Injectable Click-Crosslinked Hyaluronic Acid Depot To Prolong

Research Article Cite This: ACS Appl. Mater. Interfaces 2019, 11, 24984−24998

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Injectable Click-Crosslinked Hyaluronic Acid Depot To Prolong Therapeutic Activity in Articular Joints Affected by Rheumatoid Arthritis Jiyoung Seo,† Seung Hun Park,† Min Ju Kim,† Hyeon Jin Ju,† Xiang Yun Yin,‡ Byoung Hyun Min,†,‡ and Moon Suk Kim*,† Department of Molecular Science and Technology and ‡Department of Orthopedic Surgery, School of Medicine, Ajou University, Suwon 16499, Korea

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†

S Supporting Information *

ABSTRACT: The aim of this study was to design a click-crosslinked hyaluronic acid (HA) (Cx-HA) depot via a click crosslinking reaction between tetrazine-modified HA and trans-cyclooctene-modified HA for direct intra-articular injection into joints affected by rheumatoid arthritis (RA). The Cx-HA depot had significantly more hydrogel-like features and a longer in vivo residence time than the HA depot. Methotrexate (MTX)loaded Cx-HA (MTX-Cx-HA)easily prepared as an injectable formulationquickly formed an MTX-Cx-HA depot that persisted at the injection site for an extended period. In vivo MTX biodistribution in MTX-Cx-HA depots showed that a high concentration of MTX persisted at the intra-articular injection site for an extended period, with little distribution of MTX to normal tissues. In contrast, direct intra-articular injection of MTX alone or MTX-HA resulted in rapid clearance from the injection site. After intra-articular injection of MTX-Cx-HA into rats with RA, we noted the most significant RA reversal, measured by an articular index score, increased cartilage thickness, extensive generation of chondrocytes and glycosaminoglycan deposits, extensive new bone formation in the RA region, and suppression of tumor necrosis factor-α or interleukin-6 expression. Therefore, MTX-Cx-HA injected intra-articularly persists at the joint site in therapeutic MTX concentrations for an extended period, thus increasing the duration of RA treatment, resulting in an improved relief of RA. KEYWORDS: rheumatoid arthritis, click-crosslinking, hyaluronic acid depot, intra-articular injection, methotrexate, long-lasting release gions.6−10 However, direct administration leads to the rapid clearance of MTX from RA-affected regions,9,10 so direct MTX injections must be repeated to maintain therapeutic MTX concentrations, although adverse effects can be minimized by the use of direct intra-articular injection. Injectable in situ-forming hydrogels have been widely employed because they can be prepared as a liquid solution at room temperature, and easily injected locally to form a hydrogel depot in situ.11−15 If an MTX-loaded injectable in situ-forming hydrogel formulation is prepared as a liquid, then the formulation can be injected intra-articularly to create an MTX-loaded hydrogel depot. MTX release from the MTXloaded hydrogel depot in articular joints can be sustained over RA treatment cycles. Recently some researchers, including our group, have reported the results of direct intra-articular injection of a formulation of an MTX-loaded injectable in situ depot-forming hydrogel.16−20

1. INTRODUCTION Rheumatoid arthritis (RA) is a long-term autoimmune disorder whose cause is not completely understood.1 RA induces progressive and irreversible inflammatory damage, mainly in cartilage joints and bone, resulting in severe pain in articular joints. Most of the current treatments are intended to reduce pain in RA-afflicted patients.2 Nonsteroidal anti-inflammatory drugs, steroids, or diseasemodifying anti-rheumatic drugs (DMARDs) are available for the treatment of RA.3 DMARDs are administered at the incipient stage of the disease. Methotrexate (MTX) is an important and frequently used DMARD.4 MTX may be used to reduce or even prevent the progression of RA in its incipient stages. MTX is usually taken orally over the course of several weeks. However, repeated administration of MTX has adverse effects including vomiting, abdominal pain, and bone marrow depression, and the drug is systemically toxic to several organs.5 To develop a mode of MTX administration that minimizes these adverse effects, several research groups have directly administered MTX-only formulations to RA-affected re© 2019 American Chemical Society

Received: March 20, 2019 Accepted: June 20, 2019 Published: July 2, 2019 24984

DOI: 10.1021/acsami.9b04979 ACS Appl. Mater. Interfaces 2019, 11, 24984−24998

Research Article

ACS Applied Materials & Interfaces

Figure 1. Schematic images of (a) preparation of TET-HA and TCO-HA, (b) MTX-HA, (c) TET-HA/TCO-HA, and MTX-Cx-HA as an injectable drug depot for the sustained release of MTX from the depot and (d) the treatment of RA by intra-articular injection of an MTX-Cx-HA depot hydrogel. (The images were drawn by J.S. and M.S.K. in Adobe Photoshop 7.0 software.)

injected intra-articularly into human joints to relieve pain by attenuating the inflammatory response by the direct action of HA or by reinforcing the viscosity of joint fluid.27 Nonetheless, intra-articularly injected HA quickly disappears from articular joint sites because of the short residence time of HA.28 The residence time of HA can be significantly improved by crosslinking HA. There are several crosslinking reagents in use, such as formaldehyde, divinyl sulfone, thiols, and methacrylates.29−33 Crosslinked HA (Cx-HA) hydrogels can have a longer residence time under physiological conditions, although the above-mentioned crosslinking reagents may necessitate a complex crosslinking reaction or the use of acidic or basic reaction conditions, and require photocrosslinking; additionally, some reagents can induce the inflammatory response. On the other hand, researchers have developed a system utilizing the biorthogonal Diels−Alder click crosslinking reaction between tetrazine (TET) and trans-cyclooctene

In this study, we tested the hypothesis that the direct intraarticular injection of MTX-loaded injectable hydrogel can form an MTX-loaded hydrogel depot in situ and achieve a high local concentration inside the RA-affected region. The MTX trapped inside the hydrogel depot is expected to maintain therapeutic MTX concentrations inside the RA-affected region for the duration of the treatment period. In situ depot-forming hydrogels typically involve several natural biomaterials, including hyaluronic acid (HA), collagen, and fibrin, and several synthetic biomaterials such as block copolymers and the pluronic acid series.21−23 Among these, HA is an attractive natural biomaterial, owing to its high biocompatibility as a naturally occurring biopolymer.24 HA is also a key component of articular cartilage and is capable of water absorption. In particular, it is worth noting that HA can moderate the inflammatory response and thus contribute to the recovery of diseased tissue.25,26 Recently, HA alone was 24985


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2.3. Preparation of Near-Infrared (NIR) FluorescenceLabeled HA. IR-783 (250 mg, 0.33 mmol) was dissolved in 10 mL dimethylformamide. Under a nitrogen atmosphere, sodium azide (30 mg, 0.5 mmol) was added to the prepared IR-783 solution and then stirred at 65 °C. After 24 h, propargylamine (42.6 mg, 0.66 mmol), copper sulfate (110 mg, 0.66 mmol), and ascorbic acid (240 mg, 1.32 mmol) were sequentially added to the IR-783 solution and then stirred for 24 h at 25 °C. The reaction mixture was subsequently dried in a vacuum to produce amine-modified IR-783 (IR-783-NH2). The carboxyl group of 0.1 g of HA, TET-HA, or TCO-HA in 30 mL of DW was activated by adding DMTMM (16 mg, 0.05 mmol). IR-783-NH2 (30 mg, 0.038 mmol) was added to the activated HA, TET-HA, and TCO-HA solutions and the mixtures were stirred for 24 h at 25 °C. The reaction mixtures were added to the dialysis tubes with a molecular weight cutoff of 3.5 kDa (Spectrum Laboratories, CA). The unreacted IR-783-NH2 and DMTMM were removed through dialysis for 3 days. The solutions were then freeze-dried at −70 °C for at least 4 days to produce HA-NIR, NIR-TET-HA, and NIR-TCO-HA. 2.4. Preparation of MTX-HA and MTX-Cx-HA. The ratio of MTX to HA or Cx-HA was 37.5 μg/mL to 20 mg/mL. For the preparation of MTX-HA, MTX (37.5 μg, 0.0825 μmol) was added to 20 mg of HA in 1 mL of PBS and gently vortexed. The MTX-HA was subsequently loaded into a single-barrel syringe. To prepare MTX-Cx-HA, 18.75 μg MTX was added to 10 mg of TET-HA in 0.5 mL of PBS and to 10 mg of TCO-HA in 0.5 mL of PBS. MTX-loaded TET-HA (MTX-TET-HA) and MTX-loaded TCO-HA (MTX-TCO-HA) were loaded separately into the compartments of a dual-barrel syringe. 2.5. Cellular Uptake of Fluorescein Isothiocyanate-Conjugated MTX (FI-MTX). Confocal images were taken to evaluate the intracellular uptake of FI-MTX by RAW 264.7 or synovial cells. RAW 264.7 cells (104 per well) were cultured in the bottom chambers of 24-well Transwell plates, as described in Supporting Information, and then stimulated with 1 mg/mL lipopolysaccharides (Escherichia coli 055:B5; Sigma) for 24 h. Synovial cells (SW 982; 3 × 104 per well) were cultured in the bottom chambers of 24-well Transwell plates, and SW 982 cells were cultured as described in the Supporting Information. Formulations of FI-MTX alone (80.8 μg/mL, 0.0825 μmol), FIMTX-loaded HA (FI-MTX-HA; 80.8 μg of FI-MTX per 20 mg of HA), and FI-MTX-loaded Cx-HA (FI-MTX-Cx-HA; 80.8 μg of FIMTX per 20 mg of TET-HA and 20 mg of TCO-HA) were added to the upper chambers of 24-well Transwell plates. On days 1, 4, and 7, RAW 264.7 cells and SW 982 cells treated with each formulation were fixed with 4% paraformaldehyde for 10 min and then mounted using a ProLong Gold Antifade Reagent. Confocal laser scanning microscopy (Zeiss LSM 510, Carl Zeiss, Oberkochen, Germany) with LSM software ZEN 2009 (Carl Zeiss) was used to acquire confocal images of cellular uptake. 2.6. Induction of RA in Rats. Approval of all of the protocols used in this study was obtained from the Institutional Animal Experiment Committee of the School of Medicine of Ajou University (approval No. 2018-0021). Experiments related to the treatment and induction of RA were carried out in accordance with the approved guidelines. RA-induced rat models (RA rats) was prepared by a method similar to the one described in previous work.17−19 2.7. Synovium Hydrogel Images after Injection in Vivo. FIMTX (80.8 μg, 0.0825 μmol) was solubilized in 1.0 mL of PBS and drawn into a single-barrel syringe. FI-MTX (80.8 μg) was added to 1.0 mL of NIR-HA (20 mg), and this solution was loaded into a single-barrel syringe as well. FI-MTX (40.4 μg) was added to 0.5 mL of TET-HA (10 mg) and to TCO-HA (10 mg), and these mixtures were separately loaded into each section of a dual-barrel syringe. Next, 0.1 mL of each formulation was injected into the articular knee joints of RA rats using 26 G syringes. The articular knee joints were excised from individual rats after 1, 7, and 14 days. At each time point, fluorescence images were acquired at wavelengths of 780 and 516 nm using the FOBI imaging instrument.

(TCO) because this reaction can proceed rapidly under physiological conditions without an external catalyst.34,35 Several natural materials, such as small intestinal submucosa, HA, and collagen have been proposed as injectable clickcrosslinkable formulations. From them, we chose HA, because of the advantages described above. We designed TET-modified HA (TET-HA) and TCO-modified HA (TCO-HA) for injectable click-crosslinkable formulations as previously described.18 The injectable TET-HA and TCO-HA formulations have the merits of easy handling and reproducibility of the preparation of Cx-HA, which is formed simply by mixing TET-HA and TCO-HA. MTX can be easily loaded by mixing with TET-HA and TCO-HA solutions. Above all, the injectable Cx-HA formulation itself can induce recovery of diseased articular cartilage. Consequently, we prepared an MTX-loaded Cx-HA (MTXCx-HA) hydrogel depot in situ using the click crosslinking reaction between prepared MTX-loaded TET-HA (MTXTET-HA) and MTX-loaded TCO-HA (MTX-TCO-HA) solutions. We hypothesized that injectable MTX-Cx-HA formulations can create a Cx-HA hydrogel depot of MTX at an injected articular joint site, and maintain a therapeutic MTX concentration in the articular joint for a prolonged period (Figure 1). To the best of our knowledge, there are few studies on the intra-articular injection of MTX-Cx-HA for the treatment of RA. The objective of this study was to answer the following questions. (1) Can injectable MTX-Cx-HA formulations form a hydrogel depot for MTX at an intraarticularly injected articular joint site? (2) Can the MTX-CxHA hydrogel depot persist longer at the intra-articularly injected articular joint site in comparison with MTX-loaded HA (MTX-HA)? (3) Can therapeutic MTX concentrations released from the Cx-HA hydrogel depot persist for an extended period in the articular knee joint compared with other tissues? (4) Can the MTX-Cx-HA hydrogel depot induce significant repair of RA-affected joints? Resolving these issues will help to efficiently develop an intra-articularly injectable MTX-Cx-HA formulation that holds promise for fulfilling the unmet need for approaches to the reversal of preclinical RA.

2. MATERIALS AND METHODS 2.1. Preparation of Injectable Formulations of TetrazineModified HA (TET-HA) and trans-Cyclooctene-Modified HA (TCO-HA). One hundred milligrams of HA powder were added to 1 mL of deionized water (DW) in a series of tubes. 4-(4,6-Dimethoxy1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM) (65.7 mg, 0.25 mmol) was added to each HA solution and stirred for 30 min. To prepare the TET-HA and TCO-HA solutions, the TET hydrochloride salt (13.8 mg, 0.05 mmol) and the TCO hydrochloride salt (18.5 mg, 0.05 mmol) were added to the HA solution at 10 mg/ mL and then stirred for 24 h at 25 °C. The unreacted TET or TCO in the reaction solution was removed through dialysis for 72 h. Then, the TET-HA and TCO-HA solutions were lyophilized in a freeze dryer (FD 8508, Ilshin Lab, Daejeon, Korea). Finally, elemental analysis of HA (C: 34.7%, H: 5.2%, N: 2.9%), TET-HA (C: 43.2%, H: 6.0%, N: 5.4%), and TCO-HA (C: 37.8%, H: 5.8%, N: 3.7%) was conducted. 2.2. Preparation of HA and Cx-HA. The control preparation consisted of HA (20 mg/mL) dissolved in phosphate-buffered saline (PBS, pH 7.4). TET-HA and TCO-HA were separately mixed in PBS to prepare as a final concentration of 20 mg/mL. The TET-HA and TCO-HA solutions were loaded individually into each section of a dual-barrel syringe (donated by Ewha Biomedics, Seoul, Korea). CxHA was prepared by simultaneous injection from each part of the dual-barrel syringe into a vial for subsequent characterization. 24986


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Figure 2. Rheological properties of HA and Cx-HA hydrogels. (a) Storage and loss moduli, (b) phase angle, (c) the frequency sweep test and (d) complex viscosity (* p < 0.001), and (e) photo of ejecting TET-HA and TCO-HA through a 26 G syringe needle from each compartment of a dual-barrel syringe without clogging. 2.8. Treatment of RA Rats. RA treatment experiments were carried out in five experimental groups in accordance with approved guidelines. MTX alone (37.5 μg/mL), MTX-HA (37.5 μg of MTX per 20 mg of HA), and MTX-Cx-HA (18.8 μg of MTX per 10 mg of TET-HA and 18.8 μg of MTX per 10 mg of TCO-HA) were prepared for injection. The five experimental groups received the following treatments: untreated RA rats, MTX-treated RA rats, MTX-HAtreated RA rats, MTX-Cx-HA-treated RA rats, and normal rats. One hundred microliters of each formulation were directly injected into the articular knee joints of the RA rats. The RA rats were euthanized after 1, 3, and 6 weeks (n = 3 for each time point) to evaluate the treatments’ therapeutic effects. 2.9. Articular Index (AI) Score and Ankle Diameter Measurements in RA Rats. The hind paws of RA rats treated with one of the formulations and normal rats were examined, the ankle diameter was measured, and the AI score was calculated after 1, 2, 3, 4, 5, and 6 weeks. The circumference of the hind limb ankles was measured in at least three rats using a tape measure. AI was blind tested and scored by each observer using a method similar to the one described in previous works.36,17−19 All ankle diameters and AI scores were measured in triplicate independently. The results are presented as mean ± SD. 2.10. Biodistribution of MTX in RA Rats. To determine the in vivo distributions of FI-MTX alone, FI-MTX-HA, and FI-MTX-CxHA, each formulation-treated RA rat was euthanized on a predetermined day. The affected joint, intestines, stomach, kidneys, liver, and spleen were harvested immediately. At days 1, 3, 5, 7, and 14, fluorescence NIR images of the RAaffected joints were acquired at wavelengths of 750 nm to 825 nm using the FOBI imaging instrument as described previously. To determine the amounts of MTX remaining at days 1, 3, and 5 in the treated rats’ organs, each organ was homogenized in a tissue acetonitrile−water (9:1) solution using a T 10 basic Ultra-Turrax Homogenizer (IKA-Werke GmbH & Co., Staufen, Germany) at 4000 rpm for 10 min and incubated at 37 °C for 15 min. The homogenized tissue samples were then shaken on a NEWTRY Vortex-5 Lab Vortex

Shaker for 20 min. An equal volume of chloroform was added to the shaken samples, which were shaken for a further 30 min. The solution was centrifuged at 845g for 30 min, and then 2 mL of the resulting MTX-containing supernatant was analyzed using a spectrofluorometer (FP-8200, JASCO, software Spectra Manager, U.K.). Fluorescence signals were analyzed at an emission wavelength of 670 nm and an excitation wavelength of 516 nm. 2.11. Tumor Necrosis Factor (TNF)-α and Interleukin (IL)-6 Quantification in RA Rats. The rats were individually euthanized after 1, 3, and 6 weeks to measure TNF-α and IL-6 expression in the MTX-treated RA rats or normal rats. The articular knee joints of MTX-treated RA rats or normal rats were excised. Each joint was homogenized in a solution of 50 mM Tris−HCl buffer (pH 7.4), 0.5 mM dithiothreitol and a proteinase inhibitor cocktail (10 g/mL) at 4000 rpm for 10 min, using a T 10 basic Ultra-Turrax Homogenizer, and then incubated at 37 °C for 15 min. Rat TNF-α enzyme-linked immunosorbent assay (ELISA) Kits (R&D Systems, MN) and Rat IL6 ELISA Kits (R&D Systems, MN) were used to determine the TNFα and IL-6 concentrations in the homogenized extracts, respectively. A Coated Corning Costar 9018 ELISA plate was sealed, incubated overnight at 4 °C, and washed three times with wash buffer (1× PBS with 0.05% of Tween 20). To initiate the assay, 200 μL of ELISA/ELISPOT diluent was added to each well on ELISA Kits. The ELISA plate was incubated at room temperature for 1 h and then washed with wash buffer. 100 μL aliquots of the homogenized extracts were added to the wells and incubated at 4 °C. After 15 h, each well was treated with the corresponding antibody for 1 h, washed again with wash buffer, and then incubated with avidin−horseradish peroxidase (Invitrogen, MA). After 30 min at room temperature, each well was washed with wash buffer and incubated with a 1× 3,3′,5,5′-tetramethylbenzidine (Thermo Scientific, CA) solution. After 15 min, a stop solution (50 μL) was added to each well. The absorbance of each well was measured at 450 nm. All TNF-α and IL-6 expressions were measured in triplicate independently. 24987


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Figure 3. (a) Cumulatively released MTX amount and (b) MTX amount released at each time point from HA and Cx-HA for 14 days [(c) enlarged MTX amount released at 2 days]. Tmax, Cmax, and area under curve (AUC) values determined from the release profiles of MTX at each time point from MTX-HA and MTX-Cx-HA for 14 days (Tmax = 9 h, Cmax = 5.4 μg, AUC0−t = 13.3 μg day for MTX-HA) and (Tmax = 72 h, Cmax = 5.1 μg, AUC0−t = 38.9 μg day for MTX-Cx-HA). 2.12. Statistical Analysis. Cytotoxicity data for RAW 264.7 and synovial cells were obtained from three independent experiments for each data point. AI scores and ankle diameters of rats with RA were obtained in independent experiments (n = 3) after 1−6 weeks. The thickness of cartilage measured using haematoxylin and eosin (H&E) staining, the TNF-α-positive area measured by TNF-α staining, and the immunosuppressive effects of TNF-α and IL-6 in experimental arthritis were determined in three independent experiments after 1, 3, and 6 weeks. The results were analyzed with a one-way analysis of variance with Bonferroni’s post-hoc test using SPSS 12.0 software (SPSS Inc., Chicago, IL).

observation indicates that the TET-HA and TCO-HA formulation is suitable for injection. The structures of HA and Cx-HA hydrogels were analyzed by scanning electron microscopy (SEM) (Figure S3, Supporting Information). SEM of the HA hydrogel alone revealed the formation of a tight-packed, fibril-like morphology, with small pores. The Cx-HA hydrogel was found in SEM images to have a porous, interconnected, and predominantly polyhedral structure. The swelling ratios and degradation of HA and Cx-HA were compared (Figure S4, Supporting Information). HA was completely solubilized in PBS within 15 min. In contrast, Cx-HA maintained the shape of Cx-HA for 18 days. In the swelling test, the size of Cx-HA increased over 48 h, as it gradually absorbed approximately 6000% of its original volume in PBS. This result indicated that Cx-HA can absorb a large amount of a biological medium, but Cx-HA depot maintained in PBS without in vitro degradation over time. This result indicated that Cx-HA can be served as an MTX depot. In summary, compared with HA, the Cx-HA hydrogel manufactured by mixing TET-HA and TCO-HA had hydrogellike features and interconnected morphology and was potentially valuable as a drug depot. 3.2. In Vitro MTX Release from HA and Cx-HA Hydrogel Depots. MTX was easily mixed into the HA solution to prepare MTX-HA. MTX was also easily mixed with the TET-HA and TCO-HA solutions, separately, to prepare MTX-TET-HA and MTX-TCO-HA, respectively. The mixing of MTX-TET-HA and MTX-TCO-HA generated an MTX-CxHA hydrogel depot. An MTX release from the formed MTXHA and MTX-Cx-HA hydrogel depots in the dialysis tube was monitored at 37 °C for 14 days (Figure 3a). For MTX-HA, the MTX release was 73% at 12 h, 85% at 24 h, and almost 94% at 72 h; the 97% MTX release was followed by an additional release of the remaining MTX for 5 days. This result suggested that MTX was liberated almost completely within 3−5 days. Meanwhile, the amount of MTX released from MTX-Cx-HA was 41% at 24 h and almost 60% at 72 h, indicating an initial burst of MTX, and then around 69% liberation at 5 days. Nonetheless, compared with the HA depot, the initial burst of the MTX release at early time points was suppressed by the Cx-HA hydrogel depot. After 5 days, the MTX release was sustained for 14 days. The release of MTX from MTX-Cx-HA was observed for 14 days. Tmax, Cmax, and AUC values were determined from the release profiles (Figure 3b). The Cx-HA

3. RESULTS 3.1. Preparation and Characterization of Injectable Formulations. To prepare injectable TET-HA and TCO-HA formulations, the carboxylic group of HA activated by DMTMM was separately reacted with TET and TCO. Elemental analysis of the amine groups in TET-HA and TCO-HA identified an introduction of 81.2 and 91.5% of the TET and TCO, respectively. TET-HA and TCO-HA were obtained in almost quantitative yields. The characteristic peaks of TET and TCO in HA were observed in the 1H NMR spectra (Figure S1, Supporting Information). Mixing equal amounts of TET-HA and TCO-HA produced click crosslinking of HA. The mechanical properties of HA and Cx-HA when produced as injectable formulations were subjected to rheological analysis with changes in frequency from 0.1 to 10 Hz (Figure 2). HA alone showed little difference between loss and storage moduli. By contrast, the Cx-HA hydrogel manufactured by the mixing of TET-HA and TCO-HA revealed high storage (G′) modulus and low loss (G″) modulus (Figure S2a, Supporting Information). The small phase angle (tan δ values calculated from G″/G′) of Cx-HA indicated a more gel-like consistency compared to the HA. These results indicated that click crosslinking of HA significantly affected the hydrogel-like features of HA. In the frequency ranges tested (0.1−10 Hz), Cx-HA showed a higher storage modulus than HA, indicating higher mechanical stiffness of Cx-HA. The viscosity of Cx-HA was 4-fold higher than that of HA. Taken together, these results indicated that Cx-HA had significantly more stiffness and hydrogel-like characteristics than HA. Injectability was investigated by expelling TET-HA and TCO-HA from the compartments of a dual-barrel syringe through a 26 G needle and analyzing the formation of Cx-HA (Figures 2e and S2b). TET-HA and TCO-HA were extruded without clogging for 35 s and rapidly formed Cx-HA. This 24988


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Figure 4. Viability of (a) RAW 264.7 cells and (b) synovial cells and (c) the expression of inflammatory TNF-α in RAW 264.7 cells treated without a drug, with HA alone, Cx-HA alone, MTX alone, MTX-HA, or MTX-Cx-HA for 1, 4, and 7 days (* p < 0.001, ** p < 0.005, # p > 0.99).

depot showed significantly higher Tmax and AUC values than the HA depot. It is likely that MTX was trapped inside Cx-HA and then diffusion of the entrapped MTX from Cx-HA was sustained over 10 days, even in the dialysis tube. MTX inside Cx-HA can therefore be gradually released over an extended period, in contrast to the HA depot. 3.3. In Vitro Cell Viability on MTX-HA and MTX-Cx-HA Hydrogels. Because the injectable formulation was injected into the articular knee joints of RA rats, the viability of RAW 264.7 or synovial cells on the bottom chamber was investigated by incubation with the injectable formulation of HA alone, CxHA alone, MTX alone, MTX-HA, MTX-Cx-HA on upper chamber, and a control, in which no drug was used. All formulations were able to diffuse from the upper chamber to the bottom chamber to contact the RAW 264.7 or synovial cells. The viability of RAW 264.7 cells was evaluated at 1, 4, and 7 days (Figure 4a). The untreated RAW 264.7 control cells showed a slight increase in viability as a function of culture time. In the group treated with HA alone, the viability of the RAW 264.7 cells decreased to approximately 55% compared to the control on day 1, due to the HA hydrogel diffusing from the upper chamber to the bottom chamber, indicating that HA alone has an inhibitory effect on the viability of RAW 264.7 cells. We then observed a gradual increase in viability up to 7 days as a result of the depletion of the HA when the culture medium was refreshed. This pattern was similar to that of CxHA alone, which had a 33% inhibitory effect compared to the control on day 1, and then a gradual increase up to 7 days, indicating that some of the Cx-HA in the upper chamber diffused to the bottom chamber and had an inhibitory effect on the viability of the RAW 264.7 cells. The viability of RAW 264.7 cells and SW 982 cells did not differ greatly for MTX concentrations of 15, 30, and 37.5 μg/ mL. We therefore selected a concentration of 37.5 μg/mL, because we were seeking a sustained release of a constant MTX concentration. MTX alone inhibited the viability of RAW 264.7 cells by 85% on day 1, providing further evidence for the effectiveness of MTX. However, there was a gradual increase in RAW 264.7 cell viability over the course of the experiment, because MTX was completely removed from both chambers when the culture medium was refreshed. The viability of RAW 264.7 cells in the presence of MTXHA was reduced by 86% after 1 day and by 81% after 4 days. At 7 days, the inhibition of RAW 264.7 cell viability was 27% compared to the corresponding control cells, because MTX

was lost through the dissipation of the HA depot with the repeated replacement of the culture medium. After 1, 4, and 7 days, MTX-Cx-HA reduced cell viability to below 14−15% of that of the corresponding control cells. MTX-Cx-HA inhibited cell proliferation more effectively over a 7 day period than did MTX-HA. This suggests that MTX-CxHA acted as a hydrogel depot for MTX over the experimental period, even though the culture medium was refreshed. Next, the viability of normal synovial cells seeded on an injectable formulation of HA alone, Cx-HA alone, MTX alone, MTX-HA, or MTX-Cx-HA was examined after 1, 4, and 7 days (Figure 4b). The viability of synovial cells seeded on each formulation gradually increased up to 7 days. The viability did not differ significantly among all of the formulations. This result indicated that synovial cells grew stably on all formulations. Finally, the amount of TNF-α released by RAW 264.7 cells was measured to compare the anti-inflammatory effects of HA alone, Cx-HA alone, MTX alone, MTX-HA, and MTX-Cx-HA (Figure 4c). The amount of TNF-α released by RAW 264.7 cells in the absence of MTX, the no-treatment control, was 1600−1750 pg/mL after 1, 4, and 7 days, indicating high expression of inflammatory TNF-α in RAW 264.7 cells. In HA alone and CxHA alone groups, the amount of TNF-α was 1100 and 1130 pg/mL after 1 day, but it then increased to 1470 and 1410 pg/ mL after 4 days, and to 1610 and 1740 pg/mL after 7 days, indicating that HA alone or Cx-HA had only a slight antiinflammatory effect on RAW 264.7 cells. The concentration of TNF-α in the MTX alone group was 850 pg/mL after 1 day, increasing to 1170 pg/mL after 4 days and 1640 pg/mL after 7 days, owing to MTX loss after refreshment of the culture medium. The amount of TNF-α released after treatment with MTX-loaded HA decreased to 690 pg/mL after 1 day but increased to 970 and 1270 pg/mL after 4 and 7 days, respectively. A probable explanation for this observation is that the MTX release from the HA depot included an initial burst after 1 day as seen in the in vitro release experiment described in a previous subsection; thus, the released MTX had an anti-inflammatory effect on RAW 264.7 cells only at 1 day after the start of the experiment. Meanwhile, the amount of TNF-α released in the MTX-CxHA group was 620 pg/mL on day 1 and remained at 660 pg/ mL after 4 and 7 days. The extent of TNF-α release in the MTX-Cx-HA group was significantly lower than that in the other formulation groups and stayed similar for 7 days, indicating that this formulation had a better anti-inflammatory effect. This result indicated that MTX released from the MTXCx-HA depot kept exerting an anti-inflammatory effect on 24989


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Figure 5. (a) Confocal images of (a) RAW 264.7 cells and (b) synovial cells treated without a drug, with FI-MTX alone, FI-MTX-HA, or FI-MTXCx-HA for 1, 4, and 7 days (magnification: 400×, scale bar: 40 μm).

Figure 6. (a) NIR images during 14 days after intra-articular injection of NIR-HA or NIR-Cx-HA into the articular knee joints of RA rats (scale bars = 2 cm) and (b) release time vs signal-to-background ratio determined at each time point.

green fluorescence intensity was maintained for 7 days (Figure S6, Supporting Information). Meanwhile, no green fluorescence was observed in synovial cells in the FI-MTX, FI-MTX-HA, or FI-MTX-Cx-HA group, indicating that FI-MTX was not taken up by normal synovial cells (Figure 5b). These data explain why the synovial cells proliferated in the injectable formulation in the previous experiment throughout the experimental period. 3.5. In Vivo Persistence of NIR-HA and NIR-Cx-HA in Articular Knee Joints. We assessed in vivo hydrogel persistence using NIR fluorescence live imaging. The NIRTET-HA and NIR-TCO-HA solutions were each loaded into a compartment of a dual-barrel syringe, and the NIR-HA solution was loaded into a single-barrel syringe. The prepared NIR-HA or NIR-TET-HA and NIR-TCO-HA were individually injected intra-articularly into a joint of an RA rat (Figure 6a). An NIR-HA or NIR-Cx-HA hydrogel depot immediately formed at the injection site. NIR fluorescence images of NIR-HA were monitored for 12 h, after which this depot gradually disappeared, and it disappeared completely after 4 days. On the other hand, NIR fluorescence signals of the NIR-CxHA hydrogel depot were, however, detectable in vivo for at least 10 days (Figure 6b). This result suggested that NIR-TETHA and NIR-TCO-HA can form a NIR-Cx-HA hydrogel depot via intra-articular injection, and indicated that the NIR-

RAW 264.7 cells for the entire experimental period, owing to the synergistic anti-inflammatory effect of HA and MTX, since HA itself also had an anti-inflammatory effect. 3.4. Cell Uptake of FI-MTX from FI-MTX-HA and FIMTX-Cx-HA. To investigate the FI-MTX uptake by RAW 264.7 cells and synovial cells as a function of time, FI-MTX alone, FI-MTX-HA, and FI-MTX-Cx-HA formulations were prepared from FI-MTX. The shape of the RAW 264.7 cells in the absence of FI-MTX, the no-treatment control, was observed in optical microscope images. RAW 264.7 cells in the absence of FI-MTX emitted only blue fluorescence due to the nuclear staining of these cells (4′,6-diamidino-2-phenylindole; Figures 5a and S5). Therefore, the small size of blue fluorescence observed was due only to the nuclear size of the RAW 264.7 cells. FI-MTX, FI-MTX-HA, and FI-MTX-Cx-HA emitted the green fluorescence of FI-MTX located throughout the cytoplasm of the RAW 264.7 cells, as shown by superposition onto nuclear staining after 1 day. The green fluorescence of FIMTX disappeared after 4 days. FI-MTX-HA showed a low intensity of green fluorescence at 4 days, but this signal disappeared at 7 days after the start of the experiment (p < 0.001). FI-MTX-Cx-HA showed green fluorescence even after 7 days, implying that FI-MTX released from the FI-MTX-CxHA depot was consistently delivered to the RAW 264.7 cells. For the FI-MTX-Cx-HA depot, almost the same quantity of 24990


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Figure 7. In vivo biodistribution of FI-MTX in articular knee joints and organs [green image (FI-MTX), red image (NIR) and yellow image (merged image of green and red)]. (a) Fluorescence images acquired by an imaging instrument from the incised articular knee joint section of RA rats during 14 days after intra-articular injection with FI-MTX alone, FI-MTX-NIR-HA, or FI-MTX-NIR-Cx-HA (scale bars = 4 mm) and concentration determined by spectrofluorometer for FI-MTX in organs (the articular knee joint, stomach, liver, spleen, kidneys, and intestines) at 1, 3, and 5 days after intra-articular injection of (b) FI-MTX alone, (c) FI-MTX-HA, or (d) FI-MTX-Cx-HA (* p < 0.001, ** p < 0.005).

To assess the maintenance of MTX levels in the RA-affected region and in other tissues after intra-articular injection preparations of FI-MTX alone, FI-MTX-HA, and FI-MTXCx-HA were injected. FI-MTX levels in the RA-affected region and in other tissues were measured using a spectrofluorometer after 1, 3, and 5 days (Figure 7b−d; Materials and Methods section). One day after injection of FI-MTX alone, the articular knee joint contained approximately 5.1% of the amount of FI-MTX administered. Most of the FI-MTX was found in organs such as the stomach, liver, spleen, kidneys, and intestines. The quantity of FI-MTX rapidly decreased to approximately 0.42 and 0.14% after 3 and 5 days, respectively. This finding suggests that FI-MTX underwent fast clearance from the injected articular knee joint to other organs, even after 1 day. In the group with intra-articular injection of FI-MTX-HA, the articular knee joint contained 14.3% of the injected FIMTX after 1 day, 6.9% after 3 days, and 1.07% after 5 days, suggesting that FI-MTX persisted for only 3 days in the articular knee joint, probably owing to the dissipation of the HA hydrogel. These data suggested that HA can act as a depot for FI-MTX in the early period but has a short residence time at the intra-articularly injected site, in accordance with the results of the NIR fluorescence imaging presented in a previous subsection. One day after the injection of FI-MTX-Cx-HA, approximately 32.0% of the intra-articularly injected FI-MTX was still present in the articular knee joint, falling to 13.5 and 10.3% after 3 and 5 days respectively, and small amounts were found in the stomach, liver, spleen, kidneys, and intestines. These results indicate that directly intra-articularly injected FI-MTXCx-HA had a significantly longer residence time than did FIMTX-HA or FI-MTX in the articular knee joint, as observed using NIR imaging and a spectrofluorometer.

Cx-HA hydrogel depot persisted in vivo for a prolonged period, in contrast to HA. 3.6. In Vivo Biodistribution of FI-MTX-NIR-HA and FIMTX-NIR-HA in Articular Knee Joints and Organs. To investigate the persistence of MTX concentrations in the RAaffected region after intra-articular MTX injection, formulations of FI-MTX alone, FI-MTX-NIR-HA, and FI-MTX-NIRCx-HA were prepared and injected intra-articularly into RA rats. Fluorescence images were acquired from the incised articular knee joint section of RA rats using an imaging instrument after 1, 3, 5, 7, and 14 days (Figure 7a). A faint green fluorescence image attributable to FI-MTX was observed after 1 day, and no images were detectable after that. This finding is suggestive of fast clearance of FI-MTX from the intra-articularly injected knee joint. FI-MTX-NIR-HA produced a red NIR image attributable to NIR-HA after 1 day, and a yellow image attributable to the merging of green FI-MTX and red NIR-HA. After 5 days, negligible red and green NIR fluorescence was observed, and no fluorescence was observed after 7 days. These data indicated that NIR-HA could act as a depot for FI-MTX in the early period, but quickly disappeared from the intraarticularly injected site and thus NIR-HA had only a short residence time. Cx-HA fluorescence images acquired from the incised articular knee joint sections showed the formation of Cx-HA in the injected joint space. FI-MTX-NIR-Cx-HA produced a red NIR image attributable to NIR-Cx-HA after 1 day, and a yellow image attributable to the merging of green FI-MTX and red NIR-Cx-HA images. Dispersed green fluorescence was observed in the articular knee joint tissue after 7 days. Red, green, and yellow fluorescence were detectable even after 14 days, although the intensity was low. This result indicates that a suitable concentration of FI-MTX was maintained inside the Cx-HA depot in vivo. 24991


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ACS Applied Materials & Interfaces 3.7. Evaluation of RA Reversal via Ankle Diameter, AI, and Imaging of the Paws. To examine the alleviation RA by therapeutic MTX, injectable formulations of MTX alone, MTX-HA, or MTX-Cx-HA were intra-articularly injected into an articular knee joint of RA rats. The paw and ankle of the RA rats were monitored for 6 weeks (Figure 8a). No-treatment control and normal rats were analyzed for comparison.

score among RA rats in the no-treatment control, MTX alone, or MTX-HA group for 6 weeks. In contrast, the RA rats injected with MTX-Cx-HA showed a gradual decrease in the ankle diameter, which reached a value similar to that of normal rats after 6 weeks, although the AI score was slightly higher than that of normal rats. These results indicated that MTX-Cx-HA suppressed the inflammatory symptoms more effectively than did the other formulations and thus alleviated RA. 3.8. Confirmation of RA Relief by Histological Analysis. The therapeutic effects on intra-articularly injected RA rats were examined by H&E staining of the articular knee joint (Figure S7a, Supporting Information). Most of the hydrogel was excluded in the process of deparaffinization but left slight vestige in staining. In the images stained with H&E, normal rats had clear-cut chondrocytes within lacunae in the cartilage. RA rats that received intra-articular injection without a drug, MTX alone, or MTX-HA showed few chondrocytes in their cartilage, even after 6 weeks. The MTX-Cx-HA-treated RA rats showed a few chondrocytes after 1 week, and clear-cut chondrocytes within lacunae after 3 and 6 weeks. Cartilage thickness in sections of synovial tissue was calculated from H&E images of normal rats and RA rats intra-articularly injected with each formulation (Figure S7b, Supporting Information). The cartilage thickness in normal rats increased over time, from 160 μm, after 1 week, to 210 μm, after 3 weeks, and to 230 μm, after 6 weeks. For RA rats in the no-treatment group, the cartilage thickness decreased from 99 μm after 1 week to 69 μm after 6 weeks, indicating continuing damage to RA cartilage. RA rats treated with MTX alone also showed a decrease from 115 μm after 1 week to 83 μm after 6 weeks, although the decrease in thickness was slightly smaller than that of the no-treatment group. MTX-HA gave some increase in cartilage thickness as a function of time, from 90 μm after 1 week to 107 μm after 6 weeks, implying that there was some repair of the cartilage. The cartilage thickness of the MTX-Cx-HA-treated RA rats increased as a function of time, from 116 μm after 1 week to 160 μm after 6 weeks. The cartilage thickness increased to 70% of that measured in normal rats. This result indicated that MTX-Cx-HA had a therapeutic effect on RA via a sustained release of MTX from the Cx-HA hydrogel depot formed in the articular knee joint, according to the results of the experiment reported in a previous subsection. Glycosaminoglycans (GAGs) are important components of cartilage, acting as the synovial fluid lubricant in joints. The articular knee joint of intra-articularly injected RA rats was stained with Safranin-O (SO) to evaluate possible therapeutic effects on the cartilage (Figure 9). After SO staining, GAG deposits turned brown in the articular knee joint. Normal rats showed large, clear-cut brown-stained areas in the images of the cartilage. In contrast, RA rats that received no drug had no brown-stained areas in the images, and no lacunae shapes, even at 6 weeks. The RA rats treated with MTX alone had some brown-stained areas in the images, and some lacunae shapes after 1 week, but these signals almost disappeared after 3 and 6 weeks. MTX-HA-treated RA rats showed some brown-stained areas in the images, and some lacunae shapes after 1 week. These signals were increased slightly after 3 weeks but disappeared after 6 weeks. These data suggested that MTX from the HA depot was present in therapeutic concentrations in the early period, but the therapeutic effect disappeared after 6 weeks, owing to the

Figure 8. (a) Paw photographs of the articular knee joint, (b) ankle joint diameter, and (c) the AI score of RA rats during the 6 weeks after they received intra-articular injection without a drug, with MTX alone, MTX-HA, or MTX-Cx-HA as well as normal rats (scale bars = 10 mm) (p < 0.001 vs no treatment at time points 0 [○], 1 [△], 2 [▲], 3 [*], 4 [**], 5 [#], and 6 [##] weeks; p < 0.02 vs no treatment at 5 [△ △] and 6 weeks [▲ ▲]; p < 0.001 vs no treatment at 0 [○], 1 [△], 2 [▲], 3 [*], 4 [**], 5 [#], and 6 [##] weeks; p < 0.02 vs no treatment at 5 [△ △] and 6 [▲ ▲] weeks; + p < 0.02, + p < 0.002, and ++ p < 0.001, MTX-Cx-HA vs MTX-HA at 6 weeks).

Compared with normal rats, the rats with induced RA developed severe edema and erythema, as well as stiffness of leg movement. After intra-articular injection of each formulation, the swelling of the paws and the ankle diameter were examined after 1 week. The RA rats not treated with a drug and RA rats treated with MTX alone or MTX-HA had persistent severe edema and erythema for 6 weeks, in comparison with normal rats. The edema and erythema of the RA rats intra-articularly injected with MTX-Cx-HA decreased after 1 and 3 weeks, and then almost disappeared, to the point where they were indistinguishable from normal rats, after 6 weeks, suggesting that MTX-Cx-HA exerted significant therapeutic effects in RA rats. The ankle joint diameter and AI score of the intra-articularly injected RA rats were also determined (Figure 8b,c). There were no large improvements in the ankle joint diameter and AI 24992


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Figure 9. (a) SO staining and (b) SO positive area (determined by the corresponding (a) images) of an articular knee joint from RA rats that received intra-articular injection without a drug, with MTX alone, MTX-HA, or MTX-Cx-HA as well as normal rats at 1, 3, and 6 weeks (magnification: 200×, scale bars = 100 μm) (p < 0.001 vs no treatment at 1 [*], 3 [#], and 6 [▲] weeks).

Figure 10. (a) Micro-CT images and (b) volume ratios of new bone to tissue (BV/TV: calculated by means of the corresponding CT images) of an articular knee joint for RA rats that received intra-articular injection without a drug, with MTX alone, MTX-HA, or MTX-Cx-HA as well as normal rats at 1, 3, and 6 weeks (scale bars = 2 mm); (p < 0.001 vs no treatment at 1 [*], 3 [#], and 6 [+] weeks; p < 0.01 vs no treatment at 6 [++] weeks; ** p < 0.001, MTX-Cx-HA vs MTX-HA at 1, 3, and 6 weeks).

short residence time of MTX at the intra-articular injection site, in accord with the results of the experiments reported in a previous subsection. Meanwhile, after SO staining in the group of MTX-Cx-HAtreated RA rats, some brown-stained images and lacunae shapes were observed after 1 week, and these signals were increased after 3 and 6 weeks. In addition, chondrocytes within the rounded lacunae were detected within the cartilage of MTX-Cx-HA-treated RA rats, indicating the formation of new cartilage-like tissue. The extent of GAG deposits in sections of synovial tissue was calculated from the SO staining images from normal rats and RA rats subjected to intra-articular injections of the formulation. The GAG-positive magnitude in normal rats was 10−11% after 1−6 weeks. The proportion of GAG-positive cells in RA rats without drug treatment was close to 0% after 1−6 weeks, while that in RA rats treated with MTX alone was 1.5% after 1 week, indicating that there was an immediate effect after MTX administration, but this decreased to nearly 0% over the remainder of the study period. The proportion of GAG-positive cells in MTX-HA-treated RA rats was about 2% after 1 and 3 weeks, but this decreased to 0% after 6 weeks. In contrast to the other treatments, 3.1% of the cells in the MTX-

Cx-HA group were GAG-positive after 1 week, increasing to 5.5% after 3 weeks, and 8.6% after 6 weeks. These results indicate that the MTX-Cx-HA depot proposed in this study successfully supported the regeneration of in vivo cartilage-like tissue via the sustained release of MTX. 3.9. Confirmation of RA Reversal by Microcomputed Tomography (Micro-CT). Changes in bone volume in the articular knee joint of RA animals were evaluated by threedimensional micro-CT analysis. The normal and RA rats showed about 14 and 7%, respectively, at week 0. The formation of new bone tissue was determined in the articular knee joint among the RA rats that received intra-articular injection without a drug or MTX alone, MTX-HA, or MTXCx-HA after 1, 3, and 6 weeks (Figure 10). The untreated RA rats had an extremely low BV/TV value (7%) after 1 week, and these values increased to 9% at the 6 week time point. RA rats treated with MTX alone or MTX-HA showed slightly increased BV/TV values (10.2 and 10.8%) after 1 week and had almost similar BV/TV values (11.3 and 11.2%) even at the 6 week time point, indicating that there had been little formation of new bone tissue. Meanwhile, the MTX-Cx-HA group had the highest value after 1 week, and this value increased significantly over time. The BV/TV of the RA animals treated with MTX-Cx-HA 24993


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Figure 11. (a) TNF-α staining of articular knee joints (magnification: 200×, scale bars = 100 μm) and the expression of inflammatory cytokines (b) TNF-α and (c) IL-6 in RA rats that were treated without a drug, with MTX alone, MTX-HA, or MTX-Cx-HA as well as normal rats after 1, 3, and 6 weeks (* p < 0.001).

increased to 12.8% after 1 week and to 14.8% after 3 weeks and showed the highest value (16.3%) at the 6 week time point. This value in the MTX-Cx-HA group after 6 weeks was almost identical to the 16.8% value measured in normal rats. This result indicated that the administration of MTX-Cx-HA led to more extensive formation of new bone tissue in the articular knee joint than did any of the other formulations. 3.10. Evaluation of RA Reversal via Inflammatory Marker Assays. TNF-α plays a crucial role in the regulation of the inflammatory response in RA. Immunohistochemical staining of TNF-α in the articular knee joint was performed to investigate TNF-α expression in RA rats that received intraarticular injection without a drug, with MTX alone, MTX-HA, or MTX-Cx-HA (Figure 11a). We also stained for CD4 and ED1, to identify monocytes/macrophages and T cells in RA rats that received intra-articular injection without a drug, with MTX alone, MTX-HA, or MTX-Cx-HA (Figure S8, Supporting Information). Normal rats showed almost no reddish staining in synovial tissue, indicating the absence of expression of TNF-α, CD4, or ED1. RA rats in the no-treatment group had reddish staining in widespread areas of synovial tissue and showed a high intensity of reddish staining in these areas at 6 weeks, indicating the abundant expression of TNF-α, CD4, or ED1 and prolonged inflammation for 6 weeks. Images of RA rats treated with MTX alone or MTX-HA showed reddish-stained areas for 6 weeks although the intensity of the red areas was smaller than those of RA rats in the no-treatment group.

Images of RA rats treated with MTX-Cx-HA showed a large amount of reddish-stained areas after 1 week, but these signals almost disappeared abruptly at the 3 week time point; no reddish-stained areas were seen after 6 weeks and the images were similar to those of normal rats. These results suggested that MTX-Cx-HA suppressed inflammation almost completely in the cartilage of RA rats in vivo, thereby ameliorating RA. To investigate the expression of the inflammatory proteins TNF-α and IL-6, the magnitude of TNF-α and IL-6 expression in the articular knee joint was determined for RA rats that received intra-articular injection without a drug or with MTX alone, MTX-HA, or MTX-Cx-HA (Figure 11b,c). Normal rats were found to have very low levels of TNF-α and IL-6 in the articular knee joint up to 6 weeks. The magnitude of TNF-α expression in RA rats without a drug was 680−540 pg/mL after 1−6 weeks. The RA rats treated with MTX alone showed a decrease in the comparably high TNF-α expression of 474−360 pg/mL after 1−6 weeks, but there was a narrower range of concentrations in comparison with the no-treatment group. TNF-α expression in MTX-HA-treated RA rats was 360 pg/mL after 1 week, and this level slightly decreased and stayed at 270 pg/mL after 3 and 6 weeks. In the MTX-Cx-HA group, this level was 330 pg/mL after 1 week, gradually decreasing to 162 and 107 pg/mL after 3 and 6 weeks. Although the magnitude of TNF-α expression was higher than that observed in normal rats (5−6 pg/mL), MTX24994


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fully formed an (in vitro) in vivo MTX-Cx-HA depot at the intra-articular injection site. The advantages of the injectable formulationeasy preparation using a dual-barrel syringe, quick click-crosslinking formation at an intra-articular injection site, and an extension of the residence time of a Cx-HA depot at the intra-articular injection sitesuggest that Cx-HA is a promising candidate depot for MTX in conjunction with intraarticular injection.37,38 The MTX-HA depot almost completely released MTX within 3−5 days. This phenomenon could be attributed to the dissipation of the HA depot within a short time. Therefore, the MTX-HA depot suppressed the proliferation of, and a TNF-α release from, RAW 264.7 cells only in the early period (1 and 4 days), and the data were suggestive of rapidly increased cell proliferation and a release of the bulk of TNF-α within 7 days. Crosslinking can induce the formation of a dense hydrogel network. It is likely that MTX loads on the porous, interconnected, and predominantly polyhedral structure in the Cx-HA depot. Some of the MTX dispersed in the outer molecular Cx-HA layer can be rapidly perfused under biological conditions, indicating an initial burst of MTX. However, a large amount of MTX can load inside the molecular layer of Cx-HA and thus pass through its interconnected and predominantly polyhedral structure, which hinders the release of MTX from the Cx-HA depot. Therefore, the MTX-Cx-HA depot formed in vitro provided a sustained release of MTX for 14 days and more. A similar phenomenon has been observed by other investigators.39−42 In the Korsmeyer−Peppas semiempirical method,41 the diffusion exponent identified in an MTX releasekinetics analysis of HA and MTX-Cx-HA depots in this study was 0.68 and 0.65 (Figure S9, Supporting Information). It can be concluded that the release of MTX from a HA or Cx-HA depot follows a non-Fickian diffusion release mechanism.41,42 The sustained release of MTX from a MTX-Cx-HA depot gradually suppressed the proliferation of, and a TNF-α release from, RAW 264.7 cells for an extended period. Suppression by the MTX-Cx-HA depot was confirmed by the endocytotic uptake of MTX into the cytoplasm of RAW 264.7 cells, as evidenced by green fluorescence from FITC-MTX. These results suggest that the MTX-Cx-HA depot designed here was effective as an MTX depot for downregulation of inflammatory proteins for an extended period. MTX-HA and MTX-Cx-HA depots led to gradually increased proliferation of synovial cells over time, and no green fluorescence from FITC-MTX was detectable in the synovial cells. This result indicated that MTX inside the hydrogel depot had no significant effects on synovial cells. The direct intra-articular injection of MTX alone or MTXHA led to a significantly lower concentration of MTX at the injection site and high MTX concentrations in other organs after 1 day, indicating that there was rapid clearance from the intra-articular target site. This is a possible reason why the direct intra-articular injection of MTX has adverse systemic effects on healthy organs in RA patients. The MTX-HA group showed increased MTX concentrations at the intra-articular injection site after 1 day, which was maintained for 3 days because HA acted as a depot for MTX in the early period, but which almost disappeared after 5 days. This finding indicated that MTX stayed inside the MTX-HA depot only in the early period. Consequently, these results indicate that the short residence time of MTX in the RA articular joint means that

Cx-HA had an anti-inflammatory effect in RA rats for an extended period. The expression of IL-6 stayed within a range of 990−1096 pg/mL in RA rats without a drug and within 1024−947 pg/mL in RA rats treated with MTX alone, indicating an antiinflammatory effect in the immediate period after MTX administration. The amount of IL-6 in MTX-HA−treated RA rats decreased to 630 pg/mL after 1 week and decreased to 360 pg/mL after 3 and 6 weeks. In contrast, in the MTX-CxHA group, IL-6 concentration was 710 pg/mL after 1 week, which rapidly decreased to 260−230 pg/mL at 3 and 6 weeks after initiation of the experiment, indicating an antiinflammatory effect of MTX-Cx-HA over an extended period. These results indicate that MTX-Cx-HA suppressed the expression of the inflammatory cytokines TNF-α and IL-6 in the articular knee joint. These changes resulted in a stronger therapeutic effect against RA relative to the other formulations tested.

4. DISCUSSION HA is an important, abundant constituent of mammalian extracellular matrices, and is capable of water absorption.24 HA can moderate a cellular inflammatory response to help heal the diseased tissue. HA has excellent biocompatibility owing to its negligible toxicity and immunogenicity. Because of these properties, HA has been widely regarded as one of the best candidate biomaterials for various biomedical applications. An HA solution can be injected intra-articularly into the joints of humans to mitigate inflammation and to improve the lubrication of synovial fluid.24−26 However, intra-articularly injected HA disappears under physiological conditions because of endogenous degradation by a specific enzyme, hyaluronidase, resulting in short residence time at the site of injection.27 Strategies for crosslinking HA have been employed to prolong in vivo residence time. Of the several crosslinking methods available, for this work, we chose a biorthogonal Diels−Alder click crosslinking reaction between TET and TCO click pairs, which has an exceptionally high reaction rate.34 TET and TCO click pairs are an example of a copperfree click reaction that inhibits severe cellular cytotoxicity. A TET- and TCO-based click system provides a platform for the simultaneous crosslinking and labeling of multiple biomolecules in biological environments.34−36 Thus, the TET and TCO click system is one of the most useful reactions for biomedical applications, even though further investigation into its long-term safety in biological environments is needed. In this study, we developed an injectable click-crosslinkable formulation using TET-HA and TCO-HA to form a Cx-HA depot for the treatment of RA. MTX was easily solubilized into TET-HA and TCO-HA solutions. The MTX-TET-HA and MTX-TCO-HA solutions were easily loaded into each compartment of a dual-barrel syringe and showed good stability for several days, without precipitation of MTX before injection. The MTX-TET-HA and MTX-TCO-HA were easily injected intra-articularly into rats using a 26 G needle, and they formed an MTX-Cx-HA depot. In an experiment designed to examine residence time using NIR fluorescence imaging, NIR-Cx-HA that formed in vivo after intra-articular injection maintained its structural integrity for more than 2 weeks, while NIR-HA almost completely disappeared within 4 days. We confirmed that the injectable formulations of MTX-TET-HA and MTX-TCO-HA success24995



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exposure of the diseased tissue to the therapeutic agent will be short and thus there is a low therapeutic efficiency against RA. However, the MTX-Cx-HA formulation created a Cx-HA hydrogel depot for MTX at the articular joint injection site and maintained a therapeutic concentration of MTX over a prolonged period. The in vivo distribution of MTX in MTXCx-HA depots showed that high MTX concentrations were maintained at the intra-articular injection site for an extended period, with little or no distribution of the MTX to other organs. This pattern of distribution was probably due to the retention of MTX inside the Cx-HA depot formed at the intraarticular injection site. These results suggest that the high MTX concentrations in the articular joint arising from the use of MTX-Cx-HA can increase the duration of exposure to the drug for RA-affected tissue, thereby enhancing its therapeutic efficiency against RA. This approach should not cause adverse effects in RA patients. The injectable formulation proposed in this study retained MTX inside the Cx-HA depot that formed at the intra-articular injection site for an extended period and thus represents an efficient strategy for RA treatment. During evaluation of the treatment of RA rats with the injectable formulation described here, the RA rats had the most effective RA mitigation after the intra-articular injection of MTX-Cx-HA, in terms of AI score, greater cartilage thickness in sections of synovial tissue, extensive regeneration of chondrocytes and GAG deposits, and extensive new bone formation in the RA-affected region. The MTX-HA formulation took the second place, and MTX alone produced the weakest therapeutic effect. TNF-α overexpression was pronounced in the RA rats without drug treatment, followed by treatment with MTX alone. Treatment with MTX-HA reduced the expression of TNF-α in comparison with the RA rats of no-treatment control groups and MTX alone. Strong TNF-α or IL-6 expression during treatment with MTX alone or MTX-HA is indicative of severe RA because TNF-α and IL-6 are believed to be primarily responsible for the stimulation of systemic inflammation in RA and to play a major role in RA progression. Although some TNF-α expression in RA rats treated with MTX-Cx-HA was observed after 1 week, it had almost disappeared after 3 weeks. A similar pattern was observed for CD4 and ED1 expression following MTX-Cx-HA treatment. These observations indicate that TNF-α expression, macrophages, and T cells in RA are almost completely suppressed by the long-lasting release of MTX from the MTX-Cx-HA depot.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b04979.

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Experimental section; scheme of TET-HA and TCO-HA and 1H NMR of HA, TET-HA, and TCO-HA (Figure S1); storage and loss moduli of Cx-HA hydrogels and photo of ejecting TET-HA and TCO-HA through a 26 G syringe needle from each compartment of a dualbarrel syringe without clogging in PBS for 35 s (Figure S2); SEM of HA and Cx-HA hydrogel (Figure S3); images of swelling of Cx-HA at 1 and 48 h, swelling ratio (%) of Cx-HA vs time, images of Cx-HA at 1 day and 14 days, and mass change ratio (%) of Cx-HA vs time (Figure S4); confocal images of RAW 264.7 cells treated without and with MTX for 1, 4, and 7 days (Figure S5); fluorescence intensity of RAW 264.7 cells treated without a drug, with FI-MTX alone, FI-MTX-HA, or FI-MTX-Cx-HA for 1, 4, and 7 days (Figure S6); H&E staining and the thickness of articular knee joint for RA rats received intra-articular injection without a drug, with MTX alone, MTX-HA, or MTX-Cx-HA as well as normal rats at 1, 3, and 6 weeks (Figure S7); CD4- and ED1-staining of articular knee joint for RA rats received intra-articular injection without a drug (no treatment), with MTX alone, MTX-HA, or MTX-Cx-HA as well as normal rats at 1, 3, and 6 weeks (Figure S8); MTX released amount from HA or Cx-HA vs release time (Figure S9) (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 82-31-219-2608. Fax: 8231-219-3931. ORCID

Moon Suk Kim: 0000-0002-6689-1825 Author Contributions

J.S. and S.H.P. are equal first authors. Author Contributions

J.S., S.H.P., and M.J.K. prepared hydrogel, measured hydrogel properties, and carried out cell experiment and animal experiments. J.S., S.H.P., X.Y.Y., and M.S.K. carried out and analyzed micro-CT. J.S., S.H.P., H.J.J., and M.J.K. carried out imaging analysis for drug and hydrogel. J.S., S.H.P., H.J.J. B.H.M., and M.S.K. carried out and analyzed the stained images. M.S.K. designed all experiments and wrote the paper. All authors discussed the results and commented on the manuscript. Notes

5. CONCLUSIONS

The authors declare no competing financial interest.

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Collectively, these results support the hypothesis underlying this study that intra-articularly injected MTX-Cx-HA can persist at the joint site and maintain therapeutic MTX concentrations for an extended period, without significantly affecting other organs, and thereby induce significant repair of RA-affected joints. Additionally, direct intra-articular injection of an MTX formulation can fulfill the unmet need for the reversal of (pre)clinical RA by increasing the exposure time of the RA-affected site to a drug, with few adverse systemic effects on other organs or tissues.

ACKNOWLEDGMENTS This study was supported by a grant from the Basic Science Research Program (2016R1A2B3007448) and the Priority Research Centers Program (2010-0028294) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education and from the Korea Health Technology R&D Project (HI17C2191) through the Korea Health Industry Development Institute funded by the Ministry of Health & Welfare. 24996


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