TPCA-1 | Gamma Secretase Signal

Targeted and Combined TPCA-1-Gold Nanocage Therapy for In Vivo
Treatment of Inﬂammatory Arthritis

Ziyi Wang,1 Jing Yang,1 Yang Yang,1 Xiaorong Pu,1 Jingnan Zhao,2 and Nan Zhang1,3

Received 25 August 2020; accepted 16 October 2020

Abstract. Rheumatoid arthritis (RA) is an autoimmune disease that is currently incurable. Inhibition of inﬂammation can prevent the deterioration of RA. 2-[(Aminocarbonyl)amino]- 5-(4-ﬂuorophenyl)-3-thiophenecarboxamide (TPCA-1) suppresses inﬂammation via the inhibition of nuclear factor-κ (NF-κB) signaling pathway. Gold-based therapies have been used to treat inﬂammatory arthritis since the 1940s. Hyaluronic acid (HA) is a targeting ligand for CD44 receptors overexpressed on activated macrophages. Therefore, a combined therapy based on TPCA-1, gold, and HA was explored for the treatment of RA in this study. We used gold nanocages (AuNCs) to load TPCA-1 and modiﬁed the TPCA-1 (T) loaded AuNCs with HA and peptides (P) to construct an anti-inﬂammatory nanoparticle (HA- AuNCs/T/P). An adjuvant-induced arthritis (AIA) mice model was used to investigate the in vivo anti-inﬂammatory efﬁcacy of HA-AuNCs/T/P. In vivo distribution results showed that HA-AuNCs/T/P had increased and prolonged accumulation at the inﬂamed paws of AIA mice. Treatment by the HA-AuNCs/T/P suppressed joint swelling and alleviated cartilage and bone damage. By loading to HA-AuNCs/T/P, the effective concentration of TPCA-1 was greatly reduced from 20 to 0.016 mg/kg mice. This study demonstrated that HA-AuNCs/T/P could effectively suppress inﬂammation and alleviate the symptoms of AIA mice, suggesting a great potential of HA-AuNCs/T/P for the treatment of RA.

KEY WORDS: gold nanocages; TPCA-1; hyaluronic acid; rheumatoid arthritis.

INTRODUCTION

Rheumatoid arthritis (RA) is an incurable and chronic autoimmune disease that affects approximately 1% of popu- lation worldwide (1). The early stage of RA is characterized by inﬂammation of joints and pathological changes of cartilage and synovium (2). Deterioration of RA causes joint deformity, bone erosion, and even organ failure and death (3). Therefore, early diagnosis and treatment of RA is very important. Although the pathogenesis of RA is not completely clear at present, many studies have shown that inﬂammation suppression by interfering with the activation of the nuclear factor-κ (NF-κB) pathway can halt the progress of the disease (4–7).

Supplementary Information The online version contains supplemen- tary material available at https://doi.org/10.1208/s12249-020-01856-0.

1 Department of Pharmaceutics, School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, 450001, China.
2 Biotechnology G2018, School of International Education, Henan University of Technology, No. 100 Lianhua Street, Zhengzhou, 450001, China.
3 To whom correspondence should be addressed. (e–mail: [email protected])

NF-κB is a nuclear transcription factor expressed in various types of cells and plays an important role in the regulation of inﬂammation (8). Inhibitor of NF-κB (IκB), as an inhibitory protein, binds to NF-κB to form an inactive complex and blocks its nuclear localization sequence (NLS) to keep NF-κB in the cytoplasm in normal cells (9). However, after stimulation, the IκB protein is phosphorylated and ubiquitinated by I-κB kinase (IKK) and is ﬁnally degraded by 26S proteasomes. Subsequently, free NF-κB is translocated to the nucleus and initiates the transcription of inﬂammation-related genes, thereby releasing various inﬂammatory cytokines, adhesion molecules, chemokines, growth factors, and enzymes to aggravate the inﬂammatory response (10,11). Previous studies have shown that IKK-2, but not IKK-1, plays a key role in the regulation of NF-κB and subsequent expression of pro-inﬂammatory cytokines and growth factors (12,13). 2-[(Aminocarbonyl)amino]-5-(4- ﬂuorophenyl)-3-thiophenecarboxamide (TPCA-1) is a IKK-2 selective inhibitor that is used for anti-inﬂammatory studies (14,15). Both of prophylactic and therapeutic administration of TPCA-1 signiﬁcantly reduced the severity of the inﬂammatory symptoms of collagen-induced arthritis (CIA) mice (15). TPCA-1 attenuated the inﬂammatory symptoms mainly through the suppression of pro-inﬂammatory cytokines such as interleukin- 1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) and the inhibition of antigen- induced T cell proliferation.

Gold salts have been used to treat inﬂammatory arthritis since the 1940s (16). Compared to gold salts, gold-based nanostructures have a better targeting to inﬂamed tissues and can be used as carriers for combined therapies. Gold nanoparticles (GNPs) are spherical and solid nanogolds and have shown a higher suppression of pro-inﬂammatory pro- teins than gold salts via the reduction of expressed Toll-like receptor-4 (TLR4) and NF-κB (17). Tsai et al. also claimed that GNPs and polyethylene glycol (PEG) capped gold nanoparticles (PEG-GNPs) were antiangiogenic and amelio- rated CIA (18). It is interesting to explore the anti- inﬂammatory efﬁcacy and safety of nanogolds and their combined therapies for the treatment of inﬂammatory arthritis. Gold nanocages (AuNCs) are porous nanogold materials with high biocompatibility and have been widely used in drug delivery and imaging (19). The method is well established for AuNCs to be synthesized from silver nano- particles (AgNPs) (20,21). Hyaluronic Acid (HA) is a biodegradable material targeting CD44 receptors that are overexpressed on activated macrophages (22). In this study, we used AuNCs to load TPCA-1 (T) via a positively charged peptide (P), and the particle was then coated with HA to produce HA-AuNCs/T/ P. We also evaluated the biodistribution and in vivo efﬁcacy of HA-AuNCs/T/P.

MATERIALS AND METHODS

Reagents

2-[(Aminocarbonyl)amino]-5-(4-ﬂuorophenyl)-3- thiophenecarboxamide (TPCA-1; CAS. no. 507475-17-4) was purchased from Abmole Bioscience Inc. (Houston, TX, USA). Alfa Chemicals (Pune, India) provided sodium borohydride, chloroauric acid trihydrate (HAuCl4•3H2O), silver nitrate (AgNO3), and sodium citrate. Synthetic peptide (AP-1; MCKYFIKIVSKSAKKPVGLIGC) was obtained
from DGpeptides Co., Ltd. (Hangzhou, China). The purity of the synthetic peptide was > 99.0%. Sigma-Aldrich (St. Louis, MO, USA) provided complete Freund’s adjuvant (CFA; Lot# SLBR3877V). Indocyanine green (ICG) was purchased from Meilun Biotechnology Co. (Dalian, China). The chemical reagents were analytical grade.

Animals

Speciﬁc pathogen-free (SPF) Swiss male mice weighing 18–22 g (5–6 weeks old) were purchased from the Experimental Animal Center of Zhengzhou University (Zhengzhou, China). Experimental mice were fed with standard drinking water and normal food. They were housed in air-conditioned and light-controlled rooms to maintain their health and were acclimated for 1 week before the experiment.

Synthesis of Gold Nanocages (AuNCs)

The synthesis of AuNCs followed a previously published method (21,23). Brieﬂy, 4 mL of 1% (w/v) sodium citrate and 15 mL of dH2O were placed in a three-necked round bottom
ﬂask. This system was kept at 70°C for 15 min. Three hundred forty microliters of 1% (w/v) AgNO3 and 400 μL of 0.1% (w/v) sodium borohydride were added into the ﬂask. The mixture was then stirred at 70°C for 1 h and cooled down to obtain silver nanoparticles (AgNPs), an intermediate nano- particle for the preparation of AuNCs, with a particle size of 4 nm (20,24). Next, a water-cooled condenser was added on to the three-necked round bottom ﬂask. Under boiling and stirring, 4 mL of 1% (w/v) sodium citrate and 15 mL of dH2O were kept for 15 min, and 2 mL of AgNPs (4 nm) was added and stirred for 1 h. Three hundred forty microliters of 1% AgNO3, 400 μL of 1% (w/v) sodium citrate, and 340 μL of 1% AgNO3 were added and stirred for 1 h, and this step was repeated once to obtain AgNP seeds which could be used for the synthesis of AuNCs. Next, the condenser was removed. Ten milliliters of polyvinyl pyrrolidone (1 mg/mL) was stirred at 90°C for 1 h. Then 1 mL of AgNP seeds was added, and 4 mL of HAuCl4 (0.0711 mg/mL) was slowly added until the blue color of the reaction solution was stable. After centrifug- ing at 13,500 rpm for 15 min, free AgCl and polyvinyl pyrrolidone were discarded, and the sediment was harvested. Five milliliters of dH2O was mixed with sediment to obtain AuNCs solution at a concentration of approximately 12 μg/ mL. The accurate concentration of AuNCs solution was determined by coupled plasma atomic emission spectroscopy (iCAP 6500 DUO, Thermo Fisher Scientiﬁc, Shanghai, China) test.

Preparation of HA-AuNCs/T/P

The preparation followed a previous report (24). AuNCs were obtained by centrifuging 1.5 mL of AuNCs solution (12 μg/mL). One milliliter of AP-1 solution (50 μg/mL) was mixed with AuNCs, and the mixture was incubated for 2 h at 37°C under stirring (70 rpm). The mixture was then centrifuged and washed with water to remove free peptide residues. Then, the sediment (AuNCs/P) was suspended in
0.5 mL of dH2O, and the mixture was added to 0.2 mL of TPCA-1 (50 μg/mL) in aqueous dimethyl sulfoxide (DMSO) (DMSO/dH2O = 2:3 (v/v)). The mixture was vortexed for 3 min. Next, 0.2 mL of HA (400 μg/mL) was added into the mixture and kept at 37°C under stirring (70 rpm) for 2 h. After free TPCA-1 and HA were removed by centrifugation, the obtained sediments (HA-AuNCs/T/P) were suspended and diluted according to experimental requirements.
ICG was loaded in a similar method (25). After harvesting, AuNCs/P were suspended in 1 mL of ICG solution (1 mg/mL). The mixture was sonicated for 30 min in an ice bath to allow ICG binding. The free ICG was removed by centrifugation for three times. The sediment was mixed with 0.7 mL of dH2O and 0.2 mL of HA (400 μg/mL) and kept at 37°C for 2 h. HA-AuNCs/ICG/P were obtained after free HA was removed.

Characterization of HA-AuNCs/T/P

AuNCs, AuNCs/T/P, and HA-AuNCs/T/P were dis- persed in dH2O for further characterization. AuNCs, AuNCs/T/P, and HA-AuNCs/T/P were pipetted onto carbon-coated copper grids (Beijing Emcn Technology Co., Ltd., Beijing, China) separately and air-dried for 1 h. Themorphology images of AuNCs, AuNCs/T/P, and HA-AuNCs/ T/P were recorded using a transmission electron microscope (TEM, HT7700, FEI, USA). A Zetasizer Nano ZS (Malvern, UK) was used for characterizing the zeta potential and size distribution of AuNCs, AuNCs/T/P, and HA-AuNCs/T/P. The UV-Vis absorption spectra of AuNCs, AuNCs/T/P, and HA- AuNCs/T/P were recorded using a Shimadzu UV-2700 spectrometer (Tokyo, Japan).

Therapeutic Effect of HA-AuNCs/T/P in AIA Mice

Healthy Swiss mice were randomly divided into 6 groups: blank group, model group, TPCA-1-treated group, AuNCs- treated group, AuNCs/T/P-treated group, and HA-AuNCs/T/ P-treated group. Except for the blank group, the other 5 groups were immunized with 60 μL of complete Freund’s adjuvant (CFA) by subcutaneous injection at both hind paws of mice (26). The swelling ratio of both hind paws was recorded every 2 days. The swelling ratio was obtained by dividing the volume of the paws in real time to the initial paw volume. Clinical arthritis scores of hind paws were obtained following a standard evaluation process for each hind paw and subsequent sum of clinical scores of each hind paw (27) (Fig. 3e). (scores 0, no evidence of erythema and swelling occurred; score 1, mild erythema and mild swelling appeared; score 2, erythema and mild swelling extended from the ankle to the tarsals; score 3, erythema and moderate swelling extended from the ankle to metatarsal joints; score 4, erythema and severe swelling encompassed the ankle, paws, and digits or ankylosis of the limb).

Arthritis was successfully induced at 6 days post injec- tion. Two hundred microliters of the therapeutic agent was administered by intravenous tail vein injection every 2 days from day 6. The concentration of AuNCs in the treatment was 0.16 mg/kg mice. The model group was treated with 200 μL of saline, and mice in the TPCA-1-treated group were treated with 200 μL of TPCA-1 solution that had a concentration of 0.016 mg TPCA-1/kg mice.
After 16 days of treatment, the joints of the mice were collected. The joints were ﬁxed in 4% paraformaldehyde for 1 week and scanned by an in vivo micro-computed tomogra- phy (micro-CT, Siemens Inveon Micro-CT/PET) for 50 min with a resolution of 18 μM. The sliced samples of joints were stained with hematoxylin and eosin (H&E) and observed using a DM1000 optical microscope (Leica, Frankfurt, Germany).

In Vivo Distribution

Both hind paws of mice were immunized with 60 μL CFA by local subcutaneous injection. After 6 days, inﬂamed mice were randomly divided into 3 groups. Three groups were intravenously administered via tail vein with 200 μL of free ICG (50 μg), 200 μL of AuNCs/ICG/P (12 μg, loading ICG 50 μg), and 200 μL of HA-AuNCs/ICG/P (12 μg, loading ICG 50 μg) respectively. At the set time points (2, 4, 6, 12, and 24 h), mice were anesthetized, and the dynamic ﬂuorescence distribution was recorded using in vivo FX PRO (Bruker, Billerica, MA, USA).
Statistical Analysis

Data were expressed as mean ± standard deviation. The differences among the groups were analyzed by one-way analysis of variance (ANOVA) with LSD post-test using SPSS (SPSS, version 23.0, IBM Inc., Armonk, NY, USA).

RESULTS

Preparation and Characterization of HA-AuNCs/T/P

TEM images showed the porous round morphology of AuNCs, AuNCs/T/P, and HA-AuNCs/T/P (Fig. 1b). TPCA-1 and AP-1 loading and HA modiﬁcation also changed the diameter, surface charge, and UV absorption peak of AuNCs. AP-1 and TPCA-1 loading increased the particle size, whereas HA modiﬁcation decreased the particle size (Fig. 1c). This was because HA modiﬁcation compressed the AP-1/ TPCA-1 layer and reduced the particle size. The zeta potentials of AuNCs, AuNCs/T/P, and HA-AuNCs/T/P were
− 21.97 ± 0.23 mV, 16.80 ± 0.36 mV, and − 19.7 ± 1.57 mV,
respectively (Fig. 1d). AuNCs were negatively charged nanoparticles. Loading of positively charged AP-1 changed the AuNCs/T/P into positive charged nanoparticles, whereas negatively charged HA turned the HA-AuNCs/T/P into negatively charged nanoparticles again. The loading efﬁciency and loading capacity of TPCA-1 to AuNCs were 26.1% and
0.145 μg TPCA-1/μg AuNCs, respectively, according to our previous report (24). The absorptive peak of AuNCs shifted from 782 to 799 nm (AuNCs/T/P) and 831 nm (HA-AuNCs/ T/P), respectively, and indicated that AP-1/TPCA-1 loading and HA modiﬁcation caused different shift of UV absorptive peak of AuNCs (Fig. 1e).

HA Increases the Targeting of AuNCs to Inﬂamed Paws

A ﬂuorescent agent ICG was loaded to AuNCs for the study of in vivo distribution behavior of AuNCs/ICG/P and HA-AuNCs/ICG/P. Free ICG was adsorbed onto the surface of AuNCs/P by electrostatic reaction (28). During observa- tion, the ﬂuorescent signal in free ICG-treated mice was weak and faded quickly, suggesting a rapid clearance of ICG after injection. AuNCs/ICG/P- and HA-AuNCs/ICG/P-injected mice demonstrated signiﬁcantly stronger ﬂuorescence than free ICG-treated mice, and the accumulation of ﬂuorescence at inﬂamed paws were observed in these two groups. These results showed that AuNCs/ICG/P and HA-AuNCs/ICG/P possessed prolonged in vivo circulation and enhanced reten- tion at the inﬂamed paws. At the selected time points, the ﬂuorescent signal at the inﬂamed paws of HA-AuNCs/ICG/P injected mice was stronger than that of AuNCs/ICG/P injected mice. Because of the targeting of HA to activated macrophages (22), HA modiﬁcation further increased the accumulation of HA-AuNCs/ICG/P at the inﬂamed paws (Fig. 2).

HA-AuNCs/T/P Inhibits Inﬂammation Development in AIA Mice

After being immunized with CFA, paws of mice swelled within 24 h because of CFA-induced acute inﬂammation, and
Characterization of AuNCs, AuNCs/T/P, and HA-AuNCs/T/P. a Chemical structures of TPCA-1. b TEM images of AuNCs, AuNCs/T/P, and HA-AuNCs/T/P (the red arrows and red dotted lines indicated an additional layer on AuNCs) (scale bar: 20 nm). c Number average, d zeta potential, and e UV-Vis absorption spectra of AuNCs, AuNCs/T/P, and HA- AuNCs/T/Ppaws swelling decreased on the third day. Usually, the immunized paws swelled again on the sixth day, which revealed the chronic arthritis induced by the adjuvant (29,30). On day 6 after immunization, the AIA mice showed inﬂammatory symptoms such as swelling, redness, and erythema of toes and paws and decreased ankle mobility. TPCA-1, AuNCs, AuNCs/T/P, and HA-AuNCs/T/P were intravenously injected for treatment, respectively. During the treatment, the body weight of mice kept increasing, suggesting a good health status (Fig. 3c). On day 18, hind paws of the mice were collected. Left and right hind paws within the same experimental groups showed similar arthriticsymptoms. So, the right hind paws were selected as represen- tatives for photo shooting. Figure 3 b showed that AuNCs-, AuNCs/T/P-, and HA-AuNCs/T/P-treated groups had paws with reduced swelling. Among the treatments, HA-AuNCs/T/ P-treated mice demonstrated the highest relief of paw swelling. The clinical arthritis score of the hind paws and the swelling inhibition rate showed a similar trend with Fig. 3b (Fig. 3d, e). AuNCs/T/P and HA-AuNCs/T/P treatment signiﬁcantly reduced the clinical arthritis score and swelling of the hind paws compared with untreated and TPCA-1-treated mice. In this study, free TPCA-1 treatment demonstrated little efﬁcacy because we used a low concentration

Dynamic in vivo distribution of free ICG, AuNCs/ICG/P, and HA-AuNCs/ICG/P in anesthetized AIA mice after tail veil injection of the formulations. Fluorescent images are taken from 2 to 24 h post injection. The value of the scale bar is the ﬂuorescence signal counts calculated by the instrument. (Both hind paws are inﬂamed)

(0.016 mg/kg mice) that was equal to the amount of AuNCs loaded TPCA-1.
HA-AuNCs/T/P Shows Bone Protection and Synovial Hyperplasia Reduction

In this study, we collected the right hind paws of the treated mice and analyzed pathological changes in bones and joints via micro-CT and H&E staining, respectively. Under the same reconstruction mode, the hind paw 3D models of treated mice were established (Fig. 4). The bones in the healthy paws of mice were dense and had a smooth surface, whereas the bones in the model paw showed non-negligible bone damage. Due to inﬂammation, the phalanges, the knuckles, and the ankles in the model group had a rough surface and reduced bone density. TPCA-1- and AuNCs- treated paws demonstrated non-negligible bone damage as pointed by the red arrows in Fig. 4. But HA-AuNCs/T/P- treated paw showed less bone damage in the 3D image. Therefore, HA-AuNCs/T/P treatment possessed bone pro- tection effect, which was consistent with the data in Fig. 3.
Compared with blank group, the slice of paw in the
model group showed signiﬁcantly increased dense blue-violet nucleus from proliferated cells and thickening and invasion of synovium (Fig. 5). Untreated model group and TPCA-1- treated group still demonstrated severe synovial hyperplasia as multi-layered synovial membrane was observed. The destruction of articular cartilage was also observed near the hyperplastic synovium. After treatment with AuNCs, AuNCs/ T/P, and HA-AuNCs/T/P, the pathological symptoms of thepaws were alleviated. The slices showed less synovial hyperplasia and better protection of cartilage and bone. The HA-AuNCs/T/P-treated joint possessed the least proliferation of inﬂammatory cells and an intact structure of cartilage and bone. Therefore, HA-AuNCs/T/P can efﬁciently reduce synovial hyperplasia and protect cartilage and bone.

DISCUSSION

Since the ﬁrst report in 1956, the adjuvant-induced arthritis rodent model has been widely used in the research of rheumatoid arthritis (31,32). AIA mice developed by localized injection of CFA showed many physiological and pathological features of rheumatoid arthritis, such as joint swelling (30), paws numbness (30), erythema (33), synovial
hyperplasia (33), pannus (29), cartilage destruction (29), and bone erosion (29). The accumulation of therapeutics at inﬂamed joints is critical for anti-inﬂammatory treatment. Vascular hyperplasia causes a poorly organized structures of the blood vessels (34), and the inﬂammatory environment recruits and activates macrophages. AuNCs possess a nano- sized diameter that facilitates accumulation of therapeutics at the site of inﬂammation (35), and HA enhances the retention of therapeutics in inﬂammatory sites by interacting with activated macrophages. Therefore, HA-AuNCs/ICG/P achieved a higher and longer retention in the inﬂamed paws (Fig. 2). Local subcutaneous injection of CFA usually causes localized arthritis rather than systemic inﬂammation (36). But Zheng et al. immunized hind paws of rats with CFA and observed swelling and erythema in front paws within 14 days

a Experimental schedule of in vivo study. b Images of the right hind paws of AIA mice in each treatment group on day 18 (in each group left image: lateral view of paws, right image: front view of paws). c Body weight of AIA mice during the study. The hind paws swelling ratio e and clinical arthritis score d of AIA mice during the study. Data were represented as mean value ± SD (n = 5, **p < 0.01)(37). As we also observed ﬂuorescent accumulation in the front paws, there might be unknown mechanisms that enabled the establishment of inﬂamed front paws by the injection of CFA to hind paws of mice. This is a very interesting topic to explore in future. Nanoparticles have shown signiﬁcant accumulation in the liver in many studies because the liver is one of the largest detoxifying organs in the body. One goal of drug delivery is to minimize the accumulation of nanomedicines at the liver (38). However, based on current technology, few nanomedicines can avoid the issue. Therefore, accumulation of AuNCs/T/P and HA- AuNCs/T/P was also observed in the liver. In this study, HAwas used to enhance the targeting of AuNCs/T/P to inﬂamed paws. However, liver cells also express CD44 receptors, which led to a signiﬁcant accumulation of HA-AuNCs/T/P at the liver (39).

As an anti-inﬂammatory compound, TPCA-1 interferes with the activation of NF-κB by inhibiting IKK-2 (15,40). In the previous work, HA-AuNCs/T/P were internalized by inﬂammatory cells and inhibited the production of inﬂamma- tory factors such as IL-6, TNF-α, and reactive oxygen species (ROS) in a prophylactic treatment (15). Therefore, we applied the treatment in the early stages of inﬂammation development in AIA mice. Podolin PL et al. have reported Micro-CT images of the paws with a resolution of 18 μm. The top twelve small images showed calcaneus. The bottom six images showed metatarsal bones and phalanx. Bone erosion areas were marked with red arrows H&E stained images of ankle joints of mice on day 20 (× 100). The synovial membrane, joint cavity, cartilage, and bone of the ankle joint were indicated in blank group. The synovial hyperplasia (circle), the thickening of the synovial membrane (arrow), and the destruction of cartilage (dotted frame) were marked in model group (scale bar: 100 μm)that TPCA-1 inhibited the production of TNF-α (IC50 = 0.17 μM), IL-6 (IC50 = 0.29 μM), and IL-8 (IC50 = 0.32 μM)in lipopolysaccharide-induced human monocyte. They also showed that the effective dose of TPCA-1 for the treatment of CIA mice was 20 mg/kg mice (i.p., twice a day). At this dose, the therapeutic effect of TPCA-1 was comparable to Etanercept (12.5 mg/kg, i.p., every other day) (15). For our in vivo experiment, the dose was determined based on the concentration of AuNCs. Based on published work and our preliminary study, we determined a safe dose of HA-AuNCs/ T/P for experiments. The IC50 (the half maximal inhibitory concentration) of AuNCs and HA-AuNCs/T/P were12.3 μg/mL and 31.7 μg/mL (the concentration was calculated based on AuNCs content) (Fig. S1) in murine macrophage (Raw264.7) cells. The results also showed that intravenous injection of 200 μL 12 μg/mL HA- AuNCs/T/P could be a safe dose for experimental mice. By using AuNCs, we reduced the effective concentration of TPCA-1 from 20 to 0.016 mg/kg mice in vivo.

Therefore, intravenous injection of HA-AuNCs/T/P in the early stage of inﬂammation can effectively relief inﬂammatory symptoms of AIA mice.
RA is featured with synovial hyperplasia and pannus invasion into joints. The joint inﬂammation leads to the recruitment and inﬁltration of various cells such as macro- phages, neutrophils, T cells, and B cells. Activated macro- phages highly express IL-1 and TNF-α that stimulate synovial ﬁbroblasts and chondrocytes to overexpress matrix metallo- proteinases (MMPs) (41,42). MMPs are known to destroy extracellular matrix and lead to cartilage degradation (43). Receptor activator of NF-κB ligand (RANKL) and TNF-α from inﬂamed ﬁbroblasts also activate osteoclasts, which causes bone damage (34). Therefore, cartilage and bone protection are critical for RA treatment and should be used
as important indicators for the evaluation of anti- inﬂammatory efﬁcacy.
The mice in this study were at the young age of 5– 6 weeks old, and the immunization and treatment process ended before 10 weeks. Young and healthy mice provide an ideal model for this study. However, we have to pay attention to the age of mice when we consider further research because RA onset is related to age and human aged between 40 and 60 years old (middle-aged) have a high RA incidence (44). The immune characteristics of middle-aged mice were different from that of young mice. Research had shown that middle-aged mice exhibited a strong accumulation of T follicular helper (Tfh) cells, leading to increased arthritis and lung pathological changes in middle-aged K/BxN mice (10–15 months) (45). The age-depend accumulation of Tfh cells affected macrophages and B cells activation, thereby intensifying the inﬂammatory cascade based on B cells and macrophages (46–48). Therefore, it may be necessary to increase the dose of HA-AuNCs/T/P or introduce AuNCs- based photothermal therapy for the treatment of middle-aged arthritic mice.

CONCLUSION

In this study, HA-AuNCs/ICG/P showed prolonged in vivo circulation and enhanced accumulation in the paws of AIA mice. HA-AuNCs/T/P also alleviated the inﬂamma- tory symptoms of AIA mice. Further studies conﬁrmed that HA-AuNCs/T/P reduced synovial hyperplasia and protected cartilage and bone. By using AuNCs, we reduced the effective concentration of TPCA-1 greatly. This study indi- cates that the AuNCs-based anti-inﬂammatory therapy is efﬁcient to treat AIA mice and may hold a potential for clinical treatment of inﬂammatory arthritis like RA.

FUNDING

This research was funded by the China Postdoctoral Science Foundation (2015M582211) and Natural Science Foundation of Henan Province, China (202300410419).

COMPLIANCE WITH ETHICAL STANDARDS

Animal care and experiments were performed with the approval of the animal ethical committee of Zhengzhou University (Zhengzhou, China), according to the require- ments of the National Act on the Use of Experimental Animals (China).

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