Phosphorylation of NIR‐II emitting Au nanoclusters for targeted bone imaging and improved rheumatoid arthritis therapy

Designing a theranostic probe for noninvasive bone imaging and bone disease therapy is both challenging and desirable. Herein, an ultrasmall Au nanocluster (NC, <2 nm)‐based theranostic probe is developed to achieve highly temporospatial in vivo bone‐targeted photoluminescence (PL) imaging in the second near‐infrared window (NIR‐II) and enhanced rheumatoid arthritis (RA) therapy. The key design of the probe involves the surface phosphorylation of atomically precise NIR‐II emitting Au44 NCs. This phosphorylation enhances the bone‐targeting ability of the probe due to the highly concentrated phosphate groups, allowing the probe to realize in vivo bone‐targeted NIR‐II PL imaging. Moreover, benefiting from the enhanced bone‐targeting ability, ultrasmall hydrodynamic diameter, and excellent anti‐inflammation and immunomodulatory effects, the probe not only demonstrates superior therapeutic efficacy for RA rats, effectively restoring the destructed cartilage to nearly normal but also exhibits good renal clearance and benign biocompatibility. These favorable attributes cannot be achieved by commercial methotrexate used for RA treatment. This study presents a new design paradigm for metal NC‐based theranostic probes, offering the potential for high‐resolution bone‐targeted PL imaging and improved RA therapy.

destruction of cartilage/bone is most commonly treated with methotrexate (MTX) in clinical practice to reduce inflammation, relieve pain, and slow cartilage/bone destruction, which however may lead to serious systemic complications without a positive effect on cartilage/bone destruction if long-term use. [9,10]Overall, high-resolution noninvasive bone imaging and efficient RA therapy are both intractable on their own, let alone designing a theranostic probe integrating both, which constitutes the driving force of this study.
Herein, we report a novel Au NC-based theranostic probe for in vivo bone-targeted NIR-II PL imaging and enhanced RA therapy.Our design approach involves chemically grafting bone-targeting 3-amino-propane-1-phosphate molecules (APP) to the surface of atomically precise NIR-II emitting Au 44 MBA 26 NCs (here, MBA denotes 4mercaptobenzoic acid). [22]This results in a phosphorylated Au 44 MBA 26 -P probe (here, P denotes phosphonate) with improved bone-targeting ability due to the highly concentrated phosphate group on its surface, enabling in vivo bone-targeted NIR-II PL imaging.Moreover, benefiting from the enhanced bone-targeting ability, ultrasmall HD, and excellent anti-inflammatory and immunomodulatory effects, the Au 44 MBA 26 -P probe not only shows superior therapeutic efficacy for RA rats with obvious activity in restoring the destructed cartilage into a nearly normal one, but also exhibits good renal clearance and benign biocompatibility, which cannot be realized by commercial MTX.This study may be interesting because it offers a new design paradigm for metal NC-based theranostic probes that enable high-resolution bone-targeted PL imaging and improved RA therapy.

RESULTS AND DISCUSSION
In this study, we used NIR-II emitting Au 44 MBA 26 NCs as a model for phosphorylation (Figure 1A), which were prepared using a previously reported "NaOH-mediated NaBH 4 reduction" method. [51]The ultraviolet-visible absorption spectrum (Figure 1B) shows three optical absorption peaks at 497, 590, and 794 nm, while the electrospray ionization mass spectrum (Figure 1C) reveals a peak at m/z ∼ 6325 corresponding to [Au 44 MBA 26 ] 2-, indicating the formation of high-quality Au 44 MBA 26 NCs.Transmission electron microscopy (Figure S1) and dynamic light scattering (Figure 1D) results indicate that the core size and the HD of the Au NCs were 1.54 and ∼2.33 nm, respectively.Moreover, these NCs exhibit strong NIR-II PL with two emission peaks at 1080 and 1280 nm (Figure 1E) with the quantum yield of ∼4.5%, making them suitable for biomedical applications such as in vivo imaging with ultradeep tissue penetration and ultrahigh spatiotemporal resolution.Taken together, the as-synthesized Au 44 MBA 26 NCs possess atomically precise size, good hydrophilicity, ultrasmall HD (<3 nm), and excellent NIR-II PL properties, enabling them to be a promising molecular probe for biomedical applications.The strong affinity between the biphosphate groups of bisphosphonates and the hydroxyapatite (HA) component of bones suggests that the phosphorylation of Au 44 MBA 26 NCs could endow them with potent bone-targeting ability. [8]This is due to the highly localized concentration of phosphate groups on the NC's surface, facilitating the application of the Au 44 NC-based bone theranostic probe.To validate this assumption, we engineered the surface of the Au 44 MBA 26 NCs using a simple crosslinking strategy to conjugate APP molecules with the surface MBA ligands, which resulted in the phosphorylated Au 44 MBA 26 -P probe.As shown, the optical absorption (Figure 1B), core size (Figure S2), HD (Figure 1D), and NIR-II PL emission (Figure 1E) of the Au 44 MBA 26 NCs were all well maintained without apparent changes after phosphorylation.These results imply that the small size of the APP molecules had negligible influence on the physicochemical properties of Au 44 MBA 26 NCs or that the conjugation between NCs and APP molecules was unsuccessful.The Fourier transform infrared spectra reveal the presence of the amide bond peaks at 1535 and 1638 cm -1 after phosphorylation, confirming the successful conjugation between NCs and APP molecules.Furthermore, elemental analysis shows that each Au 44 MBA 26 NC was conjugated with approximately eight APP molecules on its surface (Supporting Information S1).
The phosphorylation of Au 44 MBA 26 NCs indeed enhances their bone-targeting ability.This finding is supported by in vitro experiments on the affinity between HA (i.e., the major component of bones) and Au 44 MBA 26 -P with Au 44 MBA 26 NCs as reference.As shown in Figure 2A, the non-luminescent insoluble HA precipitate in water solution emits strong PL after interacting with both kinds of NCs, confirming their binding affinities for HA.Time-correlated PL intensity changes of the mixed solutions of HA and NCs demonstrate that the precipitates display increased PL intensity within 6 min, with the Au 44 MBA 26 -P probe yielding a stronger response (Figure 2B).These results indicate that the HA interacts quickly with the NCs and that the Au 44 MBA 26 -P probe has stronger bone-targeting capability.
To further assess the binding ability of Au 44 MBA 26 -P and Au 44 MBA 26 with HA, we measured the PL intensities of both precipitates and supernatants of mixed solutions of HA and NCs of varying concentrations after a 2 h reaction.Figure 2C shows that the PL intensities of both precipitates increased gradually with increasing concentration of the Au NCs, indicating concentration-dependent binding of NCs with HA.In addition, we also compared the changes in PL intensity of the supernatants before and after introducing HA (Figures 2D and S3) and summarized their recession ratios in the PL intensity in Figure 2E to acquire the theoretical maximum adsorption capacity of HA for Au NCs (Figure 2F).As expected, the PL intensities of the supernatants decreased significantly after introducing HA (Figures 2D and S3), supporting the results of the PL intensity changes of corresponding precipitates (Figure 2C).To confirm the selective interaction of Au 44 MBA 26 -P with the calcium species in HA, Ethylene Diamine Tetraacetic Acid (EDTA) was added to the incubation buffer before introducing Au 44 NCs.The precipitants' PL intensity of HA decreased when preincubated with EDTA, compared to those without EDTA (Figure S4).This drop in intensity suggests that Au 44 MBA 26 -P specifically binds to calcium species in HA.Based on the results shown in Figure 2E, the Langmuir adsorption isotherms of HA for Au 44 MBA 26 -P and Au 44 MBA 26 NCs could be plotted (Figure 2F), and the maximum adsorp-tion capacities of HA for Au 44 MBA 26 -P and Au 44 MBA 26 NCs were estimated to be 54.46 μg/mg (R 2 = 0.9521) and 39.9 μg/mg (R 2 = 0.9634), respectively.Density functional theory (DFT) simulations reveal that the adsorption energies of HA with MBA-P and MBA molecules are -9.046 and -6.656 kJ/mol (Figures S5 and S6), respectively.These results further confirm the stronger binding ability of The in vivo bone-targeting and NIR-II PL imaging properties of the Au 44 MBA 26 -P probe were examined by dynamically monitoring the NIR-II imaging quality of mice in different postures after tail vein injection (p.i.; exposure time: 100 ms, 1000 nm filter), using Au 44 MBA 26 NCs as a reference.As shown in Figure 3A, rapid bone accumulation and strong NIR-II PL signals could be observed throughout the body of mice after Au 44 MBA 26 -P injection, and the uptake of Au 44 MBA 26 -P in the spine and knee was also detected 10 min after injection.Due to rapid elimination, background signals of soft tissues and kidney gradually decrease while skeletal signals increase.Inheriting the low auto-luminescence intensity and deep tissue penetration of NIL-II PL imaging, the Au 44 MBA 26 -P probe is able to realize NIR-II PL imaging for a local part of body with high spatial resolution.Specifically, all dorsal bone structures from the skull to the spine, and even ribs and femurs can be clearly identified at 12 h p.i. of Au 44 MBA 26 -P, which cannot be achieved with Au 44 MBA 26 NCs at the same dosage and test conditions where the NIR-II PL intensity decreased significantly (Figure S7).Moreover, quantitative analysis of the signal ratio of the maximum intensity of the spine to that in the surrounding soft tissue as background (SBR) revealed that the SBR reached 2.55 at 2 h p.i. of Au 44 MBA 26 -P, and did not decrease significantly until 24 h (Figure 3B).In contrast, the SBR for the Au 44 MBA 26 -injected case is 1.83, which corroborates the stronger bone-targeting ability of Au 44 MBA 26 -P.In addition, the hind paw and ribs still show intensive NIR-II PL signals even at 24 h p.i. of Au 44 MBA 26 -P (Figure 3C,D), and more importantly high-resolution imaging of ribs with an average full width at half maximum of 0.73-0.99mm could be achieved (Figure 3E).These results validate that the Au 44 MBA 26 -P probe is able to directly visualize bone structures covered by intact tissue and skin through highresolution NIR-II PL imaging, which is advantageous over other bone-targeting luminophores that require skin removal for in vivo imaging. [52]e analyzed the bone-targeting properties of the Au 44 MBA 26 -P probe and the Au 44 MBA 26 NCs by measuring NIR-II PL intensities in the rib, knee, harnpan, femur, and vertebra of injected mice under the same testing conditions at 24 h p.i.The PL intensities were significantly higher in the Au 44 MBA 26 -P-injected mice than in the Au 44 MBA 26injected mice (2.95, 2.1, 2.63, 2, and 2.61 times higher, respectively; Figure S8).Therefore, the Au 44 MBA 26 -P probe exhibits better bone-targeting NIR-II PL imaging performance than the Au 44 MBA 26 NCs.
In addition, almost all bone structures (vertebrae, ribs, joints, femurs, etc.) of skinless mice show strong PL signals at 24 h p.i. of Au 44 MBA 26 -P in the NIR-II PL imaging (Figure S9).Similarly, the PL intensities of the Au 44 MBA 26 -P-injected mice were significantly higher than those of Au 44 MBA 26 -injected ones (Figures S9 and S10).Furthermore, we also euthanized mice and performed dissection at 24 h p.i. of Au 44 MBA 26 -P through intravenous injection to evaluate its effect on major organs, such as the heart, liver, spleen, lung, and kidneys.As shown in Figure S11, no obvious NIR-II PL signals could be detected in the heart, spleen, and lungs, while the kidneys and the liver showed moderate and weak PL signals, respectively.These results indicate that the ultrasmall Au 44 MBA 26 -P probe has good renal clearance, reflecting its good biosafety for bone-targeted NIR-II PL imaging in vivo.
Based on the results, the Au 44 MBA 26 -P probe exhibits good in vivo bone-targeting properties and excellent NIR-II PL imaging performance, as well as good biocompatibility, making it a promising PL theranostic probe for the treatment of bone-related diseases such as RA.Therefore, we investigated the effects of the Au 44 MBA 26 -P probe on the treatment of RA.RAW 264.7, a commonly used mouse macrophage cell line in RA research, was used to determine its cytotoxicity and anti-inflammatory properties in vitro.The results show no significant cytotoxicity in RAW 264.7 cells treated with concentrations ranging from 0.625 to 1 mM for 24-48 h (Figure S12), indicating that Au 44 MBA 26 -P has good biocompatibility and negligible toxicity.In addition, Au 44 MBA 26 -P did not cause detectable hemolysis (Figure S13) after incubation with erythrocytes.The excellent cytocompatibility and hemocompatibility ensured the suitability for in vivo biological applications.
Next, we evaluated the effects of Au  the LPS receptor complex on the cell membrane, can induce a strong immune response and promote the secretion of various cytokines by inflammatory cells. [53]Moreover, macrophages play an important role in the pathogenesis of RA, with macrophage-derived cytokines such as tumor necrosis factoralpha (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) being relatively abundant in rheumatoid synovium.As shown in Figure 4, upon introducing different concentrations of Au 44 MBA 26 -P and Au 44 MBA 26 NCs into the culture medium of RAW 264.7 cells, both significantly inhibit LPS-induced secretion of inflammatory cytokines, including TNF-α (Figure 4A), IL-6 (Figure 4B), and IL-1β (Figure 4C).Noteworthily, TNF-α and IL-1β can regulate the expression of other cytokines and pro-inflammatory mediators, such as by controlling the expression of cyclooxygenase-2 and inducible nitric oxide synthase, thereby regulating the production of prostaglandin E2 (PGE 2 ) and nitric oxide.PGE 2 usually promotes the formation of inflammatory synovial vessels by inducing the expression of vascular endothelial growth factor, and also participates in the pathological process of RA. [54] These pro-inflammatory mediators interact with each other, playing important roles in the inflammation and bone destruction processes of RA.Therefore, we also tested the inhibitory effects of different concentrations of Au 44 MBA 26 -P and Au 44 MBA 26 on LPS-induced production of the pro-inflammatory mediator PGE 2 in the cell culture medium, and found that both significantly inhibited PGE 2 production (Figure 4D).These results manifest that both The good efficacy of Au 44 MBA 26 -P and Au 44 MBA 26 NCs in suppressing inflammation in vitro led us to investigate their in vivo anti-inflammatory and immunomodulatory effects in an RA animal model.In this study, the cattle-derived type II collagen immunization-induced RA rat model (CIA) was used in RA research because it exhibits similar inflammatory responses and other key features as those observed in human RA. [55]Besides, MTX with precise anti-inflammatory and immunomodulatory effects, as a first-line anchor drug for clinical treatment of RA, was used as a positive control for in vivo anti-inflammatory response.On the basis, CIAinduced rats were treated with Au 44 MBA 26 -P, Au 44 MBA 26 NCs, and MTX, while an equal volume of saline was administered to the healthy rat group and CIA control group rats.After 6 weeks of daily treatment with the drug, the organs were examined, and the Au content was detected, with the highest Au distribution found in the kidneys and the lower biodistribution of Au species in other major organs (heart, liver, spleen, and lung), further indicating the good biosafety of Au 44 MBA 26 -P (Figure S14).After treatment, ankle circumference and clinical arthritis score were evaluated for rats in all groups.Au 44 MBA 26 -P and Au 44 MBA 26 NCs display significant RA treatment effects similar to MTX (Figure 5A,B).It should be noted that the CIA rats could exhibit weight loss due to inflammation when compared to healthy rats.However, upon treatment with Au 44 MBA 26 -P, Au 44 MBA 26 NCs, and MTX, the CIA rats showed significant weight gain, especially those treated with Au 44 MBA 26 -P and Au 44 MBA 26 NCs, with weight gain similar to that of healthy rats after 6 weeks of treatment (Figure 5C).These results suggest that Au 44 MBA 26 -P and Au 44 MBA 26 NCs can prevent inflammation-induced weight loss in CIA rats.In addition, after 6 weeks of treatment with Au 44 MBA 26 -P and Au 44 MBA 26 NCs, blood and biochemical parameters remained within the normal range (Figure S15).There were no significant pathological abnormalities or lesions in the major organ tissues, including the heart, liver, lung, spleen, and kidneys, in any of the groups (Figure S16).Such results corroborate that Au 44 MBA 26 -P and Au 44 MBA 26 NCs did not induce significant adverse effects and can be used as a new type of nanomedicine for RA treatment.
The representative photos of the joints and paws of the five groups at the beginning, middle, and end of drug administration are shown in Figure 5D.The photos show that Au 44 MBA 26 -P and Au 44 MBA 26 NCs have a remarkable anti-inflammatory effect when compared to CIA model rats treated with saline.After 6 weeks of drug treatment, the serum levels of cytokines (i.e., TNF-α, IL-1β, and IL-6) and PGE 2 in CIA rats were detected.Compared to healthy rats, the serum levels of TNF-α, IL-1β, IL-6, and PGE 2 in CIA rats significantly increase, whereas after treatment with Au 44 MBA 26 -P and Au 44 MBA 26 NCs, the levels of TNF-α, IL-1β, IL-6, and PGE 2 decrease significantly (Figure 5E-H).Similarly, the levels of TNF-α, IL-1β, IL-6, and PGE 2 in articular tissues show the same results (Figure 5I-L).These One interesting question may arise regarding whether the Au 44 MBA 26 -P probe has better RA therapeutic efficacy than Au 44 MBA 26 , which engaged us to further evaluate their therapeutic efficacy through tissue pathological analysis and 3D micro-computed tomography (micro-CT) imaging of the joints after 6 weeks of treatment.First, joint sections were stained with hematoxylin and eosin.CIA rats exhibit severe inflammation, cartilage, and bone destruction (Figure 6A), and it is difficult to distinguish the interface between cartilage and bone due to inflammatory cell infiltration, synovial hyperplasia, and cartilage and bone destruction.The features of CIA-induced chronic inflammation were eliminated after treatment with Au 44 MBA 26 -P and Au 44 MBA 26 NCs, and a distinct interface between cartilage and bone could be observed, which resembles the tissue slices of the healthy group (Figure 6A).However, treatment with MTX only partially reduced the symptoms of inflammation.Pathological analyses of joint tissues were conducted to evaluate inflammation in the soft tissues, synovial tissue, cartilage, and bone.The results show that both Au 44 MBA 26 -P and Au 44 MBA 26 NCs are significantly effective at reducing the pathological scores compared to the CIA control group.In particular, the Au 44 MBA 26 -P probe exhibits better performance than Au 44 MBA 26 NCs with strong anti-inflammatory effects on the inflammatory reactions of cartilage and bone, and both are superior to MTX (Figure 6B).
Micro-CT was used to assess the extent of bone erosion in the hind feet of rats in different treatment groups, and the degree of bone erosion varied significantly between groups (Figure 6C).CIA rats exhibit significant bone erosion and joint deformities.However, after treatment with Au 44 MBA 26 -P and Au 44 MBA 26 NCs, bone erosion was completely eliminated, and joint deformities were signifi-cantly improved.In contrast, treatment with MTX did not dramatically improve bone erosion in CIA rats.
We also investigated the working mechanism of Au 44 MBA 26 -P for RA treatment.It is known that in immune and inflammatory responses, nuclear transcription factor kappa B (NF-κB) and mitogen-activated protein kinases play a crucial role in regulating the expression of pro-inflammatory cytokine genes. [56]NF-κB is a crucial type of nuclear transcription factors that usually remain in an inactive state in the cytoplasm but are involved in several cellular activities, including immune cell activation, T and B lymphocyte development, and apoptosis.In parallel, IκB serves as an inhibitory protein for NF-κB, and it consists of p100, p105, IκBα, IκBβ, IκBγ, IκBε, Bcl-3, and IκB-R.Under normal conditions, the p65/p50 heterodimer of NF-κB stays in the cytoplasm due to its interaction with IκBα. [57]owever, inflammatory signals could activate the IκB kinase (IKK), which in turn phosphorylates IκB at specific sites.Phosphorylation of IκB ultimately leads to its ubiquitination and degradation, which results in the activation of the two subunits of NF-κB and their translocation to the nucleus (especially the p65 subunit).Once in the nucleus, NF-κB binds to inflammation-related genes and initiates the transcription of pro-inflammatory cytokines, leading to inflammation.Immunohistochemical analysis revealed that the activated IKK complex and increased phosphorylation of IκB and p65 could be observed in the synovial and inflammatory cells of CIA rats (Figure 7).Treatment with Au 44 MBA 26 -P and Au 44 MBA 26 NCs significantly decreased the levels of phosphorylated IκB, phosphorylated p65, and activated IKK in both synovial and inflammatory cells, which are comparable to those in the normal rats.Specifically, the levels of three species in the Au 44 MBA 26 -P-treated group were lower than those in the Au 44 MBA 26 -treated group, further corroborating higher RA therapeutic efficacy of Au 44 MBA 26 -P.In contrast, MTX treatment only partially reduced the phosphorylation of IκB and p65 levels in the joint tissues of CIA rats.These findings are consistent with the general decrease in pro-inflammatory cytokine levels observed in both blood and joint tissues.

CONCLUSION
In NCs and the first-line RA drug MTX.Besides, the probe was found to have low cytotoxicity in renal clearance and excellent biocompatibility without damaging major organs or affecting blood and liver function, highlighting its potential for clinical translation in the treatment of bone diseases.

A C K N O W L E D G M E N T S
This work was supported by the National Natural Science Foundation of China (22071127), the Taishan Scholar Foundation of Shandong Province (tsqn201812074), and the Natural Science Foundation of Shandong Province (ZR2019YQ07).

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare they have no conflicts of interest.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E T H I C S S TAT E M E N T
This study was performed in strict accordance with the NIH guidelines for the care and use of laboratory animals (NIH Publication No. 85-23 Rev. 1985) and was approved by the Biomedical Ethics Committee of Qingdao Zhong Hao Biological Engineering Co., Ltd.(Qingdao, China).

F
I G U R E 1 (A) Schematic illustration of the Au 44 MBA 26 -P probe.Note: the phosphate groups are labeled with green circles, and only partial ligands are shown in the scheme.(B) Ultraviolet-visible (UV-vis) absorption spectra of Au 44 MBA 26 and Au 44 MBA 26 -P (here, the concentration of nanoclusters [NCs] was fixed to 20 μM).(C) Electrospray ionization mass spectrum (ESI-MS) and isotope patterns (inset-black curve: experimentally obtained; red curve: theoretically simulated) of Au 44 MBA 26 NCs.(D) Dynamic light scattering (DLS) profiles, (E) second near-infrared window (NIR-II) photoluminescence (PL) emission spectra (λ excitation = 808 nm; here, the concentration of NCs was fixed to 20 μM), and (F) Fourier transform infrared (FTIR) spectra of Au 44 MBA 26 and Au 44 MBA 26 -P.MBA, 4-mercaptobenzoic acid; P, phosphonate.F I G U R E 2 (A) Photographs of hydroxyapatite (HA) before (left items) and after reacting with Au 44 MBA 26 (middle items) and Au 44 MBA 26 -P (right items) under visible (upper panel) and near-infrared (NIR) light (lower panel) illumination.(B) Time-correlated changes in the photoluminescence (PL) intensity of HA precipitates (1 mg) after reacting with Au 44 MBA 26 -P and Au 44 MBA 26 (0.6 mg/mL, 0.2 mL).(C) The PL intensity changes of HA precipitates after 2 h of reaction with different concentrations of Au 44 MBA 26 -P and Au 44 MBA 26 .(D) The PL intensity changes of HA supernatants before and after 2 h of reaction with different concentrations of Au 44 MBA 26 -P.(E) The decline ratios in the PL intensities of HA supernatants after 2 h of reaction with Au 44 MBA 26 and Au 44 MBA 26 -P.(F) The Langmuir adsorption isotherms of HA for Au 44 MBA 26 -P and Au 44 MBA 26 based on the results shown in (E).Error bars, mean ± SD (n = 3), *p < 0.05.MBA, 4-mercaptobenzoic acid; P, phosphonate.

F I G U R E 3
Specific bone-targeting and high-resolution imaging of Au 44 MBA 26 -P nanoclusters (NCs) in vivo.(A) Time-course second nearinfrared window (NIR-II) photoluminescence (PL) images (exposure time 100 ms, 1000 nm long-pass filter) of a mouse in the whole body upon p.i. of Au 44 MBA 26 -P.(B) Time-course soft tissue as background (SBR) after the p.i. of Au 44 MBA 26 and Au 44 MBA 26 -P.The NIR-II PL images of the hind paw (C) and a mouse in lateral posture (D) after 24 h p.i. of Au 44 MBA 26 -P.(E) Full width at half maximum (FWHM) analysis of the ribs (red dashed line in D), based on cross-sectional intensity profiles.Error bars, mean ± SD (n = 3), *p < 0.05.MBA, 4-mercaptobenzoic acid; P, phosphonate.Au 44 MBA 26 -P for HA than that of Au 44 MBA 26 , proving the rational design of phosphorylated Au 44 MBA 26 NCs for enhanced bone-targeting ability.
Au 44 MBA 26 -P and Au 44 MBA 26 dose-dependently reduce the cellular levels of LPS-induced PGE 2 , TNF-α, IL-1β, and IL-6 and play an important role in suppressing the inflammatory response induced by LPS.

F I G U R E 6
The therapeutic effects of Au nanoclusters (NCs) in the rat collagen immunization-induced rheumatoid arthritis (CIA) model.(A) Representative histopathological images of joint sections from nonimmunized rats and CIA rats.(B) Histological scores of inflammation and cartilage and bone erosion in CIA rats.(C) Representative micro-computed tomography (micro-CT) images of metatarsal bone and ankle articulations in CIA rats.Error bars, mean ± SD (n = 3), *p < 0.05.MTX, methotrexate.results confirm that in CIA rats, the decrease in cytokine and PGE 2 levels induced by treatment with Au 44 MBA 26 -P and Au 44 MBA 26 NCs was comparable to treatment with MTX.
44 MBA 26 -P and Au 44 MBA 26 on lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 cells.LPS, an endotoxin that binds to summary, we have developed a theranostic probe based on the phosphorylation of NIR-II emitting Au 44 NCs for bonetargeted NIR II PL imaging and enhanced RA therapy.Both in vitro and in vivo experiments have confirmed the excellent bone-targeting properties of the Au 44 MBA 26 -P probe, which made in vivo bone NIR-II PL imaging possible.Moreover, the probe could also show effective anti-inflammatory activity against RA in rats by suppressing proinflammatory cytokines, resulting in almost complete restoration of the destructive bone to normal state, which endows Au 44 MBA 26 -P probe with better therapeutic efficacy than Au 44 MBA 26