Baicalin nanodelivery system based on functionalized metal-organic framework for targeted therapy of osteoarthritis by modulating macrophage polarization | Journal of Nanobiotechnology

Materials

Baicalin (Bai, 90%), Zirconium (IV) chloride (ZrCl₄, 94%), 2-aminoterephthalic acid (> 98%), N, N-dimethylformamide (DMF, ACS), CH₃COOH (ACS), folic acid (FA, ≥ 98%), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl, 98%), DMSO (≥ 99.9%), PBS buffer (pH = 7.4), HCl (ACS), LPS, N-hydroxysuccinimide (NHS, 98%) and chlorpromazine (CPZ, 95%) were purchased from Aladdin, China. All chemicals were utilized directly without the necessity for additional purification.

Preparation of UIO-66-NH₂ NPs

First, add 2.27 mmol ZrCl₄ and 2.11 mmol 2-amino terephthalic acid to the round bottom flask. N, N-dimethylformamide (DMF) and CH₃COOH were then added to the above mixture at room temperature in a volume ratio of 100: 3.5, respectively. The above mixture was then put in a high-pressure reaction kettle at 120 °C for continuous reaction. After 24 h, the precipitate obtained by centrifugation (4000 rpm, 10 min) was washed two to three times with methanol before drying overnight at room temperature.

Synthesis of FA-UIO-66-NH₂ NPs

FA-UIO-66-NH₂ was synthesized using an amidation condensation reaction in which the carboxylic acid group of FA reacted with the amino group (-NH₂) of UIO-66-NH₂ catalyzed by EDC/NHS. Under light-protected conditions, 10 mg FA, 30 mg EDC·HCl, and 10 mg NHS were activated in DMSO for 2 h. Then, 10 mg UIO-66-NH₂ nanoparticles were added to the solution mentioned above and allowed to react for 24 h. Finally, the centrifuged precipitates were dialyzed with water to remove any remaining FA, EDC, or NHS. After 72 h of dialysis, the resulting products were collected by centrifugation and named FA-UIO-66-NH₂.

Synthesis of Bai@FA-UIO-66-NH₂ NPs

In a light-protected environment at room temperature, 5 ml of Bai (2 mg/ml) was mixed with 20 ml of FA-UIO-66-NH₂ (0.1 mg/ml). The mixture was stirred using a magnetic stirrer for 24 h. After the reaction, the mixture was centrifuged at 4000 rpm for 10 min. The isolated lower precipitate was collected and centrifuged three times with deionized water, yielding the ultimate precipitate, Bai@FA-UIO-66-NH₂.

Characterization of NPs

The structural characteristics of UIO-66-NH₂ NPs and FA-UIO-66-NH₂ NPs were confirmed through a comprehensive analysis using X-ray diffraction (XRD, Rigaku, Japan), Fourier transform infrared spectroscopy (FT-IR, IRAffinity-1 S, Japan) and Ultraviolet and visible spectroscopy (UV-vis, Shimadzu, Japan). Morphological and elemental distribution of the samples were further elucidated using a Scanning electron microscope (SEM, Bruker, Germany), Transmission electron microscope (TEM, Bruker, Germany), X-ray photoelectron spectroscopy (XPS, ESCALAB 250Xi, US) and Energy dispersive X-ray spectrum (EDS). Dynamic light scattering (DLS), Zeta potential and N₂ isothermal adsorption curve contributed to the characterization of particle size, surface charge and porosity. Additionally, thermal stability was assessed through thermogravimetric analysis (TGA, Star449, Germany).

Drug loading ratio

The drug-carrying efficacy of FA-UIO-66-NH₂ NPs for Bai was determined using a centrifugal method. Specifically, Bai and FA-UIO-66-NH₂, at varying weight ratios, were combined in a certain volume solution and centrifuged. The resulting supernatant was collected, and the unloaded Bai content was measured using a UV-visible spectrophotometer. The drug loading capacity and encapsulation rate of FA-UIO-66-NH₂ NPs were then calculated. The optimal drug-to-drug ratio was determined to investigate the drug loading performance of FA-UIO-66-NH₂ nanocarriers for Bai. The applicable calculation formula is as follows:

Among them, m_Bai is the initial input of Bai, m_FU is the initial input of FA-UIO-66-NH₂, c₁ and c₂ are the concentrations of Bai in supernatant 1 obtained by the first centrifugation after the reaction and supernatant 2 collected after washing with 5 ml deionized water, V₁ and V2 are the volumes of supernatant 1 and supernatant 2, respectively.

Drug release rate

To investigate the release kinetics of Bai from the Bai@FA-UIO-66-NH₂ nanocarriers under various conditions, 5 ml Bai@FA-UIO-66-NH₂ PBS suspension of different pH was introduced into a dialysis bag. Subsequently, 95 ml of the corresponding PBS buffer solution served as the external dialysis solution. The entire dialysis lasted 48 h and was carried out at room temperature and in the dark. Samples were collected at predetermined intervals, including 0.25 h, 0.5 h, 1.0 h, 2.0 h, 6 h, 8 h, 12 h, 24 h and 48 h. Each sample consisted of 5 mL of dialysis fluid extracted and stored in a 10 mL EP tube, that was kept away from light. Simultaneously, an equivalent volume of the corresponding PBS buffer was added to the dialysis fluid, keeping the system’s total volume constant at 100 mL. A UV-visible spectrophotometer with a wavelength of 276 nm was used to measure the dialysis fluid’s absorbance at each time point. Finally, analysis was carried out using the Bai standard regression equation and the formula for calculating the cumulative drug release rate. The formula to calculate the cumulative drug release rate is as follows:

Where V_e is the volume of each sample, V₀ is the total volume of dialysate, c_i is the concentration of Bai in the samples sampled for the i time, m is the dosage of Bai, and n is the number of sampling times.

The stability study of the NPs

To assess the stability of the NPs, Bai and Bai@FA-UIO-66-NH₂ NPs were dispersed at a concentration of 2 mg/mL in various solutions (PBS = 7.2, PBS = 5.4 and DMEM medium). The images of the NPs were photographed and the diameters of Bai@FA-UIO-66-NH₂ were measured by DLS at 0, 1,3,5 and 7 d.

Hemolysis test

To analyze the hemocompatibility of the NPs, the hemolysis test was performed with fresh whole blood of SD rats. First, the whole blood of the rats was collected through the abdominal aorta with an anticoagulant tube. After five minutes of centrifuging the collected whole blood at 1000 rpm, the top layer was discarded. The underlying red blood cells were repeatedly washed with normal saline (NS) until the supernatant was colorless, and then prepared into a 2% w/v red blood cell suspension with normal saline (NS). 2 mL of NPs (FA-UIO-66-NH₂, Bai and Bai@FA-UIO-66-NH₂) with the same concentration (0.1 mg/ml) were incubated with an equal volume of the diluted red blood cell suspension at 37℃ for 2 h. Furthermore, a diluted red blood cell suspension was combined with an equal volume of distilled water and normal saline, which were designated as the positive and negative controls, respectively. Lastly, a UV-vis spectrophotometer was used to measure each sample’s hemoglobin release at 540 nm. The hemolysis percentage was calculated as follows:

ROS scavenging ability of NPs

To assess the ROS-scavenging abilities of the NPs, the SOD activity assay kit (Sigma-Aldrich, USA), CAT activity assay kit (Beyotime, China),  ABTS detection kit (Solarbio, China) and ·OH detection kit (Solarbio, China) were used following the kit instructions. The absorbance was measured at 450 nm using a microplate reader (Thermofisher, USA) and calculated the SOD enzyme activity of the various samples. Furthermore, the absorbance was respectively measured at 550 and 520 nm and 405 nm for evaluating CAT-like activity, ·OH detection ability and total antioxidant capacity.

Cell culture

The most popular in vitro model for studying inflammation and identifying anti-inflammatory active agents is the RAW264.7 cell line. The RAW 264.7 cells used in this study were commercially purchased from the American Type Culture Specimen (ATCC, USA). All cells were respectively cultured in Dulbecco-modified Eagle medium. The cultured medium was replaced every 2 days. Cells were passaged when reached 80–90% and collected for additional study.

Cell cytotoxicity assay

The cytotoxicity of Bai and NPs on RAW264.7 macrophages was assessed using the Cell Counting kit-8 (CCK-8, Biosharp). Briefly, RAW264.7 macrophages were cultured in a 96-well plate for 24 h. The cells were then subjected to varying concentrations of Bai, FA-UIO-66-NH₂ and Bai@FA-UIO-66-NH₂ for an additional 24 h. Subsequently, 100 µL DMEM medium solution containing 10% CCK-8 was added, and the samples were co-cultured for 2 h. The resulting absorbance at 450 nm was measured using a full-wavelength microplate reader (Thermo Scientific, USA) and the cell viability was calculated. Furthermore, RAW264.7 cells were pre-induced with LPS (10 ng/mL) to induce the M1 phenotype of macrophages. The subsequent investigation into the in vitro cytotoxicity of Bai@FA-UIO-66-NH₂ under OA conditions followed the aforementioned procedure.

Live and dead staining assay

The Calcein-AM/PI cell staining reagents (Beyotime, China) were used to investigate the biocompatibility. First, RAW264.7 cells (2 × 10⁵ cells) were subjected to co-cultured with Bai and different NPs for an additional 24 h following pre-treatment with LPS (10 ng/ml). Each group received an application of Calcein AM/PI dual dye, which was followed by quantitative analysis, observation, and photography under a fluorescent microscope (Olympus, Japan).

Intracellular ROS scavenging measurement

The antioxidant capacity was assessed using the Reactive Oxygen Species Assay Kit (Beyotime, China). RAW264.7 cells were initially exposed to LPS (10 ng/ml) at 37℃ for 24 h, then treated with Bai and different NPs for another 24 h. Cells were then co-cultured with 10 µM HPF (maokangbio, China) for ·OH level testing, 5 µM DHE (Beyotime Biotechnology, China) for ·O₂⁻ level testing, and 20 µM DCF (Solarbio, China) for total ROS level testing. They were then cleaned three times using PBS. Ultimately, an Olympus fluorescent microscope (Japan) was used to measure the relative fluorescent intensity, and Image J was used to perform intensity statistics.

To further quantify intracellular ROS levels, macrophages were treated in the same manner as previously described. BD Biosciences, USA) flow cytometry was used to measure the fluorescence intensity of the collected cells.

Lysosomal escape and macrophage-targeting

RAW264.7 cells were incubated with Cy5.5-labelled Bai@FA-UIO-66-NH₂ NPs for 0 h, 2 h, 4 h and 6 h. Cells were then collected, fixed and incubated with Actin-Tracker Green-488 (Beyotime, China) for 30 min, followed by a 15 min DAPI staining. Finally, the material uptake capacity of normal macrophages was observed using a confocal microscope (Leica, Germany).

RAW264.7 cells were cultured for 12 h under various conditions with or without LPS (10 ng/ml), followed by a 1 h culture under conditions with or without free FA, representing conditions -LPS (FA-), -LPS(FA+), +LPS(FA-) and + LPS(FA+). The four groups of cells were then treated with Cy5.5-labelled Bai@FA-UIO-66-NH₂ NPs for 2 h, respectively. They were then washed three times with PBS, fixed with 4% paraformaldehyde for 15 min, and incubated for 30 min with Actin-Tracker Green-488 (Beyotime, China), followed by a 15-minute DAPI staining. Finally, the cells’ targeting capacity was examined with a confocal microscope (Leica, Germany).

To further investigate the targeting mechanism, LPS-activated macrophages were only treated with chlorpromazine (CPZ, 10 µg/ml) for 1 h, then co-cultured with Cy5.5-labelled Bai@FA-UIO-66-NH₂ NPs for 2 h, followed by the previously described procedures.

To examine subcellular localization, LPS-induced RAW264.7 cells were co-incubated with Cy5.5-labeled Bai@FA-UIO-66-NH₂ NPs at various time points. After washing with PBS, the cells were fixed and stained using pre-warmed Lyso-Tracker Green (Beyotime, China) at 37℃ for 1 h. Finally, observations were conducted with a confocal laser scanning microscope (Leica, Germany).

Quantitative real-time polymerase chain reaction(qRT-PCR)

RAW264.7 cells at a density of 2 × 10⁵ were first seeded in 6-well plates and divided into different treatment groups. The total RNA of RAW264.7 cells was isolated using a total RNA extraction kit (Magen, China) and then qRT PCR with reverse transfer RNA using a qPCR detection system (Roche, Switzerland). The sequences of the required primers are shown in Table S4.

Immunofluorescence staining

RAW264.7 cells were collected after different treatments and fixed in 4% paraformaldehyde with 3% H₂O₂ (Aladdin, China). These cells were sealed with goat serum at room temperature for 30 min. CD206 and iNOS antibodies (Boster Biological, China, 1: 200) were used to incubate cells for 8 h. Staining images were observed using a fluorescence microscope.

Flow cytometry analysis of macrophage polarization

LPS-induced macrophages (2 × 10⁵ cells) were seeded into a 6-well plate overnight. the subsequent steps were consistent with the previously outlined methods. The collected cells were treated with FITC-labelled iNOS and CD206 antibodies for 30 min, respectively. The surface marker for macrophage was measured by flow cytometry.

In vivo experiments

OA model establishment

Sprague Dawley (SD) rats (170 g, 7–8 weeks old, male) were purchased from Guangxi Medical University Animal Center. In this experiment, the OA model was constructed by anterior cruciate ligament transection (ACLT). After surgery, these rats were randomly assigned to 5 groups (n = 6): 0.9% saline, Bai (100 µg/mL), FA-UIO-66-NH₂ (100 µg/mL) and Bai@FA-UIO-66-NH₂ (100 µg/mL), and received intraarticular injections with the above formulations (100 µL) once every other day for 8 weeks. Simultaneously, simple incisions of the knee skin and joint capsule were performed on the sham operation group, and no additional treatment was administered. Intra-articular injections were administered once a week, and rat samples from each group were collected 4 and 8 weeks after treatment. In addition, three independent observers were invited to evaluate and score these groups in a double-blind manner using Pelletier’s macro score.

In vivo fluorescence imaging

Further study on retention time and in vivo distribution of Bai@FA-UIO-66-NH₂ NPs in the articular cavity of SD rats using in vivo imaging. The Cy5.5-labeled Bai@FA-UIO-66-NH₂ NPs were first dispersed in a PBS buffer, forming a solution with a concentration of 100 µg/mL. Next, 100 µL of the solutions were injected into the knee joints of rats. Fluorescence signals were measured and intensity quantified using an in vivo imaging system (AniView 100, BLT, China) at different time intervals (0, 2, 6, 12, 24, 48, 72 h). Various ex vivo organs were also collected after 72 h. The fluorescence intensity was then measured and quantified using identical equipment.

Histological staining

SD rats’ knee joints from each group were collected for further evaluation, after decalcification, these samples were paraffin-embedded and stained with hematoxylin-eosin (H&E) (Solarbio, China) and Safranin O-fast green (Solarbio, China). Microscopy was used to collect photographs of these stained samples, which were then evaluated histologically.

Immunofluorescence staining

After dewaxing and hydrating, the SD rats’ knee joint slices were added with antigen repair solution and microwaved for 5 min. The slices were removed and naturally cooled to room temperature, then dipped in 3% H₂O₂ for 10 min and further sealed with goat serum for another 30 min. The knee slices were then incubated at 4℃ overnight with either iNOS (1: 100) or CD206 (1: 100). After removing the primary antibodies the next day, the FITC-labeled (1: 100) and Cy3-labeled secondary antibodies (1: 100) were incubated, stained and observed under the microscope.

Detection of ROS in articular cavity of SD rats

The levels of ROS in the articular cartilage were quantified using a tissue ROS detection kit (Bestbio, China), following the manufacturer’s instructions. In summary, 50 mg of cartilage tissue was homogenized with 1 mL of PBS buffer, then centrifuged at 1,000 revolutions per minute at 4 ℃ for 3 min to obtain the sample for subsequent analysis. Subsequently, 1 µL of the ROS probe (BBoxiProbe) was added to 200 µL of the gathered solutions, then incubated at 37℃ for 30 min. The microplate reader (BioTek, USA) was utilized to measure the absorbance of the mixture, with an excitation wavelength of 488 nm and an emission wavelength of 610 nm.

Micro-CT

The rat bone tissues were fixed in 4% formaldehyde overnight, washed with PBS, and scanned using micro-CT (voltage 90 kV, current 70 µA, resolution 10 μm). Each set of scanned images was evaluated at the same threshold to render the 3D structure for each sample. The area beneath the growth plate of the proximal tibia was chosen for three-dimensional histomorphometric analysis to determine bone mineral density (BMD).

Statistical analysis

SPP Statistics 22.0 was employed for statistical evaluation. All data are presented as mean ± standard deviation (SD). Each independent experiment was replicated at least three times. One-way ANOVA was used to assess group differences, with P < 0.05 indicating statistical significance.