In vitro investigation of anti-inflammatory activity of propolis/saffron extract/curcumin-loaded ZIF8 nanoparticles and their potential application for treating osteoarthritis

ZIF8 nanoparticles devoid of content were subjected to a pretreatment at 600°C for an hour to eliminate any moisture residing within the zeolite pores. Subsequently, these nanoparticles were employed to encapsulate bioactive substances. The process involved mixing solutions of propolis, saffron extract, and curcumin, each prepared at a concentration of 10 g per 150 mL of water (sourced from Barij Essence, Kashan, Iran), with ZIF8 nanoparticles (20 g). The mixture was then magnetically stirred for a duration of 24 hours. The resulting drug-loaded ZIF8 nanoparticles were collected through centrifugation at 15,000 × g for 30 minutes, followed by a washing step with deionized water and subsequent centrifugation. The resultant solutions were then subjected to lyophilization for 2 days to obtain the drug-loaded ZIF8 nanoparticles. These ZIF8 nanocarriers, loaded with propolis, saffron extract, and curcumin, were respectively denoted as PROZIF, SAFROZIF, and CURCUZIF. Drug-free ZIF8 nanoparticles were named ZIF.

PROZIF, SAFROZIF, ZIF and CURCUZIF were coated with gold for 250 seconds and then imaged using a scanning electron microscopy (SEM) device at 25 kV accelerating high voltage.

L929 cells, a murine fibroblast cell line obtained from ATCC, USA, were cultured in 96-well plates at a density of 4000 cells per well for 5 days. Various concentrations (1 μg/ml, 10 μg/ml, 100 μg/mL, and 1000 μg/mL) of PROZIF, SAFROZIF, and CURCUZIF nanocarriers were used. Cell viability assays were conducted on days 1, 3, and 5 using the MTT assay kit from Sigma Aldrich, USA. The assay involved replacing the culture media with 200 μL of MTT solution (0.5 mg/mL) and allowing the cells to incubate with the MTT reagent for 4 hours. This led to the conversion of MTT into formazan crystals by metabolically active cells, indicating their metabolic activity and overall health. After incubation, 200 μL of dimethyl sulfoxide (DMSO) solution was added to each well to dissolve the formazan crystals. Thorough mixing ensured complete solubilization, enabling accurate absorbance measurements at a wavelength of 570 nm.

To assess the anti-inflammatory properties of PROZIF, SAFROZIF, and CURCUZIF in vitro, the RAW264.7 macrophage cell line was utilized. RAW264.7 cells, a murine-derived macrophage cell line obtained from ATCC, were cultured in well plates at a density of 500,000 cells per well. These cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillinstreptomycin, and 1% L-glutamine. Inflammation was induced by exposing the RAW264.7 macrophages to lipopolysaccharide (LPS) at a concentration of 1 μg/mL. Following LPS exposure, various concentrations (1 μg/mL, 10 μg/mL, 100 μg/mL, and 1000 μg/ml) of PROZIF, SAFROZIF, ZIF and CURCUZIF nanocarriers were introduced to the culture wells containing the stimulated RAW264.7 macrophages. After co-culturing with the nanocarriers for 24 hours, the culture medium underwent centrifugation to remove cellular debris. The resulting supernatant was analyzed using an enzyme-linked immunosorbent assay (ELISA) kit from Abcam, USA, to accurately measure the levels of pro-inflammatory cytokines, specifically tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) present in the culture medium.

L929 cells were plated in a 24-well plate and allowed to grow until they reached 80% confluence. Subsequently, a straight incision was created at the center of the cell monolayer using a 200-μL pipette. The cellular debris was then eliminated by rinsing with phosphate-buffered saline (PBS). Following this, the cells were treated with a concentration of 100 μg/mL (determined from the results of the antiinflammatory assay) of each nanocarrier for a duration of 72 hours. The reduction in wound size was examined using a light microscope on days 0, 1, and 3, and image analysis software was employed to calculate the extent of wound size reduction.

The functional groups of PROZIF, SAFROZIF, and CURCUZIF nanocarriers were analyzed using FTIR (Bruker). FTIR is a powerful analytical technique used to identify chemical bonds and functional groups present in a sample by measuring the absorption of infrared radiation. The FTIR assay was performed within the spectral range of 400–4000 cm⁻¹, covering both the fingerprint region and functional group-specific regions of the infrared spectrum.

The release of propolis, saffron extract, and curcumin from ZIF nanocarriers was investigated using UV-visible spectroscopy at wavelengths of 356 nm, 371 nm, and 412 nm, respectively. Initially, 200 mg of each nanocarrier was loaded into 20 mL of phosphate buffer solution and maintained at 37°C for 7 days. At various time intervals, 0.5 mL of the phosphate buffer solution was sampled and replaced with an equal volume of fresh buffer solution. Subsequently, the optical density of the sampled solution was measured, and the values obtained were correlated with the standard curve of each drug. Ultimately, the cumulative drug release was determined.

The data was analyzed using GraphPad Prism software, employing Student’s t-test and one-way analysis of variance (ANOVA) methods.

Results (Fig. 1) showed that ZIF, PROZIF, SAFROZIF, and CURCUZIF nanocarriers were composed of irregularly shaped particles. The size distribution of the nanocarriers was relatively wide, and the particles exhibited a rough surface. Particle size measurements revealed that ZIF, PROZIF, SAFROZIF, and CURCUZIF nanocarriers had approximate sizes of 338.123 ± 18.29 nm, 412.72 ± 22.26 nm, 536.87 ± 17.29 nm, and 614.23 ± 29.64 nm, respectively.

Fig. 1.

Results (Fig. 2) showed that ZIF, PROZIF, SAFROZIF, and CURCUZIF nanocarriers exhibited a dose-dependent effect on the viability of L929 cells. In all groups, at all the studied time points, cells incubated with 100 and 1000 μg/mL nanocarriers displayed significantly lower viability compared to other groups. It appears that concentrations of 1 and 10 μg/mL imparted significantly lower toxicity towards cells, as evidenced by the lack of significant differences in optical density between these concentrations and the control group.

Fig. 2.

Results (Fig. 3) showed that RAW264.7 macrophage cells cultured with a concentration of 10 μg/mL had significantly lower levels of proinflammatory cytokines compared to the other concentrations. In Figure 4, the antiinflammatory activity of ZIF, PROZIF, SAFROZIF, and CUR-CUZIF was compared. Across most of the concentrations studied, PROZIF exhibited significantly stronger anti-inflammatory activity compared to the other groups.

Fig. 3.

Fig. 4.

Results (Fig. [5]) showed that on the third day, L929 cells cultured with PROZIF exhibited a significantly higher percentage of in vitro wound closure compared to the other groups. The percentage of wound closure in the ZIF group was significantly lower than in the PROZIF, SAFROZIF, and CURCUZIF groups. On the second day, no significant statistical difference was found between all groups.

Fig. 5.

Figure 6 shows the FTIR spectra of different nanocarrier systems. As shown, in the ZIF spectra, one of the prominent features is a strong peak observed in the region of 1550–1600 cm⁻¹, indicative of the C=N stretching vibration originating from the imidazole ring. This vibrational mode contributes significantly to the overall spectrum and serves as a distinguishing marker for ZIF compounds. Additionally, a medium-intensity peak typically appeared between 1400 and 1500 cm⁻¹, corresponding to the C-N stretching vibration within the imidazole ring. This feature further elucidates the presence and configuration of the imidazole moiety within the framework. Furthermore, a weaker peak in the range of 800–900 cm⁻¹ is discernible, attributed to the C-H bending vibration within the imidazole ring. Concurrently, PROZIF exhibited additional peaks or modifications in peak intensities within this range, reflecting interactions between propolis constituents and the ZIF matrix. Furthermore, characteristic peaks of propolis components, such as flavonoids and phenolic compounds, appeared in the FTIR spectra. These include peaks at approximately 1650–1750 cm⁻¹, corresponding to C=O stretching vibrations of flavonoids, and peaks around 1200–1300 cm⁻¹, attributed to C-O stretching vibrations of phenolic compounds. In the FTIR spectra of saffron-loaded ZIF nanocarriers, several peaks indicative of specific functional groups present in saffron constituents were identified. These included peaks observed around 2900–3000 cm⁻¹, representing C-H stretching vibrations, which are abundant in saffron’s organic compounds, including crocin, picrocrocin, and safranal. Carbonyl stretching vibrations, typically found in compounds like crocin and safranal, were detected in the range of 1700–1800 cm⁻¹. Additionally, peaks around 1100–1200 cm⁻¹ corresponded to C-O stretching vibrations, prevalent in saccharide molecules present in crocin, the principal carotenoid in saffron. Bending vibrations of C-H bonds, indicative of aromatic compounds such as crocin and safranal, were observed near 800–900 cm⁻¹. Furthermore, peaks in the fingerprint region (below 1500 cm⁻¹) provided insights into the overall molecular structure and composition of saffron extract, including various functional groups present in its constituents. In the FTIR spectra of CURCUZIF particles, several characteristic peaks indicative of both the ZIF framework and curcumin constituents were identified. Among these features, specific peaks associated with curcumin molecules were discernible. For instance, curcumin contains functional groups such as aromatic rings and conjugated double bonds, which give rise to characteristic peaks in the FTIR spectra. Peaks associated with aromatic C=C stretching vibrations were observed around 1600–1650 cm⁻¹. Furthermore, peaks corresponding to C=O stretching vibrations in the β-diketone moiety of curcumin appeared around 1650-1700 cm⁻¹. Additionally, peaks in the range of 1300–1400 cm⁻¹ were attributed to the C-O stretching vibrations in the methoxy groups of curcumin.

Fig. 6.

Results (Fig. 7) showed that propolis, saffron extract, and curcumin were released from the ZIF structure in a sustained manner. In the initial hours, there was a burst release profile that was followed by a sustained release phase that continued for more than 7 days. At the end of 7th day, the cumulative release percentage of propolis, saffron extract, and curcumin was measured to be around 82.71 ± 7.27%, 95.30 ± 2.60%, 84.60 ± 5.31%, respectively.

Fig. 7.