An Investigation on the Pharmacological Profile of Titanium ( IV ) and Aluminum ( III ) 8-Hydroxyquinoline Derivatives Grafted on MCM-41 Mesoporous Silica

Introduction: Regarding the increasing applications of nanomedicine in clinical practice, we aimed to assess the antibacterial and anti-fungal effects of nanocomposite complexes of titanium (IV) and aluminum (III). Methods: 8-hydroxyquinoline and 5-chloro-8-hydroxyquinoline were treated with titanium (IV) and Aluminum (III) alkoxide reagents to generate (Q)2(2-BuO) Al (Q = 8-hydroxyquinoline and 5-chloro-8-hydroxyquinoline) and (Q)2(OPr)2Ti (Q = 5-chloro-8-hydroxyquinoline) complexes. These active complexes were grafted on MCM-41 mesoporous silica in order to improve the overall toxicity and stability of them. Anti-bacterial and anti-fungal activities of the compounds


Introduction
Interest in the synthesis of purposeful metalbased composites continues to thrive due to the high demand in medicine.Among non-platinum complexes, aluminum (ΙΙΙ) and titanium (ΙV) have widely been studied due to their unique biological properties such as antitumor, 1,2 antifungal 3 and antibacterial 4 properties.][7][8][9][10][11][12][13][14][15][16] The titanium complexes bearing a quinoline moiety are well known.1][22][23][24][25] The substitution of a chlorine group on the phenolate ring of 8-hydroxyquinoline ligand results in forming biological active complexes among other substituents with subsequently reduced viability in HeLa and PC3 human tumor cells in a dose-dependent manner.Interestingly, adjoining of electron rich compounds (i.e.nitro groups and sulfonic acids) on the hydroxyquinoline ligands Fazaeli et al reduced the antineoplastic potency of complexes up to 220-fold. 9,10In spite of many therapeutic advantages of these complexes, they have some limitations including rapid hydrolysis in moist conditions, lack of stability and forming multinuclear complexes reducing their biological activity. 26n this regard, several efforts have been dedicated to improving the stability of these complexes, such as introducing nitrogen-and sulfur-based ligands. 26he overall toxicity and stability of the complexes are augmented by incorporating them in various nanomaterials. 27,28Among many nanomaterial hosts used to incorporate pharmaceutical agents, mesoporous silica (MCM-41) has attracted a great attention.For instance, MCM-41 with gold (III) pyridine complex showed a strong inhibitory effect on the growth of yeast and animal tumor cells. 29The biological evaluations of the grafted complexes; [ 64 Cu]-TPPF 20 and [ 67 Ga]-DTPA, on MCM-41 silica host showed high radiopharmaceutical uptake in fibrosarcoma tumor in Sprague-Dawley rats. 30,31Herein, in order to prevent aggregation of the complexes and improve the stability and toxicity of 8-HQ derivatives, 29 we report a simple and efficient strategy for incorporating 8-HQ derivatives into MCM-41 by using titanium (for the first time) and aluminum alkoxides, in the light of our previous study. 22

Materials
All chemicals were obtained from Sigma (Germany).Aluminum 2-butoxide and MCM-41 were synthesized and characterized according to our previous report. 22The synthesis of complexes and grafting process were in dry Argon, using standard Schlenk methods. 22eparation of (Q-Cl)2Ti@MCM-41 In order to prepare [Ti(C 9 H 5 ClNO) 2 (C 3 H 7 O) 2 ] complex (a), 5-choro-8-hydroxyquinoline (0.72 g, 4 mmol) was added to a titanium isopropoxide (0.358 g, 2 mmol) solution in toluene (10 mL, 0°C).The mixture immediately turned yellow and became transparent.The solution was stirred at room temperature overnight.Then, the solvents were evaporated under reduced pressure.The deep yellow crystals of the complex were obtained by hexane/ dichloromethane solution, m.p. 439 K. 1

Characterization of Materials
Infrared spectra (IR), 1 H and 13 C NMR spectra were recorded on a Shimadzu model FT-IR 4600 spectrometer and Bruker AVANCE 300-MHz (referenced to SiMe 4 ) respectively.Low angle X-ray diffraction patterns and thermal analysis (TGA-DSC) were obtained on a Philips-PW 17C diffractometer and a Rheometric Scientific STA-1500.
The Anti-bacterial Assay Anti-bacterial activity of the nanocomposites was assessed against Escherichia coli (ATCC 1330), Salmonella typhi (PTCC 1609) and Staphylococcus aureus (PTCC 1113) using disc diffusion method. 32All mediums were autoclaved (15 lbs pressure at 121°C) for 15 minutes and well-mixed and poured into 100 mm plates (25-30 mL/ plate) while still molten.The wells were made with the help of a metallic borer with centers at least 20 mm.The recommended concentrations of 12.5, 25, 50, 75 and 120 ppm of the test compounds were introduced into the respective wells.The plates were immediately incubated at 37°C for 24 hours.Activity against the bacterium was determined by measuring the diameter of the clearing zones (in mm) using a digital caliper and compared with standard Chloramphenicol disc (as a positive control) and substrate solution (as a negative control).The experiment was done in triplicate for each bacterium.
The Anti-fungal Assay The anti-fungal activity of the compounds was determined using the agar well diffusion assay on Sabouraud Dextrose Agar (SCC) (for yeasts) and Muller Hinton Agar medium (MA) (for filamentous fungi).In the case of yeast, stock cultures of two widely used unicellular eukaryotic model yeasts, Kluyveromyces marxianus (PTCC 5188) and Saccharomyces cerevisiae (ATCC 5027) were provided by Iran Researching Organization Collection (Tehran, Iran).In all steps of the experiment, we cultured SCC to produce new yeast cultures and then to perform anti-fungal assays.
For anti-fungal activity against filamentous fungi, the high cellulase producing fungi, Trichoderma reesei (PTCC 5142) was provided by Iran researching organization collection.A phytopathogenic soil-borne fungus M. phaseolina (Tassi) Goid, (NRCAM-27), the causal organism of charcoal rot disease, 33 was the other tested soil-born fungi in this study.The MA was prepared by dissolving 33.9 g of MA (HiMedia) powder in 1000 mL of distilled water.The concentrations of test compounds as 25, 50, 75, 120 and 150 ppm were introduced into the respective wells and then the plates were incubated at 28ºC.The plates were assessed after 24 to 120 hours of incubation for measuring inhibition zones.The results were compared with the diameter of inhibition zones of the chloramphenicol (as a positive control) and substrate solution (as a negative control).The mean value was calculated by performing the experiments in triplicates.were prepared by reacting a 1:1 equivalent ratio of alkoxide and 8-HQ derivatives in toluene at room temperature according to our previous studies with slight modifications. 19,22The labile metalloorganic complex of (Q-Cl) 2 Ti(O i Pr) 2 was characterized by recording their 1 H, 13 C NMR spectra, UV-Vis and IR spectra for the first time which confirmed the reported crystal structure.Due to the lability of these metalloorganic complexes, they were used without isolation for grafting on MCM-41(In situ reaction).

Grafting of the Aluminum and Titanium 8-HQ Complexes to MCM-41
The prepared complexes of aluminum and titanium were incorporated directly into MCM-41 silica in toluene.The facile reaction of hydroxide groups of MCM-41 with alkoxy groups of the complexes resulted in the covalently bonding of the complex to the silica.The synthesis procedure for grafting of the complexes was illustrated in Figure 1.The fluorescent color of the solution after filtration of the final compound showed the extra complexes in the solution which confirmed the maximum loading of the complex on MCM-41.As shown in Figure 2, the FT-IR spectrum of the grafted titanium 8-HQ complex on mesoporous silica showed bands in the range of 1633-1575 cm -1 and 2970 cm -1 , which revealed that the 8-HQ ligand and one of the isopropoxy groups remained intact after the reaction.Formulation of grafted 5-chloro 8-HQ titanium complexes on MCM-41 was verified by TGA analysis as 2.75(Q-Cl) 2 Ti .100SiO 2 (Figure 3).Grafting of titanium on MCM-41 was demonstrated by 17.3% weight loss in the TGA curve of (Q-Cl) 2 Ti@MCM-41 at 200 to 550°C, along with an exothermic peak in the DSC curve in the same region.This indicated lower stability of the   22 This fact lies in the presence of the isopropoxy group on the mesoporous silica which showed that the lability of the titanium (ΙV) 8-hydroxyquinoline complex was less than the aluminum (ΙΙΙ) 8-hydroxyquinoline complexes.The (100) reflection within the 2θ range of 2-3° in low angle X-ray diffraction patterns of analyses, clearly indicated that the twodimensional hexagonal mesoporous structure (P6mm) of MCM-41 was maintained after introducing titanium complex (Figure 4). 22e Anti-bacterial Activity of the Nanocomposites It was observed that (Q-Cl) 2 Ti@MCM-41 nanocomposite had anti-bacterial activity against S. aureus at concentrations of 50, 75 and 120 ppm and its activity was dose-dependent.However, the anti-bacterial effect of this nanomaterial on E. coli and S. typhi was started at the concentration of 75 ppm.Q 2 Al@MCM-41 weakly inhibited the bacterial growth of E. coli at 75 and 120 ppm concentrations (Table 1).(Q-Cl) 2 Al@MCM-41 did not show any anti-bacterial activity at any concentration (Figure 5).
The Anti-fungal Activity of the Nanocomposites As shown in Table 2, all materials had anti-yeast activity and (Q-Cl) 2 Ti@MCM-41 was the strongest, showing yeast inhibition activity at 50 ppm concentration.Q 2 Al@ MCM-41 showed the minimum anti-fungal activity.The (Q-Cl) 2 Ti@MCM-41 had considerable anti-yeast  activity in comparison with other tested compounds.K. marxianus presented more resistance than the S. cerevisiae in exposure to all the compounds.(Q-Cl) 2 Al@ MCM-41 showed anti-fungal potential (in both yeast and filamentous fungi), however, no anti-bacterial potency was observed.M. phaseolina completely resisted against Q 2 Al@MCM-41 and had less sensitivity than T. reesei to (Q-Cl) 2 Al@MCM-41 and (Q-Cl) 2 Ti@MCM-41 (Figure 6).

Discussion
Complexes of 8-hydroxyquinoline derivatives of titanium and aluminum were adjoined with MCM-41 nano silica using condensation of hydroxide groups of the MCM-41 beds modified by alkoxy groups.The concentrations of complexes were adjusted to maximum to achieve the highest possible availability and stability of antimicrobial material on MCM-41 beds.Therefore, after grafting of complexes on MCM-41 nano silica, the color of solutions was still visible.Notably, no leaching was noted in the grafted nano silica complexes in exposure to polar or non-polar solvents.The anti-microbial (bacterium and fungi) effects of the tested nanoparticles used in this study were found to be very potent.5][36] The antimicrobial effects of the agents were dose-dependent and more pronounced against gram-positive bacteria than gramnegative organisms, 37 however, Shrivastava et al reported that gram-positive bacteria had higher resistance against silver nanoparticles. 38n the case of anti-fungal activity, all the tested isolates belonged to the Ascomycota family.The yeasts (S. cerevisiae and K. marxianus) showed more sensitivity to these compounds than the filamentous fungi (T.reesei and M. phaseolina) and this may present the role of mycelial body in resistance against antibiotic stresses.The less sensitivity of M. phaseolina than the T. reesei was observed in all treatments that may be related to the ability of M. phaseolina to produce more pigments.5][36] In addition, the resistance of K. marxianus and M. phaseolina against nanomaterial make them suitable candidates for bio-   synthesis of nanoparticles.Bare complexes of aluminum and titanium are not stable in aqueous medium, therefore, their MICs (minimum inhibitory concentration) could not be determined accurately.It was already shown that similar complexes of titanium alkoxides with moisturestable ligands also had no antifungal activity. 26Our study showed that the substitution of the metallic core from aluminum (III) to titanium (IV) improved the cytotoxicity of compounds.

Conclusion
This study showed that it is possible to prepare moisturestable titanium (IV) and aluminum (III) 8-HQ derivatives complexes with direct grafting on MCM-41 mesoporous nanotube.The complexes were biocompatible and showed anti-microbial (bacterium and fungi) activities.The prepared materials were stable both under air and high temperature (at least up to 200°C).These nanocomposites were well dispersed in water supporting their potential chemotherapeutic application in clinical practice.

Conflict of interests
There is no conflict of interests in this study.

Ethical Issues
There is no experiment that needed approval.

Figure 5 .
Figure5.The antibacterial activity index was the average diameter of the clearing zones (mm) presented in 50 ppm (A), 75 ppm (B) and 120 ppm (C) concentrations of (Q-Cl) 2 Ti@MCM-41 against E. coli, and 75 ppm (D) and 120 ppm (E) concentrations of (Q-Cl) 2 Ti@MCM-41 against S. aureus subsp.areas using the digital caliper in three replicates.

Figure 6 .
Figure 6.The antifungal activity index was the average diameter of the clearing zones (mm) presented in 500 ppm (A), 750 ppm (B) 1200 ppm (C) and 1500 ppm (D) concentrations of (Q-Cl) 2 Al@MCM-41 against S. cerevisiae.

Table 1 .
The Results of Antibacterial Activity of Compounds by Disc Diffusion Method *n: negative, a-e: Different superscript small letters within a row denote significant differences (P<0.01,Duncan's multiple range test) in each nanocompound concentration.

Table 2 .
The Results of Fungicidal Activity of Compounds by Agar Well Diffusion Assay Method Different superscript small letters within a row denote significant differences (P<0.01,Duncan's multiple range test) in each nanocompound concentration.

Table 1 .
The Results of Antibacterial Activity of Compounds by Disc Diffusion Method *n: negative, a-e: Different superscript small letters within a row denote significant differences (P<0.01,258 Duncan's multiple range test) in each nano-compound concentration.