Smart osteoclasts targeted nanomedicine based on amorphous CaCO3 for effective osteoporosis reversal | Journal of Nanobiotechnology

Materials

Ammonium Carbonate ((NH₄)₂CO₃, 99%), Calcium Chloride (CaCl₂, 99%), and polyvinyl pyrrolidone (PVP, K30) were purchased from Shanghai Tian Scientific Co., Ltd. Oroxylin A (ORO) was purchased from Shanghai Standard Technology Co., Ltd.

DPPC and cholesterol were purchased from AVT (Shanghai) Pharmaceutical Tech Co., Ltd. Receptor activator of nuclear factor-κB ligand (RANKL, 95%) was purchased from R&D systems Co., Ltd. Macrophage colony-stimulating factor (M-CSF, 98%) were purchased from PeproTech Co., Ltd. DSPE-PEG₂₀₀₀-DGlu6 was customized by ChinaPeptides Co., Ltd. CTX-I ELISA Kit and PINP ELISA Kit were purchased from Shanghai Lengton Bioscience Co., Ltd. All primers were synthesized by Sangon Biotech Co., Ltd.

Synthesis of DSPE-PEG₂₀₀₀-DGlu6

DSPE-PEG₂₀₀₀-DGlu6 was obtained through a conjugation reaction between DSPE-PEG₂₀₀₀-Mal and DCys-DGlu6. In brief, DCys-DGlu6 and DSPE-PEG₂₀₀₀-Mal (4:1, mol/mol) were dissolved in a solution of ACN/H₂O (2:1, v/v). The mixture was stirred, and then 0.2 M PBS and 6 M HCl solution (pH = 7.0) were added. Nitrogen gas was used to purge the mixture, replacing oxygen, and the reaction was conducted for two hours in a nitrogen environment at room temperature. The remaining unreacted DCys-DGlu6 peptide was subsequently removed by dialysis (MWCO, 1000 Da). The resulting product, DSPE-PEG₂₀₀₀-DGlu6, was then freeze-dried to obtain the final product.

Preparation of OAPLG

ACC was prepared using a steam diffusion method [48]. Initially, 200 mg of CaCl₂ was placed in a 200 ml round-bottom flask. After completely dissolving the CaCl₂ in 300 μl of distilled water, 100 mL of anhydrous ethanol was added. The flask was tightly sealed with a film and punctured with a needle to create small holes. The round-bottomed flask was placed in a desiccator along with glass vials containing (NH₄)₂CO₃ and reacted at 37 °C for 24 h. Afterward, the product was purified by centrifugation (8000 rpm,10 min), washed several times with anhydrous ethanol, and then dispersed in anhydrous ethanol for storage at 4 °C. To prepare the OCA nano-core, an ORO ethanol solution (24 mg, 1 mL) was added to a CaCO₃ ethanol solution (6 mg, 2 mL) containing PVP molecules (20 mg). The mixture was agitated at 25 °C for 4 h, and the resulting drug-coated CaCO₃, referred to as the OCA nano-core, was collected.

The ethanol solution of the OCA nano-core (20 mg, 5 mL) was mixed with a chloroform solution of DOPA (2 mg, 1 mL), which was purchased from AVT (Shanghai) Pharmaceutical Tech Co. The mixture was then subjected to 40 min of water bath sonication. The DOPA-coated nanoparticles were then purified through centrifugation. The resulting particles were re-suspended in a chloroform solution (6 mL) containing cholesterol (2 mg) (AVT (Shanghai) Pharmaceutical Tech Co.), DPPC (4 mg), and DSPE-PEG₂₀₀₀-DGlu6(8 mg) and then stirred overnight. The chloroform was removed using a rotary evaporator, and the particles were rehydrated with PBS (2 mL) under the influence of ultrasonic waves. The polyethylene glycol-coated nanoparticles were collected and purified through centrifugation (8000 rpm, 10 min) before being stored at 4 °C for further experimentation.

Drug loading content

The drug loading of OAPLG was determined using UV-Vis spectrophotometry. The measurement was performed at a wavelength of 272 nm. To disrupt the OAPLG dispersion, a 1:1 mixture of 1 M HCl and ethanol was added, effectively breaking down the OAPLG particles. The free ORO was obtained by high-speed centrifugation (20,000 rpm, 15 min). The drug loading percentage (DL%) of ORO was calculated as follows:

Here, W_A represents the weight of the free drug in the supernatant, while W represents the total weight of the system.

Drug release

To investigate drug release, 2 ml of OAPLG solution containing a consistent ORO concentration of 200 μg was placed in a dialysis bag with a molecular weight cut-off of 7,000 Da, which was then immersed in a centrifuge tube containing 25 ml of phosphate buffer solution of different pH (pH 4.5/6.5/7.4). Samples of 0.5 ml were collected at different time points under constant temperature conditions at 37 °C and a rotational speed of 100 rpm. After each sampling, an equal volume of the release medium at the same temperature was promptly replenished. The concentration of ORO was quantified with a UV-Vis spectrophotometer.

pH responsiveness on bone surface

The DiD-labeled OAPLG was added to a 96-well plate containing bone slices, incubated in complete medium for 12 h at 37 °C, and then the medium was removed; after three washes with physiological saline, the samples were replenished with complete α-MEM medium. At 1, 3, and 7 days, the DiD fluorescence signal was measured. The co-incubation was then continued by replacing the medium with an acidic buffer solution (pH 4.5). A series of images were taken over a period of 5 min to observe changes in DiD fluorescence.

Extraction and cultivation of BMMs

BMMs were extracted from the femur and tibia of C57BL/6 mice (4 weeks, female). The mice were euthanized using 3% sodium pentobarbital, and femurs and tibiae were obtained from the hind limbs in a sterile environment after 10 min of immersion in 75% ethanol. The ends of the long bones were cut to expose the marrow cavity. Subsequently, the femurs and tibiae were washed 2–3 times in a cell culture dish containing PBS and transferred to another cell culture dish containing complete growth medium. The bone ends of the femur and tibia were opened using ophthalmic scissors, and 1 mL of complete growth medium was aspirated with a syringe to flush the bone marrow cells from one end of the bones into a sterile 50 mL centrifuge tube. This process was repeated multiple times until the bones turned white. The erythrocytes were lysed with erythrocyte lysis buffer, and the cell pellet was collected by centrifugation, resuspended in α-MEM cell culture medium, and filtered through a 200-mesh sieve. The cells were subsequently suspended in 6 mL of α-MEM growth medium containing 25 ng/mL macrophage colony-stimulating factor (M-CSF; R&D Systems) for 3 days. BMMs were the only cells able to adhere and survive under M-CSF stimulation, so the culture flask’s adherent cells at the base were recognized as BMMs. When the adherent cells reached approximately 90% confluence, they were harvested by digesting with trypsin (Gibco) for 15 min and used for subsequent in vitro experiments.

Cell toxicity assay

BMMs were added to 96-well cell culture plates containing complete α-MEM medium at a density of 1 × 10⁴ cells/well and cultured for 24 h to ensure complete spreading. Subsequently, different concentrations of ORO and the nanocarrier were introduced to the cells, and they were cultured for either 24 or 72 h. Then, 10 μl of CCK-8 was added to each well and incubated at a constant temperature for a period of time to measure the absorbance of the plate using a multifunctional microplate reader. The absorbance value for the blank medium group was set to 100%.

TRAP staining

BMMs were cultured in 96-well plates (8000 cells/well) in complete growth medium containing both 25 ng/mL M-CSF and 50 ng/mL RANKL. To promote osteoclast maturation, the culture medium was changed every two days. After 5 days of induction culture, cells were fixed and stained using a TRAP staining kit (Sigma, St. Louis, USA). Mature osteoclasts were identified as cells with three or more nuclei, and Image J software was utilized for further image analysis.

Bone resorption assay

BMMs (1 × 10⁴ cells/well) were seeded onto a 96-well plate containing small bovine bone slices and induced for 10 days as previously described. Following that, osteoclasts were removed using 5% sodium hypochlorite, and the bone slices were subsequently stained with 1% toluidine blue for 2 min. An optical microscope was used to examine the absorption area of the bone slices. Image J software was utilized for further image analysis.

Fluorescent staining of F-actin rings

As described above, osteoclasts were induced in different treatment groups for 5 days. The cells were fixed and permeabilized with Triton-X100. To visualize F-actin within the cells, Fluorescein isothiocyanate (FITC)-labeled phalloidin staining was carried out. Additionally, the nuclei were stained with DAPI. Confocal microscopy images of F-actin rings were acquired (FITC Ex/Em = 488/525 nm, DAPI Ex/Em = 340/488 nm). Image J software was utilized for further image analysis.

Real-time quantitative PCR

BMMs (4 × 10⁵ cells/well) were seeded evenly in a 6-well plate with α-MEM complete medium containing RANKL. The cells were treated with ORO, APLG, OAPL, and OAPLG, respectively, and blank and positive controls were included. After 5 days of incubation with medium changes every two days, total RNA was extracted from the cells utilizing RNAiso Plus (TaKaRa, Japan). Reverse transcription was performed using PrimeScript™ RT Master Mix (TaKaRa, Japan), and finally, qPCR was performed using MonAmp™ SYBR® Green qPCR Mix (Monad, China). The primers used were those reported in previous studies and their sequences are presented in Supplementary Table S1 [44].

Western blotting

The protein levels of NFATc1 (sc-7294, Santa), c-fos (2250, CTST), Ctsk (4980, CTST), and GAPDH (ab181602, Abcam) were detected using Western blotting. To extract the proteins, cells were lysis by adding RIPA Lysis Buffer (Beyotime, China). SDS-PAGE was used to separate the extracted proteins. The proteins were transferred onto a PVDF membrane after electrophoresis. Skim milk was then used to block the membrane. The membrane is washed and incubated with primary antibody overnight with gentle shaking, then incubated with secondary antibody for 2 h at room temperature. An enhanced chemiluminescence substrate was used to detect the antibodies’ chemiluminescent signal. ImageQuant LAS 4000 system (GE Healthcare, Silverwater, Australia) was used to capture images, which were then analyzed with ImageJ software.

In vitro bone targeting of OAPLG

To examine the binding capacity of bone matrix and bone-targeting delivery systems in vitro, a bone slice adsorption experiment was conducted. In simple terms, DiD-labeled OAPL and OAPLG were added to a 96-well plate containing bone slices. The plate was gently oscillated at room temperature to facilitate the adsorption of DiD-labeled OAPL and OAPLG onto the bone slices. The adsorption of DiD-labeled OAPL and OAPLG was quantified by measuring the difference in fluorescence intensity between the beginning stock solution and the supernatant after adsorption, using the following formula:

In this equation, I₀ refers to the initial fluorescence intensity of the stock solution, while I_a refers to the fluorescence intensity of the supernatant after 1 h of incubation.

In vivo bone targeting of OAPLG

All experiments related to the animals involved in this study were conducted in strict accordance with the Guidelines for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of Shanghai University. C57BL/6 mice were used as the experimental subjects to evaluate the bone-targeting ability of OAPLG. The mice were given an intravenous injection of either 100 μL of PBS or a solution containing nanoparticles labeled with DiD, with or without Dlu6 surface modification (DiD concentration of 20 μg/mL). At 4 and 8 h after nanoparticle administration, the mice were euthanized under anesthesia. Tissue samples were collected for in vivo analysis to investigate the biodistribution of the DiD-labeled nanoparticles.

Establishment and treatment of osteoporosis mouse model(OVX)

A total of 30 female C57BL/6 mice (6 weeks) were purchased from Changzhou Cavens Experimental Animals Co., Ltd. (Changzhou, China). After a two-week acclimation period, 25 mice were randomly selected to undergo ovariectomy surgery. Following anesthesia, the dorsal fur was shaved, and the mice were placed in a supine position. The dorsal skin was disinfected, and a 1 cm longitudinal incision was made along the midline. Careful dissection was performed to expose the ovaries by removing the surrounding adipose tissue. After ligating the fallopian tubes, both ovaries were completely excised, and the incision was closed and disinfected with iodine. The remaining 5 mice underwent the sham surgery group, where the same procedure was performed, but the ovaries were preserved. After one month post-surgery, 25 OVX mice were randomly assigned to 5 groups. Finally, a total of 6 groups were established: sham surgery group, OVX group, OVX + ORO group, OVX + APLG group, OVX + OAPL group, and OVX + OAPLG group. ORO, APLG, OAPL, and OAPLG solutions were administered every two weeks at a controlled drug concentration of 0.1 mg/kg. The sham surgery group and OVX group received intravenous injections of normal saline. The treatment duration was 2 months. Upon completing the experiment, peripheral blood samples were obtained through enucleation under anesthesia. Subsequently, the mice were euthanized to retrieve bilateral femurs as well as major organs. All collected tissues were subsequently fixed in 4% PFA for further experiments. Micro-CT analysis (Bruker micro CT, Belgium) was performed to assess alterations in femoral trabecular bone. The SkyScan-1176 system was used with a voxel size of 13 μm, 90 kV, 278 μA, exposure time of 230 ms, 0.5 mm aluminum filter, and a rotation step of 180°. Three-dimensional reconstruction and image visualization were conducted using NR Economy software version 1.6. After three-dimensional reconstruction, bone analysis was performed using CT software version 1.13. The assessed parameters encompassed BMD, BV/TV, Tb. Th, Tb. N, and Tb. Sp, serving as indicators for evaluating bone repair conditions.

Tissue histological analysis

The femur samples from the mice were placed in a 10% EDTA solution for the purpose of decalcification, with regular solution changes occurring every 3 days. After decalcification, the samples underwent gradient ethanol dehydration and were then made transparent by exposure to xylene solution. Later, the samples were immersed in liquid paraffin and left to solidify before obtaining 4 μm thick sections along the longitudinal axis. These sections were subjected to staining using a combination of H&E reagents, Masson’s reagent, and a TRAP staining kit. The major organs of the mice were fixed and H&E stained to further evaluate the safety of various drug treatments.