Comparative Study of Bi2 O3 , MgO and ZrO2 Nanomaterials designed by Polymer Sacrificial Method for Amoxicillin delivery and Bone Regeneration: In-Vitro Studies

doi:10.21203/rs.3.rs-3146890/v1

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Comparative Study of Bi2 O3 , MgO and ZrO2 Nanomaterials designed by Polymer Sacrificial Method for Amoxicillin delivery and Bone Regeneration: In-Vitro Studies

https://doi.org/10.21203/rs.3.rs-3146890/v1

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Hard tissue scientists face many difficulties, including persistent osteomyelitis and diseased bone abnormalities. Inorganic mesoporous nanomaterials are excellent candidates for the adsorption and loading of bioactive medicinal substances because to their chemical-physical characteristics. Recently, zirconium oxide, magnesium oxide and bismuth oxide nanoparticles are of great surface area and biocompatibility, and they have been described as a new drug delivery carrier. In this study, amoxicillin antibiotic was loaded into the prepared mesoporous nanomaterials (ZrO₂, MgO and Bi₂O₃) to form a local antibiotic delivery system. The prepared mesoporous nanomaterials were investigated by XRD, FTIR, TEM, zeta potential and BET surface area measurements. Amoxicillin antibiotic was released from the prepared mesoporous nanomaterials in PBS. The effectiveness of the antibacterial study against several gram-positive and gram-negative bacterial strains was assessed. The cytotoxicity study of the human osteoblast-like cells (MG-63) was tested for all prepared mesoporous nanomaterials utilizing MTT assay. ZrO₂ demonstrated particle diameters in the range of (5.26– 11.47nm), MgO was (70–80nm) and Bi₂O₃ was (9.79– 13.7nm). The greater surface area was confirmed for Bi₂O₃ sample (3.99 m²g^− 1) by BET surface area. Amoxicillin loaded mesoporous nano powders exhibited impressive antibacterial and antifungal activities. MgO and Bi₂O₃ mesoporous nanoparticles exhibited better antimicrobial activities compared to ZrO₂ sample. The proliferation for all samples gave good results especially for MgO and Bi₂O₃. As a result, the produced mesoporous nanomaterials have a significant potential for use as medicine delivery systems for bone regeneration and for enhancing the properties of other products in medical applications.

Mesoporous nanomaterials

amoxicillin antibiotic

drug delivery

cell variability

bone regeneration

Bone is normally supplied and repaired by its own remodeling process, in which there are interactions between osteoclasts and osteoblasts cells and signaling molecules to remove old and damaged tissue and form new bone [1]. The modern age of nanotechnology has altered medicine delivery and targeting methods, as well as the landscape of the pharmaceutical sector. The integration of nanomaterials with porous structures is the most exciting of the rapidly growing industries. Porous materials comprise solid composites with microporous (≥ 2 nm), mesoporous (2–50 nm), and macroporous (> 50 nm) pore networks. Particularly, porous materials could be natural or artificial materials [2]. The mesoporous nanomaterials are characterized by large surface area, large pore volume, biocompatible, high loading capacity of drug, high degree of tenability, ease of synthesis and surface functionality [2].

Concerns about human health have recently increased. Pharmaceutical companies and the scientific community have pledged to increase both understanding and on-field deployment of newly designed medications and processes. Despite the numerous proven treatments available, there is still an urgent need to create new and novel technologies that could aid in the restoration of injured tissues [1–3]. Furthermore, the development of novel antimicrobial drugs capable of combating rising antibiotic resistance has become crucial [4].

Recently, the use of artificial nanoparticles (NPs) has grown in a variety of industries, including electronics, biological applications, and the pharmaceutical industry. Zirconia (ZrO2) nanoparticles are a key nanomaterial utilized in the production of refractories, foundry sands, and ceramics. ZrO₂ is also used in biomedical fields like bone and dental implants, cancer treatment, along with joint endoprostheses, due to its superior mechanical strength [1–3]. However, the widespread use of particles has prompted concerns about their possible health and environmental dangers, with workplace and patient safety being a primary worry. There have been few toxicological investigations on ZrO₂ NPs so far, which demonstrated mixed findings results.

Earlier investigations have found that ZrO₂ NPs are more biocompatible than other nanomaterials such as Fe₂O₃, TiO₂, and ZnO [4–6]. Others have observed ZrO₂ NP could generate minor [3, 7] or no cytotoxic effects [8–10], which is consistent with these findings, and only a few investigations suggested a mild cytotoxic potential. Previous research has shown that nanoparticles (such as mesoporous silica) are frequently utilized as tissue-engineered materials and have the capacity to promote osteogenic differentiation of osteoblasts [11–17].

MgO and ZnO NPs have high antibacterial and anti-biofilm activity, as well as antioxidant properties [16, 17]. MgO NPs supply mineral nutrients that are needed for most biological activities, such as bone reformation, after dissolution by promoting bone-like mineral deposition, osteogenic differentiation, and proliferation [18, 19]. Mesoporous MgO nanostructures with a high surface area were created using a combination surfactant-templating technique. Using the least inhibitory concentration (MIC) method, MgO was examined for antibacterial activity against E. coli and B. subtilis, and the IC50 that exhibited promising activities [20].

Bismuth nanoparticles (Bi-NPs), because of their low toxicity and environmentally friendly qualities, have gained greater interest for biomedical applications [21]. Furthermore, the relatively inexpensive cost and abundance of Bi make it appealing for large-scale applications [22]. Bi chalcogenide nanostructures have intrinsic electrical and optical characteristics, making them appropriate for a wide range of biomedical applications [23]. The antibacterial impact of metal-based NPs is influenced by the formation of ROS, cation release from the NPs, ATP depletion, membrane impairment, protein malfunction, and interference with nutritional assimilation [24, 25]. Nevertheless, applying the same preparation conditions to the above-mentioned nanoparticles (ZrO₂, MgO and Bi₂O₃) were not unified and studied in a comparative manure before. Therefore, this study aimed to design these mesoporous nanomaterials using the same method and explore the behavior of each nanoparticle on the antimicrobial activity, drug delivery profiles, and cell viability. Particularly, mesoporous MBiNs, MMgNs, and MZrNs nanomaterials were synthesized and examined, and their physicochemical and morphological properties were elucidated and correlated to their possible application.

Starting ingredients of pure analytical quality were purchased from Sigma Aldrich and other multinational chemistry firms. In details, polyacrylamide (C₃H₅NO)n, average Mn 150,000 g/ mol, CAS Number: 9003-05-8. Zirconium (IV) oxychloride octahydrate, Linear Formula: ZrOCl₂⋅8H₂O, CAS No.: 13520-92-8, Molecular Weight: 322.25 g/mol, bismuth oxychloride (BiOCl), Magnesium chloride (MgCl₂).

2.1. Preparation method of mesoporous nanoparticles

The MZrNs, MMgNs and MBiNs have been developed according to prior research with some alterations [3]. Initially, 400 ml of distilled water and 1 g of polyacrylamide were combined with a magnetic stirrer at a temperature of 80°C. Separately, the oxide precursors were individually thoroughly dissolved in 1200 ml of the appropriate medium (distilled water or ethanol) using a magnetic stirrer according to the molar weights of zirconyl chloride octahydrate, magnesium chloride, or bismuth chloride. The zirconia, magnesia, and bismuth oxide solutions were then combined with the polymer solution while being continuously stirred. The combinations were then left to dry overnight at 100°C in an oven dryer. For determining the morphology of the produced nanoparticles, the dry powders were calcined at 600°C.

2.2. Morphological properties

The produced mesoporous nanomaterials' shape and particle size were assessed by HR-TEM. Specifications of utilized TEM (JEOL, Japan, JEM2100, Electron Microscope, and HR-TEM). The produced samples were dispersed in a little amount of ethanol. The produced suspensions were then applied on copper grids, which were then drying is permitted at ambient temperature before being inspected using a TEM machine.

2.3. Physicochemical characterizations

2.3.1. XRD analysis

The created mesoporous nanomaterials have been examined using a German-made X-ray diffraction (XRD) model Bruker axes, D8 ADVANCE, which was equipped with a Ni-filter for Cu-K irradiation, 40Kv, and 40 mA. This method was used to assess the samples under investigation's crystallinity and amorphousness. JCPDS chemical database was used to match the XRD recorded phases.

2.3.2. FTIR analysis

Using a Fourier Transformer Infrared spectrophotometer (FT-IR) (model FT/IR-6100 type A) with a measurement range of 400–4000 cm-1, the chemical groups of the produced mesoporous nanomaterials were studied. Prior to testing, the sample powder was mixed with KBr powder in a ratio of 2:100.

2.4. BET- surface area parameters

An automatic analyzer (Quantachrome Nova Automated Gas Sorption System Version 1.12) was used to determine the specific surface area and pore-size distribution of the produced mesoporous nanomaterials. It typically operates using the Brunauer-Emmett-Teller (BET) technique. At 77 K, pore-size distribution was determined using indirect molecular adsorption methods such as nonlocal density functional theory (NLDFT) and N₂ isotherms.

2.5. Distribution of particle diameters and Zeta potential characterizations

The zetasizer (Nano ZS, Malvern Instruments, UK) was used to evaluate the particle diameter and surface charge of the mesoporous nanomaterials. Individually, the mesoporous nanoparticles were suspended in distilled water (5 mg in 5 ml of dispersion media). The created suspensions were transferred to Malvern DTS1060 measuring cells after filtration, and data analysis software (Version 4.0) was used. Appling electrical filed to the measurement cell to quantify particle surface charges spontaneously.

2.6. Drug delivery

Drug loading

In this experiment, amoxicillin sodium (Amox) (EIPICO Egyptian INT Pharmaceutical industries CO, Cairo, Egypt) was employed. Because of its broad-spectrum antimicrobial activity, amoxicillin was chosen. The mesoporous nanomaterials (100 mg) were soaked in 50 ml of ethanol containing 200 mg of amoxicillin powder, yielding a concentration of (2 mg/ml). To ensure drug loading homogeneity within the MZrNs, MMgNs, and MBiNs porous structures, a shaker incubator (200 rpm/18 h) was used. Finally, the powders were allowed to dry at 37°C, and the Amox loading efficiency was evaluated using Eq. 1.

$$\text{%} \text{l}\text{o}\text{a}\text{d}\text{i}\text{n}\text{g} \text{e}\text{f}\text{f}\text{i}\text{c}\text{i}\text{e}\text{n}\text{c}\text{y}=\frac{\text{A}\text{m}\text{o}\text{u}\text{n}\text{t} \text{o}\text{f} \text{A}\text{m}\text{o}\text{x} \text{i}\text{n} \text{n}\text{a}\text{n}\text{o}\text{p}\text{a}\text{r}\text{t}\text{i}\text{c}\text{l}\text{e}\text{s}}{\text{T}\text{h}\text{e}\text{o}\text{r}\text{i}\text{t}\text{i}\text{c}\text{a}\text{l} \text{A}\text{m}\text{o}\text{x} \text{i}\text{n} \text{n}\text{a}\text{n}\text{o}\text{p}\text{a}\text{r}\text{t}\text{i}\text{c}\text{l}\text{e}\text{s}}\times 100 \left(1\right)$$

2.7. Drug release profiles

Amox release profiles were determined by submersing the drug-loaded nanoparticles in PBS at pH 7.4 and 37^oC. To begin, the drug-loaded samples were put in 50 ml of PBS. At defined intervals, 3 ml of the solution was taken and replaced with 3 ml of a new PBS. The obtained solutions were maintained in the freezer for future measurements. A UV spectrophotometer with a 275 nm wavelength was used to quantify the amount of Amox released into the solution. A previously defined standard curve was used to calculate the medication concentrations.

2.8. Antibacterial activities

Nanoparticle disc sample in vitro antibacterial activity was investigated. Gram-positive Bacillus subtlus ATCC-6633, Gram-negative Escherichia coli ATCC-25922, and yeast were the types of bacteria used in this investigation. (Candida albicans ATCC-1023). These bacteria were obtained from the United States Department of Agriculture's Northern Utilization Research and Development Division in Peoria, Illinois, and the American Type Culture Collection (ATCC, Rockville, Maryland, USA). (NRRL). The bacteria were revived for the bioassay by sub-culturing them in fresh nutrient broth medium for 24 h before the test. Fungi were grown on potato dextrose agar (PDA) for 7 days prior to the experiment at 28°C.

The antimicrobial activity of the nano powders was assessed utilizing the agar plug diffusion method. In sterile Petri dishes that had already been marked with the test bacterium, a 0.1 ml aliquot of an 18-hour broth culture of the previously stated bacteria was applied. To ensure that the bacteria were evenly disseminated throughout the medium, molten, sterile Muller-Hinton was aseptically poured into the plates. Sample discs of equal weight were placed on the agar surfaces and allowed to diffuse for 2 h at 4°C. The plates were incubated at 37°C for 24 h for bacterial strains and 28–30°C for 48 h for fungal strains in triplicate. The bacterial inhibition areas were marked and measured in millimeters around the wells.

2.9. The cytotoxicity evaluation

The cytotoxicity of synthesized mesoporous nano powders on human normal cell lines was investigated in this study. Human osteoblast-like cells (MG-63) were obtained frozen in liquid nitrogen (-180 ^oC) and were kept at the NRC through successive sub-culturing. The 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent was used to assess cell cytotoxicity. The mitochondrial succinate dehydrogenase (present only in living cells) can decrease the yellow MTT salt to generate purple formazan crystals. When formazan crystals dissolve in dimethyl sulfoxide (DMSO), a colored solution is formed. The experiment was carried out over a period of 24 h. In particular, 200 µl of pre-warmed medium and 200 µl of MTT stock solution (2 mg/m in PBS) were added to the samples, which were then incubated at 37 ^oC in a humidified atmosphere away from light. The surplus media was removed after 4 h, and the formazan crystals were dissolved in 2 ml of DMSO for 1 h. This was followed by the addition of 200 µl of formazan solution, after which they were transferred from each well to a 96-well plate. Finally, the optical densities (ODs) were assessed using a micro-plate reader/spectrophotometer at 570 nm. Five parallel trials have been conducted for each nanomaterial. Individual cells (1104 cells) were cultivated on cell culture slides (SPL, South Korea) with the produced nano powders for 72 hours before being dissected. Slides were flushed with PBS and stained for 10 minutes in the dark with ethidium bromide/acridine orange (EB/AO; 100 g/ml of each in 100 l PBS) before being examined with a fluorescence camera (Axio Imager Z2, Zeiss, Jena, Germany) and ZEN 11 blue version programming for Image Analysis (Zeiss, Jena, Germany). For each mesoporous nano powder, the percentages of surviving, early and late apoptotic, as well as necrotic cells, were measured throughout time.

2.10. Statistical analysis

Data management and statistical analysis were performed using SPSS version 20 (Statistical Package for Social Sciences). The mean, standard deviation, and confidence interval were used to summarize numerical data. The data was tested for normality by inspecting the distribution and performing the Kolmogorov-Smirnov and Shapiro-Wilk tests. To compare groups, Tukey's post hoc test was utilized, and one-way ANOVA tests were used to compare groups regarding normally distributed numerical variables (parametric). P-values are bi-polar. P-values of less than 0.05 were considered significant.

3.1. TEM analysis

Figure 1 represented TEM images of the prepared mesoporous ZrO₂, MgO and Bi₂O₃ nanomaterials (MZrNs, MMgNs and MBiNs) samples to obtain the shape, size, the distribution, and morphology of the mesoporous nanoparticles. TEM analysis of MZrNs nanoparticles showed uniform nanostructures of semi-spheres with an average diameter of 5.26–11.47 nm with porous structure on their surfaces (Fig. 1 (a)). The obtained result matches the MZrNs nanoparticles early reported by [3]. The morphology of sample MMgNs nanoparticles demonstrated the agglomeration of a high heterogeneity of irregular -shaped nanoparticles coated with water layer on their surfaces to be in a diameter in the range of 70–80 nm denoting the MgO observed hydroxyl group layer on its surface during the preparation for TEM process (Fig. 1 (b)). This result agreed with the previous study [20]. The morphology study of both Bi₂O₃ sample (MBiNs) is shown in Fig. 1 (c). The TEM of MBiNs sample possessed a heterogeneity of semi-round nanoparticles with size diameter 9.79–13.70 nm with clear lattice fringes of in nm interplanar spacing, which is in nanoscale accordance of Bi2O3 mesoporous structure [26]. All samples were in crystalline nature of nanostructures as observed from the TEM analysis.

3.2. Physicochemical characterizations

3.2.1. XRD analysis

X-ray diffraction (XRD) curves of the prepared mesoporous Bi₂O₃, MgO and ZrO₂ nanomaterials free and loaded with Amox are illustrated in Fig. 2. Figure 2.a. represented the XRD of Bi₂O₃ and Bi₂O₃/ Amox. In which observed peaks indicating that the Bi₂O₃ nanoparticles are crystalline for sample Bi₂O₃ free drug. The peaks are associated with the monoclinic crystal phase of Bi₂O₃ (COD: No-96-152-6459) at the 2θ values of 15^°, 28.56^°, 34.35^°, 42.52^°, 46.50^° and 58.58^° [27]. The XRD of Bi₂O₃ loaded with drug (Bi₂O₃/Amox) reveals the crystalline structure of Bi₂O₃ with the characteristic peaks of amoxicillin trihydrate at 2θ = 12.11, 14.73, 17.57, 18.43, 25.48, and 27.38 in low intensity due to the small amount of drug loading [28]. Figure 2(b) represented the XRD of MgO and MgO loaded Amox. The XRD pattern showed sharp lines at 2θ = 37.8°, 42.5° and 63.54° indexed to (111), (200), (220) planes respectively, confirming crystalline nature of MgO by comparison with the JCPDS card no. 45–0946 [20]. The XRD of MgO loaded with drug reveals the crystalline structure of MgO with the characteristic peaks of amoxicillin trihydrate at 2θ = 12.11, 14.73, 17.57, 18.43, 25.48, and 27.38 in low intensity due to the small amount of drug loading [28].

The produced MZrNs' XRD pattern revealed a highly crystalline phase with extremely sharp peaks (Fig. 2(c)). It was confirmed that there were 70.3% monoclinic phase MZrNs and 29.7% tetragonal phase MZrNs. The primary peaks of the monoclinic phase of MZrNs, which corresponded to the JCPDS XRD card (COD 1521753), were at 2 = 28.81, 31.46, 34.15, 35.3, 40.77, 45.28, 50.14, and 50.62. The synthesized mesoporous MZrNs nano powders produced cubic phase XRD peaks at 2 = 30.49, 35.17, 50.65, 60.29, and 63.17 that matched the JCPDS XRD card (COD 1522143). The XRD of ZrO₂ loaded with drug reveals the crystalline structure of ZrO₂ with the characteristic peaks of amoxicillin trihydrate at 2θ = 12.11, 14.73, 17.57, 18.43, 25.48, and 27.38 in low intensity due to the small amount of drug loading [28].

3.2.2. FTIR analysis

Figure 3 (a) shows the FT-IR spectrum of the prepared mesoporous Bi₂O₃, MgO and ZrO₂ nanomaterials free and loaded with Amox. The FTIR absorption spectrum of the bismuth oxide nanoparticle (Fig. 3 (a)) possessed O–H stretching vibrations appear at 3421cm^− 1. The peak at 1400cm^− 1 is related to C-O vibrations, which could be gained from the air. The peak at 435 ~ 505cm^− 1 is originated from the metal-oxygen (Bi-O) bond. Furthermore, absorption bands emerged at 600 cm^− 1 because of the stretching mode of Bi-O, the peak of stretching vibration of O-H appeared at 3429 cm-1. Bands at 2900 cm-1, 2800 cm-1, 1113 cm-1, and 1046 cm-1 are associated to vibrations of the CH2 aliphatic and C-O bonds of beta vulgaris precursor. [29].

The FTIR spectrum of magnesium oxide nanoparticles had bands at 3440 cm^− 1, indicating O-H group adsorption, and vibrations in the absorption band of 1642 cm^− 1, which are most likely attributable to (O-H) vibration. A very broad peak at 500 cm^− 1 can be assigned to Mg–O stretching vibration. The peak observed at 434 cm^− 1 confirmed the formation of MgO-NPs. Furthermore, the large absorption band at 578 cm^− 1 confirms the Mg-O bond's symmetric stretching. The presence of hydroxyl groups (OH) is shown by the absorption bands at 3345 cm^− 1 (stretching) and 1431.7 cm^− 1 (bending), which is attributable to the presence of Mg (OH)₂, while, MgO stretching causes the bands at 1095 cm^− 1 [20].

For ZrO₂ nanoparticles, the strong absorption band identified in the region of 3200 to 3800 cm^− 1 is due to the stretching vibration of the -OH group of water molecules adsorbed on the surface of ZrO₂ nanoparticles, and the peak at 1635 cm^− 1 is due to the typical bending vibration of the -OH group of water molecules. Sharp peaks at 500 cm^− 1 correspond to the stretching vibration of Zr-O in the ZrO₂ phase, confirming the development of ZrO₂ as a crystalline phase [3]. The intensity of the upper absorption band decreases with calcination temperature, implying that water molecules are removed from the sample and particle development occurs.

The FTIR of the prepared mesoporous nanoparticles with drug (Amox) revealed presence of the characteristic peaks of amoxicillin trihydrate in low intensity due to the small amount of drug loading or maximum inclusion within the nanoparticle's pores (see Fig. 3 (b)) [28–30]. Infrared spectrum of amoxicillin trihydrate sample presented bands between 3525 and 557 cm ^− 1. Referring to Min et al. [31], two small bands at 3525 and 3461 cm^–1 refer to crystallization water (-OH stretch) and amide (-NH stretching), respectively, followed by bands at 3178 and 3039 cm^–1, characteristic of phenol (-OH stretching) and aromatic (-CH stretching), respectively. Carbonyl (-C = O) in the beta-lactam ring and amide are indicated by prominent distinctive bands between 1800 and 1650 cm^− 1 (1776 and 1687 cm^− 1). Carboxylate asymmetric stretching is responsible for the 1581 cm^− 1 band. Bands in 1484 and 1120 cm^− 1 are assigned to N-C lengths of primary amine and O-C, respectively, according to Zha et al. [32]. Multiple bands between 1150 and 950 cm^− 1 represented typical aromatic compound in-plane CH bending vibrations. No more information about the other bands was found in the literature. Both XRD and FTIR results support the maximum and safe inclusion of Amox within the microstructure of fabricated nanoparticles without altering the physicochemical features of both carrier and drug.

3.3. BET surface area analysis.

The microstructure features of the fabricated nanomaterials in this research work were assessed through recording their isothermal curves alongside the pore diameters distribution as represented Fig. 4. The pore width, which is the separation between the two opposing walls, is a frequent way to characterize the pore size. Evidently, only when the geometric shape is precisely determined does the pore size have a defined meaning. The porosity of a material is often defined as the volume of pores and gaps in relation to the volume occupied by the solid [33–36]. Another way to categories porous materials is according to their adsorption traits. Since a gas condenses on a free surface as opposed to being absorbed into the bulk, the term "adsorption" originally referred to this process. The uptake of a gas by porous materials is usually referred to as adsorption or simply sorption, even though this distinction is frequently not detected, regardless of the underlying physical mechanism. The quantitative explanation for the adsorption of a gas by a porous material is an adsorption isotherm, which shows the amount of gas adsorbed by the material at a specific temperature as a function of pressure. IUPAC conventions have been established to classify pore diameters and gas sorption isotherms, which indicate the relationship between porosity and sorption [33–35]. Most frequently, the pore diameters of porous materials are categorized using data on gas sorption.

Figure 4 depicts the detected isotherms of produced nanomaterials. In general, the isotherm curves of all scaffolds showed IV and V classes. The adsorption hysteresis (IV and V) shows a link between the texture of a mesoporous material, including pore size distribution, pore geometry, and connectivity, and the shape of the hysteresis loop. Based on de Boer's earlier classification of hysteresis, the IUPAC proposed an empirical classification of hysteresis loops [33, 34]. Some of the isotherm curves are not closed (Fig. 4 (Bi₂O₃ and MgO)). However, it is worth highlighting that, this phenomenon (non-closed desorption isotherm) is not only related to pore size, but rather to the adsorptive physical property at the adsorption temperature. The adsorption phase cavitation in the pores is responsible for this behavior. Since this pore is not only unrelated to the pore distribution on the material, but it must also be disregarded when considering desorption part of N₂ isotherm at 77 K.

Moreover, the fabricated nanomaterials demonstrated isotherm curves H3 type. The limiting adsorption at high P/Po, which is seen with non-rigid aggregates of plate-like particles, is not visible in the isotherms showing type H3. The desorption curve of H3 hysteresis has a slope due to the so-called tensile strength effect, which corresponds to a force acting on the hysteresis loop (these phenomena may occur for nitrogen at 77 K in the relative pressure range between 0.4 and 0.45) [35]. The BET surface area values of the obtained nanomaterials (3.99 ± 0.1731, 1.44 ± 0.1556, and 2.23m²g^− 1) for MBiNs, MMgNs and MZrNs, respectively. This means that the nano powders possessed acceptable surface areas for drug careers. Moreover, the pore distribution curves of both MBiNs and MZrNs possessed mesoporous structures as they were in the range of 2-50nm. Nevertheless, MgO did not possess mesoporous pore structure as it demonstrated pore sizes below 2nm (microporous nanomaterials). This difference in the microstructure of MMgNs could be owed to higher hydration ability of MgO nano powders.

3.4. Zeta potential characterization

Particle sizes and zeta potential measurements confirmed the prepared mesoporous nanoparticles were stable colloids nano powders of MBiN, MMgNs and MZrNs as shown in Figs. 5 and 6. The particle size distribution of mesoporous MBiN nanoparticles determined an average dynamic particle size of 305 nm in 100% Fig. 5(a). The average dynamic particles size of sample MMgNs showed an average particle size of 684nm in 100% Fig. 5(b). Figure 5(c) represented average dynamic particles size of sample MZrNs with 288nm.

The zeta potential of mesoporous MBiNs was illustrated in Fig. 6 (a), which demonstrated to values of zeta potential − 7.12 and − 23.5 mV. Figure 6 (b), showed the zeta potential of sample MMgNs, observed at -16.8 mV with the area % value 100%. The change in the charges of the MZrNs may be due to the presence of Zr which moved the particle charge toward neutrality with slight negatively − 19 mV. However, all the prepared nanoparticles showed negative charges on their surfaces, which is favorable for cell internalization [37]. This in turn would enhance the medication delivery within the targeted cells to maximize the patient benefit and decrease the side effects.

3.5. Drug release behaviors

The loading efficiencies of Amox within the fabricated mesoporous nanoparticles were 98.15, 96.47 and 99.79% for MBiNs, MMgNs and MZrNs samples, respectively. A UV spectrophotometer set at = 275nm was used to measure the amount of drug released into the PBS buffer solution. Figure 7 depicts the cumulative drug release (%CDR) patterns of amoxicillin released from prepared mesoporous (MBiNs, MMgNs, and MZrNs samples) suspensions. According to the release profiles, there is a significant variance in the release rates of all drug-loaded samples. The release profiles of MgO, Bi₂O₃, and ZrO₂ samples show protracted burst release, respectively of 15% to 20 within 60h, and it was linear up to 100h for the three samples. About 23, 30 and 35% of the drug was released from mesoporous MgO, Bi₂O₃ and ZrO₂ samples respectively, within approximately 700 h. The observed difference in the release profiles between MMgNs and MZrNs and MBiN is that MgO nanoparticles downregulated the Amox release up to 23%. This could be owed to the adsorbed water on the surface of MgO nanoparticles as it was confirmed by the TEM results, which in turn slows the Amox release from MMgNs [38, 39].

3.6. Antibacterial tests

All the Amox loaded nanoparticles demonstrated antimicrobial effect against the tested microorganisms as shown in Fig. 8. The higher antimicrobial activity was recorded for MBiNs and MMgNs, which could be explained by the antibacterial effect of the Bi₂O₃ and MgO nanoparticles. The possible antibacterial activity of MgONs loaded with Hydrangea paniculate for over streptomycin against E. coli and S. aureus was mentioned by Karunakaran et al. in 2016 [40]. Their ability as antioxidants, which in turn prevented bacterial enzymes from working, was directly related to their potential action. In a recent study, Abdallah et al., 2019 [41] confirmed the rosemary flowers' MgONPs' promising antibacterial effect by inhibiting bacterial biofilm formation and swimming motility. Using pharmacology and metalloproteomics methods, Li et al. [42] investigated the action mechanism of bismuth-based medicines for treating Helicobacter Pylori infection. The authors explained the efficacy of bismuth-based medications because of bismuth's capacity to disrupt various biological pathways by perturbing the activity of important enzymes. The authors proposed that bismuth materials inhibited metallo-lactamase by displacing Zn (II) cofactors and were useful in the treatment of Helicobacter pylori infection.

3.7. Cytotoxicity evaluation

MG63 cells human osteoblast cells were seeded with the prepared mesoporous nanoparticles (MBiNs, MMgNs and MZrNs) for periods of 1 and 3 days to establish the cytotoxic effects of the prepared mesoporous nanomaterials on cells. Figure 9 represented cell viability test of the oxides with the MG63 cell line after 1- and 3-days incubation. The cell viability percentages after 1- and 3-days incubation of sample MBiNs showed enhanced cell viability (130 ± 10 and 520 ± 8%), respectively. For MMgNs after 1- and 3-days incubation (530 ± 12 and 250 ± 13%), respectively and for MZrNs after 1- and 3-days incubation (130 ± 12 and 100 ± 9%), respectively. The cell viability percentages for all prepared mesoporous samples revealed excellent results of the cell proliferation and growth denoting the high safety for using these samples in bone grafting or as drug delivery carriers for other medical applications.

A micrograph of cell numbers with the prepared nanomaterials after 3 days of culture is shown in Fig. 10. Cells populations were increased and observed attached with the prepared mesoporous nano powders MBiNS sample (Fig. 10 (a)) it showed that cells with stretched, flat, long filopodia, confluent and crowded suggesting healthy cells especially after 3 days incubation, denoting the healthy cells and well cell proliferation. Figure 10 (b) showed the round cells with the prepared mesoporous nano powders MMgNs sample and high number of the cells denoting the good proliferation and the decline in the cell count could be related to the over-proliferation of cells that cause a non-suitable environment for their viability. The micrographs show cells increased after 3 days Fig. 10 (c), forming healthy cells for the prepared mesoporous nano powders MZrNs. Closer examination of the cells (Fig. 10 (a and c)) showed that the cells have anchored firmly together, cells confluent distribution and were well spread out with filopodia stretched out, suggesting that the cells are in the process of proliferating and confirmed the positive cellular response of samples MBiNs and MZrNs as compared with sample MMgNs. These preliminary results demonstrate the non-cytotoxic effect of all prepared mesoporous samples (MBiNs, MMgNs and MZrNs) and they created a favorable microenvironment for cell proliferation and growth.

Patients must take large doses of the medication to get the intended therapeutic benefits for the treatment of disorders due to the drug molecules' lack of specificity and solubility. There are several drug carriers available in pharmaceuticals that can be utilized to deliver therapeutic substances to the body's target place to address these issues. Mesoporous nanomaterials have established themselves as a strong contender for solving the concerns highlighted previously and generating consequences in a controllable and sustained manner. Mesoporous nanoparticles are employed in targeted and regulated drug delivery systems because of their high drug loading capacity and minimal toxicity. The current study compares the first time of three different mesoporous nanoparticles (MBiNs, MMgNs and MZrNs) prepared by the same method (polymer sacrificial method) to define their suitability for drug loading, release, and bone regeneration. Although, the preparation technique did not alert their physicochemical features, they possessed different microstructural properties. This difference changed their behaviors of drug release and antimicrobial properties alongside cells growth (cytocompatibility and proliferation). Further investigations are still needed to get the full image about these nanoparticles and their potential applications in the medical field.

Funding: No funds, grants, or other support was received.

Availability of Data and Material Not applicable.

Acknowledgements Not applicable.

Consent for publication Not applicable.

Conflict of interest the authors declare that they have no conflict of interest.

Ethical approval Not applicable.

Author Contributions Statement

A. Yasser Soliman (Yasser M. Soliman), Co-authors.

B. Mostafa Mabrouk (Mostafa Mabrouk), Co-authors.

C. Ahmed Abdraboh (A. S. Abdraboh), Corresponding authors.

D. Khairy Tohamy Ereiba (Khairy T. Ereiba), Co-auther.

E. Hanan El-Beherei (Hanan H. Beherei), Co-authors.

"A.C. and D.E. wrote the main manuscript text and B.C. prepared figures 1-10."

"A.C.D. Analysis and/or interpretation of data and A.C. Drafting the manuscript."

"All authors reviewed the manuscript."

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No competing interests reported.

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Comparative Study of Bi2 O3 , MgO and ZrO2 Nanomaterials designed by Polymer Sacrificial Method for Amoxicillin delivery and Bone Regeneration: In-Vitro Studies

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Abstract

Figures

1. Introduction

2. Materials and method

2.1. Preparation method of mesoporous nanoparticles

2.2. Morphological properties

2.3. Physicochemical characterizations

2.3.1. XRD analysis

2.3.2. FTIR analysis

2.4. BET- surface area parameters

2.5. Distribution of particle diameters and Zeta potential characterizations

2.6. Drug delivery

2.7. Drug release profiles

2.8. Antibacterial activities

2.9. The cytotoxicity evaluation

2.10. Statistical analysis

3. Results and discussion

3.1. TEM analysis

3.2. Physicochemical characterizations

3.2.1. XRD analysis

3.2.2. FTIR analysis

3.3. BET surface area analysis.

3.4. Zeta potential characterization

3.5. Drug release behaviors

3.6. Antibacterial tests

3.7. Cytotoxicity evaluation

Conclusions

Declarations

References

Additional Declarations

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