Antibacterial Activity and Iron Release of Organic-Inorganic Hybrid Biomaterials Synthesized via the Sol-Gel Route

: The aim of this work was the synthesis of hybrid materials of iron (II)-based therapeu-tic systems via the sol-gel method. Increasing amounts of polyethylene glycol (PEG 6, 12, 24, 50 wt%) were added to SiO 2 /Fe20 wt% to modulate the release kinetics of the drug from the systems. Fourier-transform infrared (FTIR) spectroscopy was used to study the interactions between different components in the hybrid materials. The release kinetics in a simulated body ﬂuid (SBF) were investigated, and the amount of Fe 2+ released was detected via ultraviolet-visible spectroscopy (UV-Vis) after reaction with ortho-phenanthroline. Furthermore, biological characterization was carried out. The bioactivity of the synthesized hybrid materials was evaluated via the formation of a layer of hydroxyapatite on the surface of samples soaked in SBF using spectroscopy. Finally, the potential antibacterial properties of seven different materials against two different bacteria— E. coli and S. aureus —were investigated.


Introduction
The iron (II) citrate complex represents an effective solution for resolving iron-deficiency anaemia in people with chronic kidney conditions. This complex is usually used to treat low levels of iron in the blood. Iron is an important metal for human life because it is a cofactor of many types of enzymes and cytochromes of the mitochondrial electron transport chain, and it is part of the haemoglobin and myoglobin structures, which act as oxygen transporters. The use of ferrous organic complexes, such as iron (II) citrate, represents an excellent option to improve health care, since they increase bioavailability and solubility, solving the iron deficiency problem [1][2][3][4]. At the physiological pH of blood (7.4), high levels of Fe 2+ (aq) would theoretically result in the precipitation of iron (II) hydroxide. Complexation by citrate allows an almost 30-fold increase in the amount of Fe 2+ that can be dissolved in the blood. Iron-based systems are also proven to be effective for antitumor treatment. Daily uptake of iron through food is not sufficient to provide an adequate level of this metal in the blood. Some nonspecific symptoms linked to iron deficiency are impaired concentration, loss of energy, and fatigue. However, the most serious effects of iron deficiency, such as anaemia, are associated with loss of hair, angular stomatitis, and premature birth and bone resorption in pregnant women [5].
Iron deficiency can occur in surgical patients due to blood loss or pre-existing anaemia. The encapsulation of the iron (II) citrate complex in porous silica biomaterials allows a slow release of iron, thus resolving problems linked to the lowering of the iron levels in patients' blood [6].
Iron can also be toxic in excess; high concentrations of iron can promote pathogenic bacterial growth and modify their metabolic pattern [7]. Therefore, by modulating the iron concentration, it is possible to reduce bacterial growth.
Silica biomaterials have high biocompatibility, avoiding negative outcomes as a result of their implantation in bodily tissues; in addition, it is known from the literature [8][9][10] and from medical practice [11] that silica-based materials are able to form a hydroxyapatite layer on their surface that improves the interaction between medical devices and tissue. Drug trapping in its structure is enabled by its porosity [12].
One of the techniques that allow the synthesis of a hybrid biomaterial is the sol-gel method. It is well known that the characteristics of the synthesis methodology have a great influence on the structure, morphology, and chemical composition, as well as on the mechanical, thermal, and optical properties of the resultant product.
The formation of a sol followed by gelation, starting from inorganic materials as precursors, is the basis of the sol-gel technique. A three-dimensional network gel is formed via the condensation of hydrolysed species in solution. Parameters such as temperature, pH, water content, and precursor concentrations affect the structure of the initial gel and the properties of the material [13]. The main advantage of using the sol-gel method for biomaterial synthesis is the possibility of encapsulating different organic compounds and other bioactive molecules that can be used as carriers for drug delivery [14][15][16]. Indeed, it has been reported that gels can release drugs (such as anti-inflammatory agents, antibiotics, and antitumor agents) in a controlled manner [17][18][19][20]. Sol-gel preparation can be done with a wide variety of precursors, but silica-based carriers are preferred because they are a well-known system, inexpensive, and biodegradable [21]. Moreover, it has also been reported that silica/PEG hybrids allow us to obtain greater elasticity, to avoid particle agglomeration, and to create a network that entraps many kinds of drugs and compounds in only one step [22].
In this work, iron (II)-based biomaterials were synthesized via the sol-gel route. The aim was the synthesis of several hybrid materials in which increasing amounts of polyethylene glycol (PEG 6, 12, 24, 50 wt%) were added to SiO 2 /Fe20 wt%, while materials with different percentages of ferrous citrate complex (Fe(II)C 5, 10, 15, 20 wt%) were added to the silica matrix with 50 wt% of PEG.

Preparation of Materials
First, 25.0 g of citric acid monohydrate (>99.5%, Fluka, Munich, Germany) was dissolved in 500 mL of Milli-Q water under continuous stirring and heating. After reaching 90 • C, 6.0 g of iron powder (>99%, Sigma-Aldrich, St. Louis, MO, USA) was added to the solution. After the complete iron reaction, the solution was cooled down to room temperature, obtaining a grey/pearly precipitate that was washed with water, filtered under vacuum, and freeze-dried at −20 • C [1]. The selectivity of Fe 2+ was assessed by means of the colorimetric assay with 1,10-phenantroline and sodium thiocyanate, as reported elsewhere in [23].
Fe(II)C and PEG were added to silica solution (the molar ratios for the silica matrix were H 2 O/TEOS = 26.6 and EtOH/TEOS = 6). After the gelation process was complete, samples were dried at 50 • C for 24 h, obtaining glass materials that were finely grounded. Figure 1 shows a flow chart of the sol-gel procedure used to synthesize the hybrid materials, which are summarized in Table 1. During the synthesis, the ferrous ions are stable, as the authors have shown in a previous paper [23]. Figure 1 shows a flow chart of the sol-gel procedure used to synthesize the hybrid mate-96 rials, which are summarized in Table 1. During the synthesis, the ferrous ions are stable, 97 as the authors have shown in a previous paper [23].

FTIR Analysis
Fourier-transform infrared (FTIR) analysis was carried out to investigate specific intermolecular interactions between organic and inorganic components. Transmittance spectra of samples were obtained using a Prestige 21 Shimadzu (Japan) instrument equipped with a DTGS KBr (deuterated triglycine sulphate with potassium bromide windows) detector, with a resolution of 2 cm −1 (45 scans). FTIR spectra were recorded in the 400-4000 cm −1 range, using a disk consisting of 2 mg of sample and 198 mg of KBr. The FTIR spectra were processed using Prestige software (IR solution).

In Vitro Release of Fe 2+
The study of in vitro release was carried out to investigate the amounts of Fe 2+ released from the hybrid glass materials. In particular, the release of Fe 2+ from different materials was analysed at different release times using the normative reference A3500-Fe B, phenanthroline method. Powders of hybrid materials were soaked in 10 mL of SBF solution. Afterwards, 1.00 mL of this mixture containing the hybrid materials was added to a solution composed of 10% hydroxylamine hydrochloride, 10% acetate sodium, 0.1% 1,10-phenanthroline, and water. Release measurements, taken by means of a Shimadzu UV 700 Double-Beam Scanning UV-Vis spectrophotometer (Shimadzu, Kyoto, Japan), were repeated three times on each hybrid. The absorbance of Fe 2+ released was obtained at a wavelength of λ = 520.0 nm. Means ± standard deviation were calculated for each measure. The calibration curve of absorbance versus concentration was determined in the range 0-4.99 mg L −1 . Over this interval, the data fitted the Beer-Lambert law: where A is absorbance, ε is the molar absorptivity coefficient (M −1 cm −1 ), l is the path length in cm, and C is concentration (M).

Bioactivity Test
To investigate the formation of a hydroxyapatite layer on glass materials, 250 mg of powder and 250 mg of disk (obtained from the powders) were soaked in 50 mL of simulated body fluid (SBF) at 37 • C for 21 days. The SBF solution was replaced every two days to avoid the depletion of ionic species. Finally, exposed samples were dried and analysed using a Quanta 200 scanning electron microscope (SEM) equipped with electron dispersion spectroscopy (EDS) and X-ray diffraction (XRD) analysis, along with a Philips 139 diffractometer equipped with a PW 1830 generator, tungsten lamp, and Cu anode, where the source of the X-rays was given by Cu-Kα radiation (λ = 0.15418 nm). The ratio between the total exposed surface and the volume of solution was chosen according to the literature [24].

Antibacterial Activity
The Kirby-Bauer test was performed on the glass hybrid materials to evaluate their possible use in preventing the proliferation of pathogenic bacteria. Escherichia coli (ATCC 25922) as Gram-negative bacteria (−) and Staphylococcus aureus (ATCC 25923) as Grampositive bacteria (+) were grown in the absence and presence of specimens. A total of 100 mg of each of the glasses were finely ground to a powder and irradiated with UV light for 1 h for sterilization. Bacterial suspensions of 10 9 CFU/mL were obtained by diluting pellet strains in distilled saline water (0.9% NaCl). E. coli was plated on TBX medium (Tryptone Bile X-Gluc) (Liofilchem, Italy), while S. aureus was plated on Baird-Parker agar (Liofilchem, Italy). Both media were sterilized at 120 • C for 15 min. After autoclaving, Baird-Parker agar was cooled to 50 • C, and an emulsion of egg yolk containing potassium tellurite was added. After placing the materials on the centre of Petri plates, E. coli plates were grown at 44 • C for 24 h, while S. aureus plates were grown at 36 • C for 24 h. The diameter of inhibition halos (IDs) in relation to the Petri dish diameter (PPD) (6 cm) was calculated. Four measurements were carried out for each specimen to obtain the mean standard deviation. Data are expressed as bacterial viability (BV) (%).
where 100% BV represents the bacteria viability without samples. The mean standard deviation is expressed as a relative standard deviation (RSD). A flow chart of antibacterial detection is represented in Figure S1.

FTIR Analysis
FTIR analysis of hybrid materials allowed us to obtain information about interactions between the individual components. Figure 2 shows the FTIR spectra of SiO 2 hybrid materials with increasing amounts of PEG. The silica spectrum in curve (a) shows asymmetric and symmetric stretching of Si-O-Si at 1080 cm −1 , with a shoulder at 1200 cm −1 . In addition, we can see the bending vibrations of Si-O-Si at 800 and 460 cm −1 , whereas the band at 948 cm −1 is assigned to Si-OH bond vibrations. The intense band at 3450 cm −1 and the peak at 1640 cm −1 are due to stretching and bending vibrations of the OH of water. The broadness of the OH vibration suggests H-bonding between the OH of the solvent molecules and Si atoms. The addition of PEG to the silica matrix is confirmed by the increase in the intensity of the peaks at 2927 cm −1 and 1454 cm −1 that are associated with C-H asymmetric stretching and bending, respectively. In addition, we observe a shift of this peak in pure PEG to 2870 cm −1 [25][26][27]. The presence of Fe(II)C (Figure 3) is detectable in curves (b-d) by an increase in the intensity of the peak at 1722 cm −1 , which is due to C=O stretching [28]. In addition, the increase in the intensity of the peak at 1560 cm −1 , ascribed to the COO − group, is another sign of the encapsulation of Fe(II) citrate in the system [23].
Appl. Sci. 2021, 11, x FOR PEER REVIEW 5 of 12 (b-d) by an increase in the intensity of the peak at 1722 cm −1 , which is due to C=O stretch-170 ing [28]. In addition, the increase in the intensity of the peak at 1560 cm −1 , ascribed to the 171 COO − group, is another sign of the encapsulation of Fe(II) citrate in the system [23].   (b-d) by an increase in the intensity of the peak at 1722 cm −1 , which is due to C=O stretch-170 ing [28]. In addition, the increase in the intensity of the peak at 1560 cm −1 , ascribed to the 171 COO − group, is another sign of the encapsulation of Fe(II) citrate in the system [23].

In Vitro Release
Controlled Fe 2+ release is essential to evaluate the continuity and efficacy of treatments. The release was assayed by measuring the UV absorbance of Fe 2+ at λ = 520.0 nm as a function of the concentration of the solution. A calibration curve was drawn using standard solutions with concentrations from 0 to 4.99 mg/L ( Figure S2). Figures 4 and 5 show the Fe 2+ release rates of the compound delivered.
then it slowed down, ending after 8 h. Furthermore, the 12 wt% PEG system released a 188 higher concentration of Fe(II)C, likely because this is incorporated into the porous matrix 189 of the system and is more easily released than when the PEG concentration is greater, and 190 forms stronger interactions within the system. In particular, the amount of Fe(II)C released was very fast in the first 5 hours, and 187 then it slowed down, ending after 8 h. Furthermore, the 12 wt% PEG system released a 188 higher concentration of Fe(II)C, likely because this is incorporated into the porous matrix 189 of the system and is more easily released than when the PEG concentration is greater, and 190 forms stronger interactions within the system. Regarding Fe(II)C 20 wt% with various amounts of PEG, the trend is quite similar for all of the systems.
In particular, the amount of Fe(II)C released was very fast in the first 5 h, and then it slowed down, ending after 8 h. Furthermore, the 12 wt% PEG system released a higher concentration of Fe(II)C, likely because this is incorporated into the porous matrix of the system and is more easily released than when the PEG concentration is greater, and forms stronger interactions within the system. Instead, in the hybrid systems with a constant amount of PEG (50 wt%) and varying Fe(II)C content (5, 10, 15, or 20 wt%), the release of Fe(II)C was fast up to 4 h, and then it slowed down, ending after 8 h.

Bioactivity
Glasses exposed to SBF solution for 21 days were observed via SEM. In Figure 6 it is possible to see some globules on disks that are soaked in SBF. This shape is typical of hydroxyapatite formed on a glass surface. Those particles were visible over the entire surface of all materials. The EDS analysis (Figure 6) of the observed crystals shows their chemical composition. The atomic content ratio between Ca and P is 1.6 and is in agreement with the chemical formula of hydroxyapatite [29]. The mechanism of the hydroxyapatite formation is explained in Figure 7: the Si-OH groups present on the surface of the material combine with the positively charged Ca 2 + ions present in the SBF fluid, forming calcium silicate. The accumulation of calcium ions allows the increase in the positive charge on the surface which, in turn, combines with the negative charge of the phosphate ions to form amorphous calcium phosphate, which spontaneously transforms into hydroxyapatite [Ca 10 (PO 4 ) 6 (OH) 2 ] [30].
Appl. Sci. 2021, 11, x FOR PEER REVIEW 7 of 12 chemical composition. The atomic content ratio between Ca and P is 1.6 and is in agree-204 ment with the chemical formula of hydroxyapatite [29]. The mechanism of the hydroxy-205 apatite formation is explained in Figure 7:  Figure   218 8 ( ), confirming that the material surface was covered by a hydroxyapatite layer after 21 219 days of soaking in SBF. In Figure S3, XRD measurements show the amorphous structure 220 Figure 6. SEM micrograph of globular hydroxyapatite crystals after the bioactivity test, and EDS elementary analyses of crystals.
Appl. Sci. 2021, 11, x FOR PEER REVIEW 7 of 12 chemical composition. The atomic content ratio between Ca and P is 1.6 and is in agree-204 ment with the chemical formula of hydroxyapatite [29]. The mechanism of the hydroxy-205 apatite formation is explained in Figure 7: Furthermore, XRD measurement was used to identify the presence of hydroxyapatite on the hybrid surface. The intense peaks of crystalline hydroxyapatite are visible in Figure 8 ( ), confirming that the material surface was covered by a hydroxyapatite layer after 21 days of soaking in SBF. In Figure S3, XRD measurements show the amorphous structure of the sample before being soaked in SBF. Moreover, this result suggests that the hydroxyapatite layer is too thick to allow the detection of XRD signals from the hybrids' surface [24,[31][32][33][34].  [24,[31][32][33][34].

Antibacterial Activity
The antimicrobial properties of these materials were investigated via the Kirby-Bauer Test ( Figure S4) to validate their safety by the ability to inhibit the growth of pathogenic bacterial strains.
Representative images ( Figure 9A) of antibacterial tests of SiO 2 /PEG50% with increasing amounts of Fe(II)C in the hybrid matrix show that iron has an inhibitory effect on both bacterial strains tested. Indeed, the higher the iron concentration in the silica matrix, the higher the inhibitory effect on bacterial growth, as shown in ( Figure 9B). In the literature, it is reported that despite iron being an essential nutrient for bacterial growth, thanks to its main role as an electron transporter, excessive concentrations can become toxic to most bacterial strains. Frawley et al. reported that alterations of the H 2 O 2 and iron concentrations can produce high amounts of free radical species that damage the DNA, proteins, and lipids of bacterial cells [35]. The bacterial inhibitory effects of the increasing amounts of PEG in the SiO2 matrix 248 when 20% of Fe(II)C was encapsulated were evaluated, as shown in Figure 10. The in-249 creasing amounts of PEG had an effect on E. coli and S. aureus growth, as can be seen from 250 the Petri dish images in (Figure 10a). Thus, the highest concentration of PEG allows bac- In accordance with Kalantari et al. [7], low concentrations of iron did not show a significant inhibition, while the bacterial growth of S. aureus and E. coli decreased when 20 wt% of Fe(II)C was encapsulated ( Figure 9C).
The bacterial inhibitory effects of the increasing amounts of PEG in the SiO 2 matrix when 20% of Fe(II)C was encapsulated were evaluated, as shown in Figure 10. The increasing amounts of PEG had an effect on E. coli and S. aureus growth, as can be seen from the Petri dish images in ( Figure 10A). Thus, the highest concentration of PEG allows bacterial inhibition. The bacterial inhibitory effects of the increasing amounts of PEG in the SiO2 matrix when 20% of Fe(II)C was encapsulated were evaluated, as shown in Figure 10. The increasing amounts of PEG had an effect on E. coli and S. aureus growth, as can be seen from the Petri dish images in (Figure 10a). Thus, the highest concentration of PEG allows bacterial inhibition.

Conclusions
The iron (II) citrate complex represents a solution for resolving iron-deficiency anaemia and improving health care, since it increases the bioavailability and solubility of iron. The sol-gel route allowed the synthesis of organic/inorganic hybrid materials consisting of a SiO 2 inorganic matrix, in which different percentages of a polymer (PEG) and a compound (Fe(II)C) were entrapped.
The FTIR spectra of the hybrids showed the typical bands of the polymer and silica, along with the bands of citrate, indicating encapsulation of Fe(II)C in the system. Fe(II)C release measurements were carried out to evaluate the hypothetical controlled release of the compound. Indeed, the results confirm that this system is able to release Fe(II)C in a controlled manner, because it was fast in the first few hours, and then it slowed down, ending after 8 h.
Furthermore, SEM and XRD analysis showed that the hybrids are bioactive, because they are able to form a hydroxyapatite layer on their surface.
Finally, the antibacterial activity of the materials was tested. E. coli and S. aureus were inoculated with the hybrid materials. According to the results obtained, a high concentration of iron is able to inhibit microbial growth, while increasing amounts of PEG do not show a significant inhibition of microbial growth.