies.

Self-Assembly of Luminescent Gold Nanoparticles with Sensitive pH-Stimulated Structure Transformation and Emission Response toward Lysosome Escape and Intracellular Imaging.

Ultrasmall luminescent gold nanoparticles (AuNPs, d < 3.0 nm) with distinct optical properties and good biocompatibilities hold enormous promise for advanced disease theranostics. However, ultrasmall AuNPs generally show low cellular interaction and are hardly ever transported into the specific subcellular compartments, hampering their further biomedical use in cellular delivery and intracellular tracking. Using a conventional cationic polymer chitosan (CS) with the isoelectric point of 6.5 as a template, ultrasmall luminescent AuNPs can be easily formed into self-assembled nanostructures (AuNPs@CS) with significantly enhanced cellular interaction capability and sensitive emission response toward subcellular location. The self-assembled AuNPs@CS become compacted nanostructures (∼23.5 nm) with high luminescence at low pH values (e.g., pH < 6.5) but reversibly transform to swelled structures with weak luminescence at high pH values (e.g., pH 7.4). The self-assembly of AuNPs not only improves the emission properties but also alters the surface charge and assembly size, resulting in both enhanced cellular internalization and effective endosomal escape capability. More importantly, the sensitive luminescence response of the AuNPs@CS from the acidic organelle lysosome to the neutral cytoplasm demonstrates the great potential in optical intracellular tracking.