Anti-inflammation and joint lubrication dual effects of a novel

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Biological and Medical Applications of Materials and Interfaces

Anti-inflammation and joint lubrication dual effects of a novel hyaluronic acid/ curcumin nanomicelle improve the efficacy of rheumatoid arthritis therapy Zengjie Fan, Jie Li, Jianli Liu, Hongjing Jiao, and Bin Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b06236 • Publication Date (Web): 19 Jun 2018 Downloaded from http://pubs.acs.org on June 21, 2018

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ACS Applied Materials & Interfaces

Anti-inflammation and joint lubrication dual effects of a novel hyaluronic acid/curcumin nanomicelle improve the efficacy of rheumatoid arthritis therapy Zengjie Fana*, Jie Lia, Jianli Liub, Hongjing Jiaoa, Bin Liua* a School of the stomatology, Lanzhou University, Lanzhou 730000, PR China b Second Hospital, Lanzhou University, Lanzhou 730000, PR China

Corresponding Authors: Dr. Z. F. Email: [email protected] Prof. B. Liu Email: [email protected]

Keywords: Rheumatoid Arthritis, Curcumin, Joint Lubrication, Anti-inflammatory, Nanomicelle, Hyaluronic acid

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ABSTRACT: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease, which can cause endless suffering to the patients and severely impact their normal lives. To treat RA, the drugs in use have many serious side effects, high cost or only focus on their anti-inflammatory mechanisms without taking joint lubrication into consideration. Therefore, in this study, we aim to construct a novel anti-RA drug composed of hyaluronic acid/curcumin (HA/Cur) nanomicelle to resolve these problems. Characterizations show that Cur is bound to HA by ester linkages, and self-assembles to form a spherical nanomicelle with a diameter of around 164 nm under the main driving of the hydrophilic and hydrophobic forces. The nanomicelle enjoys excellent biocompatibility that effectively promotes the proliferation of chondrocytes. When injected to the RA rats, the nanomicelle significantly lowers the edema degree of the arthritic rats compared to other groups; more critically, a dramatic decrease in friction between the surfaces of cartilage around the joints has been found, which protects the cartilage from the RA-induced damage. Additionally, systematic mechanism investigation indicates that the nanomicelle diminishes the expression of related cytokines and vascular endothelial growth factor (VEGF), finally leading to the excellent performance. The newfound nanomicelle has potential for clinical practice of RA therapy, which will contribute significantly to alleviating the pain of patients and improving the quality of life for them. ■ INTRODUCTION Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that results in hyperplasia of the synovial membrane, degradation of cartilage, destruction of bones in diarthrodial joints, ultimately joint dysfunction.1 Approximately 1% of the population in the world have suffered from this disease, and more than 3,000,000 new cases are being diagnosed each year.2 While the etiology of RA remains unclear, it has been postulated that various cytokines (IL-1, TNF-α, IL-6, and IL-8, et al.) play a critical role in the pathogenesis of RA; besides, the causes of RA could be closely related to chemokines, cell adhesion molecules, and matrix metalloproteinase.3,4 The therapies for RA are mainly focused on minimizing the symptoms e.g., pain and swelling, decreasing the bone deformity, and maintaining the normal physiological ACS Paragon Plus Environment

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function of joints. The drugs used for treating RA can be divided into two types of medications: ion non-steroidal anti-inflammatory drugs (NSAIDs) and disease modifying anti-rheumatic drugs (DMARDs).5 Unfortunately, the majority of these drugs can cause numerous serious side effects.3, 6 In addition, biological therapies have been recently adopted for the treatment of RA, such as tumor necrosis factor (TNF) blockers (infliximab, etanercept, and adalimumab), IL-1 (anakinra) and IL-6 (tocilizumab) inhibitors. However, the exorbitant price limits their practical applications.6 Accordingly, an effective, low-cost, and safe anti-RA drug is urgently required. Curcumin (Cur), a major extraction derived from turmeric, has excellent antitumor,

anti-microbial,

and

anti-oxidant

activities.7,8

Owing

to

the

above-mentioned properties, Cur has been used as therapeutic agents for various diseases such as diabetes, chronic inflammatory, cutaneous diseases, Alzheimer’s disease, and so forth.9-11 Nonetheless, Cur has poor bioavailability due to its poor solubility, poor absorption, rapid metabolism, and quick systemic elimination, all of which limit its therapeutic effect in vivo.12,13 In order to resolve the aforementioned problems, various nanomicelles, including encapsulation of Cur in polymeric nanoparticles and phospholipids, have been extensively investigated, aiming to construct a protective structure which can not only protect the Cur from enzymatic and pH degradation to prolong its circulation inside the body but also enhance its solubility.14,15 So far, various attempts to enhance the bioavailability of Cur have been made by encapsulating Cur into the inner core of carries, such as chitosan,16 phospholipids,17 bovine serum albumin,18 poly (lactic-co-glycolic acid) (PLGA),19 and so on. The synthesized nanomicelles have been mainly used as antitumor drugs, ulcerative colitis therapy, neurons protection, and wound healing.20-22 However, there have been relatively few reports devoted to the applications of Cur in treatment of RA. For example, K. Coradini et al. prepared resveratrol and Cur co-encapsulated in lipid-core nanocapsules and improved their efficacy as oedematogenic agents, with no evidence of hepatotoxic effects.23 M. K. Jeengar et al. mixed Cur with emu oil and obtained a nanoemulgel formulation. They found that the nanoemulgel showed a ACS Paragon Plus Environment


significant improvement in anti-inflammatory activity.24 R. Arora et al. synthesized Cur loaded solid lipid nanoparticles finding that the drug can ameliorate CFA-induced arthritis in rats through attenuation of oxido-inflammatory and immunomodulatory cascade.25 However, these studies merely focus on anti-inflammatory activity, and ignore the joint wear protection between two joints after bone deficiency formation caused by RA. Furthermore, RA often causes the degradation of cartilage and destruction of bones in knee joints, causing a high friction between opposing surfaces of articular cartilage, which further worsens the RA condition. Hence, joint wear is a crucial issue, which definitely deserves serious consideration, yet has often been ignored in the present studies. In healthy weight-bearing joints, there is full of lubricant synovial fluid, composed of hyaluronic acid or hyaluronan (HA), lubricin, and surface-active phospholipids.26,27 Among synovial fluid is the main extracellular matrix component, HA, which plays a key role in sustaining the normal physiological function of joints, like tissue hydration, lubrication, and the interactions between proteins and proteoglycans of the extracellular matrix.28 For its lubrication role, it has been validated by many studies that HA can effectively reduce friction, protect the surfaces of joints, and alleviate the pains in the long run.29-32 Owing to its excellent lubrication role, HA was chosen as a carrier of Cur to prepare HA/Cur nanomicelles. Bearing the advantages of Cur and HA in minds, we aim to construct a new type of nanomicelles composed of Cur and HA. Not only could the new nanomicelles overcome the poor bioavailability of Cur, but they also could exert a lubricating action between joints. The healing effect of the nanomicelle was systematically evaluated in a RA animal model induced by Complete Freund’s adjuvant (CFA) and collagen Ⅱ by biochemical and histological analyses, energy spectrum detection, for the purpose of providing an affordable, efficient, and user-friendly anti-RA drug. ■ EXPERIMENTAL SECTION Materials and Reagents. Sodium hyaluronate (HA, weight-average molecular weights 10 and 25 kDa) was purchased from Guanglong (Shandong, China), Curcumin, 4-dimethylaminopyridine (DMAP), and dicyclohexylcarbodiimide (DCCI) ACS Paragon Plus Environment

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were purchased from Aladdin. Complete Freund's adjuvant (CFA) and Bovine Collagen Ⅱ were obtained from Sigma-Aldrich Co. Formamide and dimethyl sulphoxide (DMSO) were purchased from Tianjin No.1 Chemical Reagent Factory. Nanomicelles Preparation. The whole experimental process was conducted in dark to avoid the degradation of Cur. 100 mg HA was dissolved in 30 mL formamide solution with the aid of magnetic stirring under 60 oC thermostatic waterbath overnight, following the addition of 25 mL DMSO. After the obtained solution was purged with N2 for 30 min to remove O2, 203 mg DCCI and 120.6 mg DMAP were added to the above-mentioned solution. When DCCI and DMAP were completely dissolved, 68 mg Cur dissolved in 10 mL DMSO was added dropwise and stirred for 24 h in dark under the protection of N2. The solution was dialyzed against water, and the water was changed every day until the unreacted and organic reagents were completely removed. The resultant solution was frozen at -55 oC for 2h and then dried by sublimation in a vacuum chamber (1 Pa) for 24 h. The dry sample was stored in a refrigerator at 4 °C for further study. Cur Concentration in Nanomicelles. Cur concentration was determined by using a UV-Vis spectrometer. A standard curve was plotted by testing the OD value of different known Cur concentration at a constant wavelength of 429 nm. The OD value of Cur concentration in nanomicelles can also be determined at 429 nm, and then the Cur concentration in nanomicelles can be calculated according to the standard curve equation. Nanomicelles Characterizations. The morphologies of the synthesized samples were characterized by transmission electron microscopy (TEM, JEOL JEM-2010). The chemical composition was analyzed by fourier transform infrared spectroscopy (FTIR, Bruker, IFS120HR). The mean particle size and size distribution, and zeta potential were determined by dynamic light scattering (DLS) using a particle size analyzer (Zetasizer Nano ZS, Malvern Instruments, UK) equipped with a 632.8 nm He–Ne laser. Prior to the experiment, the particles were diluted in distilled water tol mg mL−1. Cellular Toxic Determination. Bovine articular chondrocytes were isolated by enzymatic digestion as described previously.33,34 The obtained chondrocytes were ACS Paragon Plus Environment


seeded into a culture petri dish containing DMEM/F-12 supplemented with 10% (v/v) fetal bovine serum. The cells were kept under aseptic conditions at 37 °C and 5% CO2. The media were refreshed every 2 days until the cells reached confluence. The cultured cells were collected and diluted into a solution with a concentration of 5×103 cells/mL. The cells were added to a 96-well plate with a volume of 100 µL per well, and cultured for 24 h. After that, 100 µL nanomicelles with five different concentrations were added to the 96-well plate, and the wells only had culture medium as the control group. At a given time (24, 48, and 72 h), 20 µL MTT solution was added into each well and cultured for another 4 h, and the upper solution was discarded. Subsequently, the blue formazan reaction product was dissolved by adding 20 µL of DMSO. The absorbance of the dissolvable solution was recorded by using a microplate reader. Data were averaged from three parallel experiments. RA Introduction. Male Wistar rats in 6 to 8 weeks with body weight ranging from 150 to 180 g were obtained from the Biomedical Experiment Center of Lanzhou University. All of the animal procedures were reviewed and approved by the Ethics Committee and the Institutional Animal Care and Use Committee of Lanzhou University. The RA animal model was prepared by using a collagen-induced approach.35 An emulsion composed of 2 mg/mL collagenⅡand CFA at a volume ratio of 1:1 was used to induce RA. The rats were anaesthetized with isoflurane, and 250 µL emulsion was injected into three different locations, including voix pedis, the tail root, and dorsum subcutaneous tissues. A booster injection was performed after one week by injecting 100 µL same emulsion into the voix pedis subcutaneous tissue. According to the suggested scoring criterion, the RA model can be classified into the scale of 0-4. For score 0, the paw of the rats is normal. For score 1, the rats show erythema and weak swelling limit to one toe. For score 2, the erythema and weak swelling appear more than one toe, but not entire paw. For score 3, the erythema and weak swelling extend the whole paw. For score 4, the severe erythema and swelling appear on the whole paw ankle or ankylosed paw.36 In this study, rats with score 4 were chosen as the RA animal model for further studies. Treatment of RA animals. We chose three different concentrations of the synthesized ACS Paragon Plus Environment

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HA/Cur nanomicelles for pre-experiment to determine what was the optimal concentration for RA therapy, including 336, 168, and 84 µg/mL, and the results showed that 336 µg/mL of HA/Cur nanomicelles had a higher inhibited effect on edema than the other two, so we used 336 µg/mL of drug for the following experiment (Supporting information, SI, Figure S-1). In order to verify the therapeutic effect of Cur nanomicelles, the RA model rats were randomly divided into five groups, and each group had six animals, including HA/Cur nanomicelles treatment for group 1, HA treatment for group 2, Cur treatment for group 3, normal saline treatment for group 4 and without treatment for group 5. The HA/Cur nanomicelles, HA, Cur, and normal saline were administered by arthritic ankle joint injection at a dose of 100 µL. Arthritis Assessment. Paw edema was evaluated by measuring the volume of the hind paw. In detail, the hind paws of the rats were immersed into a beaker full of water, making some water overflow, and then we could measure of the excess water from the overflowing beaker, and finally get the paw volume. Measurements were taken daily from the 14th day after administration of CFA to the 28th day after treatment with drugs. Changes in paw edema (%) were calculated using the following formula: ௏௣௢௦௧ି௏௡௢௥௠௔௟

Edema (%)=

௏௡௢௥௠௔௟

× 100%

Vpost denotes the paw volume of post-treatment; Vnormal denotes the paw volume of the normal animal. In addition to measuring the volume change, local CT scanning (HD750 CT scanner, GE Healthcare, Little Chalfont, UK) was performed as well to detect the change of joints before and after treatment. To be specific, scanning was performed in the axial mode, using the following parameters: gantry rotation time, 0.5 s; tube voltage, 80/140 kVp, fast switching; tube current, 630 mAs; pitch, 1.375:1; detector coverage, 20 mm; scan field of view (SFOV), small head; display field of view (DFOV), 9 cm; reconstruction type, standard; matrix size, 512; adaptive statistical iterative reconstruction (ASIR), 30%. On completion of the relevant RA assessment, the blood samples were collected



with a 5 mL injection syringe from the aortaventralis after the heart of the rat was exposed. The collected blood samples were put in a 4 oC refrigerator for 2 h, and then the samples were centrifuged for isolating the serum, which will be used for the further biochemical assay. When the above-mentioned processes were done, the rats were sacrificed and the knee joints of both hind legs were removed and fixed in a 10 % formalin (pH 7.4) at room temperature. Biochemical Assay. The serum IL-1, TNF-α, VEGF levels were measured using Enzyme Linked Immunosorbent Assay Kit (ELISA) (R&D Biosystems). To evaluate the possible hepatotoxic effects of the treatment with HA/Cur nanomicelles, the serum collected beforehand was used to assay the enzymatic activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP). All enzymes were calorimetrically assayed by using a commercial diagnostic kit (Doles Reagents, Goiânia, GO, Brazil). The biochemical assays were estimated for the following groups (n = 6): healthy animals, which did not receive CFA (control), arthritic rats injected with normal saline, Cur, HA, and HA/Cur nanomicelles. Histological Analysis. The fixed knee joints of the rats were dehydrated and embedded in paraffin. The embedded samples were sectioned into 5 µm slices, and then the sections were stained with hematoxylin and eosin (H&E). Lubrication Effect. The lubrication effect or frictional reduction of the prepared nanomicelles was systematically evaluated on a CETR UMT-2MT tribometer in a ball-on-plate contact configuration. Articular cartilages were collected from bovine femoral condyles and the moral-patella articulating surfaces. The cartilages were separated from the subchondral bones by using a surgical knife blade, and then cut into a square form of 1 × 1 cm with an average thickness of about 1.5 mm. The peeling cartilages were stuck to a metal disk with a 2 cm diameter. The cartilages were immersed in a PBS solution for 30 min so as to let them rehydrate and restore their original structures before the test. The Si3N4 balls (ϕ = 6 mm) were employed as the stationary upper counter friction pair. The applied load was 10 N, and the invariable sliding rate was 600 rev/min. In order to observe the lubrication effect of different samples, the four samples were added to the samples chamber respectively, ACS Paragon Plus Environment

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and made the samples completely submerge the cartilages (Detailed test, please refer to Supporting information, Figure S-2). Each experiment was repeated three times under the same condition. The samples after being tested on the tribometer were dried by means of freeze-drying and observed by SEM to quantify the degree of wear. ■ RESULTS AND DISCUSSION Considering the insolubility of Cur, we use the main component of extracellular matrix, HA, as the drug carrier to synthesize the nanomicelles to improve its solubility. The entire experimental process is illustrated in Figure 1, which can be divided into three parts, including the nanomicelles preparation, RA animal model construction, and healing effect assessment. Cur molecule has one hydroxyl attached to each of the two benzene rings as depicted in Figure 2. Therefore, the hydroxyl can readily react with carboxyl in HA under special conditions, and form ester linkages, ensuring that Cur binds with HA firmly. As shown in Figure 1, the Cur is insoluble in water, and it could be observed that some tiny particles either floated on the surface or sank to the bottom of the bottle (Figure 1b). When it reacted with HA, nanomicelles could be formed, yielding a transparent and yellow solution (Figure 1c).

Figure 1. Schematic illustration of the whole experimental process, including sample preparation and animal experiments. The light photos of HA (a), Cur (b) and HA-Cur (c).



Figure 2. Schematic illustration of the chemical reaction process between HA and Cur.

To verify the reaction, FTIR spectra of HA, Cur, and HA/Cur were measured and illustrated in Figure 3a. For HA, the band around 3426 cm-1 was assigned to the –OH groups, while the band around 1412 cm-1 was associated with the C–O stretching vibration of –COOH groups.37-38 In addition, the characteristic bands of glucose units around 1087, 1035, and 940 cm-1 appeared in the spectrum of HA.39 With respect to Cur, a relatively sharp band around 3514 cm-1 was assigned to phenolic OH groups of Cur. Besides, the four characteristic bands appeared around 1634, 1428, 1284, and 729 cm-1, which can be assigned to C=O stretching vibration, Phenolic C–O stretching vibration, enolic C–O stretching vibration and the benzene ring CH2 rocking vibration, respectively.40,41 Having reacted with Cur, the –COOH groups peak of HA vanished, and a new band around 1735 cm-1 appeared in the spectra of HA/Cur, which could be assigned to C–O stretching vibration of ester linkages.42,43 Additionally, the characteristic bands of Cur around 1284 and 729 cm-1 also appeared in the spectra of HA/Cur, indicating that Cur was successfully linked to HA by the ester linkages. Cur exhibited an intense absorption peak at 420 nm (Figure 3b), which shifted to 398 nm after conjugating with HA. It should be caused by the ester linkage formation and the introduction of numerous of hydroxyl.44 Cur shows a low solubility in water, (around 0.6 µg/mL). After conjugation with HA, the concentration of Cur among the ACS Paragon Plus Environment

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composite can attain ca. 30 µg/mL determined by UV-vis absorption spectra, which is 50 times higher than pristine Cur, implying that this method can effectively enhance the solubility of Cur. HA is a hydrophilic polymer, and its Zeta potential was about -69.4 mV. After conjugation, the Zeta potential of the composite was reduced to -45.5 mV (SI, Figure S-3), indicating the reduction of the number of carboxyl groups owing to the reaction with hydroxyl of Cur. Meanwhile, the remaining carboxyl ensured the solubility of the composite, and the concentration can attain ca. 30 µg/mL. No precipitation can be observed even in such a high concentration. The previous reports have shown Cur has poor stability in water solution, and nearly 90% of Cur can degrade into different products within 30 min owing to its protonation and fragmentation.45,46 The instability of Cur has also been observed in our experiment and found that 80% of Cur was decomposed. However, the degradation rate of Cur was reduced because of its conjugation with HA, and only 20% of Cur was decomposed owing to the protective effect of HA (SI, Figure S-4), which can protect the Cur from the protonation and subsequent fragmentation.

Figure 3. The FTIR spectra of HA, Cur and HA/Cur (a), the four purple rectangles denote the special peaks and their relative changes, and the inset is the magnified curve of partial HA/Cur spectra. The UV-vis absorption spectrum of Cur and HA/Cur (b).

The underlying mechanisms for the formation of nanomicelles when Cur binds with HA by esterification reaction are discussed as well (Figure 4). The hydrophilic and hydrophobic interaction forces should play a key role. Driven by the forces,



hydrophobic Cur will form the inner core, while the hydrophilic HA will form the outer layer. As a result, an assembly process occurred which was similar to liposome formation. In order to demonstrate the aforesaid postulation, here we employed TEM to directly observe the nanomicelles structure (Figure 4). In Figure 4a, we could see that nanomicelles were in a spherical shape with a diameter of 62 nm, which was smaller than that determined by dynamic light scattering (DLS) measurement (164 nm). The different diameter between two differently observed methods may be caused by the expanding of HA in a water solution owing to its hydrophilicity.47 By further magnifying the nanomicelles, it showed a clear, hierarchical, and circular ring structure. The HA chain rolled up and self-assembled to form multilayer circle structure under the driver of the hydrophilic and hydrophobic interaction force, thereby verifying our postulation (Figure 4b).

Figure 4. TEM images of nanomicelles (a) and (b). The insert in (b) is a magnified image of a single nanomicelle. Particle size and size distribution of HA/Cur in aqueous solution determined by DLS measurement (c) and size distribution obtained from TEM (d).


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MTT assay is a commonly used method to quantify the toxic effect of materials on cells by analyzing cellular mitochondria activity.48,49 As the major component of knee joints cells, chondrocytes were isolated and cultured.50 Figure 5 shows the cellular viability of the nanomicelles assessed by MTT assay by co-culturing with chondrocytes for 1, 2, and 4 days. It is shown that the OD value of experimental groups was far higher than the control groups during the entire experimental processes, suggesting that these materials were nontoxic to chondrocytes, and even enhanced the proliferation of chondrocytes. On the fourth day, the OD value was lower than that on the second day except for the concentration of 336 µg/mL. It should be attributed to the excessive proliferation of chondrocytes, leading to a transient shortage of nutrition supply. Compared to the other four samples, the 336 µg/mL showed the highest OD value, implying that this material was more favorable for the proliferation of chondrocytes.

Figure 5. The OD values of cells, the cells were incubated for 1, 2, and 4 days.



Figure 6. The virtual reality (VR) reconstruction photograph of ankle joints of rats by energy spectrum CT. (a) Normal control group, represented the healthy ankle in rat. (b) Animal models of RA group, representing swollen joints of the rat. (c) Treatment with HA/Cur nanomicelles group, showing a similar photograph to that of the normal control group, significantly reduced edema. Treatment of rats with Cur (d), HA(e), and normal saline (f), showing similar photographs to that of animal models of RA group, did not significantly reduce swelling.

The ultimate purpose of the synthesized drug was to assess the therapeutic effect on RA. The RA animal model was prepared by using a collagen-induced method. The edema in the hind paw of the animals was severe after two weeks’ injection of CFA and collagenⅡ(Figure 6b). It remained visibly swelling by the end of the experiments in the RA animals treated with Cur, HA, and normal saline (Figure 6d-f). The paw volume of CFA-induced arthritis in rats was similar to those of the rats treated with Cur, HA, and normal saline until the fourth week (p > 0.05) (Figure 7). However, two weeks later, the rats treated with HA/Cur nanomicelles showed a marked inhibition of the paw edema when compared to the control group (Figure 6c) (p0.05) (Figure 7).

Figure 7. Influence of Cur, HA, normal saline, and HA/Cur nanomicelles on rat paw edema (%) before and after treatment. Notes: Values are expressed as mean ± S.E.M. (n = 6 animals/group). ﹡denotes a statistically significant difference, (p < 0.05).

Energy spectrum CT was used to observe the anatomical structures of the joints of the rats because this approach can offer the 3-dimensional imaging with temporal and spatial information about ankles in healthy or CFA-induced arthritic rats. In addition, a detailed anatomic information can be provided by a noninvasive way in vivo. The joints of healthy rats had no swelling soft tissue, and still kept complete joint structure and clear joint space（Figure 8a). However, the joints of CFA-induced arthritic rats were characterized by blurred articular surfaces, swelling soft tissue, and widened articular space (Figure 8b). Having been treated with HA/Cur nanomicelles for two weeks, it showed slightly blurred articular surfaces and the swelling of soft tissue subsided, which was almost similar to the normal control group (Figure 8c). It is revealed that the HA/Cur nanomicelles could increase the antioedematogenic activity and significantly reduce edema. The treatment of rats with Cur, HA and normal saline did not obviously reduce swelling, but showed similar photographs to that of animal models of RA group (Figure 8d-f).



Figure 8. The Multiple Planar Reconstruction (MPR) photographs of ankle joints of rats by energy spectrum CT. (a) Normal control group, representing the healthy joints of rats. There is no swelling in soft tissue, and remains complete joint structure and clear joint space. (b) Animal models of RA group, characterized by blurred articular surface, swelling in soft tissue and widened articular space. (c) Treatment with HA/Cur nanomicelles group, showing slightly blurred articular surface and the reduced swelling of soft tissue, similar to the normal control group. Treatment of rats with Cur (d), HA (e), and saline (f) showed similar photographs to that of the animals of RA group.

The fixed hind paws of five groups in a 10 % formalin were sectioned, stained with H&E, and observed by light microscopy. The corresponding results were shown in Figure 9. For normal group, there were no signs of inflammation in the synovial and perisynovial soft tissue. The joint space kept its original structure and the surface of cartilage remained smooth (Figure 9a). However, having been treated with Cur (Figure 9d), HA (Figure 9e), and normal saline (Figure 9f), all rats showed a similar histology to the rats of RA group (Figure 9b). All of these groups showed a dense inflammatory infiltration in the synovial and pericapsular tissue, rough surface of cartilage, enlarged joint space, and partial loss of cartilage. In addition, the rats treated


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with Cur showed coarser surfaces of cartilage with a sawtooth-like structure than those of the other four groups caused by the insoluble Cur particles, which would increase friction between both surfaces of cartilages, accelerating the damage of cartilage. Completely different from the other experimental groups, HA/Cur nanomicelles can restore the RA to normal status. No evident inflammation appeared in the synovial and perisynovial soft tissue, and the surfaces of cartilage remained smooth as well. The reason may be that two important roles are played by HA/Cur nanomicelles,

including

anti-edema

and

lubricating

action.

The

excellent

anti-inflammatory effect of Cur has been validated by many studies, which may involve several cytokines and growth factors, such IL-1, TNF-α, IL-6, and VEGF (vascular endothelial growth factor).51-53 Cur can effectively inhibit the expression of these cytokines and growth factors to decrease the negative impact of the inflammatory cells, thereby protecting the normal tissues from being damaged. As for these two important functions of HA/Cur nanomicelles, we will thoroughly discuss them in the following ELISA and tribological experiments.

Figure 9. Representative histological images of the hind paw joints (H&E ×40) of rats. (a) Normal control group, (b) RA group, (c) HA/Cur nanomicelles group, (d) Cur group, (e) HA group, and (f) normal saline group. Blue arrow denotes the bone deficiency and S denotes the synovitis.



The biosafety in vivo of HA/Cur nanomicelles were also systematically assessed by assaying the enzymatic activities in serum, including AST, ALT, and ALP. The enzymes of AST and ALT are liver abnormality markers, while the enzyme of ALP is the marker of bone resorption. The corresponding in Figure 10a showed that for the ALT level, there was no obvious difference among the normal, RA, and HA/Cur groups. However, for the AST level, the value of RA group was much higher than that of the normal group, while the value of HA/Cur group was slightly higher than that of the normal group, indicating the HA/Cur nanomicelles showed a certain restoration effect on AST level. For ALP, the ALP level of RA group was higher than that of control and HA/Cur groups, implying the occurrence of bone resorption. In addition, the influence of Cur, HA, and the HA/Cur nanomicelles on TNF-α, IL-1, and VEGF in the serum of CFA-induced arthritic rats was determined by ELISA kit. As shown in Figure 10b-d, the TNF-α, IL-1 levels in the serum of HA/Cur group were slightly higher than that of the normal groups, while the VEGF levels were lower than those of the normal groups (P

Anti-inflammation and joint lubrication dual effects of a novel

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