Supplies
Iron acetylacetonate, N,N-carbonyl diimidazole, oleylamine, oleic acid, calcium hydroxide, polyethyleneimine (Mw ~ 1800), hydroxy polyethylene glycol (Mw ~ 5000), sodium hyaluronate, and 3-aminobenzeneboronic acid have been bought from Aladdin Bioscience Co. (Shanghai, China). Fetal bovine serum, DMEM excessive glucose medium and different biologically associated reagents or kits have been bought from KeyGen Biotechnology Co. (Nanjing, China).
Preparation of Fe3O4 NPs
To synthesize iron nanoparticles, initially, 1.4 g of iron acetylacetonate was weighed and blended with 8 mL of oleic acid and 12 mL of oleylamine. The response commenced by purging with N2 at room temperature for 15 min, adopted by vacuuming utilizing an oil pump for 15 min whereas sustaining the nitrogen purge. The response combination was heated to 120 °C and held for two h. Subsequently, the temperature was raised to 220 °C and maintained for 30 min, then elevated at a charge of two °C/min to 300 °C and held for 30 min. The answer was cooled to 120 °C and stored for an extra 30 min. After cooling to 120 °C, nitrogen move was stopped, and air was launched to proceed the response for 90 min. Upon completion, the heating was turned off, and the liquid was transferred to a 50 mL centrifuge tube. Acetone (50 mL) was added, and centrifugation at 8000 rpm for five min separated the supernatant, which was discarded. The precipitate was dissolved in 5–10 mL of hexane, adopted by addition of 40 mL of ethanol and repeated centrifugation. This washing course of was repeated twice. Lastly, the nanoparticles have been dissolved in 5 mL of hexane, sealed, and saved at room temperature.
Preparation of PEI-PEG
Anhydrous and alcohol-free chloroform solvent was ready by initially mixing chloroform with deionized water, subjecting it to triple extraction, and treating it with calcium hydride below reflux at 65 ℃ for 4 h to acquire redistilled chloroform. Within the subsequent step, HO-PEG2000-OH was activated by combining 2 g of HO-PEG2000-OH with 0.821 g of CDI in 20 mL of the ready chloroform solvent, stirring in a single day below a nitrogen ambiance, and precipitating the ensuing PEG-CDI with chilly ether adopted by vacuum drying. Additional, 1.4 g of PEG-CDI and 1.9 g of PEI (Mw ≈ 1800 Da) have been dissolved in 7.5 mL of chloroform, blended, and reacted for 12 h below nitrogen. The product, PEI-PEG, was obtained after precipitation with chilly ether, washing, and vacuum drying, and saved at −20 ℃. Structural affirmation was carried out by dissolving 5 mg of PEI-PEG in 500 μL of deuterated chloroform (CDCl3), transferring it to an NMR tube, and analyzing its construction utilizing 1H NMR spectroscopy.
Preparation of Fe3O4-PEI-PEG NPs
Fe3O4 nanoparticles (20–30 μL), dispersed in hexane, have been transferred to an EP tube and ethanol was added to induce precipitation. After centrifugation, the supernatant was discarded, and the precipitate was purged with nitrogen, dried, and weighed to find out the nanoparticle mass. The nanoparticles have been then redispersed in chloroform and blended with a PEI-PEG answer, adopted by sonication for 30 min to 1 h and in a single day shaking. The ensuing combination was dried utilizing rotary evaporation and additional subjected to hoover drying for twenty-four h. The product was subsequently dispersed in water by way of sonication and subjected to dialysis for 3 days with three modifications of dialysate. Lastly, the product was saved below sealed situations.
Willpower of whole iron focus in Fe3O4-PEI-PEG NPs
A small quantity (roughly 10 μL) of nanoparticles was handled with nitric acid and heated to 200 °C, with steady shaking to stop evaporation. Upon evaporation of a lot of the nitric acid, the residue was diluted with water, and the dilution issue was recorded. Phenanthroline and hydroxylamine hydrochloride have been dissolved in acetic acid-ammonium acetate answer and 2M hydrochloric acid, and added to an EP tube. After incubating for two h in the dead of night, the answer was transferred to a 96-well plate and the absorbance was measured. The iron focus of the pattern was decided from a regular curve, and multiplied by the dilution issue to acquire the entire iron focus of the unique pattern.
Fe3O4-PEI-PEG NPs loaded siRNA methodology
An answer of 1 OD260 of siRNA (roughly 33 μg) (Shanghai GenePharma Co., Ltd.) was dissolved in 40 μL DEPC water. The focus of siRNA was exactly measured utilizing a μDrop Plate (Thermo Scientific™, USA) to find out the nucleic acid focus. Fe3O4-PEI-PEG NPs have been diluted accordingly, and a proportional quantity was aspirated utilizing a pipette. The NP answer was then added dropwise to the siRNA answer whereas gently mixing with a pipette gun. The combination was completely blended and allowed to face at room temperature for 30 min to type complexes of siRNA and Fe3O4-PEI-PEG NPs (si-Fe NPs).
Analysis of the flexibility of Fe3O4-PEI-PEG NPs to guard siRNAs
Free siRNA and si-Fe NPs have been blended with numerous concentrations of RNA degrading enzyme (RNase) in 0.2 mL EP tubes, sealed with movie, and incubated for 1 h in a 37 °C water tub. Subsequently, gel electrophoresis was carried out.
Analysis of mobile uptake capability of si-Fe NPs
The C28/I2 cell line was divided into two teams in a 24-well plate: the bare siRNA group and the siRNA-Fe NPs group. For the siRNA-Fe NPs group (siRNA: 80 pmol/mL), FAM-siNC-Fe NPs have been ready, whereas equal quantities of free FAM-siNC have been used for the free siRNA group. Cells have been incubated in 24-well plates for 12 h, adopted by medium aspiration and two PBS washes to take away extracellular free siRNA or nanoparticles. Subsequent, 200 μl of 4% paraformaldehyde was added for fixation. After fixation, the cells have been washed twice with PBS and stained with 200 μl of DAPI answer. Following 2–3 PBS washes, fluorescence microscopy was used for commentary.
Preparation of HA-PBA polymers
HA (2 g) was dissolved in 200 mL of deionized water and stirred in a single day till absolutely dissolved, yielding a transparent and viscous liquid answer. Subsequently, PBA (940 mg) was dissolved in 20 mL of deionized water below sonication till full dissolution. This answer was then mixed with the HA answer. The pH of the ensuing answer was adjusted to six.5~7 utilizing 1 M NaOH and 1 M hydrochloric acid options. DMTMM (1.6 g) was then dissolved in 10 mL of deionized water by ultrasonication, adopted by mixing with the HA + PBA answer. The response combination was stirred at room temperature below a nitrogen ambiance for 72 h.
Preparation of HPP hydrogels in several ratios
Firstly, PVA options of various concentrations have been ready. PVA (9 g) was added to 100 mL of deionized water and heated to 90 ℃ to dissolve. After cooling to room temperature, the answer was transferred to a pattern bottle, leading to a 9% (w/v) PVA answer. Parts of this answer have been diluted to acquire 3 and 6% PVA options with deionized water. Subsequent, HA-PBA options of various concentrations have been ready. HA-PBA samples (2, 4, and 6 mg) have been individually weighed into 1.5 mL EP tubes. To dissolve the samples, 200 μL of PBS was added to every tube, adopted by steady sonication. This course of yielded 1, 2, and three% (w/v) HA-PBA options in whole. Subsequently, 66.7 μL of three, 6, and 9% PVA answer was added to corresponding parts of the HA-PBA options. A pipette gun was used to combine the PVA answer into the HA-PBA answer whereas stirring, guaranteeing thorough mixing earlier than persevering with stirring to keep up uniformity. After stirring for five–10 s, the liquid within the EP tube condensed right into a viscous stable. The combination was then centrifuged at 10,000 rpm for 3 min to take away air bubbles. Profitable formation of the HPP hydrogel was confirmed by inverting the EP tube; no liquid move indicated profitable gelation.
Hydrogel rheology testing
The HPP and si-Fe-HPP hydrogel have been molded into cylinders with a diameter of 8 mm and top of two mm utilizing a PTFE mildew. These cylinders have been rigorously eliminated and positioned on the bottom plate of a rheometer. Subsequently, a collection of rheological checks have been carried out utilizing the rheometer. A pressure sweep was carried out to guage the response over a pressure vary of 0.1–1000% at a set angular velocity frequency of 1 Hz. An angular velocity frequency sweep adopted, sustaining a pressure of 1% whereas scanning angular velocities from 0.1 to twenty Hz. A time sweep was carried out at 1% pressure and 1 Hz frequency, spanning 120 s with time because the x-axis. Lastly, a step pressure take a look at concerned various strains (1–1000%) at 1 Hz frequency, with every pressure step lasting 2 min. These checks aimed to characterize the mechanical properties and viscoelastic conduct of the hydrogel formulations below completely different situations, offering insights into their potential functions in biomedical settings.
In vitro degradation of hydrogels with completely different ratios of HPP
9 completely different hydrogel formulations have been ready with various ratios of HA-PBA and PVA: 1% HA-PBA + 3% PVA (denoted as 1% + 3%, and so forth), as much as 3% HA-PBA + 9% PVA. Every formulation was duplicated, leading to a complete of 18 hydrogel samples. Initially, every hydrogel pattern was immersed in 2 mL of PBS to achieve swelling equilibrium, and the preliminary weight was recorded. Subsequently, pairs of hydrogels with an identical compositions have been individually incubated in 2 mL of PBS and a couple of mL of 100 μM H2O2 answer at 37 °C. Each 48 h, the incubation medium was changed, and the hydrogels have been gently centrifuged at low velocity. After eradicating the liquid, the floor of every hydrogel was dried with filter paper, reweighed, and the load recorded. This course of was repeated till full degradation of the hydrogel occurred within the PBS medium. These experiments have been carried out to evaluate the degradation conduct of the HPP hydrogels below oxidative situations, mimicking environments related to biomedical functions.
Investigation of ROS-responsive degradation conduct of HPP hydrogels
4 an identical HPP hydrogel samples (2% + 9%) have been ready and individually positioned into separate 2 mL EP tubes. Every hydrogel pattern was initially immersed in 2 mL of PBS till swelling equilibrium was reached. The preliminary weight of every hydrogel was recorded as 100%. Subsequently, the PBS was changed with options containing 50 μM H2O2, 100 μM H2O2, and 500 μM H2O2 in separate EP tubes containing one hydrogel pattern every. The samples have been then incubated at 37 °C, and each 48 h, they have been gently centrifuged at low velocity. After eradicating the supernatant, the floor of every HPP hydrogel was rigorously dried with filter paper, reweighed, and the load recorded. The incubation medium (PBS or H2O2 answer) was replenished after every measurement, and the method was repeated till full degradation of the HPP hydrogel was noticed below the respective situations. This experimental setup aimed to guage the degradation kinetics of the HPP hydrogel below oxidative stress situations induced by various concentrations of H2O2, simulating environments related to biomedical functions.
Investigation of H2O2 scavenging capability of HPP hydrogel in vitro
4 completely different concentrations of H2O2 answer (50, 100, 500 μM, 1 mM) have been ready in duplicate in 1.5 mL EP tubes, totaling eight samples, every with a quantity of 500 μL. 100 microliters (100 μL) of HPP hydrogel was immersed in every H2O2 answer and incubated at room temperature for two h in darkness. Subsequently, the hydrogel samples have been centrifuged to gather 100 μL of the supernatant, which was then transferred to a 96-well plate. To this, an equal quantity of 1 M aqueous sodium iodide answer was added and allowed to react for 5 minutes within the absence of sunshine. UV absorption spectra have been then measured utilizing a spectrophotometer inside the wavelength vary of 300~500 nm to investigate the response merchandise shaped below oxidative situations induced by completely different concentrations of H2O2. This methodological strategy aimed to evaluate the oxidative degradation conduct of the HPP hydrogel and characterize its response to various ranges of oxidative stress.
Cell compatibility
C28/I2 cells have been seeded into 96-well plates. Si-Fe nanoparticles (NPs) have been ready at numerous concentrations primarily based on iron mass, dispersed in full medium, and added to the 96-well plates. Ultimate concentrations of iron (Fe) within the wells have been adjusted to 0, 0.049, 0.088, 0.195, 0.390, and 0.781 μg/mL, with every focus examined in triplicate wells. The identical experimental setup was utilized to HPP and si-Fe-HPP hydrogels. This strategy ensured constant testing throughout completely different supplies (Si-Fe NPs, HPP, and si-Fe-HPP hydrogels).
RT-qPCR
Mobile mRNA was extracted from chondrocytes following the producer’s directions utilizing the RNA-quick Purification Package (#RN001, ES Science, Shanghai, China). The HiScript-TS 5′/3′ RACE Package (RA101, Vazyme, China) was utilized for the detection of MMP-13, IL-1β, ACAN, and Col II expression ranges. RT-qPCR evaluation was carried out on a LightCycler 480 PCR system (Roche, Switzerland) utilizing ChamQ Common SYBR qPCR Grasp Combine (Q711, Vazyme, China). Primer sequences for the assays are offered in Desk S2.
Animal experiments
All animal experiments have been carried out with the approval of the Ethics Committee of Drum Tower Hospital affiliated with Nanjing College. The experiments adhered strictly to the ARRIVE pointers and have been carried out in compliance with the U.Ok. Animals (Scientific Procedures) Act of 1986 and related pointers, EU Directive 2010/63/EU for animal experiments, and the Nationwide Institutes of Well being Information for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). Male 12-week-old C57BL/6 mice have been procured from the Animal Mannequin Analysis Middle of Nanjing College and have been housed below particular pathogen-free situations with advert libitum entry to meals and water. The OA mouse mannequin was induced by surgical destabilization of the medial meniscus below isoflurane anesthesia. Twelve-week-old male C57BL/6 mice have been prepped by sterilizing the pores and skin with iodophor. Throughout surgical procedure, the joint cavity of the precise knee was opened, and the medial meniscotibial ligament was severed to destabilize the medial meniscus from the tibial plateau below a stereomicroscope. Loosening was confirmed utilizing microscopic forceps, after which the incision was sutured layer by layer and sterilized once more. The identical surgical process was carried out on the Sham group with out ligament resection. Following Sham or DMM surgical procedure on the precise knee joint, mice have been randomly allotted into 5 teams: (1) Sham group; (2) DMM group; (3) DMM with HPP injection group; (4) DMM with si-Fe NPs injection group; and (5) DMM with si-Fe-HPP injection group. One week post-surgery, the si-Fe-HPP group acquired intra-articular injections of 10 μL si-Fe-HPP (siMMP-13: 1.5 nmol/10 μL) as soon as per thirty days for a interval of 12 weeks. The HPP and si-Fe NPs teams have been administered equal quantities of their respective elements present in si-Fe-HPP, whereas the Sham and DMM management teams acquired an equal quantity of PBS. After 12 weeks of therapy, mice have been euthanized, and joint tissues, main organs, and serum samples have been collected for efficacy and security analysis. Motor operate was assessed utilizing the open area take a look at, gait evaluation, and Von Frey take a look at earlier than the animals have been sacrificed for sampling (Fig. 5a).
Histological evaluation
A ten% ethylenediaminetetraacetic acid (EDTA) answer (#1340, Biofroxx, Germany) was utilized for decalcifying mouse knee joints, adopted by embedding in paraffin blocks. The knee joints have been sectioned coronally into steady 5 μm slices utilizing a microtome (Thermo, Germany). Sections have been subjected to H&E staining (#C0105S, Beyotime) and S.O. staining (#G1371, Solarbio) to guage synovitis and cartilage lesions, respectively. Synovitis severity was assessed utilizing a scoring system starting from 0 to three, and cartilage degeneration was evaluated utilizing the Osteoarthritis Analysis Society Worldwide (OARSI) grading system (0–6) by two unbiased blinded observers. The utmost scores for synovitis and OARSI have been recorded, and their common values have been calculated. Moreover, to evaluate the in vivo biocompatibility of si-Fe-HPP, sections of main organs (coronary heart, liver, spleen, lungs, and kidneys) have been stained with H&E (#C0105S, Beyotime). Serum biochemical indicators have been additionally analyzed to guage security profiles.
Immunohistochemical staining
Endogenous peroxidase exercise was quenched utilizing 3% (v/v) H2O2. Horseradish peroxidase-conjugated secondary antibodies, particularly goat anti-rabbit or anti-mouse immunoglobulin G (IgG) (Biosharp, Shanghai, China), have been used. Immunohistochemical staining was visualized utilizing the Ultrasensitive DAB equipment (#1205250, Typing, Nanjing, China). Non-immune IgG served as a unfavourable management throughout the immunohistochemical staining course of.
Micro-CT evaluation
Micro-CT scans have been carried out utilizing a VivaCT 80 scanner (Scanco Medical AG, Switzerland) geared up with a 70 kVp mild supply. 3D reconstructions of mouse knee joints have been generated utilizing Scanco Medical software program (Scanco, Switzerland) with a threshold set at 220 to guage bone morphology. Quantitative evaluation of osteophyte formation was carried out primarily based on the 3D reconstructed pictures.
Habits checks
The open area take a look at was carried out in a 50 cm × 50 cm sq. enviornment with 25 cm excessive partitions below quiet, dimly-lit situations. Mice motion trajectories have been recorded utilizing a monitoring system (Zhenghua Know-how). For gait evaluation, mice freely walked on a 70 cm × 20 cm white runway. Previous to testing, the forepaws have been marked with pink ink and hindpaws with blue ink to report footprints. Measurements have been analyzed by two unbiased observers blinded to the experimental situations. Ache sensitivity was assessed utilizing an digital von Frey anesthesiometer (IITC, Woodland Hills, USA), recording the mechanical paw withdrawal threshold.
Statistical evaluation
Statistical analyses have been carried out utilizing GraphPad Prism software program (model 8.0) and SPSS software program (model 25.0). Quantitative outcomes characterize findings from not less than three unbiased experiments. Graphical Evaluation Utilizing GraphPad Prism 8.0 and Origin 2022. No samples or animals have been excluded from the evaluation. To evaluate knowledge variance equality and regular distribution, Levene’s take a look at and Shapiro–Wilk’s take a look at have been utilized, respectively. For comparisons amongst a number of teams, both one-way or two-way evaluation of variance (ANOVA) adopted by Tukey’s publish hoc take a look at was carried out. Knowledge are introduced as imply ± commonplace deviation, and statistical significance was set at P < 0.05.
