A smart hypochlorous acid fluorescent probe enabling Ibuprofen-release for osteoarthritis theranostics

Background: Osteoarthritis (OA) standing as the most prevalent form of arthritis, closely associates with heightened levels of reactive oxygen species, particularly hypochlorous acid (HOCl). Although there are numerous probes available for detecting HOCl in the OA region, probes with dual functions of diagnostic and therapeutic capabilities are still significantly lacking. While this type of probe can reduce the time gap between diagnosis and treatment, which is clinically needed. Methods: We developed a fluorescent probe (DHU-CBA1) toward HOCl with theranostics functions through the release of methylene blue (MB) and ibuprofen (IBP) in this work. DHU-CBA1 can detect HOCl with high specificity and sensitivity, releasing MB and IBP with an impressive efficiency of ≥ 95% in vitro. Results: DHU-CBA1 exhibits good biosafety, enabling in vivo imaging of endogenous HOCl, along with reducing arthritis scores, improving synovitis and cartilage damage, and maintaining catabolic balance while alleviating senescence in cartilage. Conclusions: This study proposes a novel approach to enhance osteoarthritis therapy by releasing IBP via a smart HOCl-enabled fluorescent probe.


Instruments
1 H NMR (400 MHz) and 13 C NMR (100 MHz) spectra were recorded with a Bruker AV400 nuclear magnetic resonance spectrometer with DMSO-d6 as the solvent.Proton chemical shifts are reported in parts per million downfield from tetramethylsilane (TMS), with tetramethylsilane (δ = 0.0 ppm) or DMSO-d6 (39.52 ppm for 13 C) as the chemical shift standard.High-resolution mass spectra (HRMS) were observed on Bruker Micro TOF II instrument using electrospray ionization (ESI) or Bruker Compact QTOF-HRMS.High-performance liquid chromatography (HPLC) was acquired utilizing an Agilent 1200 Series system.UV-vis absorption spectra were obtained using a Shimadzu UV-2600 spectrophotometer at a medium scanning rate and quartz cuvettes with a path length of 1 cm.Fluorescence spectra were recorded at room temperature using an Edinburgh FLS 1000 spectrometer with an Xe lamp as the excitation source.The lived / dead cytotoxicity assay images were taken by a confocal laser scanning microscope (CLSM, FV3000, Olympus, JPN).The fluorescence imaging were carried out on a commercial IVIS Lumina Ⅲ small animal in vivo fluorescence imaging system (PerkinElmer, USA).

Preparation of probes and analytes
Reserve solutions of probes (5 mM) were prepared in dimethyl formamide (DMF), and then diluting with PBS buffer (10 mM, pH 7.4) to the test concentration.All the characterizations are managed at room temperature.Other analytes were made in ddH2O on the basis of our reported method and specifics were as follows [1, 4, 5].HOCl was obtained from sodium hypochlorite solution (0.1 M, H2O-medium).H2O2 was taken by a 30% H2O2 solution.• OH (Hydroxyl radical) was carried out on Fenton reaction (H2O2: FeCl2 = 1: 10) and the concentration of • OH was equivalent to the concentration of H2O2.TBHP (tertbutyl hydroperoxide) was produced from 70% TBHP solution in ddH2O.ROO • was acquired from dissolving 2, 2'-azobis (2-amidinopropane) dihydrochloride in ddH2O.NO was conducted with dissolving SNP (sodium nitroferricyanide (III) dihydrate) in ddH2O.O2 − was produced by dissolving KO2 (potassium superoxide) in DMSO.ONOO − was obtained from 3-morpholinosydnonimine hydrochloride.t-BuOO • was taken by adding TBHP within 10 equiv. of ferrous chloride and the concentration of t-BuOO • was equal to the TBHP concentration.

Procedure of selectivity studies in vitro
The probes DHU-CBA1 and DHU-CBA3 were treated with various concentrations of ROS in PBS (1‰ DMF) for an adequate 30 min along with the published methods.Unless otherwise noted, for all fluorescent measurements, the excitation wavelength was 620 nm and the emission wavelength was 686 nm and collected from 630 nm to 850 nm.The relevant parameters were set as the same as the fluorescence response experiment.

Detection limit
The detection limit (3σ/k) was calculated based on the linear relationship between the fluorescence intensity at 686 nm and the concentration of HOCl.σ is the standard deviation of the blank measurement (n = 15) and k is the slope of the fluorescence intensity versus HOCl concentration.

Drug release of HPLC analysis
HPLC was acquired from an Agilent 1200 Series system utilizing a C18 column at 30 ℃.The eluant was a mixture of HPLC-grade CH3CN and H2O (1‰ trifluoroacetic acid).The relevant parameters were set as follows: flow rate 1 mL/min.Solvent A (CH3CN) and solvent B (H2O) were washed within 30 min according to different gradients.Absorbance wavelengths were 210 nm, 254 nm, 290 nm, and 664 nm, successively.HPLC experiments were conducted after the MB for 11.26 min, IBP for 16.02 min, and p-pTA for 11.79 min.

Primary articular chondrocytes culture
Primary articular chondrocytes were extracted from the articular surface of knee joint of 1-week-old Sprague Dawley (SD) rats.Articular cartilage was shredded and then digested in 0.2% collagenase II (Sigma-Aldrich) at cell incubator (5% CO2, 37 ℃) for 4 h.After filtration and centrifugation, the chondrocytes were cultured with F12 Ham medium containing 10% fetal bovine serum (FBS) as well as 1% of antibiotics, followed by incubation in the incubator.
The A, A0, and As respectively represented the A450 value of only medium group, the experimental group, and control group.

Cell live/dead assay
After being cultured with DHU-CBA3 and DHU-CBA1 for 12 h and 24 h, the cells were stained with calcein-AM and PI solutions for 30 min at 37 ℃ using a live/dead cytotoxicity assay kit (Invitrogen).
After washing three times (5 min each time) with PBS, the samples were observed under a laser confocal microscope.

Probes response in vitro
The MB release properties of DHU-CBA1 and DHU-CBA3 in vitro were studied in chondrocytes cells.Cells (5 × 10 8 per mL) were separately plated on 20 mm glass bottom cell culture dish and allowed to adhere for 12 h.The stock solution of DHU-CBA1 in DMF (10 mM) was diluted with basic F12 Ham medium with final concentration of 10 μM.The cells were then incubated with different analytes for a pre-set time at 37 °C.After incubation 1 h, the cells were washed one time with PBS.CLSM imaging was performed on Leica SP 8 Confocal Laser Scanning Microscope with a 63 × oil-immersion objective lens for cells.λex = 633 nm; red channel: 700 ± 50 nm.
1.12 Blood routine assay PBS, DHU-CBA1, or DHU-CBA3 (0.5 mM, 100 μL) were injected into the intraperitoneal of healthy mice (n = 3).After every three days for one month of injection, ocular vein blood was taken for a blood routine.

Animal model construction
Guiding principles used for animal treatment and care closely followed the recommendations of the animal ethics committee of Zhongshan Hospital at Fu Dan University (Shanghai, China, No: 2024-036).All C57BL/6 mice (aged eight weeks old, female) were purchased from JSJ company (Shanghai, China).
Osteoarthritis (OA) mouse models were established by surgical destabilization of medial meniscus (DMM).Briefly, after anesthesia with pentobarbital, left knee joint capsules were incised medial to the patellar tendon, and then transecting medial meniscotibial ligaments using microsurgical scissors.The joint capsule and skin were accordingly closed.The mice with sham surgery were conducted with arthrotomy, followed by suture without meniscotibial ligament transection.

Probes response in OA model
The drug release behavior of the probes DHU-CBA1 and DHU-CBA3 was detected in OA mice.The left knee joint was conducted with DMM surgery, and the right knee joint was conducted with sham surgery.
After one week, the left and right knee joint were injected nearly 10 μL of DHU-CBA1 (0.5 mM) and DHU-CBA3 (0.5 mM), respectively.Subsequently, the fluorescence of every mouse was collected by in vivo bioluminescence imaging.

Histological analysis
To evaluate the morphology of articular surface and subchondral bone and proteoglycan loss, the slices were stained by safranin/F fast green staining and hematoxylin and eosin (HE) staining in accordance with the manufacturer's protocol.Then, the slices were imaged via an automatic digital slide scanning system, which were finally evaluated by several well-accepted osteoarthritis and synovitis scoring system.

Immunohistochemistry
Sections were incubated in 0.1% trypsin for 20 min, followed by blocking with goat serum albumin (10%) for 1 h.After co-incubation with primary antibodies against Aggrecan, MMP13, collagen II, and P21 overnight at 4 °C, the slices were incubated with the HRP-conjugated secondary antibody for 2 h.The slices were imaged, and quantitatively by positive cell rate.

Statistical analysis
Data were presented as mean ± standard deviation (SD) for n ≥ 3 independent experiments.Statistical significance was calculated via one-way ANOVA with Tukey's test.