s2fieldsofstudy: Biology Chemistry

text:
Further Exploration of the Benzimidazole Scaffold as TRPC5 Inhibitors: Identification of 1‐Alkyl‐2‐(pyrrolidin‐1‐yl)‐1H‐benzo[d]imidazoles as Potent and Selective Inhibitors

Abstract The transient receptor potential cation channel 5 (TRPC5) plays an important role in numerous cellular processes. Due to this, it has gained considerable attention over the past few years as a potential therapeutic target. Recently, TRPC5 has been shown to be involved in the regulation of podocyte survival, indicating a potential treatment option for chronic kidney disease. In addition, a recent study has shown TRPC5 to be expressed in human sensory neurons and suggests that TRPC5 inhibition could be an effective treatment for spontaneous and tactile pain. To understand these processes more fully, potent and selective tool compounds are needed. Herein we report further exploration of the 2‐aminobenzimidazole scaffold as a potent TRPC5 inhibitor, culminating in the discovery of 16 f as a potent and selective TRPC5 inhibitor.

Introduction
The transient receptor potential (TRP) channel family act as molecular sensors and are engaged in various roles. This larger TRP class is further subdivided into six subgroups: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRP (polycystin) and TRPML (mucolipin). [1,2] The TRPC channels are Ca 2 + permeable nonselective cation channels and are a collection of seven different proteins. These channels are subdivided into four subclasses: TRPC1, TRPC4/5, TRPC3/6/7, and TRPC2 based on their sequence similarity. [1,2] The TRPC4/5 and TRPC3/6/7 subclasses share~80 % amino acid homology. Individual channels can function as either homo-or heterotetramers consisting of TRPC5 family channels and other TRP channels. This family of proteins is also referred to as receptoroperated channels due to their activation by PLC after stimulation with receptor tyrosine kinases or GPCRs. Recently, the TRPC3, TRPC4, TRPC5, and TRPC6 structures have been solved using Cryo-EM which will provide valuable information for the structural basis for understanding the mechanism of these channels. [3][4][5] Recently, our laboratory and others have developed new TRPC5 tool compounds and have shown the role that TRPC5 plays in chronic kidney disease (CKD), specifically focal segmental glomerulosclerosis (FSGS). [6,7] AC1903, 1, was the first reported selective TRPC5 antagonist and this was shown to block TRPC5 channel activity in glomeruli of proteinuric rats ( Figure 1). [8] Administration of 1 was able to suppress proteinuria, prevent podocyte loss in an animal model of FSGS, and provide benefit in a rat model of hypertensive proteinuric kidney disease. [8] This was the first small molecule TRPC5 antagonist to provide in vivo benefit in an animal model. [9] Next, the discovery and characterization of GFB-8438, 2, from Gold-finch Bio, was reported and was shown to reduce both total protein and albumin concentrations in urine, a key biomarker for CKD. [10] Goldfinch has since advanced another compound, GFB-887, into Phase 2 clinical trials. More recently, 3 was discovered and it also showed benefit against protamine sulfate (PS)-induced podocyte injury in vitro. [11] In addition to CKD, TRPC5 antagonists have shown activity in animal models of depression and pain. Namely, 1 was active in vivo against inflammatory mechanical and spontaneous pain in mice. [12] And, HC-070, 4, was active in animal models of anxiety and depression. [13] The furan bearing benzimidazole compound AC1903, 1, played a significant role in establishing that TRPC5 inhibition prevents and reverses podocyte injury in FSGS. [8] However, much improvement needs to be made toward its potency and pharmacokinetic liabilities owing to the presence of the furan ring. In this study to identify new inhibitors, we focused our attention on additional analogs around the benzimidazole core. These compounds included a 2-position N-linked alkyl or a saturated N-heterocycle containing benzimidazole. We started our exploration on an N-1-benzyl containing benzimidazole and tried various 2-position substitutions.

Chemical synthesis of benzimidazole analogs
The synthesis of the target compounds was achieved following a procedure that we previously published. [14] The commercially available 2-chlorobenzimidazole, 5, was alkylated with an appropriately substituted benzyl bromide (DMF, NaH, 0°C) to yield 6 (Scheme 1). Next, the amino compounds were reacted with the substituted 2-chlorobenzimidazole under microwave conditions (μW, 200°C, 30 min) to obtain the final targets 7 a-j. Similarly, the reaction sequence could be reversed to explore the southern portion of the molecule.
The 5-and 6-substituted analogs were synthesized as outlined in Scheme 2. To this end, the 2-nitroaniline, 10, was alkylated with BnBr to yield 11. Next, the nitro group was reduced (H 2 /Pd) and then cyclized with 1,1'-carbonyldimidazole (THF, rt) to yield the benzimidazole-2-one, 12, which can be converted to the 2-chloro derivative, 13 (POCl 3 , PCl 3 , 90°C). [15] The final targets, 14 a-c,i-o, were completed following the procedure outlined in Scheme 1. The O-linked benzimidazole analogs, 14 d-k were accessed after demethylation of 15 (BBr 3 ) followed by alkylation (Cs 2 CO 3 , RÀ Br, DMF, 60°C). The synthetic procedures outlined above allow for modular synthesis of the final targets and easy access to multiple points of diversification.

Biological activity of benzimidazole derivatives
Previously, our work centered around replacing the furan with other heteroaryl moieties [14] ; however, this work looks to find a replacement of the furan with alkyl or cyclic alkyl groups ( Table 1). The previously identified, 1, was used as the standard control and the % inhibition at 3 μM was normalized to 100 %. We triaged compounds by assessing % inhibition of the TRPC5 channel using the Syncropatch instrument to have a higher throughput capacity for SAR, which is then followed up with IC 50 determinations of active compounds. Replacing the methylfuran moiety with six-membered compounds 4-methylpiperidine, 7 a, and piperidine, 7 b, were only moderately active (~50 % inhibition) compared to 1. Reducing the ring size to the 5-membered pyrrolidine, 7 c, resulted in a compound equipotent to 1 with an IC 50 = 4.30 μM. Since pyrrolidine can also be susceptible to oxidative metabolism, we tried to incorporate a 2-methyl substitution, which provided compounds that were equipotent with 1 (100 % inhibition at 3 μM) with the (S)-7 d or (R)-7 e stereoisomers showed a slight preference for 7 e. Any attempt to elongate the right hand side with the help of even bulkier heterocycles such as N-4-(1methylpiperidin-4-yl)piperazine, 7 h, and diazaspiro [3.5]nonane, 7 i, were not productive. Adding a two-carbon spacer to 7 c resulted in a compound, 7 j, with moderate activity. However, the addition of straight chain alkyl groups proved to be a productive change as N-propyl, 7 f, and N-isopentyl, 7 g, were potent with an IC 50 of 0.72 μM and 1.3 μM, respectively.
Moving forward, we explored the southern benzylic substitution on the active pyrrolidine moiety, 7 c. The pyrrolidine 7 c was chosen as this provided an easier synthetic handle to explore the southern portion. Active compounds would then be followed up with an additional SAR around the 2-position. We started with evaluating the southern portion using electronwithdrawing and electron-donating groups ( Table 2). The fluoro and chloro compounds (9 a-e) provided potent compounds and no discernible difference between the halogens (i. e., F and Cl with same substitution). The 4-substitution appears to be more potent (9 b, 1.49 μM vs. 9 c, 0.59 μM and 9 d, 0.72 μM vs. 9 e, 0.44 μM) and each were better than the 2-substituted analog. A variety of substitutions on the benzyl system provided more potent compounds than 1 with 4-methyl (9 k, 0.90 μM), 4chloro-2-fluoro (9 n, 0.84 μM), and 3-cyano-4-fluoro (9 o, 0.54 μM) being of interest. Interestingly, switching the chloro and fluoro to the 2-chloro-4-fluoro derivative, 9 m, was not active. Addition of a heteroaryl in the form of pyridine (9 q-s) was not active, although addition of a 2-chloro (9 t, 3.11 μM) did regain some potency which may indicate the basicity of the pyridine nitrogen is important. Any attempt to mask the benzylic methylene with a carbonyl or sulfone, 9 u and 9 v, yielded inactive compounds.
Next, we tried to diversify the benzimidazole core itself by incorporating various substituents on the C-5 and C-6 positions of the phenyl ring in the benzimidazole compound. The 5,6dimethylbenzimidazole (14 b, 101 % inhibition, IC 50 = 12.3 μM) had moderate activity (Table 3). Unfortunately, this portion of the molecule did not allow for modification as nearly all the compounds evaluated were not active. Free hydroxy provided an excellent handle to make ethers of varied length; however, elongation beyond a simple methoxy resulted in a reduction of activity. Next, we tried to incorporate the chloro pyridazinone moiety from the Goldfinch Bio compounds to see if it would be tolerated. We attached it to C-5 via a CÀ C bond as in 14 n but the activity was not as active as 1 or the reported Goldfinch compounds. [10] Finally, we wanted to merge the active N-1 substitution 4chlorobenzyl with some active and diverse C-2 substitution ( Table 4). The 2-or 4-methylpiperidine, 16 a and 16 b, were not active, nor was the thiomorpholine-1,1-dioxide, 16 c. Reinvestigating the 2-methyl pyrrolidine, the (R) stereoisomer, 16 e, (IC 50 = 2.89 μM) was more active compared to (S)-enantiomer, 13 d. The most significant improvement was observed with the N-propyl containing moiety, 16 f, which turned out to be the most active compound in the series with an IC 50 = 0.068 μM. 16 f is the first sub 100 nanomolar compound in the benzimidazole core containing series of molecules.
Next, we further evaluated selected compounds based on liver microsome stability and plasma protein binding (Table 5). [16][17][18] Most of the compounds tested were not stable in either human or mouse liver microsomes. Even addition of the 4-chlorine to block the para-oxidation of the phenyl ring system did not provide any beneficial effects. This could be due to oxidation of the pyrrolidine moiety. Addition of the 3-cyano-4fluoro groups in 9 o did improve the stability in both human and mouse, although only modestly. All the compounds tested displayed good free fraction (> 2%) in both human and mice, except for 9 e and 9 n. In addition, we profiled select compounds (9 o and 16 f) for selectivity against the closely related TRPC4 channel and we tested them against the Psychoactive Drug Screening Panel at the University of North Carolina, Chapel Hill. [19] This panel consists of 45 receptors and transporters that are of importance in the CNS. 9 o was active against PBR (Ki = 399 nM) with no other receptor Ki < 7.0 μM. 16 f was active against H1 (Ki = 6.2 nM), 5-HT 2B (Ki = 249 nM), α 2B (Ki = 677 nM) and α 2C (Ki = 434 nM) showing these compounds are predominantly selective for TRPC5, except for the noted exceptions above (Supporting Information Table 1).
Lastly, we evaluated a few key compounds in an in vivo cassette experiment utilizing a 4-in-1 experiment design (Table 6). [20,21] In order to conserve resources as well as animals, we chose to do the in vivo PK experiments in rats so that we could serially sample as well as utilize the cassette design. Based on the balance of the potency and in vitro PK properties, we chose 9 o and 16 f for the cassette. As can be seen in Table 6, both compounds displayed high clearance with short half-lives (CL > 55 mL/min/kg; t 1/2 < 1 hr). The compounds also had good V ss (1.8-2.3 L/kg) and good exposure. As TRPC5 is of interest for a multitude of CNS disorders, we also profiled the Table 1. SAR of the right-hand portion.

Syncropatch pharmacology
The potency of the compounds was measured by inhibition of riluzole-induced TRPC5 currents as measured on the automated patch clamp platform Syncropatch 384PE (Nanion). Extracellular solution was composed of: 140 mM NaCl, 5 mM KCl, 10 mM HEPES, 2 mM MgCl 2 , 2 mM CaCl 2 and 10 mM glucose. Intracellular solution was composed of: 20 mM CsCl, 90 mM CsSO 4 , 5 mM EGTA, 2 mM CaCl 2 , 10 mM HEPES, 10 mM glucose, 4 mM Na 2 -ATP and 0.5 mM Na-GTP. A ramp protocol of 200 ms from À 100 mV to + 100 mV was pulsed at 5-10s intervals to measure TRPC5 currents. Baseline currents were recorded followed by addition of riluzole to a final concentration of 30 μM to activate currents. Only cells that developed the characteristic doubly-rectifying current of TRPC5 homomers were taken for subsequent analysis. The compound being tested was then added while maintaining 30 μM of riluzole in solution. Cells were discarded if there was noticeable runup or rundown in the current generated from the last 3 voltage ramps before compound addition. Percent inhibition was calculated as the average of the 3 ramps after compound addition (once the current had stabilized) divided by the average of the 3 ramps before compound addition. IC 50 's were calculated by using the three parameters nonlinear regression (curve fit) function of Prism 7 (GraphPad).


Heterogeneous Photocatalysis of Amoxicillin under Natural Conditions and High-Intensity Light: Fate, Transformation, and Mineralogical Impacts

The β-Lactam antibiotic amoxicillin is among the most widely used antibiotics in human and veterinary medicine. Consequently, amoxicillin is abundant in natural waters and can undergo diverse abiotic reactions to form degradation compounds under environmental conditions. Yet, little is known about these decay pathways and mineralogical impacts on environmental amoxicillin degradation. The current study focuses on understanding the mineralogical influences of amoxicillin degradation under ecological conditions. We studied the role of anatase and kaolinite on amoxicillin degradation under irradiated and non-irradiated conditions. Anatase increases amoxicillin degradation by 4.5-fold in the presence of light compared to just being exposed to sunlight. Interestingly, anatase also showed a higher degradation rate under dark than light controls. Conversely, kaolinite diminishes the amoxicillin degradation under irradiation. The formation of degradation compounds was mineralogy-controlled, while no mineralization was observed. Further, we irradiated amoxicillin with a high-intensity light to evaluate its removal from wastewater. The formation of varying amoxicillin degradation products with high-intensity light will limit its removal from wastewater. Our study emphasizes that the mineralogical impact on amoxicillin degradation is diverse, and the role of anatase is significant. Consequently, the increased addition of manufactured titanium nanoparticles to the environment can further enhance these effects.

Introduction
Pharmaceuticals in the environment are a recurring problem that threatens human health and ecosystem balance due to their widespread availability. Pharmaceuticals are designed for specific therapeutic functions and may cause unexpected physiological effects on non-target species [1]. The potential consequences of these compounds in the environment have to be considered seriously, and growing attention has been directed to pharmaceuticals in ecological systems. The primary pathway for pharmaceuticals to penetrate the environment is direct disposal by hospitals, industrial, and domestic sewage. Additional sources of drug pollution are represented by metabolites, mainly via human and veterinary excretion [2][3][4][5]. Given that only about 25% of antibiotics are effectively used in aquaculture, the animal body poorly absorbs most antibiotics, and 30-90% of antibiotics are excreted via urine and feces into the environment. Consequently, antibiotics are frequently detected in surface water, groundwater, soils, and sediments [1,6,7].
Amoxicillin, a semi-synthetic broad-spectrum β-lactam antibiotic, is one of the most widely used antibiotics in the world. This drug is widely used in veterinary practices and can reach the environment primarily via aquaculture, livestock breeding, or human and veterinary excretion. It has been used extensively for disease control and livestock feed for several decades due to its excellent therapeutic value [1,4,7,8]. As a result, livestock agriculture is considered a significant source of amoxicillin pollution. Human excretion is recognized as another major entry point. Previous reports indicate that amoxicillin is poorly metabolized in the human body, and consequently, 40-80% of unmetabolized amoxicillin is excreted after oral application [2,6,9].
The continuous input of amoxicillin and poor metabolism in the human body should result in a relatively high concentration of amoxicillin in wastewater and surface water. Conversely, the reported amoxicillin concentrations in wastewater are at low levels [10,11]. Previous reports indicate that the environmental concentrations of amoxicillin are typically low in raw sewage, which can vary from several ng L −1 to μg L −1 [1,9,12,13], except some rare cases showed mg L −1 levels [12]. The infrequent detection of amoxicillin may be explained by a short retention time of amoxicillin in the environment due to the highly reactive β-lactam ring, which can be broken under environmental conditions and transformed into different products [4,6,[9][10][11]14]. On the other hand, these compounds are tremendously challenging to trace due to the too low sensitivity of the analytical methods and lack of standards [4,10,15].
The fate of amoxicillin in the environment can be influenced by various factors, such as the physicochemical properties of the amoxicillin and the characteristics of the receiving environment. It can thus undergo degradation via various biotic, involves microorganisms mediated degradation of the organic compound, and abiotic pathways. Photolysis, hydrolysis, and chemical oxidation in surface waters are the most critical abiotic processes to convert amoxicillin into various secondary compounds [4]. Certain degradation products of amoxicillin have been found in the natural environment. A major degradation product, amoxicillin diketopiperazine, was confirmed in Israel wastewater effluents [10] and Spain river water [15] samples. These degradation compounds may exhibit more persistent and more hazardous than amoxicillin. For example, amoxicilloic acid, one of the primary degradation products of amoxicillin, retains allergenic properties that can cause adverse reactions in sensitive individuals [9].
A handful of studies have reported their attempts to identify amoxicillin degradation compounds under various simulated environmental conditions [14,16]. These studies have no or limited focus on the mineralogical impacts of amoxicillin degradation. The environmental minerals contain catalytic surfaces, which can result in different degradation products and decay rates of amoxicillin in the environment in combination with sunlight and water. Therefore, the current work's most considerable interest is understanding potential mineralogical impacts on environmental amoxicillin degradation. Here, we selected two common environmental minerals, anatase and kaolinite. Anatase (TiO 2 ) is a semiconductor mineral, present in suspended soil particles, with high photocatalytic activity [17,18]. On the other hand, kaolinite was selected as a proxy for the soil clay minerals [7,[19][20][21][22].
Continuous exposure to low, subtoxic concentrations of amoxicillin can cause unexpected consequences and unintended effects on non-target species, such as the development of antibiotic-resistant microbial strains [8,13] and some negative impacts on the growth of several algal species in the environment [23]. Chronic exposure to antibiotics or other toxicants, along with antibiotics, may exert pressure on the evolution of antibiotic-resistant bacteria, which has become a significant environmental concern [3,4,7]. Furthermore, the bioaccumulation of small quantities of antibiotics may cause severe problems for humans and animals in the long term [4]. Therefore, amoxicillin can be recognized as a significant environmental contaminant. Due to its high usage in food-producing animals, amoxicillin residues can be present in food, leading to long-term health problems in humans and the spread of drug-resistant microorganisms. To ensure the safety of food for consumers, the European Union (EU) Commission has laid down maximum residue limits of 50 ppb of amoxicillin in animal tissues and four ppb in milk [24]. Additionally, degradation products of amoxicillin can enhance different interactions among diverse species within the ecosystems. These interactions can have more adverse effects on humans than parent amoxicillin. Yet, the amoxicillin degradation products' occurrence, persistence, and toxicological effects are poorly understood. Although our current work does not focus on reporting such toxicological impacts, the results of this study can be exploited to make confident predictions of those issues in future studies.
Several chemical and biological methods for antibiotic removal from wastewater sludge have been proposed. These approaches include chemical processes such as ozonation, solid-phase separation, flocculation, coagulation, ultrasonic degradation, photodegradation, electrochemical degradation, and advanced oxidation processes [25,26]. Antibiotic degradation via biological processes has also been proposed [25]. These technologies suffer from various flaws as they are technically sophisticated or economically unfavorable. Moreover, these processes can result in antibiotic degradation products, potentially more toxic than the parent compound [14]. Therefore, the current work further focuses on mineralogy-controlled or mineralogy-dependent amoxicillin degradation with a highintensity light to access such methods to remove amoxicillin from wastewater. Overall, the current study reports the photodegradation of environmental amoxicillin and the formation of degradation products, which are highly mineralogy-dependent.

Materials
Amoxicillin (98% purity) was purchased from the TCI chemicals and used without further purification. Amoxicillin solution of 1 mM was prepared using double DI Milli-Q water (Res > 18.2 MΩ, Millipore Advance A10, MilliporeSigma, Burlington, VT, USA). Anatase and kaolinite KGa-1b were purchased from the Source Clay Repository in Washington County, Georgia, USA, as proxy soil minerals.

Particle Characterization
The shape and size of mineral particles were determined from single particle analysis with a scanning electron microscope (SEM) and a transmission electron microscope (TEM). The detailed experimental protocols are provided in our previous work [27][28][29]. The size distribution was determined by analyzing ~800 particles using the software package ImageJ.
Surface areas of mineral samples were measured in a seven-point N 2 -Brunauer-Emmet-Teller (BET) isotherm using a Quantachrome Autosorb-1 surface area analyzer. Samples were outgassed overnight (~24 h) at a temperature of 105 °C prior to the BET analysis. X-ray diffraction (XRD) of mineral proxies was measured in an X'Pert Pro Diffractometer (Malvern Panalytical, Westborough, MA, USA) equipped with a Cu source. Sample spectra were compared with reference XRD using ICDD database provided by International Centre for Diffraction Data. Attenuated total reflectance-Fourier transform infrared (ATR)-FTIR spectra of samples were obtained using a Nicolet IS50 spectropho-tometer (Thermo Fisher Scientific, Waltham, MX, USA), equipped with a liquid-nitrogen cooled mercury-cadmiumtelluride (MCT) detector and Ge ATR element.

Batch Reactor Studies-
The experiments were conducted under a nitrogen environment in custom-built glass reactors with a suspension capacity of 100 mL and a removable air-tight cap with a quartz window on the top. The quartz window (12.5 cm 2 ) permitted light entry during the solar experiments. During light experiments, the solution surface received a solar flux of ~105 mW/cm 2 and remained constant throughout reaction time. During the experiments, 1 mM amoxicillin solutions were used. Due to the difficulty in detecting transformed products in the final mixture, a high initial concentration of amoxicillin was used. On the other hand, these compounds are tremendously challenging to trace due to the low sensitivity of the analytical methods and lack of standards. Further, the added amoxicillin can be continuously transformed into other products leading to lower reported values. Therefore, concentrations of amoxicillin in the natural environment can be at higher levels than we used in the current study. These experiments were conducted in the presence and absence of a solar simulator (150 W xenon lamp, New Port Corporation, Irvine, USA) with Air Mass 1.5G and Air Mass 0 filters. The temperature was kept constant throughout the experiment using a water jacket. The particle loading was maintained at 0.1 g L −1 of anatase and kaolinite in solutions of 1 mM amoxicillin. The pH of the amoxicillin solution was allowed to vary freely during the experiment and was measured every 24 h (Thermo Orion, model 9272, Thermo Fisher Scientific, Beverly, MA, USA, equipped with an Orion Ross combination electrode). Over 14 days, samples were removed at 24 h intervals from the reactor using a disposable syringe connected to 12 cm of Teflon tubing. Aliquots (1.5 mL) were collected into HPLC vials after passing through a 0.2 μm PTFE filter (Cole-Parmer, Vernon Hills, USA). The filtered extracts were analyzed by reverse-phase HPLC (Agilent HPLC 1200 series, Eclipse Plus C18 Column, UV detector). The HPLC analyses were performed in isocratic mode with mobile phase 15% acetonitrile and 85% sodium phosphate buffer at 1 mL min −1 flow rate. The wavelength of the UV detector was set to 220 nm. The HPLC validation data for linearity is provided in Figure S5 in Supporting Information.

Liquid Chromatography-Mass Spectroscopy (LC-MS)-Filtered samples
collected at the end of the batch reactor experiments were analyzed using LC-MS to identify the amoxicillin degradation products in the reaction mixture. All LC-MS experiments were performed at the Montana State University Proteomics, Metabolomics, and Mass Spectrometry Facility. An Agilent 1290 LC was coupled to an Agilent 6538 quadrupole time-of-flight mass spectrometer. Liquid chromatography was performed on Waters Acquity C 18 HSST3 (1.8 μm particle size, 2.1 × 100 mm) column maintained at 50 °C. The mobile phases consisted of water (0.1% formic acid) as solvent A and acetonitrile (0.1% formic acid) as solvent B. The initial mobile phase composition of 95% A was maintained for 1 min, followed by a linear gradient to 95% B over 4 min, held at 95% B for 4.5 min, and returning to 95% A for 0.5 min. A flow rate of 0.6 mL min −1 was used. The LC eluent was coupled to an electrospray ionization source operating in the positive-ion mode. The mass spectrometer was operated at 1 Hz over a mass range of 50-1000 m/z.

Statistical Analysis
All dark and light experiments were conducted at least in triplicate with average measurements reported. The kinetic of amoxicillin degradation is modeled as the pseudofirst-order using the equation ln C/C 0 = −kt.

Particle Characterization
Surface and bulk characterization of the mineral particles provides essential information on their particle size, shapes, crystal phases, and purity. As reported in our previous work [27,28], anatase (TiO 2 ) has an average particle size of 25 (±3) nm, determined from SEM and TEM images. That of kaolinite was 46 (±5) μm. The specific surface areas of anatase and kaolinite were 59 (±2) m 2 g -1 and 10.1 (±0.1) m 2 g -1 , respectively. SEM image showed that the clays are highly irregular in shape. XRD patterns of mineral samples are in good agreement with their respective reference XRD patterns, confirming no phase change or Ellepola and Rubasinghege Page 5 Environments. Author manuscript; available in PMC 2022 September 22. contamination. ATR-FTIR spectrum of kaolinite showed the absorption bands at 3750-3550 cm −1 , confirming the presence of surface hydroxyl groups [29].

Photo-Degradation Studies
The photodegradation of amoxicillin was measured in the presence of anatase and kaolinite under dark and irradiated conditions. A solar simulator was used as the light source. Samples were irradiated with terrestrial solar spectrum using AM 1.5G filter and highintensity light using AM 0 filter. The solar spectrum corresponding to each filter is provided in Figure S6 in Supporting Information. The kinetics of amoxicillin degradation is modeled as the pseudo-first-order using the equation ln C/C 0 = −kt to calculate initial decay rates for the first 150 h [30][31][32]. All the observed rate constants, half-life, and R 2 values are given in Table 1. The kinetic data with fitted lines are provided in Figure S2 in Supporting Information. It is essential to mention that heterogeneous processes can be more complex than a simple first-order reaction, including the formation of adsorption complexes, equilibrium reactions among adsorbed and "free" chemical species, desorption, and many more. The observed minor deviations from the linearity for some experimental conditions in Figure S2 could be due to these processes, which are unaccounted for in the pseudo-first-order model. The reactions among adsorbed complexes are challenging to evaluate, and we suggest them for future studies.

Amoxicillin Degradation with Terrestrial Solar Spectrum (AM 1.5G Filter)
The photodegradation of amoxicillin under terrestrial solar light is shown in Figure 1A.
The contribution of the direct photolysis on the amoxicillin degradation in the experimental conditions adopted was relatively low. In the "Light Control," which does not contain any mineral, only 30% of amoxicillin was degraded after 14 days of irradiation ( Figure 1A). The rate constant and half-life for the Light Control were 0.00174 h −1 (R2 = 0.994) and 398 h, respectively. The observed low photodegradation of amoxicillin can be attributed to little or no overlapping between the amoxicillin light absorption profile (<280 nm) and the effective near UV/vis irradiation reaching the aqueous solution (>300 nm). Previous reports claim that amoxicillin is relatively stable and shows resistance to photolytic transformation in the presence of wavelengths above 300 nm [23,32,33]. Besides direct photolysis, amoxicillin can degrade via hydrolysis. This pathway proceeds via an attack by H 2 O, acting as a nucleophile to the β-lactam ring, followed by ring-opening [32,34]. Thus, a mechanism combining direct and indirect photodegradation of amoxicillin is proposed in the current study. For direct photodegradation, amoxicillin absorbs irradiation to reach a singlet-excited state. It then undergoes intersystem crossing to a triplet excited state, which leads to the transformation of amoxicillin molecules to degradation products, as shown in Equations (1) and (2). For indirect photodegradation, amoxicillin molecules are supposed to react with hydroxyl radicals according to Equations (3)-(5).

AMO*
Products (2) Indirect photodegradation: (5) where AMO is the ground state of pollutant, 1 AMO * is the singlet-excited state of AMO, 3 AMO * is the triplet excited state of AMO [35].

AMO+ O • H Products
It is also important to mention that the Dark Control showed 10% amoxicillin degradation ( Figure 1A), which could be attributed to the hydrolysis reaction taken place by the nucleophilic attack on the beta-lactam ring from water molecules. Interestingly, after introducing the anatase into the reaction mixture with light, the degradation rate of amoxicillin increased 4.4-fold compared to the Light Control, showing the degradation rate 0.00773 h −1 (R 2 = 0.992) with a half-life of 89.6 h. Rapid decay of more than 70% of the initial concentration of amoxicillin ( Figure 1A) was observed during the first 150 h of irradiation with anatase. The increased degradation rate reveals the "catalytic effect" of anatase surface during the amoxicillin degradation.
Anatase is a semiconductor having a bandgap of 3.2 eV. The bandgap is the energy difference between the valence band (VB) and the conduction band (CB). It relates to the photon energy required to excite the electron from the VB to the CB to create an electronhole pair (e − and h + ) [12,32,33,36,37]. During photocatalytic reactions, electrons and holes on the surface of a semiconductor participate in redox reactions that produce reactive species (ROS) such as hydroxyl radicals ( • OH) and superoxide anion radicals ( • O 2 − ) [32,34,36,37].
ROS reacts readily with surface adsorbed organic molecules, either by electron or hydrogen atom abstraction forming organic radical cations or by adding reactions to unsaturated bonds. Moreover, photogenerated holes can directly decompose the organic compounds [32,33,38]. The primary photocatalytic and redox reaction mechanisms are summarized in Equations (6)- (16) [32,36,39].
Photoexcitation: TiO 2 + hv TiO 2 e − + h +  (9) Direct photo holes degradation: organics +h + Degradation Products (10) Photo-excited electron scavenging: Electron-hole recombination e − TR + h + (VB) e − (CB) + heat (16) The current study was carried out under reduced conditions, and thus it is hard to claim the formation of superoxide radicals. Conversely, our experiments cannot expect Equations (11)-(15) in the above photocatalytic oxidation and reduction mechanism. The photodegradation of amoxicillin in the presence of TiO 2 can be thus attributed to the reactive species, i.e., hydroxyl radicals and holes produced [32].
The inherent limitations of the broad bandgap for TiO 2 (3.2 eV) will facilitate the electronhole recombination according to Equation (16), which yields the low photo quantum efficiency, typically limiting its catalytic activity under visible light irradiation. In the absence of an electron scavenger, the photoexcited electron recombines with the valence band hole in nanoseconds [12,32,37]. Electron scavengers are vital in the photocatalysis of TiO 2 to retard recombination. Often, oxygen acts as an electron scavenger, prevents the recombination of the electron-hole pair, and allows superoxide formation (O 2 − ). Superoxide radicals can be protonated to form hydroperoxyl radicals ( • HO 2 ) that can react further to form H 2 O 2 [32,36]. Interestingly, irradiation of amoxicillin with anatase showed a rapid decay even in the reduced environment. The pH of the reaction is considered an essential factor since it influences the surface charge properties of both anatase and amoxicillin.
Amoxicillin molecule possesses amphoteric properties ( Figure S1) due to three main functional groups; COOH (pK a1 = 2.7), NH 2 (pK a2 = 7.4), and OH (pK a3 = 9.6) [31,32,37,40,41]. Similarly, pH affects the surface charge of the catalyst, making it one of the most significant operating parameters in heterogeneous photocatalysis. The net surface charge of the catalyst can be positive or negative due to the amphoteric behavior of the metal oxide catalyst. The pH of the point of zero charge (pH PZC ), the pH at which the Ellepola  surface charge of the catalyst is zero, helps define the effect of pH on the surface charge of the catalyst. Several studies have reported that the pH PZC of TiO 2 ranges between pH 6 and 8 [36]. For acidic conditions (pH < pH PZC ), the surface charge of anatase is positive and increases the attraction for negatively charged contaminants. The anatase surface charge is negative for basic conditions (pH > pH PZC ), attracting positively charged pollutants.
Equations (17) and (18) Figure 1B shows that the pH of the reaction mixture of amoxicillin in the presence of anatase varies from 4.7 to 4.2. The acidic environment is favorable for forming a positive charge at the surface. Meanwhile, amoxicillin owns both positive and negative charges in its structure, allowing more amoxicillin molecules to be adsorbed onto the photocatalyst surface. As a result, photogenerated holes will have less chance to undergo recombination. A large amount of amoxicillin adsorbed onto the anatase surface should be effectively degraded by • OH radicals and photogenerated holes (h + ). Previous reports claim that photogenerated holes (h + ) are the dominant oxidizing species at low pH, where hydroxyl radicals play the dominant role in pollutant oxidation while under neutral or alkaline solutions [30,36,39,42]. Thus, photogenerated holes may play a significant role in the degradation of amoxicillin on the anatase surface. Besides, it is well known that TiO 2 particles tend to form agglomerates when dispersed in aqueous media. Such agglomeration strongly depends on parameters such as ionic strength and the pH of the suspension. It has been reported that agglomeration of TiO 2 particles in water decreases at acidic conditions compared to neutral or alkaline conditions, thus increasing the effective surface area of the catalyst [31,37].
Another possible pathway to retard the electron-hole recombination in an acidic medium is capturing electrons by hydrogen ions according to Equation (19) [27]. It further helps to prevent electron-hole recombination. These factors can collectively enhance the catalytic activity of anatase for amoxicillin degradation.
The overall process of heterogeneous photocatalysis can be decomposed into five steps. First, amoxicillin moves from the fluid phase to the interface region of the catalyst by diffusion. Second, amoxicillin is adsorbed onto the surface of the photocatalyst. Third, a chemical reaction occurs with oxidizing and/or reducing species in the vicinity of the surface. Fourth, by-products desorb. Finally, products are removed from the interface region into the bulk fluid [36].
It is worth highlighting that the catalytic effect of anatase for amoxicillin degradation was also significant under dark conditions. Figure 1A shows that adding anatase to the reaction Ellepola Figure 1B shows that the pH of the Anatase_Dark reaction changed from 4.4 to 4.0, during which the anatase surface was positively charged. Amoxicillin owns both positive and negative charges in its structure at this pH. This will lead more amoxicillin molecules to adsorb to the anatase surface. Further, hydroxyl radicals can rapidly react with the aromatic ring in the side chain of amoxicillin [42]. Hence, the observed rapid degradation of amoxicillin could be attributed to the catalytic effect of surface hydroxyl groups of anatase.
Conversely, the amoxicillin degradation was lower in kaolinite (band gap = 4.99 eV) [43] presence under irradiated conditions than the Light Control. The observed rate constant was 0.00105 h −1 (R 2 = 0.997) with a half-life of 660 h. The photocatalytic effect of the kaolinite for amoxicillin degradation is insignificant due to limited light penetration in the presence of clay particles. However, the observed 20% amoxicillin loss over the 14 days could be attributed to the hydrolysis of amoxicillin via hydroxyl groups on the kaolinite surface. The pH of the point of zero charge (pH PZC ) of kaolinite lies between 6 and 6.5 [44]. Figure 1B shows that pH of the kaolinite reaction under light varies from 4.5 to 3.8, and the kaolinite surface will be positively charged. Amoxicillin owns both negative and positive charges in its structure at this pH. Hence, amoxicillin can be adsorbed to the positively charged kaolinite surface via an ionized carboxylate group. This is further confirmed by the 20% amoxicillin loss in the presence of kaolinite under dark conditions ( Figure 1A). Moreover, we observed a 5-fold increase in amoxicillin degradation compared to the Dark Control, which reveals the catalytic effect of the kaolinite surface to increase the hydrolysis of the β-lactam ring.

Amoxicillin Degradation Mechanism under Terrestrial Solar Radiation
The proposed degradation pathways of amoxicillin degradation under dark and light conditions are discussed below. Our results highlighted that the formation of degradation products is highly mineralogy-controlled. Here, we will compare all the proposed degradation mechanisms with the Light Control. The complete degradation pathway of amoxicillin with the proposed degradation products in the absence of a mineral surface is summarized in Figure 2.
The degradation of amoxicillin in an acidic pH starts with the opening of the strained four-membered beta-lactam ring to yield the hydrolysis by-product 1, amoxicillin penicilloic acid [14,16,34,41,[45][46][47][48][49]. Compound 1 can undergo two possible degradation pathways to yield several different transformed compounds. In one path, the decarboxylation of the free carboxylic acid of compound 1 leads to the formation of stereoisomeric compound 2, amoxicillin penilloic acid [14,16,34,41,[46][47][48]. The subsequent release of the carboxylic group from the penilloic acid generates the degradation compound 3. Another possible degradation pathway of compound 1 is forming a new, stable, six-membered ring, amoxicillin diketopiperazine, compound 4 via cyclization reaction after removing a water molecule [14,16,34,45,49,50]. Compound 2, amoxicillin penilloic acid, can undergo Ellepola and Rubasinghege Page 10 Environments. Author manuscript; available in PMC 2022 September 22. hydrolysis at the amide bond, leading to the formation of another two amoxicillin degradation products, 5 [49] and 6. Several of the above degradation products have been previously reported for actual environmental samples. For example, amoxicillin penicilloic acid was detected in Israel's secondary effluents, and amoxicillin diketopiperazine was detected in groundwater in Israel [14].
After introducing kaolinite into the reaction mixture, we observed the formation of a fewer number of degradation products compared to the Light Control. According to Figure S3, irradiation of amoxicillin in the presence of kaolinite produced only compounds 1, 2, and 3. Here, kaolinite did not facilitate the cyclization reaction of compound 1, amoxicillin penicilloic acid, to form amoxicillin diketopiperazine, compound 4. Further, we did not observe the hydrolysis of compound 2, amoxicillin penilloic acid, to form compounds 5 and 6. As explained above, light penetration is limited after introducing kaolinite compared to the Light Control. Hence, limited energy for amoxicillin molecules to undergo further degradation could be the reason for forming fewer transformed products.
Interestingly, we observed significant differences in the formation of amoxicillin degradation products after introducing anatase into the reaction mixture ( Figure 3). It is worth highlighting that anatase facilitated a different amoxicillin degradation pathway that was not observed in the Light Control or kaolinite reaction. Hydrolysis of amoxicillin amide bond was observed to produce intermediate 7, and subsequent decarboxylation of intermediate 7 produced the degradation compound 8. However, intermediate 7 was not detected in our LC-MS analysis due to the rapid degradation in the presence of the anatase surface. Apart from the amoxicillin amide bond hydrolysis pathway, amoxicillin degradation via hydrolysis of the β-lactam ring was also observed, similar to the Light Control and kaolinite reactions Only compound 3 was observed on the 14th day, while compounds 1 and 2 disappeared. Though the hydrolysis of the amide bond of compound 2 was observed, only compound 5 was detected in the reaction mixture on the 14th day, while compound 6 disappeared. Furthermore, compound 4, amoxicillin diketopiperazine, was not observed. The high catalytic effect of anatase leads to fast degradation of these intermediates, which could be attributed to the disappearance of some intermediates.

Amoxicillin Degradation under Dark Conditions
In the Dark Control, compound 1, amoxicillin penicilloic acid, was the only degradation product observed. Similar to the Dark Control, the kaolinite reaction in the dark also showed the formation of only compound 1 as an amoxicillin degradation compound.
Interestingly, anatase showed significant differences in the formation of amoxicillin degradation products under dark conditions compared to the Dark Control and kaolinite dark reactions. The proposed reaction mechanism is illustrated in Figure S4. We observed that anatase could facilitate the formation of compounds 1, 2, 3, 5, and 6 under nonirradiated conditions. The catalytic effect of the anatase surface via surface hydroxyl groups could facilitate the formation of these compounds. However, it did not form compound 4, amoxicillin diketopiperazine, the cyclization product of compound 1. It should be highlighted that anatase can produce similar amoxicillin degradation compounds under dark Ellepola and Rubasinghege Page 11 Environments. Author manuscript; available in PMC 2022 September 22. conditions similar to the Light Control except for compound 4. These observations further reveal the significant impact of the anatase on environmental amoxicillin degradation.

Amoxicillin Degradation with High-Intensity Light (AM 0 Filter)
Further, we continued our experiments by irradiating amoxicillin under high-intensity light using AM 0 filter in the solar simulator. Our primary focus in these experiments was to understand the possibility of using high-intensity light to remove amoxicillin from wastewater. These decay curves and kinetics of amoxicillin degradation under these conditions are shown in Figure 4. Here, the Dark Control, Kaolinite Dark, and Anatase Dark are the same decay curves presented in Figure 1 and Table 1, and included in Figure 4 for comparison.
We observed that amoxicillin degradation rates were significantly increased in every case. Similar to the terrestrial light, anatase showed the highest degradation rate of amoxicillin (k = 0.0206 h −1 ). This is a 2.7-fold increase compared to amoxicillin degradation under terrestrial light when mixed with anatase. The rate of degradation of amoxicillin in the Light Control was 0.0140 h −1 which is an 8-fold increase from the terrestrial light. In the presence of kaolinite, the amoxicillin degradation rate showed a 12.8-fold increase compared to the AM 1.5G filter light. Even though the degradation rate of amoxicillin was significantly increased with high-intensity light, we did not observe mineralization of amoxicillin in any case. Interestingly, LC-MS analysis conformed formation of degradation products for high-intensity light different from terrestrial light. These degradation compounds' ecological impacts and human health effects are not well understood yet, and thus it will limit the use of high-intensity light in removing amoxicillin from wastewater. The observed rate constants, R 2 , and half-life values for amoxicillin with high-intensity light are given in Table  2.

Amoxicillin Degradation Mechanisms with High-Intensity Light
Our results highlight the formation of several degradation products of amoxicillin under high-intensity light ( Figure 5). When high-intensity light was used in the Light Control, the amoxicillin degradation rate increased significantly compared to the terrestrial light. However, we did not observe differences in the formation of degradation products with a high-intensity light. Instead, the exact degradation mechanism for amoxicillin was observed to be similar to the terrestrial solar spectrum, as depicted in Figure 2.
In contrast, irradiation of amoxicillin with high-intensity light and kaolinite showed extended degradation compared to terrestrial light. Apart from degradation compounds 1, 2, and 3 here, we observed the formation of compounds 4, 5, and 6, which were not detected under terrestrial light ( Figure S3). The amount of available photons for amoxicillin molecules to degrade is higher with the AM 0 filter than with the AM 1.5G filter, and that could be the reason for the formation of compounds 4, 5, and 6 under AM 0 filter.
Excitingly, in the presence of anatase, we observed significant differences in the formation of degradation compounds with a high-intensity light. The proposed degradation mechanism in the presence of anatase and high-intensity light (AM 0 filter) is provided in Figure  5. Amoxicillin was able to undergo deamination reactions with AM 0 filter light which were not observed in any of the other cases. Here, we observed the formation of two new degradation compounds, 9 and 10, via deamination reactions. Hence, compound 5 undergoes deamination to produce compound 9. Additionally, compound 3 yields compound 10 after a deamination reaction.
Similar to the anatase reaction with terrestrial light, amoxicillin undergoes a hydrolysis reaction at the amide bond to form intermediate 7 and subsequent decarboxylation to produce degradation compound 8. Here, amoxicillin also undergoes the usual β-lactam ring hydrolysis pathway. Due to amoxicillin's significantly high degradation rate with anatase and high-intensity light, we did not observe compounds 1, 2, 3, 5, and 7 in the final reaction mixture. Overall, we observed amoxicillin degradation compounds 8, 9, and 10 in the final reaction mixture in the presence of anatase with a high-intensity light. It should be highlighted that compounds 9 and 10 formed via deamination reactions were only observed in high-intensity light and anatase.
Based on our results, we confirm that amoxicillin can degrade significantly faster with a high-intensity light. However, the use of high-intensity light to remove amoxicillin from wastewater is still a concern due to the formation of considerable degradation products in high-intensity light and minerals. Besides, the toxicological impacts of the observed amoxicillin degradation products are not well understood yet and are strongly recommended for future studies.

Conclusions
Our study investigated the photolytic degradation of β-lactam antibiotic amoxicillin upon exposure to light and ecological minerals anatase and kaolinite. Here, we proposed a combination of direct photolysis and indirect photodegradation mechanisms for amoxicillin in the environment. Our study highlighted for the first time that the rate of amoxicillin degradation could be varied with the exposed mineral surface, while anatase caused the highest degradation rate. On the other hand, solar flux under ecological conditions was not sufficient to activate the kaolinite as photocatalyst. Thus, the material acted only as an adsorbent. Moreover, the formation of degradation compounds also varied with the available mineral surface. Light Control formed the compounds 1, 2, 3, 4, 5, and 6, and kaolinite formed only compounds 1, 2, and 3. Anatase formed compounds 3, 5, and 8.
In dark reactions, the effect of anatase was significant on amoxicillin degradation. However, the Light Control and kaolinite mineral presence caused a slow degradation. In terms of amoxicillin degradation compounds, anatase formed compounds 1, 2, 3, 5, and 6, while Light Control and kaolinite reactions produced only compound 1. It reveals that environmental mineralogy can play a significant role in the degradation of amoxicillin and the formation of degradation products regardless of the light availability.
Further, we irradiated amoxicillin with a high-intensity light to understand the possible method to remove amoxicillin from wastewater. Here, we observed that amoxicillin degraded significantly faster with high-intensity light than in terrestrial light-however, the Ellepola  formation of different amoxicillin degradation products limits high-intensity light to remove amoxicillin from wastewater. Here, anatase formed compounds 8, 9, and 10. Light Control and kaolinite reactions formed 1, 2, 3, 4, 5, and 6. The toxicological impacts of these degradation compounds can be very different from the parent pharmaceutical amoxicillin on human health and environmental species. In further research, we suggest investigating the effects of amoxicillin degradation products in the short term and long term.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.  Proposed degradation mechanism of amoxicillin in the absence of mineral surface under dark and terrestrial solar radiation (AM 1.5G filter) Ellepola   Proposed degradation mechanism of amoxicillin in the presence of anatase under irradiated conditions using AM 1.5G filter. Red-colored compounds were not detected in this work. Ellepola Figure 1 and Table 1, and included here for comparison.  Proposed degradation mechanism of amoxicillin in the presence of anatase and highintensity light (AM 0 filter). Red-colored compounds were not detected in this reaction. Ellepola and Rubasinghege Page 22 Environments. Author manuscript; available in PMC 2022 September 22.


s2fieldsofstudy: Biology Medicine

text:
The Therapeutic Effect of iMSC-Derived Small Extracellular Vesicles on Tendinopathy Related Pain Through Alleviating Inflammation: An in vivo and in vitro Study

Background Tendinopathy is a common cause of tendon pain. However, there is a lack of effective therapies for managing tendinopathy pain, despite the pain being the most common complaint of patients. This study aimed to evaluate the therapeutic effect of small extracellular vesicles released from induced pluripotent stem cell-derived mesenchymal stem cells (iMSC-sEVs) on tendinopathy pain and explore the underlying mechanisms. Methods Rat tendinopathy model was established and underwent the injection of iMSC-sEVs to the quadriceps tendon one week after modeling. Pain-related behaviors were measured for the following four weeks. Tendon histology was assessed four weeks after the injection. To further investigate the potential mechanism, tenocytes were stimulated with IL-1β to mimic tendinopathy in vitro. The effect of iMSC-sEVs on tenocyte proliferation and the expression of proinflammatory cytokines were measured by CCK-8, RT-qPCR, and ELISA. RNA-seq was further performed to systematically analyze the related global changes and underlying mechanisms. Results Local injection of iMSC-sEVs was effective in alleviating pain in the tendinopathy rats compared with the vehicle group. Tendon histology showed ameliorated tendinopathy characteristics. Upon iMSC-sEVs treatment, significantly increased tenocyte proliferation and less expression of proinflammatory cytokines were observed. Transcriptome analysis revealed that iMSC-sEVs treatment upregulated the expression of genes involved in cell proliferation and downregulated the expression of genes involved in inflammation and collagen degeneration. Conclusion Collectively, this study demonstrated iMSC-sEVs protect tenocytes from inflammatory stimulation and promote cell proliferation as well as collagen synthesis, thereby relieving pain derived from tendinopathy. As a cell-free regenerative treatment, iMSC-sEVs might be a promising therapeutic candidate for tendinopathy.

Introduction
Tendinopathy is a prevalent musculoskeletal disorder in athlete and non-athlete populations characterized by pain, swelling, and restricted movement. 1 It frequently occurs in the knee, shoulder, as well as hand. Tendinopathy has a high prevalence and incidence rate in general practice, resulting in detrimental effects on patient's daily life. 1,2 Among various symptoms of tendinopathy, pain is the most prominent and disabling one. Rest, oral nonsteroidal anti-inflammatory drugs (NSAIDs), and physical therapy are frequently used to alleviate pain derived from tendinopathy. 3 However, in some rare cases, more invasive interventions may need to be considered when the patient has failed rehabilitation. 4 Besides, most researches regarding tendinopathy focus on promoting tendon regeneration or anti-inflammation, [5][6][7] which lacks direct evidence for alleviating pain. Thus, it is of great value to develop a novel strategy for tendinopathy-related pain.
Inflammation plays a vital role in tendinopathy 8 and is an important mechanism underlying chronic musculoskeletal pain. 9 Traditionally, tendinopathy is considered to be a degenerative disease without inflammation. However, recent research suggests that inflammatory cytokines and immune cells could play critical roles in the pathogenesis of tendinopathy. 10,11 Increasing studies have demonstrated the presence of macrophages, mast cells, T and B lymphocytes in tendinopathy. 12,13 Macrophage-driven inflammatory reaction is an important feature of tendon repair in the early stage, but excessive inflammation is not conducive to tissue repair. 14 Tumor necrosis factor ɑ (TNF-ɑ) is a proinflammatory cytokine involved in the degradation of type I collagen and contributes to tendon degeneration. [15][16][17] Upon stimulation with TNF-ɑ, production of proinflammation cytokines is elevated, including interleukin (IL)-1β, IL-6, substance P, and nerve growth factor (NGF); these further contribute to tendon degeneration and pain. 18,19 NGF is an essential factor in inducing osteoarthritis and lower back pain. 20,21 Taken together, we hypothesize that inflammation plays a crucial role in the pathogenesis of tendinopathy and related pain. Alleviating inflammation is of great value in the management of tendinopathy pain.
Mesenchymal stem cells (MSCs) have been well investigated in the field of inflammatory diseases as well as autoimmune disorders. 22,23 We recently derived MSC from induced pluripotent stem cell (iPSC) and proved this iMSC to have the same character as adult MSC in function. 24 As iMSC overcomes the limitation of tissue-derived MSC, it is a promising source for stem cell therapy. In recent years, MSCs derived small extracellular vesicles (MSCs-sEVs) have attracted much attention as a novel cell-free strategy in tissue repairing. These 30-150 nm vesicles contain various proteins, nucleic acid, and other biomolecules secreted from parent cells and can transport them to the recipient cells. 25 In our previous research, iMSCs-sEVs were observed to attenuate osteoarthritis by alleviating inflammation and promoting chondrocyte proliferation. 26,27 In addition, MSCs-sEVs have been reported to attenuate inflammatory response after tendon injury and promote tendon regeneration by facilitating tendon stem/progenitor cell proliferation and migration. 28,29 However, there is no report on the application of iMSC-sEVs to alleviate pain derived from tendinopathy to date.
In the present study, we investigated the analgesic effect of iMSCs-sEVs on a rat tendinopathy model and further investigated the potential functional mechanism on IL-1β-induced tenocyte inflammation. It turns out that iMSC-sEVs alleviate inflammation both in vitro and in vivo. iMSC-sEVs treatment was found to upregulate the expression of antiinflammation-related genes and those that regulate cell proliferation and tenogenic differentiation under high-throughput transcriptome analysis. Here, we show for the first time that iMSC-sEVs possess the potential to ameliorate pain derived from tendinopathy via protecting tenocytes from inflammatory stimulation, at least in part, by inhibiting inflammation and promoting tenocyte proliferation.

Derivation and Characterization of Induced MSCs
Three human iPSC lines were provided by the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences and the South China Institute for Stem Cell Biology and Regenerative Medicine Group of the Chinese Academy of Sciences and used in generation of mesenchymal stem cells. The local ethics committee approved the use of human iPSC in this study of the Shanghai Sixth People's Hospital affiliated with Shanghai Jiao Tong University. The generation of mesenchymal stem cells from human induced pluripotent stem cells as previously described. 30 Surface antigens of iMSCs were analyzed by flow cytometry. Single-cell suspension was collected and incubated with 1% bovine serum albumin (BSA, Gibco). The iMSCs were then stained with the following monoclonal antibodies (BD Bioscience) against CD73, CD90, CD105, CD34, CD45, CD146and HLA-DR. These cells were further analyzed by a CytoFLEX flow cytometer (Beckman Coulter Life Science, USA). Isolation of iMSC-sEVs iMSC-sEVs were extracted by centrifugation protocols as previously described 31 from the conditioned medium of iMSC. Briefly, the conditioned medium was collected and centrifuged at 300g for 10 min and 2000g for 20 min to remove cells, dead cells, respectively. Afterward, the supernatant was centrifuged at 10,000g for 30 min and filtered through a 0.22-μm filter sterilize Steritop™ (Millipore) to remove cellular debris and microvesicles. This collected supernatant was subsequently subjected to ultracentrifugation at 100,000 × g for 70 min in an SW 32 Ti Rotor Swinging Bucket rotor (k factor of 256.8, 28536 rpm, Beckman Coulter, Fullerton, CA) to pellet sEVs. After removing the supernatant, the pellet was resuspended in phosphate buffer saline (PBS), followed by another ultracentrifugation at 100,000g for 70 min. Finally, pelleted sEVs were resuspended in PBS stored at −80°C. All steps were performed at 4°C under sterile conditions.

Nano-Flow Analysis of the Size and Concentration of iMSC-sEVs
The size and concentration of iMSC-sEVs were assessed using a nano-flow cytometer (N30 Nanoflow Analyzer, NanofCM Inc., Xiamen, China), as described previously. 32 Briefly, the side scatter intensity (SSI) was measured by loading the standard polystyrene nanoparticles (200 nm) with a concentration of 1.58 × 108/mL to the nano-flow cytometer. Then, the isolated iMSC-sEVs sample was diluted with 1000-fold PBS (the nanoparticle concentration was about 5 × 109/mL) and loaded into the nanofiltration to measure SSI. Finally, the concentration of sEVs was calculated according to the ratio of SSI to particle concentration in standard polystyrene nanoparticles. For size measurement, standard silica nanoparticles of mixed sizes (68 nm, 91 nm, 113 nm, 155 nm) were loaded into a nanometer flow cytometer to generate a standard solution, which was then loaded into the sEVs sample. Size distribution is calculated according to the standard curve.

Establishment of Rat Tendinopathy Model
A total of 20 adult female Sprague Dawley rats (200-300 g) were used to develop the tendinopathy model. Rats were randomly assigned to sham or tendinopathy groups with different treatments (PBS, steroid or iMSC-sEVs local injection). The rat tendinopathy model was established as previously reported. 33,34 For this rat tendinopathy model for evaluating pain, we used a variation of carrageenan-induced rat paw edema model in which a single injection of carrageenan solution was made around the quadriceps tendon. The procedure included the following. The carrageenan solution was made by dissolving carrageenan powder (Sigma-Aldrich/Merck KGaA, Darmstadt, Germany) into sterile PBS to make a 4% solution. Briefly, rats were anesthetized with isoflurane, positioned supine, and the fur above the right quadriceps was shaved clean. Then, 100 μL of the 4% carrageenan solution was injected in a single bolus around the right quadriceps tendon with a 27-gauge needle under ultrasound imaging guidance (Fujifilm VisualSonics; Bothell, Washington, USA). The ultrasound probe was a high-frequency 40-MHz linear array transducer positioned parallel to the femur. We were careful not to damage the tendon with the injection needle physically.
Pain-related behaviors (below) were assessed one week after carrageenan injection and once per week for the following 4 weeks. After 4 weeks of treatment, both quadriceps tendons were harvested following histological evaluation after a lethal overdose of anesthetic. The animal research committee approved animal care and experimental procedures of the Hospital. All the rats were housed under specific-pathogen-free conditions, 12/12-hour light/dark cycle, and free access to food and water.

Hind-Paw Withdrawal Threshold
Reflexive measures of pain using stimulus-evoked responses, such as hind-paw withdrawal threshold, are commonly used in rats to assess potential hyperalgesia, and these are thought to reflect clinical expressions of the neuropathic pain. 35 Hind-paw withdrawal thresholds were measured according to a previous report. 36 Briefly, the rats were placed individually in an elevated metal grid cage with sufficient space for them to move their paws while the rest of their body was restricted with plastic plates. After the rats acclimated to the apparatus, the rat's right hind paw withdrawal threshold (PWT) was assessed by an electronic von Frey instrument (model BIO-EVF4; Bioseb, Vitrolles France). The probe tip of the instrument was gently placed perpendicularly into the mid-plantar surface of the paw, and steadily increasing pressure (between 0 and 100 grams) was applied until the hind paw was first lifted. This force is independent of the movements of the limb. The PWT was recorded as the required pressure to first lift the paw. The data were expressed as PWT in grams (g). Lower PWT values (g) were taken as indicators of pain.

Static Weight Bearing
The static weight-bearing (SWB) distribution over the right and left knee was assessed by measuring postural equilibrium between the injected and non-injected leg. 36,37 Briefly, a rat was placed in the chamber of a weight-bearing measuring device (model #BIO-SWB-TOUCH-M; Bioseb) and allowed to acclimate before regular testing. The force applied through each hind limb to the paw resting on the floor of the chamber was measured in grams (g), and an SWB index was calculated as follows:

SWB index ¼
Force applied to right limb Force applied to right limbþ Force applied to left limb The right side received the carrageenan injection. SWBs can vary between 0 and 1, with values closer to zero indicating that postural equilibrium favors the rat's left non-injected side. For each rat, the test was given three times at each assessment period, and the mean value was taken as the SWB at that time point.
iMSC-sEVs (1 × 10 10 particles of iMSC-sEVs in 100 μL PBS) or vehicle (PBS alone, 100 μL) were administered via local injection once a week for 4 weeks. Steroids were prepared by mixing 0.4 mg triamcinolone acetonide (Kunming Jida Pharmaceutical Co., Ltd, Kunming, China) with 100 μL of lidocaine (Shandong Hualu Pharmaceutical Co., Ltd, Shandong, China), which was then injected into the peritendon space of the quadriceps tendon under the ultrasound guidance once a week for 4 weeks. The groups were blinded to the investigators performing behavioral tests and histological assessments. Pain-related behaviors were analyzed each week for the following 4 weeks, after which histological assessment was performed. Reversal (%) of pain-related behaviors was calculated as follows:

Histology and Immunohistochemistry
For histological analysis, the rat tendon samples were fixed en bloc in 4% PFA for 24 hours, dehydrated with a graded ethanol series, embedded in paraffin, and sectioned (5 μm thick) parallel to the long axis of the tendon. One series of sections was mounted on glass slides, cleared with xylene, then hydrated in a graded-ethanol series, stained with hematoxylin and eosin (H&E), and coverslipped. Another series of sections were prepared for immunohistochemical analysis.
Histological and immunohistochemical staining was evaluated and photo-documented digitally with Leica digital camera. Image J and modified Bonar system were used for semiquantitative histological analysis as previously reported. 38,39 Isolation and Culture of Rat Primary Tenocytes In this study, rat primary tenocytes were isolated from rat Achilles tendon as previously described with minor modification. 40 Briefly, The Achilles tendon was detached from the rat and washed with PBS; the tendon sheath and surrounding paratenon were carefully removed. Afterward, the tendon tissues were cut into small pieces and followed by digestion with 3mg/mL type I collagenase (Invitrogen, Carlsbad, CA, USA) in low-glucose Dulbecco's Modified Eagle's Medium (LG-DMEM) for 3-4h at 37°C. After centrifugation at 1000 revolutions per minute (rpm) for 5 min at room temperature, the digested cells were then suspended in LG-DMEM containing 10% (v/v) fetal bovine serum (FBS, Gibco Life Technologies), 100 U/mL penicillin and 100 mg/mL streptomycin. The cells were incubated in humidified conditions at 37°C and 5% CO2, with media changes every 2 to 3 days. After 10 days, cells started to adhere to the tissue culture flask. When tenocytes reached 80% to 90% confluency, they were treated with 0.05% trypsin-ethylene diamine tetra-acetic acid (EDTA) and passaged. Cells from passage 3 to 5 were used for the subsequent experiments.

Uptake of iMSC-sEVs by Tenocytes in vitro
In the in vitro experiment, iMSC-sEVs were labeled with DiO fluorochrome (Thermo Fisher, USA) according to the protocol as previously described 41 with minor modification. Briefly, sEVs were incubated with DiO fluorescent dye under room temperature for 15 min, followed by ultracentrifugation at 100,000g in PBS to get rid of the unlabeled dye. Next, DiO-labeled sEVs were added into the culture medium and incubated with rat tenocytes for 12h. In the control group, the same volume and concentration of DiO dye without iMSC-sEVs labeling was ultracentrifuged and washed as mentioned above, followed by incubation with tenocytes. Next, the culture medium was discarded, and the cells were rinsed twice with PBS and fixed with 4% PFA. After being stained with DAPI solution, the cells were observed with a fluorescence microscope (Leica, DM6B, Germany).

Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) Analysis of Tenocytes
Following treatment of IL-1β with or without iMSC-sEVs for 24h as described above, targeted gene expression analyses were performed by RT-qPCR. Briefly, the total RNA of samples was extracted using EZ-press RNA Purification Kit (EZBioscience, USA). RNA quantity and purity were confirmed with a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE). A 4× Reverse Transcription Master Mix (EZBioscience, USA) was used for reverse transcription reaction. PCR reactions were run using the ABI Prism 7900HT Real-Time System (Applied Biosystems, Carlsbad, CA) with 2× SYBR Green qPCR Master Mix (EZBioscience, USA). The primer sequences used in this study are listed in Supplementary Table 1.

Evaluation of Tenocyte Proliferation
The CCK8 assay was used to determine cell viability under iMSC-sEVs treatment. Rat tenocytes (5000 cells/well) were seeded in 96-well plates and cultured with various treatments for 72 hours. At 24, 48 and 72h after the treatments, the cell viability was measured. Briefly, the culture medium was replaced with a medium containing 10% CCK-8 solution. After incubation with CCK-8 solution for 1h at 37°C, absorbance in each well was measured at 450 nm in a microplate reader.

Enzyme-Linked Immunosorbent Assay (ELISA)
We performed ELISA of the culture medium to evaluate the inflammatory factors secreted by the tenocytes. Following treatment of IL-1β with or without iMSC-sEVs for 48h as described above, the culture medium was collected. Interleukin (IL)-1β, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and nerve growth factor (NGF) concentrations were measured by using a rat ELISA kit (Shanghai Westang Bio-Tech Co., LTD., Shanghai, China) according to the manufacturer's instructions. The absorbance was measured by a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm.

RNA-Seq Analysis
Before constructing the RNA-seq libraries, the total RNA samples (1 μg) of tenocytes were treated with VAHTS mRNA Capture Beads (Vazyme) to enrich polyA+ RNA. RNA-seq libraries were prepared using VAHTS mRNA-seq v2 Library Prep Kit for Illumina (Vazyme) according to the manufacturer's instructions. Briefly, polyA+ RNA samples were fragmented and then used for first-and second-strand cDNA synthesis with random hexamer primers. DNA End Repair Kit was used to repair the ends of the cDNA fragments. The cDNA fragments were then modified with Klenow to add an A at the 3' end of the DNA fragments and ligated to adapters. Twelve cycles of PCR amplification were performed to the purified dsDNA. The libraries were then sequenced by the Illumina sequencing platform on a 150 bp paired-end run. Sequencing reads from RNA-seq data were aligned using the spliced read aligner HISAT2, supplied with the Nov. 2020 (mRatBN7.2/rn7) assembly of the rat genome (rn7, Wellcome Sanger Institute) from UCSC Genome Browser. FPKM (fragments per kilobase of transcript per million mapped reads) was used to calculate the gene expression levels. Gene Set Enrichment Analysis (GSEA) pre-ranked was run on the ranked list using the Molecular Signatures Database (MSigDB) as the gene sets.

Statistical Analysis
All numerical data are presented as means ± SD. Student's t-test was used to evaluate paired group differences, and Wilcoxon signed-rank test was used to evaluate nonpaired groups. Statistical analyses were performed with GraphPad Prism version 8.0.0 for Windows (GraphPad Software, San Diego, California, USA). P value <0.05 was considered significantly different.

Characterization of iMSC and iMSC-sEVs
In the present study, iPSCs-derived MSC (iMSC) was used to produce therapeutic sEVs in alleviating tendinopathy and related pain. Flow cytometry was applied to evaluate the surface antigen profile of these cells to identify iMSC. iMSC highly expresses antigen markers, including CD73, CD29, CD44, and CD146, but does not express CD34, CD45, CD133, and HLA-DR ( Figure 1A). sEVs were isolated and collected from the serum-free culture medium of iMSC by DovePress standard differential centrifugation protocol. The iMSC-sEVs pellets were subsequently resuspended with PBS at an approximate concentration of 2×10 11 particles/mL measured by nano-flow cytometry. After collection, systematic characterization was performed to evaluate the quality of isolated sEVs. Western blot analysis determined the presence of exosomal markers, such as CD9, TSG101, and CD63, whereas the cis-Golgi matrix protein GM130 was not detected ( Figure 1B). The morphology of iMSC-sEVs was typical cup-shaped vesicles, as shown by TEM analysis ( Figure 1C). Nano-flow analysis revealed that the average diameter of collected sEVs ranges from 60 to 160 nm ( Figure 1D).

Analgesic Effect of iMSC-sEVs on a Rat Tendinopathy Model
In order to assess the analgesic effect of iMSC-sEVs on tendinopathy, a rat tendinopathy model was established and subjected to iMSC-sEVs injection a week after the model establishment. Pain-related behaviors were assessed for a total of 5 weeks (Figure 2A). Static weight bearing (SWB) and hind-paw withdrawal thresholds (PWT) are proxy measures of limb pain widely used to quantify knee pain and hyperalgesia in rat models of osteoarthritis. 44 We used these measures to assess the analgesic effect of iMSC-sEVs on pain-related behaviors.
First, in order to inject drugs into the peri-tendon space of the quadriceps tendon, we subjected the following injection procedure under the guidance of ultrasound ( Figure 2B and C). After one week after injecting iMSC-sEVs around the tendon, significant PWT and SWB reversal was observed in the iMSC-sEVs group compared with the PBS group. Both SWB and PWT improved gradually within 4 weeks after iMSC-sEVs treatment, and most SWB and PWT measures of pain had significant long-term analgesia, except for SWB at 1 week after treatment ( Figure 2D and E).
We further compared the analgesic effect of iMSC-sEVs with an anesthetic combined with a steroid, which are conventional drugs frequently used as a conservative treatment for tendinopathy. Anesthetic and steroids were injected under the same injection protocol as iMSC-sEVs. Both groups have a significant effect in alleviating pain over four weeks. The reversal of hind paw withdrawal threshold and static weight-bearing of the two groups ( Figure 2F and G) were statistically indistinguishable.
Thus, chronic pain derived from tendinopathy could be significantly reversed by an injection of iMSC-sEVs solution. The analgesic effect is comparable to anesthetic and steroid injection.

iMSC-sEVs Alleviate Inflammatory Cytokine Infiltration in Tendinopathy Rat Model
In order to investigate the potential mechanism of iMSC-sEVs alleviating pain in the rat tendinopathy model, we performed a complementary experiment with the tendons of rats. Five weeks after model establishment, rat tendon showed the characteristic tendinopathy features, including undulating, disorganized collagen fibers, hypercellularity, increased numbers of round-shaped, darkly stained nuclei, and clusters of capillaries. In contrast, the iMSC-sEVs group appeared less collagen degeneration and cellularity with H&E staining ( Figure 3A). Semiquantitative histological analysis showed that the total Bonar score, which includes cell morphology and cellularity was significantly lower in the iMSC-sEVs group than the PBS group ( Figure 3B). CD31 and CD86 is a key marker of vascularity 45 and macrophage (proinflammatory phenotype, M1), 46 respectively. Upon visual inspection, we also observed fewer (visually compared to the PBS group) positively immunostained cells for IL-1β, TNF-α, IL-6, NGF, CD86 and CD31 ( Figure 3A). Moreover, quantitative histological analysis of positively stained area percent showed the same trend ( Figure 3B). In conclusion, iMSC-sEVs can alleviate inflammation infiltration (inflammatory cytokines and cells), inhibit capillary proliferation and rescue tendon from degenerative status in the rat tendinopathy model according to the histological analysis of the tendon.

iMSC-sEVs Protect Tenocyte from Inflammatory Stimulation
First of all, primary tenocytes were isolated from the rat Achilles tendon and further identified by the expression of Scleraxis and Col I ( Figure 4A). In order to determine whether iMSC-sEVs could be internalized by tenocytes, iMSC-sEVs were labeled with Dil fluorescent dye and cocultured with tenocytes isolated above. After 12 hours of incubation, Dil-labeled iMSC-sEVs were efficiently up-taken by tenocytes ( Figure 4B).
To further explore the effect of iMSC-sEVs on the function of tenocytes under inflammation conditions, an in vitro tendinopathy model was established by stimulating rat tenocytes with IL-1β. Tenocytes subjected to IL-1β stimulation exhibited significantly decreased ability of cell proliferation and increased expression of inflammatory mediators as compared to the vehicle group ( Figure 4C and D). iMSC-sEVs significantly enhanced the proliferation capability of tenocytes at three days after the iMSC-sEVs intervention when compared to the vehicle treatment ( Figure 4C). Besides, decreased expression of inflammatory factors including IL-1β, TNF-α, IL-6, and NGF was also observed in the iMSC-sEVs group, compared to that in the vehicle group 24 h after administration, as shown in Figure 4D.
The secretion of inflammatory factors into the culture medium was also decreased in the iMSC-sEVs group. Upon IL-1β stimulation, significantly increased expression of IL-1β, TNF-α, IL-6, and NGF could be observed in the culture medium of the tenocyte compared with the vehicle group. Compared to the vehicle group, the concentration of TNF-α in the iMSC-sEVs group was significantly decreased (18.1 ± 0.4 pg/mL vs 22.5 ± 0.9 pg/mL [vehicle], P < 0.01). The concentration of IL-1β was statistically indistinguishable from the mean concentration in the vehicle group (45.0 ± 1.0 pg/mL vs 52.9 ± 2.5 pg/mL [vehicle]). The concentration of IL-6 significantly decreased (483.3 ± 9.1 pg/mL vs 532.6 ± 22.1 pg/mL [vehicle], P < 0.01). The concentration of NGF also significantly decreased (788.6 ± 8.6 pg/mL vs 844.4 ± 27.5 pg/mL [vehicle], P < 0.01) ( Figure 4E). Collectively, these data indicated that iMSC-sEVs could protect tenocytes from IL-1β stimulation by promoting cell proliferation and resisting the secretion of inflammatory cytokines of tenocytes.

iMSC-sEVs Modulate the Gene Expression Pattern of Rat Tenocytes Under the Stimulation of IL-1β
To explore the underlying molecular mechanism by which iMSC-sEVs rescue tenocytes function, we performed RNAseq analysis to clarify the changes in gene expression in tenocytes. We applied IL-1β-treated tenocytes with iMSC-sEVs

1431
(1 × 10 9 particles/mL) or PBS for 48 hours. We identified 485 up-regulated genes (> 2-fold, p < 0.05) and 379 downregulated genes (<0.5-fold, p < 0.05) after sEVs treatment ( Figure 5A). Next, we performed gene set enrichment analysis (GSEA) according to Reactome Knowledgebase (https://reactome.org). The results revealed that many differentially expressed genes were involved in cell proliferation and inflammation ( Figure 5B). Specifically, iMSC-sEVs exposure significantly increased the enrichment score for the module of "cell proliferation" ( Figure 5C) and "DNA replication" (Figure 5D), while decreased the enrichment score for the module of "interleukin-2 family signaling" ( Figure 5E) and "collagen degradation" (Figure 5F) in tenocytes. Genes that showed a significant difference in expression (fold change >2) in the GSEA analysis were visualized by heatmap ( Figure 5G and H). Furthermore, RT-qPCR analysis confirmed that the expression of proliferative and anti-inflammatory genes in tenocytes was increased after iMSC-sEVs treatment ( Figure 5I). Together, these data clearly revealed that alleviation of inflammation phenotypes in tenocytes after iMSC-sEVs treatment is associated with genetic programs and pathways that control critical aspects of the anti-inflammatory process, cell proliferation, and collagen degradation.

Discussion
In this study, we found that sEVs isolated from iPS-MSCs significantly alleviate pain derived from tendinopathy and reduce inflammation infiltration in a rat tendinopathy model. We further demonstrated that iMSC-sEVs could promote tenocyte proliferation and modulate inflammation conditions under the stimulation of IL-1β. Mechanistically, iMSC-sEVs alleviate pain, in part, via modulation of inflammation, cell proliferation, and collagen degeneration in tendinopathy. Our present study is the first to report that iMSC-sEVs modulate inflammation and alleviate pain derived from tendinopathy.
To date, most efforts have focused on repairing and antiinflammation in tendinopathy. Less attention has been directly paid to the management of pain, which is the most common complaint of patients with tendinopathy. 47 Steroid injections may have good short-term effects in tendinopathy but are known to have side-effect on the long-term perspective, 48 and may even result in tendon rupture after steroid use. 49 In this study, we aimed to investigate novel therapy for pain derived from tendinopathy and further explore the potential mechanism. Firstly, we expanded the use of carrageenan to develop painful quadriceps tendinopathy and observed remarkable increases in pain-related behaviors.
Rats possess advantages over other animals used to model tendinopathy. The rat quadriceps tendon is large enough to be operated on and harvested efficiently, and the limb anatomy of rats is similar to that of humans. 50 In addition, wellestablished pain-related behaviors can be measured with various inexpensive apparatuses. Carrageenan is a proinflammatory substance that has generally been used in generating acute and chronic pain in the muscle 51 and joints 52 and subcutaneous pain. 53 It represents a consistent and convenient tool in developing animal models of pain and is well studied. In terms of tendinopathy, carrageenan has been applied to induce patella, 33 deep digital flexor, 54 and Achilles 55 tendinopathy. It leads to the deteriorative mechanical and histological changes in the affected tendons. 33,34 Thus, the application of this carrageenan-induced quadriceps tendinopathy model and the pain-related behavior could be generalized in research regarding novel therapies for tendinopathy pain.
iMSC-sEVs have a high potential as cell-free factors for pain management because of their superior functions in the modulation of cell fate and immune response. It has been reported that MSC has an analgesic effect in osteoarthritis 56 and neuropathic pain. 57,58 MSC-derived sEVs were also demonstrated to exert an analgesic effect as they can transport bioactive components, including lipid, protein, as well as RNAs, from the sourced cells to recipient cells. He et al reported that exosomes derived from bone marrow MSC could relieve pain derived from knee OA and protect cartilage damage. Consistently, in this research, iMSC-sEVs were demonstrated to alleviate pain derived from the rat tendinopathy model. Compared with steroids, iMSC-sEV may not have these harmful side effects and can therefore be promising new therapeutics.
An increasing number of studies have confirmed the presence of inflammatory cells and other mediators in chronic tendinopathy. The presence of mediators like MMPs, COX, VEGF, and inflammatory cells indicate that anti-inflammatory compounds may have potential in managing chronic tendinopathy and related pain. 16 MSC-derived sEVs possess the ability to regulate inflammation and immune response. Zhang et al reported that exosomes derived from MSC could alleviate pain in temporomandibular joint osteoarthritis by regulating signal pathways including AKT, ERK, and AMPK. 59 Previously, our team reported the effect of MSC-derived sEVs could attenuate chronic prostatitis/pelvic pain syndrome in the rat by immunoregulation. 60 In terms of osteoarthritis, we demonstrated that iMSCs-sEVs could  The experiment was repeated three times independently. P-value is indicated as sEVs group versus ctrl group. **P < 0.01, ***P < 0.001. significantly ameliorate osteoarthritis by suppressing inflammation and promote matrix synthesis. 26 In our present study, significantly decreased expression of inflammatory cytokines and cells was observed in vivo and in vitro after iMSC-sEVs treatment, indicating that iMSC-sEVs may exert an analgesic effect on the tendinopathy rat model mainly by regulating the inflammatory environment within the tendon.
RNA-seq and bioinformatics analysis of sequencing data after iMSC-sEVs treatment identified upregulated genes involved in tenocyte proliferation collagen synthesis, as well as downregulated genes involved in inflammation. This finding is consistent with the therapeutic effect of iMSC-sEVs in protecting tenocytes from inflammatory stimulation and promoting tenocyte proliferation. Moreover, GSEA analysis showed that iMSC-sEVs activated the expression of genes involved in anti-inflammation, cell proliferation and collagen synthesis. Among these genes, Stat5a, Stat5b, Sos2, Sos1, Jak1, Il3ra, Jak2 are associated with the IL-2 family signaling pathway and have been reported to inhibit inflammation of tenocytes. [61][62][63] Ccnb1, Nek2, Hmmr, Birc5 are cell cycle-related genes that have been demonstrated to promote cell cycle progression, suppress apoptosis and stimulate tendon regeneration. [64][65][66] In addition, MMP2, MMP9, MMP13 are known to be essential for the collagen degradation of tenocytes and extracellular matrix. 67,68 In conclusion, this evidence suggests that the therapeutic effect of iMSC-sEVs in protecting tenocytes from inflammatory stimulation is credited to the activation of these anti-inflammation, proliferation, and collagen synthesis associated genes.
On the whole, iMSC-sEVs can significantly ameliorate pain derived from tendinopathy, which is attributed to the potency of anti-inflammation, proliferation, and collagen synthesis. iMSC-sEVs have the potential to be translated into the treatment for tendinopathy patients.

Ethical Review Committee Statement
The study protocol was approved by the Ethics Committee of Shanghai JiaoTong University Affiliated Sixth People's Hospital (DWLL2020-0583), and the experimental procedure followed the ethical standards in the Institutional Animal Care and Use Committee of our hospital. All efforts were made to minimize the number and suffering of animals used in this study.