The role of echinacoside-based cross-linker nanoparticles in the treatment of osteoporosis

Reagents

Dulbecco’s Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were commercially purchased from Gibco Inc. (Themo Fisher Scientific, Waltham, MA, USA). Echinacoside, cross-linker, and esterase were purchased from MedChemExpress (Monmouth Junction, NJ, USA), Source Leaf (China), and Maclean (China), respectively. M-CSF and RANKL were acquired from PeproTech Inc. (Themo Fisher Scientific).

BMDMs and BMSCs

Following the description in a previous study (Marim et al., 2010), BMDMs were extracted from the femurs and tibiae of 6- to 7-week-old C57BL/6 mice purchased from Shanghai Laboratory Animal Center (Shanghai, China). DMEM was used to culture the BMDMs, and 10% heat-inactivated FBS and 1% penicillin-streptomycin were added as supplements.

CL-ECH nanoparticle preparation and characterization

ECH (2 mg) and CL (cat no. S26218; Source Leaf; 1:4; 1:2; and 1:0.5 molar ratios, respectively) were dissolved in dimethyl sulfoxide (DMSO) solvent (100 µL) overnight. The solution was added dropwise to deionized water (five mL) and stirred for 2 h. The CL-ECH nanoparticles were obtained by ultrafiltration (8,000 rpm, 30 min), resuspended in one mL of deionized water, and filtrated through a filter (0.22 µm). The size of the nanoparticles was measured with dynamic light scattering and the morphology was observed under a transmission electron microscopy (TEM). Nanoparticles were measured under an ultraviolet (UV)-visible spectroscopy (Thermo Fisher Scientific, Waltham, MA, USA), and the content of ECH was calculated using the standard curve of ECH (Fig. S1).

After co-culturing the cells with nanoparticles for 4 h, the uptake of Cy5-loaded CL-ECH nanoparticles in BMDMs and BMSCs was detected using flow cytometry (Beckman Coulter, Danaher Corporation, Brea, CA, USA).

Intracellular ROS measurement

DCFH-DA (20,70-dichlorodihydrofluorescein-diacetate) stock solution (Calbiochem, Germany) was prepared using DMSO and kept in the dark at 20 °C. On culture dishes (60-mm), BMDMs were primed with 50 µM of ECH or CL-ECH (5, 10, 25, 50 µM) for 6 h and then added with 100 ng/mL of LPS (lipopolysaccharides from Salmonella enterica serotype Minnesota; Sigma-Aldrich, St. Louis, MO, USA) when the confluence reached 90%. After 24-hour LPS stimulation, the cells were treated with 10 M of DCFH-DA for 30 min. The green fluorescence of DCFH-DA was recorded at 515 nm with flow cytometry (Beckman Coulter).

Visualization of NRF2 nuclear translocation with confocal microscopy

BMDMs (0.1 × 10⁶) were plated on a 24-well chamber slide and treated by ECH (50 µM) or CL-ECH (10 µM) for 6 h at 37 °C and then by LPS stimulation for 18 h. Next, the cells were fixed with 4% paraformaldehyde and permanently fixed with 0.3% Triton X-100 in phosphate-buffered saline (PBS). The cells were first probed by Anti-pNRF2 primary antibody (DF7519; Affinity) and then by anti-rabbit Alexa Fluor 488 secondary antibody (Life Technologies, Thermo Fisher Scientific). DAPI dye was used to mount the cells, and the cells were photographed using a confocal microscope (Leica, Wetlzar, Germany).

Real-time polymerase chain reaction (qRT-PCR)

The total RNA was extracted using TRIzol solution (Invitrogen). A Primer Script RT Reagent Kit (Takara Bio, Tokyo, Japan) was used to perform reverse transcription of the RNA samples into complement DNA (cDNA). Three independent PCR amplifications were carried out using RT-PCR system (Roche Diagnostics, Basel, Switzerland) with specific primers and iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA). The PCR cycling was set at 95 °C for 3 min, followed by 39 cycles at 95 °C for 10 s, at 57 °C for 10 s, and at 72 °C for 30 s.

ALP staining

After osteogenic differentiation for 7 days, the cells were fixed by 4% polyoxymethylene for 30 min and then washed by PBS 3 times. ALP staining solution (BCIP/NBT alkaline phosphatase kit; Beyotime, Shanghai, China) was used to incubate with the cells together at 37 °C for 10 min and then the cells were rinsed in distilled water 3 times.

Alizarin red S staining

After osteogenic differentiation induction, the cells were fixed with 4% polyoxymethylene for 30 min and washed with PBS 3 times. Next, the cells were stained with 1% Alizarin red S (pH 4.2; Sigma-Aldrich, St. Louis, MO, USA) for 15 min. Histomorphometry was observed and photographed using Nikon Eclipse optical microscope (Nikon, Tokyo, Japan) and Nikon D7000 camera (Nikon).

TRAP and phalloidin staining

BMDMs were plated onto 24-well plates (5 × 10³ cells/well) in the presence or absence of recombinant RANKL (100 ng/mL) with or without 50 µM ECH or 10 µM of CL-ECH. During 6 days of culture, all media were refreshed every 3 days. The cells were stained for TRAP using an acid phosphatase kit (Sigma-Aldrich) and fluorescein isothiocyanate (FITC) phalloidin (#p5282; Sigma-Aldrich). TRAP-positive, multi-nucleated cells (over 3 nuclei) were defined as dark-red multinucleated cells.

Hematoxylin and eosin (HE) staining and Masson tissue staining

All the animal experiments were officially approved by the Animal Ethics Committee of Tongji University (Approval number: (2022)- DW- 02) and conducted following the National Institutes of Health guide for the Care and Use of Laboratory Animals. An osteoporotic mouse model was established using 20 eight-week-old female C57BL/6 mice (approximately 25 g, Shanghai Laboratory Animal Center). The mice were randomly divided into 4 groups (n = 5) as follows: (I) control group with sham surgery, (II) OVX group, (III) ECH group with free ECH, and (IV) CL-ECH group with CL-ECH nanoparticles. ECH and CL-ECH nanoparticles (400 µg ECH/mL in 0.9% NaCl solution) at a dose of 1 mg/kg were injected into the mice via tail vein every 3 days for 8 weeks. Five animals were kept in a cage, fed with standard laboratory food, and provided with access to tap water in a climate-controlled environment (55% humidity, 12 h of light and 12 h of darkness at 25 °C).

The mice were euthanized by cervical dislocation. The femurs were removed bilaterally, and the soft tissues on the surface were collected and then fixed in 4% polyethylene. HE and Masson staining was performed on the tissue sections after decalcification, paraffin embedding, sectioning, and dewaxing.

Statistical analysis

SPSS 20.0 software (IBM Corp., Armonk, NY, USA) was used for statistical analysis. Data were shown as the mean ± standard deviation. When two independent groups were compared, Student’s t-test was used. P < 0.05 was defined as statistically significant difference.

Characterization and cellular uptake of CL-ECH

CL-ECH was prepared by mixing cross-linker and ECH at different ratios, and the particle size and UV level were measured. The results showed that the particle size was larger than 100 nm when the ratio was 1:4 and 1:2 and the size became smaller than 100 nm when the ratio was 1:0.5 (Fig. 1A). The TEM results showed a spherical morphology (Fig. 1B). The drug loading rate was measured to be 8.4 weight (wt) % according to the UV results (Fig. 1C) and the standard curve of ECH (Fig. S1). In the presence of esterase, CL-ECH was rapidly released (Fig. 1D).

After 4 h of co-incubation with the cells, flow cytometry analysis showed that Cy5-loaded CL-ECH nanoparticles were endocytosed into BMDMs, with around 15.05% of cells (and 9.02% of BMSC) showing Cy5-positive (Fig. 1E).

CL-ECH pretreatment promoted the antioxidant effect of BMDMs

Identification of BMDMs

After induction of mouse monocytes, 97.9% of the cells differentiated into mature macrophages expressing F4/80 marker (Fig. 2A), indicating that the mouse-derived macrophages were successfully cultured and could be used for subsequent experiments.

CL-ECH and ECH had similar osteogenic effects on BMSC cells

ALP staining and ARS staining in the CL-ECH group showed similar results when compared to the ECH group (Figs. 3A and 3B). Additionally, except for the RANKL genes, whose expression was reduced and significantly different between the two groups, the messenger RNA (mRNA) expression levels of osteogenic-related genes (RUNX-2, ALP, COL, OCN, and OPG) manifested a slight increase but the difference was not statistically significant (Fig. 3C). In conclusion, CL-ECH and ECH shared a potential and similar ability to promote osteogenesis in BMSC cells.

CL-ECH pretreatment inhibited RANKL-induced osteoclastogenesis in BMDMs

TRAP staining and ghost pen cyclic peptide staining were performed during RANKL-induced osteoclastogenesis, and the results demonstrated significantly inhibited osteoclastogenesis in both ECH and CL-ECH groups (Fig. 4A). Through the detection of osteoclast-related gene expression, we found that the mRNA expression levels of NF-κB, C-FOS, and MMP-9 were further reduced in CL-ECH group but those of TRAP genes remained unchanged (Fig. 4B). This revealed that CL-ECH inhibited osteoclastogenesis from decreasing osteoclast inflammation levels.

Evaluation of HE and MASSON tissue staining

Observation of the tissue sections under an inverted light microscope showed that compared to the control group (Fig. 5), the number of bone trabeculae was significantly reduced and the distribution of bone trabeculae was sparser in the OVX group, indicating that the osteoporosis model was established successfully. After the ECH intervention, we also found that the number of trabeculae increased and the density increased and bone loss was inhibited in the ECH administration group. Interestingly, CL-ECH further reduced the loss of bone trabeculae.

Based on these findings, a molecular mechanism diagram was developed, as shown in Fig. S2. Cross-linked nanoparticles based on ECH had unique nano-biological effects by promoting the antioxidant effect and inhibiting osteoclast generation of macrophages regulated by delivered ECH.