3.1. Synthesis of nanomaterials
In this study, Glab@ZIF-8 and ZIF-8 materials were synthesized through a greener approach, the “one-pot technique”, as illustrated in Fig. 1. In comparison to the other typical methods, the one-pot synthesis method is more convenient for handling raw materials at a smaller scale and is feasible for obtaining the resultant material at low cost, energy and time [41]. In addition, the one-pot strategy significantly reduces the number of reactions [41]. In the case of environmentally friendly ZIF-8, this method requires only zinc metal ions and 2-methylimidazole (as an organic linker) solutions in deionized water. During the mixing of these two solutions at room temperature (27°C) and at the specified times, porous structures with hollow cavities are generated [41]. For Glab@ZIF-8, the drug solution was made into ethanol, which was subsequently added to the ZIF-8 solution system. This approach allows guest molecules to be trapped in cavities of ZIF-8 core shells, which may cause stretching and an increase in the size of the core shell structure of the composite (Glab@ZIF-8) compared to that of pure ZIF-8 [41]. In this study, ZIF-8 or Glab@ZIF-8 nanocomposite synthesis was observed within a few seconds (~ 15), as the transparent solutions were immediately turned into milky solutions [18, 19]. The yield of Glab@ZIF-8 based on dry weight was 38.5%. However, in the single-step synthesis of Glab@ZIF-8, a 28.44% loading efficiency was found for glabridin, and the drug loading encapsulation efficiency was ~ 98.7%. The high drug loading of glabridin into ZIF-8 frameworks could be attributed to the large pore size [27], robust porosity [25] of ZIF-8 and robust contact between the guest (glabridin drug) and host (ZIF-8) [42].
3.2. Characterization of ZIF-8 and Glab@ZIF-8 nanomaterials
The encapsulation of glabridin within the ZIF-8 i.e., Glab@ZIF-8 nanomaterial was confirmed through UV‒Vis spectroscopy and FTIR spectroscopy. Figure 2 (a) shows that glabridin exhibited a maximum UV‒Vis absorption peak at 283 nm, which is consistent with the findings of previous studies [38, 43]. ZIF-8 had a maximum UV‒Vis absorption peak at a wavelength of 211 nm [44, 45]. Compared with that of ZIF-8, the UV‒Vis absorption peak of Glab@ZIF-8 was redshifted at 222 nm, and the characteristic absorption peak of glabridin was masked. This confirmed the encapsulation of the drug (glabridin) into the ZIF-8 framework [18–20, 46]. The entrapment and interaction between the drug and ZIF-8 are subjected to various forces, such as electrostatic interactions, π-π interactions or strong hydrogen bonds between guest (glabridin) and host (ZIF-8) molecules [18, 19, 42]. FTIR analysis of pure glabridin (Fig. 2b) revealed O − H stretching in the IR spectrum, with a maximum absorption peak at 3390 cm− 1 [38]. The peak at 2974 − 2922 cm− 1 represents the CH3 group [38]. The peak at 1744 − 1463 cm− 1 might be attributed to the C–C stretching vibration of the aromatic ring [38]. The peak at 1370 − 1329 cm− 1 is due to deformation vibration, and the peak at 1215 − 1152 cm− 1 is attributed to C-O-C stretching. It has also been reported that the peak at 1080 − 945 cm− 1 corresponds to C-O stretching vibrations and that the peak at 851 − 706 cm− 1 may correspond to O-H bending [38]. The ZIF-8 IR spectrum, as presented in Fig. 2 (b), depicted N-H stretching and C-H aromatic stretching at absorption peaks from 3431 − 3099 cm− 1 [47] and C─H stretching [18] within 2916–2922 cm− 1. However, a peak at 2844 cm− 1 is assigned to the vibrational stretch of the methyl group contained in 2-methylimidazole [19]. Moreover, representative peaks at 1744 cm− 1, 1679 − 1656 cm− 1 and 1562 − 1428 cm− 1 attributed to ZIF-8 were attributed to the C-C and C–N stretching of imidazole and entire ring stretching, respectively [18, 19, 47]. The peak at 1386 − 681 cm− 1 is also attributed to the 2-MIM ring [47, 48]. The Glab@ZIF-8 composite material exhibited similar peaks to those of ZIF-8 (Fig. 2b). However, compared with those of the pure ZIF-8 peak, the broad peak at 3503 − 3410 cm − 1 is blueshifted (3746 cm − 1, 3431 cm − 1), and a redshift can be observed compared to the broad absorption peak at 3390 cm− 1. Consequently, this pattern showed the encapsulation of glabridin in ZIF-8 frameworks [18, 38, 49]. Furthermore, other distinctive peaks of pure glabridin were obscured by the Glab@ZIF-8 material [38].
The DLS study showed that the mean hydrodynamic diameter sizes of ZIF-8 and Glab@ZIF-8 were 103.19 nm and 146.03 nm, respectively (Fig. 2c, d). The diminished dimensions of nanoparticles (NPs) confer significant advantages in biomedical applications, particularly due to their enhanced suitability and efficacy [50]. On the other hand, the increase in the size of the Glab@ZIF-8 material to that of ZIF-8 [29] also confirmed drug encapsulation. Furthermore, ZIF-8 exhibited a + 35.17 mV zeta potential (ζ), and Glab@ZIF-8 also exhibited a + 41.9 mV zeta potential (Fig. 2e). Therefore, the absence of any electrical charge and a slightly increased zeta potential also indicate the encapsulation of glabridin in ZIF-8 [46]. High zeta potential values indicate that these nanoparticles are well dispersed and stable in water and PBS (pH 7.4) [46]. Additionally, it has been reported that ζ-potentials of NPs within ± 30 to ± 40 mV indicate moderate electrostatic stability, although ζ-potentials above ± 40 mV reveal strong electrostatic stability [51]. Subsequently, in the present study, Glab@ZIF-8 NPs were prepared with increased stability and could also be stored in suspension at room temperature [51]. Moreover, TEM analysis based nanoparticle size of ZIF-8 was 91.69 ± 6.2 nm (Fig. 2f). However, Glab@ZIF-8 had an average size of 138.62 ± 5.4 nm (Fig. 2f). An increase in size results from the loading of drug molecules into MOF cavities.
The morphology of the prepared materials was studied through TEM and SEM. The nanosized ZIF-8 structure as developed by TEM and SEM is homogeneous, uniform and hexagonal as shown in Fig. 3 (a, c). Similarly, the morphology of Glab@ZIF-8 as depicted in Fig. 3 (b, d) is analogous to that of ZIF-8. Furthermore, the TEM image of Gla@ZIF-8 revealed that the material has sharp edges, which are highly favorable for certain antibacterial applications [52–54]. The elemental composition of ZIF-8 and Glab@ZIF-8 nanomaterials was analyzed EDX via SEM technique using EDAX software. It can be seen in Fig. 4 (a) that ZIF-8 has an elemental composition of 21.95 % zinc(Zn), 34.82 % carbn (C), 17.37 % nitrgen (N) and 19.01 % oxygn (O). In contrast, Glab@ZIF-8 as shown in Fig. 4 (b) has 19.09 % Zn, 9.61 % C, 1.27 % N an 14.24 % O inrespect of elemental composition. It is supposed that after loading of glabridin into ZIF-8 frameworks (Glab@ZIF-8), the weight % of Zn was reduced, while, the weight % of C was increased to that of ZIF-8. Yet the weight % of O and N was also oscillated. This phenonmenon also demonstrated the loading of drug into ZIF-8 nanoparticles [55, 56].
The loading of glabridin into ZIF-8 was also explored by comparing the powdered XRD pattern of the pure materials with that of the synthesized material (Fig. 5). XRD is widely applied as an absolute technique for observing the crystal structure of certain materials [57]. The XRD pattern of ZIF-8 showed 2θ clear sharp characteristic peaks at 7, 9, 10, 11, 11.6, 12.4, 13, 16, 17, 18, and 18.6 diffraction angles, which are similar to reported works [18, 58] and are inconsistent with the planes indices 110, 200, 112, 022, 013 and 222, respectively [18, 19]. Other 2θ peaks in ZIF-8 were observed at 20, 22, 22.7, 23.3, 24.6, 25.9, 27, 27.7, 28, 29.3, 30, 32, 33, 34, 35, 36.6, 38, and 40 cm− 1 diffraction angles. Thus, the material ZIF-8 is in its pure phase and crystalline [18]. Here, the XRD pattern of pure glabridin was consistent with that of previously reported studies [38]. The characteristic 2θ peak values were observed at 8.5 10, 12, and 18 [38], where diffraction angles appeared. However, other 2θ peaks at 43.28 and 44.4° were also noted. Moreover, similar 2θ peaks were observed for Glab@ZIF-8, although the 2θ peaks at 8.5 10, 12, and 18 cm were masked by the Glab@ZIF-8 composite. This could result from the loading of drugs into the cavities of ZIF-8 frameworks [20, 42, 59]. Likewise, the XRD pattern of Glab@ZIF-8 was consistent with that of pure ZIF-8; henceforth, the composite material had a crystalline structure and was found to be a pure phase [18, 60].
3.3. Thermal stability of ZIF-8 and Glab@ZIF-8 nanomaterials
The Fig. 6 illustrates the thermal stability of Glab@ZIF-8, ZIF-8, and glabridin. Glab@ZIF-8 exhibited a 1.43% initial weight loss at 145°C, which was due to the removal of any solvent [18, 19, 61]. Afterwards, an 8.86% (at 214°C) weight loss was attributed to the primary decomposition of the MOF ring structure [18, 19, 61]. Moreover, Glab@ZIF-8 degraded 12.03%, 18.65% and 32.14% at 251°C, 450°C and 644°C, respectively. Henceforth, 67.86% of the Glab@ZIF-8 particles were estimated to remain stable even at the highest temperature. It is assumed that at high temperatures, 2-methylimidazolate is completely burned; therefore, the decomposition of ZIF-8 occurs. In contrast, glabridin gradually degraded at 50°C, 92°C and 293°C, as indicated by weight loss values of 1.87%, 4.96% and 5.85%, respectively. Complete degradation of glabridin was considered up to ≥ 365°C, as the weight loss was 79.12%. In contrast, ZIF-8 remained stable between 76°C and 260°C, so minimal weight losses of 1.84% and 10.71%, respectively, were observed. However, gradual degradation of ZIF-8 occurred at 286°C (23.53%), 466°C (28.36%) and 598°C (30.57%). In conclusion, the encapsulation of biomaterials in ZIF-8 frameworks could be considered to modify and improve their thermal stability, and their shelf life could also be extended for a longer time at room temperature.
3.4. Study of antibacterial activity
Humans are easily infected by many pathogenic organisms in communal settings and via the consumption of contaminated food. Among human pathogens, Bacillus cereus and Escherichia coli cause gastroenteritis, Staphylococcus aureus is responsible for skin and soft tissue infections, and Pseudomonas aeruginosa causes infections in burn patients. It has been documented that these bacteria may produce resistant biofilms; colonize wounds, implants, and medical equipment; and frequently cause morbidity and death [62]. The overprescription and excessive use of antibiotics have led to multidrug resistance among bacteria. Henceforth, the ongoing development of substitute antibacterial agents for traditional antibiotics is a major task. Natural antibacterial agents obtained from animals, plants, algae, fungi and bacteria are of great interest as substitutes for typical antibiotics [63]. Additionally, natural bioactive compounds are “generally acknowledged as secure” and have a “greener” impact on consumers. Plant-originated antimicrobial agents have long-term value in the healthcare market because they satisfy customers' growing need for environmentally friendly products, hence enhancing the importance of researching new, promising biopharmaceutical products [63]. For example, glabridin is a crystalline compound enriched in numerous biological applications, including antibacterial potential [64]. The suboptimal pharmacokinetic characteristics and inadequate stability of glabridin restrict its potential therapeutic applications. Consequently, in response to advancements in nanotechnology, medicinal and synthetic chemists have embraced the strategy of developing drug-based metal-organic frameworks (MOFs) to address the limitations associated with naturally occurring bioactive compounds. Previous research has documented the effectiveness of natural drug-based nanomaterials in combating drug-resistant bacteria [18, 19].
The antibacterial activity of the tested materials is given in Fig. 7 (a, b, c). The findings of this study illustrate that 25 µg/50 µL of Glab@ZIF-8 had additional comprehensible growth inhibition effects on S. aureus, followed by E. coli and Bacillus subtilis, with growth inhibition zones of 26 ± 0.01 mm, 23 ± 0.02 mm and 11 ± 0.01 mm, respectively. However, compared with Glab@ZIF-8, glabridin and ZIF-8 (50 µg/100 µL) exhibited almost half the restriction on the growth of S. aureus, E. coli and Bacillus subtilis (table S1). Subsequently, growth inhibition zones of 15 ± 0.03 mm and 17 ± 0.01 mm, 12 ± 0.01 mm and 13 ± 0.03 mm and 7 ± 0.01 mm and 7.9 ± 0.01 mm were noted for glabridin and ZIF-8, respectively. Nevertheless, 25 µg/50 µL of ampicillin suspension had 27 ± 0.03 mm, 23 ± 0.03 mm and 20 ± 0.01 mm growth inhibition zones for S. aureus, E. coli (BW25113/F), and Bacillus subtilis (JCL16), respectively. In conclusion, the improved antibacterial efficacy of Glab@ZIF-8 compared to that of the natural compounds glabridin and pure ZIF-8 is attributed to the synergistic mechanism of action. Furthermore, encapsulation of similar drugs in MOFs could be used to formulate new alternative antibacterial materials, and this strategy would be highly helpful for reducing the prevalence of MDR bacteria. Nevertheless, an important benefit would be that microbes should not be as likely to become resistant to these newly synthesized nanomaterials, as they might harbor more than one bactericidal target [63]. The MIC values for S. aureus and E. coli were 31.2 µg (Fig. 7d) and 62.5 µg (Fig. 7e), respectively. However, 125 µg was the MIC against Bacillus subtilis (Fig. 7f). The MIC assay results are in agreement with the antibacterial growth inhibition assay results. The results of this study revealed that Glab@ZIF-8, ZIF-8 and glabridin were more effective against gram-positive S. aureus bacteria than against gram-negative E. coli bacteria. As far as Bacillus subtilis, which is reported to be a highly drug-resistant bacterium [65], high doses of the tested materials were effective compared to those of S. aureus and E. coli.
Somewhere, it was stated that glabridin exhibited antimicrobial activity against Helicobacter pylori and methicillin-resistant Staphylococcus aureus [64]. Furthermore, 29.16 g (1 mL) showed antitubercular activity against Mycobacterium [64]. Glabridin was also more effective against gram-positive microbes than against gram-negative microbes [64]. These findings support and authenticate the results of the present study. Moreover, glabridin contains two free phenolic hydroxyl groups at the 1,3-position, which may strengthen its antibacterial potential [64]. On the other hand, ZIF-8 contains Zn2+ and 2-methylimidazole, so Zn2+ may penetrate bacterial cell walls easily [66, 67] or 2. Methylimidazole can attach to the cell wall, causing the membrane to tear, distort, and release cytoplasmic substances [18, 19]. However, in the case of MOF interactions with the cell matrix, Zn2+, drugs and 2-methylimidazole released from MOFs could intermingle with bacterial enzymes and may inhibit respiratory reactions [18, 19]. Additionally, 2-methylimidazole can attach to protein-SH groups, which can cause bacterial cell death [18, 19]. Currently, Zn2+ composites containing imidazole linker derivatives have gained considerable recognition for their antibacterial properties [18, 19]. On the other hand, metallic nanomaterials may cause pits or holes in the bacterial cell wall, which in turn permits the entry of NPs into the cell matrix or the rupture of the cell membrane, allowing integral material to leak and cause cell death. In recent years, nanoantimicrobials have been shown to have different mechanisms of action for both types of bacteria. Researchers have documented low drug resistance in S. aureus following infection with E. coli due to the cell wall composition [68]. S. aureus has only a single layer of cytoplasmic membrane attached to peptidoglycan sheets, which exhibit chains of linear polysaccharides divided by brief peptides, where lipoteichoic acids adhere. The difference in cell walls could also be attributed to differences in antibacterial activity [69]. As a result, cell membranes become anionic and amphiphilic. Henceforth, metal ions and bioactive compounds can easily bind to the cell membrane or enter the cytoplasm and can produce reactive oxygen species or bind to SH- groups of genetic material, ultimately resulting in cell death [19, 69]. Considering these findings, S. aureus is considerably less drug resistant than is E. coli, whereas the E. coli peptidoglycan layer between the cell wall and cytoplasm contains lipopolysaccharides, which hinder the entry of certain chemicals [19, 68], and the additional periplasmic space between layers contains few enzymes that may also breakdown extracellular substances [68, 70]. E. coli has a high level of drug resistance because different outflow pumps in cell membranes effectively lower the concentration of such medicines within the cell [68]. In addition, it is accepted that smaller metallic nanoparticles provide better interactions between NPs and bacterial cell walls, thus allowing them to rupture membranes and develop the highest antibacterial efficacy [71]. In this study, the nanosized prepared material (as shown in Fig. 5) exhibited distinct sharp edges attributed to the presence of zinc metal. Consequently, it is postulated that this particular morphology holds the greatest potential for exerting antibacterial activity. Mechanically, as bacteria interact with materials, sharp edges will destroy cell walls [52–54]. In conclusion, Glab@ZIF-8 demonstrated the highest level of antibacterial activity against microorganisms, suggesting that this medication may be useful for treating illnesses related to microbial infections and may be helpful in biomedical applications based on nanomaterials.
3.5. In vitro pH-dependent drug release profile
This study investigated the release profile of the drug glabridin from Glab@ZIF-8 in different pH environments and in tween-80 solutions (Fig. 8). The results showed that the highest drug release (89.76%) occurred at pH 5 after 72 hours, followed by 81.23% at pH 5.5 and 24.78% at pH 7.4. This high drug release under acidic conditions is attributed to the protonation of the imidazolate ring of ZIF-8 [18, 19, 32], which breaks the coordination of the metal framework and allows the drug to be released [18]. In contrast, under basic conditions, ZIF-8 remains stable, resulting in slower drug release [18, 19, 32]. The drug release process is also time dependent, further confirming the stability of the MOF. The study also revealed minimal drug release at pH 5 and pH 5.5 during the first hour, while no drug was released at physiological pH 7.4 after 4 hours, indicating that long-term exposure to ZIF-8 at alkaline pH can lead to gradual release of the loaded material [24, 72, 73]. It was concluded that ZIF-8 shows promise as a material for drug protection and delivery, and encapsulated ZIF-8 materials can be stored in normal or physiological media for a specified time.