Conversion of senescent cartilage right into a pro-chondrogenic microenvironment with antibody-functionalized copper sulfate nanoparticles for environment friendly osteoarthritis remedy | Journal of Nanobiotechnology

August 8, 2023

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Supplies

Anhydrous copper chloride (CuCl₂), 3,3’,5,5’-tetramethylbenzidine (TMB), bovine serum albumin (BSA), and sodium sulfide nonahydrate (Na₂S·9H₂O) have been from Macklin Biochemical (Shanghai, China). Doxorubicin (Dox), N-(3-Dimethylaminopropyl)-N’-ethylcabodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) have been from Sigma-Aldrich (USA). Antibodies in opposition to p16^ink4a (polyclonal, ab108349), B2M (monoclonal, ab75853), excessive mobility group protein 1 (HMGB-1) (ab228624), matrix metalloprotein 13 (MMP-13) (ab39012), and kind II collagen (Col-2) (ab34712) have been from Abcam (UK). The mobile senescence β-galactosidase staining package and cell counting package (CCK-8) have been from Beyotime Biotechnology (Shanghai, China). FITC-xtra and MitoROS™ 580 have been from ATT Bioquest (USA). The Evo M-MLV Reverse Transcription Reagent and SYBR Inexperienced Professional Taq HS qPCR Equipment have been from Correct Biology (China). The chondrogenic differentiation package was from Cyagen Biosciences (USA).

Synthesis of CuS NPs and B2M-CuS NPs

CuS NPs have been synthesized by a beforehand reported methodology, with some modifications [29]. Briefly, 100 µL of CuCl₂ (1 M, 13.445 mg) answer was added to 10 mL of BSA (10 g L^− 1) answer and stirred at room temperature (RT, 25 °C) for five min. Then, 100 µL of Na₂S answer (1 M, 7.804 mg) was added dropwise over 5 min into the above combination beneath steady stirring. The combination was stirred at 90 °C till a darkish inexperienced shade fashioned, and free BSA and ions have been eliminated by centrifugation and dialysis in opposition to ddH₂O.

To organize the B2M-CuS NPs, B2M antibody was conjugated to CuS NPs by an EDC/NHS response [30, 31] with a 1:3 antibody:NP molar ratio. Unmodified B2M antibody was eliminated by repeated centrifugation for 10 min at 10,000 rpm. The resultant B2M-CuS NPs have been saved at -20 °C earlier than use.

Characterization

The morphology of CuS NPs was noticed by high-resolution transmission electron microscopy (HRTEM; JEOL JEM 2100 F, Japan). The crystalline construction of CuS NPs was decided by X-ray diffraction (XRD; Rigaku Smartlab 9KW, Japan), and absorption spectra have been obtained by a spectrophotometer (Thermo Scientific Multiskan GO, USA). The valence states of Cu in NPs have been studied by X-ray photoelectron spectroscopy (XPS; Thermo Scientific Ok-Alpha). The zeta potential and hydrated particle measurement of CuS NPs and B2M-CuS NPs have been decided utilizing a nano-analyzer (Malvern Zetasizer Nano ZS90, UK).

Peroxidase-like exercise

The peroxidase-like exercise of CuS NPs was evaluated as beforehand reported utilizing TMB as a substrate [24]. For the detection of H₂O₂-dependent oxidation of TMB, totally different reagent mixtures have been set, together with (1) TMB + H₂O₂, (2) TMB + NPs, and (3) TMB + H₂O₂ + NPs. CuS NP options (100 µL, 100 µg mL^− 1) have been diluted with HAc/NaAc buffer (0.2 M:0.2 M, pH = 4.6). Then, 100 µL of TMB answer and 100 µL of H₂O₂ (10 mM) in dimethyl sulfoxide (2 mM) have been added to the NP options. After incubation at RT for 30 min, the absorbance of the samples was recorded (BioTek Synergy H1, USA).

Institution of senescent chondrocytes

The mouse embryonic carcinoma-derived chondrogenic cell line ATDC5 was used for in vitro research. ATDC5 cells have been cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; VivaCell, Shanghai, China) and 1% penicillin/streptomycin at 37 ℃ beneath 5% CO₂. Senescent chondrocytes have been established by Dox remedy in keeping with earlier studies [32, 33]. Briefly, ATDC5 cells have been handled twice with 0.1 µM Dox (48 h remedy interval), and senescent chondrocytes have been established after 4 days of stimulation.

Immunofluorescence staining of senescent cells

Regular and DOX-induced senescent ATDC5 cells have been fastened with 4% paraformaldehyde for 15 min, permeabilized with phosphate-buffered saline (PBS) containing 0.25% Triton X-100 for 10 min, after which blocked with 4% (v/v) BSA for 30 min at RT. Cells have been then incubated with main antibody in a single day at 4 ℃, washed thrice with PBS, after which incubated with the corresponding secondary antibody for two h. After washing with PBS, cell nuclei have been stained with 4,6-diamino-2-phenyl indole (DAPI) for five min, after which cells have been imaged by fluorescence microscopy.

SA-β-galactosidase staining

To measure the marker senescence-associated β-galactosidase (SA-β-gal) [34, 35], each regular and senescent ATDC5 cells have been stained utilizing a SA-β-gal staining package (Beyotime Institute of Biotechnology, Nanjing, China) in keeping with the producer’s directions. Cells have been washed with PBS, fastened with paraformaldehyde for 15 min at RT, after which stained in a single day at 37 ℃. Photographs have been acquired utilizing a microscope.

Cytotoxicity of the NPs

The cytotoxicity of CuS and B2M-CuS NPs was investigated by the usual CCK-8 methodology. Regular ATDC5 cells have been seeded in a 96-well plate (1 × 10⁴ cells per nicely) and cultured with DMEM containing 10% FBS for twenty-four h at 37 ℃. Cells have been then cultured with CuS and B2M-CuS NPs at a number of concentrations (100, 50, 25, and 10 µg mL^− 1) for twenty-four h. Thereafter, cells have been incubated with 10% CCK-8 answer for an extra 2 h and optical density (OD) values have been measured at 450 nm utilizing a microplate reader.

Hemocompatibility of NPs

The hemocompatibility of CuS and B2M-CuS NPs was evaluated. Purple blood cells (RBCs) derived from wholesome C57BL/6 mouse blood have been diluted with PBS (pH 7.4, 4:5 v/v). The NPs have been suspended in PBS and incubated at 37 ℃ for 30 min. After incubation, 200 µL of diluted RBCs have been added to the samples and incubated at 37 ℃ for an extra 1 h. PBS and ddH₂O have been unfavorable and constructive management samples, respectively. After centrifugation at 3000 rpm for five min, the supernatant OD545 values have been measured on a microplate reader. The hemolysis ratio was calculated by the next components: Hemolysis ratio (%) = (OD_pattern – OD_unfavorable) / (OD_constructive – OD_unfavorable) × 100.

Concentrating on impact of B2M-CuS NPs on senescent chondrocytes

To organize FITC-labeled NPs, 5 µL of FITC-xtra answer (20 mM) was added to five mL of CuS and B2M-CuS NPs options (5 mg). After stirring on ice for 60 min, free FITC-xtra was eliminated by centrifugation for five min at 10,000 rpm.

To research the concentrating on specificity of B2M-CuS NPs, senescent ATDC5 cells and regular ATDC5 have been seeded in a 48-well cell tradition plate and grown for twenty-four h. Subsequently, FITC-labeled CuS and B2M-CuS NPs (50 µg mL^− 1) have been added and cultured with cells for an extra 4 h. After eradicating the supernatant, cells have been washed with PBS, fastened with 4% paraformaldehyde for 15 min, stained with DAPI for five min, and visualized beneath a confocal laser scanning microscope (LSCM; Nikon ECLIPSE Ti2-E, Japan). As a management, senescent cells have been first handled with free B2M antibody for six h, after which B2M-CuS NPs have been added. After 24 h, cells have been washed and noticed. Moreover, circulation cytometry was carried out to detect the fluorescence depth of handled senescent cells.

Particular elimination of senescent chondrocytes by NPs

To check the precise elimination of senescent chondrocytes by the NPs, DOX-induced senescent ATDC5 cells have been seeded on a 48-well cell tradition plate (5 × 10⁴ cells per nicely). CuS and B2M-CuS NPs (50 µg mL^− 1) have been added to the plate and cultured with cells for twenty-four h, and cell viability was assayed with CCK-8. Stay/lifeless staining was additionally carried out. The above-treated cells have been gently washed with PBS and stained with 200 µL of Calcein AM and propidium iodide (PI; BestBio, China) for 30 min at 37 ℃ at the hours of darkness. Cells have been imaged with a fluorescence microscope.

Detection of intracellular ROS and mitochondrial ROS

Senescent ATDC5 cells have been cultured with CuS and B2M-CuS NPs (50 µg mL^− 1) for twenty-four h, and intracellular ROS was detected through a 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) methodology as we beforehand reported [36, 37]. Following remedy, cells have been washed with PBS after which incubated with 500 µL DCFH-DA working answer (20 µM) at 37 °C for 30 min. Nuclei have been Hoechst stained for five min. Mitochondrial ROS was detected by MitoROS™ 580 staining in keeping with the producer’s directions. MitoROS™ 580 working answer (200 µL) was added to every nicely and incubated for 10 min. Cells have been washed with PBS twice and noticed utilizing LSCM. As a reference, intracellular ROS and mitochondrial ROS ranges of regular chondrocytes have been measured.

Apoptosis and necrosis of senescent chondrocytes

After culturing with CuS and B2M-CuS NPs (50 µg mL^− 1) for twenty-four h, senescent ATDC5 cells have been collected and incubated with 100 µL of Annexin V-FITC and PI answer (BD, USA) for 20 min at RT. Cell apoptosis and necrosis have been measured by circulation cytometry and the outcomes have been analyzed with FlowJo X. To research the elimination of senescent cells by NPs, handled cells have been stained with 7-aminoactinomycin D (7-AAD) for 30 min after which analyzed by circulation cytometry.

The expression ranges of apoptosis-related genes (e.g., Bax, Bcl-2, caspase-3, and caspase-9) have been decided by real-time quantitative PCR (RT-qPCR). Complete RNA from handled senescent ATDC5 cells was extracted with Trizol (Thermo Fisher Scientific), and RNA concentrations have been decided by spectrophotometry (Nanodrop 2000; Thermo Fisher Scientific). RNA was reversed transcribed into cDNA utilizing the Evo M-MLV reagent package in keeping with the producer’s directions. The biking protocol was: 95 ℃ for 15 min, adopted by 45 cycles of 95 ℃ for five s and 60 ℃ for 30 s. The outcomes have been analyzed with Rotor-Gene Actual-Time software program. Relative mRNA expression ranges of every gene have been normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ranges utilizing the two^−ΔΔCT methodology. Primers used for RT-qPCR are listed in Desk S1.

Chondrogenic inductivity of the NPs

To guage the in vitro chondrogenesis induced by NPs, ATDC5 cells have been cultured with chondrogenic medium (MUXMX-90,041, Cyagen Bioscience, USA) containing CuS NPs or B2M-CuS NPs (50 µg mL^− 1). After 7 days, cells have been washed with PBS, fastened with 4% paraformaldehyde for 15 min, and stained with alcian blue 8GX answer for 30 min at RT to detect intracellular glycosaminoglycan (GAG). After washing with PBS thrice, cells have been noticed with a microscope.

To find out the expression stage of chondrogenesis-related genes aggrecan (Acan) and Col-2, cells have been collected after 7 and 14 days of chondrogenic induction and RT-qPCR was carried out.

As well as, ATDC5 cells have been induced into cell pellets, as beforehand reported, and cultured in chondrogenic medium with NPs (50 µg mL^− 1) [38]. The medium was modified each 3 days. After 21 days, cell pellets have been collected and glued in 4% paraformaldehyde, embedded in paraffin, and lower into sections. Col-2 expression was detected by immunohistochemistry.

Therapeutic results of the NPs in an OA mouse mannequin

All animal research have been accredited by the Institutional Animal Care and Use Committee (IACUC) of the Normal Hospital of Southern Theater Command of PLA (2021032401). Grownup male C57BL/6 mice (20 g, 12 weeks) have been anesthetized and induced into surgical destabilization of the medial meniscus (DMM) [38]. Briefly, the medial meniscus supporting ligament (MMSL) was resected after a medial capsular incision was made on the suitable knee joint of mice. After 4 weeks, OA mice fashions have been established. The management group acquired sham surgical procedure (opening of the joint capsule with out MMSL resection; n = 6).

The OA mice have been randomly divided into three remedy teams (n = 6 per group): (1) PBS, (2) CuS NPs, and (3) B2M-CuS NPs. The NPs (10 µL, 2.5 mg kg^− 1) and PBS (10 µL) have been delivered by intra-articular injection into the suitable knee of the OA mice at 4 weeks and 6 weeks post-surgery, respectively. Mice have been sacrificed at 8 weeks post-surgery and the suitable knee joints have been collected for additional analysis.

Histological evaluation and biodistribution

For histological evaluation, the suitable knee joint samples have been fastened in 4% paraformaldehyde in a single day, decalcified in an EDTA answer, and embedded in paraffin blocks. Sections have been lower into 5 μm-thick slices and stained with hematoxylin and eosin (H&E) and Safranin O/quick inexperienced. All sections have been visualized with an inverted microscope (Nikon TS100, Japan). The Osteoarthritis Analysis Society Worldwide (OARSI) scores have been evaluated in keeping with a earlier report [39]. Immunofluorescence staining for protein expression (Col-2, MMP-13, p16^ink4a, and HMGB-1) was carried out following our beforehand reported strategies [38, 40]. Photographs have been obtained utilizing an inverted fluorescent microscope. The fluorescence depth was calculated utilizing ImageJ software program.

To guage the biodistribution of B2M-CuS NPs, mice have been intra-articularly injected with the NPs. After 24 h, mice have been sacrificed and the principle organs (coronary heart, liver, spleen, lung, kidney, and proper knee joint) have been collected and weighed. Samples have been digested with aqua regia and Cu²⁺ content material was decided by ICP-MS (Agilent 7700, Germany).

Statistical evaluation

Information are introduced as imply ± commonplace deviation (SD). Statistical comparisons amongst totally different teams have been carried out by utilizing one-way evaluation of variance (ANOVA) adopted by posthoc a number of comparisons (LSD take a look at). A p-value < 0.05 was thought-about statistically vital and a p-value < 0.01 was thought-about extremely statistically vital.