Characteristic of Ag/TiO
2–SiO
2bionanocatalysts prepared by sol–gel method as potential antineoplastic compounds
TESSY LO´ PEZ-GOERNE1,* , ALINA GRACIA1, FRANCISCO J PADILLA-GODI´NEZ1,2, PAOLO LOTTICI3and ANA M SILVESTRE-ALBERO4
1Department of Health Care, Autonomous Metropolitan University - Xochimilco, 04960 Mexico City, Mexico
2Institute of Cellular Physiology, National Autonomous University of Mexico, 04510 Mexico City, Mexico
3Department of Mathematical, Physical and Computer Sciences, University of Parma, 43121 Parma, Italy
4Advanced Materials Laboratory, Alicante University, 03690 Alicante, Spain
*Author for correspondence (tessy3@prodigy.net.mx) MS received 21 June 2021; accepted 6 August 2021
Abstract. In this study, bionanocatalysts composed of a titanosilicate oxide matrix (TiO2–SiO2) impregnated with different concentrations of silver (0.1, 0.5, 1.0, 5.0 and 10 wt%) were prepared by the sol–gel method using silver nitrate (AgNO3) as silver (Ag) source. The physicochemical properties were evaluated by scanning and transmission electron microscopies, Raman and Fourier transform infrared spectra, X-ray photoelectron spectra, and UV–visible spectroscopy.
Furthermore, we studied the cytotoxic effect on a cervical uterine cancer cell line as a first approach attending to the potential antineoplastic properties of the Ag/TiO2–SiO2bionanocatalysts. The bionanocatalysts exhibited no significant differences in morphology or composition independently of the amount of Ag stabilized, with a reduction in particle size proportional to Ag concentration. All samples could achieve cell mortality rates above 91%, obtaining results similar to those of pure AgNO3(considered as 100% Ag) from the sample loaded with 1% Ag. The results in this study suggest the possibility of using bionanocatalysts to enhance the intrinsic cytotoxic properties of silver such that a minimum con- centration is required to achieve desired results.
Keywords. Bionanocatalyst; silver; characterization; sol–gel; antineoplastic.
1. Introduction
Bionanocatalysts are nanostructured inorganic catalysts whose surface is covered with organic agents that mimic cellular conditions [1]. In recent years, bionanocatalysts have attracted wide attention due to their excellent prop- erties, such as high specific surface area, thermal stability, mesoporous structure, selective catalytic activity and par- ticle sizes below 100 nm [2–8], which can be obtained by the modification of variables involved in the synthesis technique used: the sol–gel method [9]. Bionanocatalysts exhibit a wide range of actual and potential applications in various fields [10]. Regarding the last-mentioned, in pre- vious work, we reported the case of a pediatric ependy- moma which was treated with a titanosilicate bionanocatalyst, where we achieved total elimination of the tumour, clinical improvement and no tumour recurrence or adverse effects [11]. This occurs due to the mechanism of action of the bionanocatalysts: by directly catalysing the breakage of carbon-carbon and carbon-nitrogen bonds present in the DNA molecule instead of generating sec- ondary compounds that would carry out the cytotoxic out- come [11,12], the bionanocatalysts can inhibit cellular
growth despite the nucleotide sequence in the genetic material.
Bionanocatalysts have been observed to enhance the intrinsic properties of metallic species when stabilized in the catalytic matrices composed of titania [13] and silica [14]. Particularly, platinum has exhibited both antineo- plastic and antimicrobial effects [15,16]. Notwithstanding, the high costs associated with platinum compounds used as precursors makes it necessary to search for substitute metals with equal efficacy and greater selectivity and biocompat- ibility. Regarding the foregoing, silver has been amply studied for its bactericidal activity against Gram-positive and Gram-negative bacteria [17]. Moreover, anti-cancer properties have also been associated recently with nanos- tructured silver [18]. Nonetheless, the medical conditions and environmental concerns related with silver accumula- tion [19,20] compel for the research on strategies to opti- mize the properties of the material in such a way that smaller concentrations are needed to exert the same thera- peutic effects. Thus, in this present work, we have evaluated the physicochemical properties of a bionanocatalyst composed of a titanosilicate oxide matrix (TiO2–SiO2) impregnated with different concentrations of Ag (Ag/TiO2–SiO2) to https://doi.org/10.1007/s12034-021-02570-8
identify potential structural or functional modifications regarding the addition of Ag. Furthermore, we studied the cytotoxic effect on a cervical uterine cancer cell line as a first approach attending the potential antineoplastic properties of the Ag/TiO2–SiO2 bionanocatalysts and the minimal amount of stabilized Ag required to achieve the cytotoxic effect.
2. Experimental
2.1 Material
Silver nitrate (AgNO3, 99.9%), and titania and silica precursors were purchased from Sigma-Aldrich Co. Abso- lute ethanol was purchased from J T BakerTM. All chemi- cals were of analytical grade and were used without further purification.
2.2 Preparation
In this study, bionanocatalysts were prepared using the sol–gel method, following the procedure described by Lo´pez et al [21]. Five bionanocatalysts were prepared at five chemistry-concentrations (w/w) of Ag with respect to the TiO2–SiO2matrix: 0.1, 0.5, 1.0, 5.0 and 10% w/w, and were labelled according to each concentration of Ag: Ag 0.1%, Ag 0.5%, Ag 1.0%, Ag 5.0% and Ag 10%, respec- tively. As the silver precursor, the proper amount of AgNO3 was dissolved in deionized water. Both solutions were mixed and stirred in a three-neck flask at room temperature until homogenization. Then, the titania and silica precursors were added dropwise. The biphasic solution was kept under constant stirring. The catalysts were dried, and the samples were manually ground until a fine powder was obtained.
2.3 Characterization
The particle size, grain size, morphology and texture were observed by scanning electron microscopy (SEM, JSM- 6010LV, JEOL, USA) working at 10–20 kV, and dark-field transmission electron microscopy (TEM; JEM-2100F, JEOL, USA) working at 200 kV. The samples were placed on copper grids covered with holey carbon films.
The vibrational state and bond types were identified with Fourier-transform infrared spectroscopy (FTIR). FTIR spectra of the samples were recorded at room temperature on a 40% sample–KBr transparent pellet using a Shimadzu IRAffinity-1 spectrometer in the 4000–400 cm-1range.
Non-polarized Raman spectra on the powdered Ag/TiO2– SiO2 samples were recorded at 632.8 and 473.1 nm in a nearly backscattered geometry with a Horiba-Jobin Yvon LabRam micro-spectrometer (300 mm focal length spec- trograph) equipped with an integrated Olympus BX40
microscope. The Rayleigh radiation was blocked by an edge filter and the backscattered Raman light was dispersed by an 1800 grooves per mm holographic grating on a Peltier cooled CCD, consisting of an array of 1024/256 pixels. The entrance slit width was fixed at 100 lm. The spectral res- olution was about 1.3–2.2 cm-1according to the excitation light. The laser power was adjusted by means of density filters to check thermal effects. Spectra were collected using both 9100 and long working distance 950 microscope objectives. Typical exposures were 10–60 s with 5–9 rep- etitions. The system was regularly calibrated using the 520.6 cm-1Raman band of silicon or by means of reference emission lines of Ne or by Cd light sources. The data analysis was performed by using the LabSpec built-in software.
X-ray photoelectron spectra (XPS) were acquired with a Physical Electronics PHI 5700 spectrometer equipped with a non-monochromatic MgK radiation source (300 W, 15 kV,h= 1253.6 eV). Spectra were recorded at a 45take-off angle by a concentric hemispherical analyzer operating in the constant pass energy mode at 25.9 eV, using a 720 mm diameter analysis area. The C 1s, O 1s, Ti 2p, Si 2p and Ag 3d core level signals were recorded. Charge referencing was done against the adventitious carbon (C 1s at 284.8 eV).
Solids were mounted on a Cu sample holder without adhesive tape and kept overnight in a high vacuum chamber before they were transferred inside the analysis chamber of the spectrometer (\10-7 Pa). The PHI ACCESS ESCA- V6.0 F and Multipack 8.2b software packages were used for acquisition and data analysis. A Shirley-type background was subtracted from the signals. Recorded spectra were always fitted using symmetrical 70%–30% mixed Gaussian–Lorentzian peak shapes to determine the binding energy of the different element core levels more accurately.
The accuracy of the binding energy (BE) values was within
±0.2 eV.
2.4 In vitrocytotoxicity
2.4a Cell culture: The cell line for cervical-uterine cancer (HeLa (ATCC CCL-2)) was cultured to generate a cell bank to place them in ELISA boxes. The cell lines were main- tained with Dulbecco’s modified Eagle medium (DMEM) (Caisson DML 19-400 ML) enriched with 4 mM L-glutamine and 12.5 ml or HEPES 25 mM and a phosphate buffer (PBS1X). For the cell growth medium, we used 90 ml of DMEM added with 10% fetal bovine serum, filtrates and 1% antibiotic (penicillin at 10,000 U ml-1and strep- tomycin (CAISSON) at 10,000 lg ml-1). Cell growth was performed at 37C in a 5% CO2incubator.
2.4b Bionanocatalysts dissolutions: The dissolutions of the bionanocatalysts were prepared with DMEM growth medium (90 ml) and kept under refrigeration. The culture medium in the ELISA boxes was removed with a Pasteur
pipette connected to a Bu¨chner flask with a vacuum. The bottom of the boxes was not touched as cells were adhered to it. The cells were rinsed with 200ll of PBS and extracted with the Pasteur pipette. The cells were exposed to the bionanocatalysts at concentrations of 125, 250, 500 and 1000lg ml-1with DMEM for 24 h at 37C in a 5% CO2
atmosphere. The bionanocatalysts were dissolved in the growth medium because of their easy-suspension formation.
The study was carried out by triplicates. The control alone contained 200ll of the growth medium.
2.4c MTT cell viability test: The cytotoxic effect of the bionanocatalysts in the cancerous cell line was evaluated through the MTT assay which is used to quantify the cell viability by colorimetry because of the amount of formazan produced by the living cells. The assay was conducted in triplicates. After 24 h, the medium and bionanocatalysts were removed with the Pasteur pipette. Hundred microlitres of growth medium with 10 ll of MTT dissolved in 5 mg ml-1of PBS and 100ll of DMSO was added on each cell with the unwashed cells and incubated for formazan syn- thesis for 4 h at 37C in a 5% CO2 atmosphere. The supernatant was put in a new ELISA box, and UV-Vis study was carried out with a spectrophotometer adjusted to a wavelength of 595 nm (680 model, Bio-Rad brand) per each plate.
3. Results and discussion
3.1 Characterization of bionanocatalysts
Common nanostructured Ag tends to present identifiable morphologies, such as seeds [22], nanorods and nanowires [23], triangular nanoprisms [24], nanocubes [25], nanodisks [26], and nanospheres [27]. As shown in figure1, our bio- nanocatalysts exhibit no recognizable crystalline structures attributable to the incorporation of the Ag into the TiO2– SiO2catalytic matrix, which, on the contrary, presents an amorphous structure associated with the conjunction of silica and titania, as described in previous works [28]. All samples, independent of the concentration of Ag stabilized, show the formation of conglomerates similar in size (*50 lm) and the presence of submicron grains\500 nm in diameter with spherical shapes. Since the incorporation of Ag into the catalytic matrix does not generate significant changes in the surface morphology of the bionanocatalysts, regardless of the concentration, it can be inferred that the Ag particles are distributed homogeneously throughout the matrix without forming agglomerates that could exhibit structures associated with unattached nanostructured Ag0. The aforementioned was corroborated by TEM (figure 2).
The dark-field micrographs show the amorphous aggregates that make up the bionanocatalysts with sizes of\500 nm and the homogeneous (*100% dispersion) deposition of silver particles (bright spots) with sizes of the order of 25
nm. This phenomenon is observed for all the samples (fig- ure2c, e, g and i), except for the one loaded with 0.1% Ag w/w (figure2a). This could be due to the fact that the silver particles generated by such a low precursor concentration are so few that the TEM equipment used is unable to detect them, hence, it would be necessary to use equipment with higher resolution for their observation. The positions of the silver particles, exclusively on the surface of the matrix, suggest that they interact with the TiO2–SiO2 catalytic matrix without being free. Regarding the size distribution of the bionanocatalysts, it is observed that the size of the conglomerates decreases as the silver deposition increases (figure2b, d, f and h). The conglomerate size of the 0.1%
Ag bionanocatalysts shown in figure2b ranges from 200 to 650 nm, and its average size is 425.44 nm. The conglom- erate size of the 0.5% Ag sample (figure2d) is 384.25 nm, and the size ranges from\75 to 675 nm. It can be seen from figure 2f that the size distribution of the 1.0% Ag bionanocatalysts is between\50 to 650 nm with an average size of 271.32 nm. Finally, figure2i shows that for the 5.0%
Ag sample, the size ranges from\50 to 650 nm with an average size of 258.17 nm. The size distributions observed for all the samples correspond to the conglomerates (as seen in the dark-field TEM micrographs). Although the TiO2– SiO2 matrix presents aggregates with sizes\50 nm [29], the adhesion of such aggregates into bigger conglomerates, as observed in the case of our bionanocatalysts, indicates the need for the addition of dispersants that allow the nanoparticles to be observed individually. This will be studied in further work.
The obtained Raman spectra are reported in figure3 for the red, 632.8 nm (figure 3a) and the blue, 473.1 nm, (fig- ure3b) excitation lights. The spectra are with fluorescence background for all compositions. The TiO2–SiO2reference compound show two characteristic features at 490 and 980 cm-1[30] and a broad band with a maximum at about 770 cm-1 corresponding to the Si–OH flexion bonding [30].
Contrary to what is expected, there is no evidence, for both laser lines, of the strong anatase peak at about 143–152 cm-1characteristic of nanocrystalline titania usually found for nanoparticles sized below 3–5 nm [31]. Other crystalline forms, rutile or brookite, give no characteristic features in the spectra. With increasing Ag content, the SiO2features progressively become less evident and two sharp peaks emerge, for both excitations at 968 and at 1046 cm-1. The first signal corresponds to the symmetric stretching vibra- tion of the organic groups present on the surface of the bionanocatalysts [32], which, for the case of TiO2–SiO2 reference, is overlapped by the 980 cm-1 peak character- istic of the SiO2matrix [33]; as silver is added to the matrix, a rearrangement of the lattice occurs, allowing the organic groups to be exposed, hence, increasing the intensity of the peak. On the other hand, the peak at 1046 cm-1is due to silver nitrate (AgNO3). This feature is the main symmetric stretching vibration of the NO3groups [34]. The intensity of
Figure 1. SEM of bionanocatalysts synthesized with different concentrations of Ag: (a, b) Ag 0.1%; (c,d) Ag 0.5%; (e,f) Ag 1.0%; (g,h) Ag 5.0%; (i,j) Ag 10%.
Figure 2. Dark-field TEM of bionanocatalysts synthesized with different concentrations of Ag and its histogram of particle size distribution: (a–b) Ag 0.1%; (c–d) Ag 0.5%; (e–f) Ag 1.0%; (g–h) Ag 5.0%; and (i) Ag 10%.
both peaks increases with the Ag concentration, even if the measurements taken on different points are not uniform.
To further analyse the bonds present in the bionanocat- alysts, an FTIR spectrophotometer was adopted to study vibrational modes of the samples (figure 4). Peaks in the spectra were assigned according to previous works [10,35].
Peak assignation is summarized in table 1. The band observed at 3712 cm-1 (figure 4a) corresponds to the stretching (m) mode of free surface hydroxyl groups (non- hydrogen-bonded) [10]. These groups are very acidic and endow the sample with Brønsted and Lewis sites [36]. The wide band centred at 3454 cm-1corresponds to the O–Hm mode of bonded hydrogen, which is characteristic of sol–gel materials [11]. The little shoulder observed at 3196 cm-1 corresponds to hydroxyl groups, such as Si–OH and Ti–OH located at the surface of the network [10]. The little band at 1708 cm-1(figure4b) may correspond to C=O due to the atmospheric CO2. The small peak at 1639 cm-1 can be associated with the bending (d) mode of H–O–H in water
[10] and with thedmode of N–H in the organic molecules.
At 1394 cm-1, the small peak corresponds to the O–H m mode of humidity; for the AgNO3and the sample Ag 10%, the peak is shifted to 1382 cm-1, possibly due to the hydrophilicity of the silver surface [37]. The tiny shoulder at 1219 cm-1could be associated with themmode of C–O.
The asymmetric stretching mode of the Si–O–Si bond is located at 1091 cm-1 [38]; the peak flattens as the silver concentration increases. The wide feature at 968 cm-1 is associated with the asymmetric stretching mode of Si–O–Ti [39], probing for the interaction of both oxides as a single mixed-oxide matrix. The characteristic peak of TiO2 at 690 cm-1 corresponding to Ti–O stretching band [40] is overlapped by the wide peak ofd Si–O at 808 cm-1. This peak may also be overlapping the characteristic band at 784 cm-1associated with organic groups [41]. The band at around 617 cm-1 is attributed to the vibrational state of anionic -O–Si–O- species [10]; the presence of such anionic species contributes to the catalytic activity of the Figure 3. Raman spectra of the bionanocatalysts at different Ag concentrations, taken with the (a) red, 632.8 nm and
the (b) blue, 473.1 nm excitation laser lights.
Figure 4. FTIR of bionanocatalysts at different Ag concentrations, TiO2–SiO2 reference and AgNO3 from (a) 4000–2800 cm-1and (b) 2000–400 cm-1.
TiO2–SiO2matrix. Finally, the intense band at 470 cm-1is due to Si–O-[10].
In figure5, the narrow windows at C 1s, O 1s, Si 2p, Ti 2p and Ag 3d core levels are shown. At the energy of C 1s core orbital, three contributions were found: carbonyl
groups (C=O) at 289.3 eV, alkoxy ligands (C–O) at 286.8 eV and alkyl substituents (C–H) at 285.2 eV [42]. At the O 1s region, the main peak observed at 533.4 eV corresponds to the TiO2 overlapping carbonyl groups [43]. Hydroxyl HO–Ti–O components were found at 531.1 eV [44]. At the Table 1. FTIR band assignation for bionanocatalysts.
Wavenumber (cm-1)
Assigned vibrations
TiO2–SiO2 Ag 0.1% Ag 0.5% Ag 1.0% Ag 5.0% Ag 10% AgNO3
3712 3712 3712 3712 3712 3712 — m(O–H, surf.)
3475 3475 3475 3475 3475 3475 — m(O–H, H2O)
3196 3196 3196 3196 3196 3196 — m(M–O–H)
1708 1708 1708 1708 1708 1708 — m(C=O)
1639 1639 1639 1639 1639 1639 — d(H–O–H)
1394 1394 1394 1394 — — — m(O–H, H2O)
— — — — 1382 1382 1382 m(O–H, H2O)
1219 1219 1219 1219 1219 1219 — m(C–O)
1091 1091 1091 1091 1091 1091 — mas(Si–O–Si)
968 968 968 968 968 968 — mas(Si–O–Ti)
808 808 808 808 808 808 — d(Si–O)
617 617 617 617 617 617 — d(–O–Si–O–)
470 470 470 470 470 470 — d(Si–O–)
m= stretching,mas= asymmetric stretching,d= bending, M = Ti, Si.
Figure 5. XPS narrow windows of the TiO2–SiO2matrix loaded with Ag at the (a) C 1s, (b) O 1s, (c) Si 2p, (d) Ti-2p and (e) Ag-3d core levels. (f) Schematic representation of the bionanocatalyst.
energy of Si 2p core orbital, the Ag/TiO2–SiO2 material showed a peak at 104.2 eV, which is attributed to titanium–
silicate species (TiSiOx) [45]. The characteristic Ti-2p1/2 and Ti-2p3/2 spin-orbit components of TiO2 were clearly observed respectively, at 465.1 and 459.4 eV [42]. The Ag was incorporated into the titania–silica mixed oxide net as a metallic agent Ag0. The Ag-3d3/2 and Ag-3d5/2 spin-orbit signals were found respectively, at 374.6 and 386.6 eV [46].
The XPS data is summarized in table 2.
Finally, figure 6 shows the main UV–visible bands observed for the samples. Ag/TiO2–SiO2 bionanocatalysts have a doublet at 315 and 318 nm due to the transfer of charge betweensandporbitals in the compounds. Samples Ag 5.0% and Ag 10% exhibit a shift in this doublet due to the increase in silver concentration. At 363 nm, a very intense peak is seen, which may be indicative of a silver reduction process. At higher concentrations, the peak of absorption energy has a strong relationship with the struc- ture, components and concentration of these components, as well as with particle size.
3.2 In vitrocytotoxicity
Once the physicochemical characteristics of the bionanocatalysts were evidenced, their potential antineo- plastic properties were evaluated by the MTT assay on a cervical-uterine cancer cell line (HeLa (ATCC CCL-2)). As a first approach in the study of the cytotoxicity of the bio- nanocatalysts, four concentrations (125, 250, 500 and 1000 lg ml-1) were used, so as to evaluate the cytotoxic beha- viour from a relative low concentration to a high concen- tration. HeLa cells with no treatment showed a normal morphology (in spindle) and were confluent and adhered (figure not shown for the sake of brevity) [47]. Figure 6 shows the cytotoxic behaviour of the samples against HeLa cells. The treatment with the bionanocatalysts at different silver concentrations exhibited a general drastic increase in
cell mortality as early as 125 lg ml-1with a reduction in formazan production, achieving virtually no synthesis from 250 lg ml-1. The morphology (not shown) was also affected: the cells were visualized as amorphous, small and nonadhesive. A clear trend of increasing cytotoxicity (figure 7a) was observed with respect to the increasing concentration of silver loaded in the TiO2–SiO2matrix. The minimum observed concentration of silver capable of exerting significant cytotoxicity (P\0.05) from 125 lg ml-1 was the one corresponding to the sample Ag 0.5%
with a maximum mortality rate (MMR) of 94.96 ±9.5%
(figure7b). Notwithstanding, sample Ag 0.1% was the one that exhibited the highest MMR at 95.50 ± 9.5%, per- centage even higher than that of AgNO3(94.15±9.1%). As shown in figure7a (right panel), the fitted curves show that AgNO3 exhibited the lowest concentration required to achieve its MMR, followed by Ag 5.0%, Ag 10% (MMR = 94.89 ± 9.5%), Ag 1.0% and Ag 0.5%. On the contrary, when treated with the TiO2–SiO2matrix, cells displayed no visible change in formazan concentration, which indicates a low cytotoxic effect with a MMR of 28.75 ± 2.8%
Table 2. XPS data obtained for TiO2–SiO2matrix loaded with Ag.
Core level Binding energy (eV) Assignment FWHM (eV) Atomic composition (%)
C 1s 285.2 C–H/C–C 1.70 10.5
286.8 C–O/C–N 1.95
289.3 C=O 1.96
O 1s 531.1 O–H 2.03 59.1
533.4 TiO2 1.96
Si 2p 104.2 SiO2 1.84 28.7
Ti 2p1/2 465.1 TiO2 2.27 0.8
Ti 2p3/2 459.4 TiO2 1.75
Ag 3d3/2 374.6 Ag0 1.43 1.0
Ag 3d5/2 368.6 Ag0 1.45
FWHM = full width at half maximum.
Figure 6. UV–Vis spectra obtained for bionanocatalysts, the TiO2–SiO2reference and AgNO3.
(figure 7a); a significant reduction (P\0.05) in cell via- bility was not observed until 250 lg ml-1 (figure 7b).
Sample Ag 0.1% did not show a significant (P\0.05) reduction in cell viability until 250 lg ml-1 (figure 7b), with even poorer results at 125lg ml-1than those observed for the TiO2–SiO2matrix.
The cytotoxicity profile observed for the bionanocatalysts with relatively low concentrations of silver (as compared to the precursor considered as 100%) demonstrates the effi- cacy of the catalytic TiO2–SiO2 matrix to enhance the intrinsic cytotoxic properties of nanostructured silver.
3.3 Hypothesis on cytotoxic mechanism and further perspectives
Nanostructured Ag is currently being investigated for its cytotoxicity on both cancer and normal cell lines [48].
However, questions on how to contain and control the
toxicity effects of nanostructured Ag and how much we know about its toxicological profile, still remain [48].
Hence, it is necessary to develop new techniques that allow the enhancement of intrinsic properties of the metal in such a way that lower concentrations are required to achieve effects similar to those desired. The bionanocatalysts rep- resent an interesting approach as the incorporation of noble metals in such structures has been identified to catalytically enhance their intrinsic features [49]. The bionanocatalysts are believed to exhibit cytotoxicity because of the silver atoms distributed in the TiO2–SiO2 matrix (occupying oxygen vacancy sites or attached to the matrix), which have been reported to induce cellular damage in terms of loss of cell membrane integrity, oxidative stress and apoptosis [50].
Nonetheless, the cytotoxic profile observed for the pure matrix suggests that the alterations in the cell are also related with the catalytic properties of the matrix; the silver atoms not only act as the active compound, but also as enhancers of the matrix [49]. The sol–gel TiO2–SiO2matrix Figure 7. (a) Mortality fitted curves for HeLa cells in contact with bionanocatalysts from 0–100% (left) and from 92–97% (right);
and (b) cell viability histograms for each sample compared with silver precursor (pure silver). Significant differences were marked as
*P\0.05vs. control.
acquires Lewis and Brønsted acid properties (electron–pair acceptors and hydrogen donors) as metallic ions (particu- larly transition metal ions which have empty valence orbi- tals) act as Brønsted and Lewis acids [12,51,52]; the presence of such Brønsted and Lewis sites increases the catalytic properties of the matrix. In previous works [1,11], we have shown that similar catalytic matrices adhere to the surface of the membrane of cancerous cells and trigger endocytosis through a theorized ligand–receptor interaction carried out by the organic agents present in the matrix. This endocytosis process allows for the uptake of bionanocata- lysts [11]. Once inside the cancerous cell, the acid sites in the catalytic structure destabilize DNA by electron heist and breaking of hydrogen bonds [53]. Furthermore, TiO2–SiO2 nanoparticles have been observed to locate in the peri- region of the nucleus as aggregated particles, increase ROS, decrease reduced glutathione and induce cell death by apoptosis [54]. Such interactions are directly related with the catalytic properties of the TiO2–SiO2structure.
Though the experiments carried out for this work rep- resent only a first approach regarding the antineoplastic properties of Ag/TiO2–SiO2 bionanocatalysts, the results are quite promising. Further studies must be done to evaluate their effect on different cancerous cell lines and at more concentrations (specially, concentrations lower than 125 lg ml-1) to fully elucidate the minimal con- centration of bionanocatalysts required to achieve a mor- tality rate comparable to current anti-tumoural compounds, such as cisplatin. Similarly, cytotoxic assays must be carried out to evaluate the effect of these Ag/TiO2–SiO2 bionanocatalysts on normal cells. Previous works have demonstrated high selectivity and biocompatibility in non- cancerous cell lines (fibroblasts) for similar bionanocata- lysts composed of platinum stabilized in titania [1,55].
Although still under investigation, the main hypothesis is based on a possible ligand–receptor interaction carried out by functionalization agents present on the surface of bio- nanocatalysts, which trigger cellular uptake by endocytosis.
Indeed, drugs often include components that interact with antigens or receptors on target cells [1,56]. Hence, consid- ering the results observed for similar bionanocatalysts in cancer and normal cell lines [1,11,55], this hypothesis could explain in terms of presence or absence of such specific receptors, why cellular uptake is only observed in damaged cells, while normal cells remain intact.
In advance, regarding cytocompatibility, Jadhav et al [57] reported C95% cell viability when evaluating silver nanoparticles (synthesized with silver nitrate) against fibroblast cell lines at concentrations as high as 78.62 ppm.
Similarly, Dulski et al [58] recently reported viability of normal dermal fibroblast and osteoblast cells when inter- acting with Ag–SiO2/TiO2 nanostructures. Furthermore, biocompatibility of both nanostructured SiO2and TiO2(as well as mixed matrices of both) is widely known [59–65].
Both our previous results and current data in the literature support the biocompatibility of silver nanoparticles at such
low concentrations as those used for our bionanocatatalysts, as well as for the TiO2–SiO2 matrix that composes them.
Nonetheless, regarding the concern on silver accumulation and toxicity, assays will be done with these silver bio- nanocatalysts in a future study. When fully proved for biocompatibility and efficacy, these compounds could rep- resent an effective, biocompatible and low-cost alternative to common chemotherapeutics.
4. Conclusion
In this study, bionanocatalysts composed of a titanosilicate oxide matrix (TiO2–SiO2) impregnated with different con- centrations of silver (0.1, 0.5, 1.0, 5.0 and 10 wt%) were synthesized, characterized and evaluated for cytotoxicity in a cancerous cell line. The bionanocatalysts exhibited no significant differences in morphology or in composition independently of the amount of Ag stabilized, with a reduction in particle size proportional to Ag concentration.
All samples could achieve cell mortality rates above 91%, obtaining results similar to those of pure AgNO3(consid- ered as 100% Ag) from the sample loaded with 1% Ag. The results in this study suggest the possibility of using bio- nanocatalysts to enhance the intrinsic cytotoxic properties of silver such that a minimum concentration is required to achieve the desired results. Further research must be done to elucidate the minimal concentration of bionanocatalysts required to achieve mortality rates comparable to current anti- tumoural compounds, bothin vitroandin vivo. Similarly, the biocompatibility must be assessed in normal cells andin vivo to evaluated possible adverse effects. These silver bio- nanocatalysts could represent an effective, biocompatible and low-cost alternative to common chemotherapeutics.
Acknowledgements
FJPG (CVU 1037918) is supported by a grant from the National Council of Science and Technology. We would like to thank Enrique Rodrı´guez-Castello´n, PhD and Francisco Rodrı´guez-Reinoso, PhD ( ) for their valuable collaboration to this paper. The author FJPG is especially grateful to Melissa Escarpita Go´mez for the technical assistance offered.
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