Antimicrobial Activity of Hippurate Nanocomposite and Its Cytotoxicity Effect in Combination with Cytarabine against HL-60 Samer

1 Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia 2 Laboratory of Vaccines and Immunotherapeutics, Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia Materials Synhesis and Characterization Laboratory (MSCL), Institute of Advanced Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia Department of Nutrition and Dietetics, Faculty of Medicine and Health Science, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia


Introduction
Metal hydroxide compounds can be classi�ed according to their structures and chemical compositions into two groups.e �rst group is the layered double hydroxide (LDH) derived from the brucite structure (Mg(OH) 2 ) through substitution part of the divalent cations by the trivalent ones.As a result, the layers acquire excess positive charge, which is balanced by the incorporation of anions into the interlayer space.e general formula may be thus given as (LDH − M 2+  1− M 3+  (OH) 2 (A − )  ⋅ H 2 O), where M 2+ is divalent cations, M 3+ is trivalent cations, and A − is exchangeable anion with a charge (m − ) [1]. e second group is the metal hydroxide compounds which are layered metal hydroxide salt (LHS) that can be represented by the general formula (LHS − M 2+ (OH) 2− (A − )  ⋅ H 2 O) [2].e interlayer anion exchangeable capability of LDH and LHS meets the requirement of inorganic layers for encapsulating organic drugs with negative charge and is used in drug delivery applications [3], as well as controlled release systems [4][5][6][7].
Zinc layered hydroxide (ZLH) is an example of the layered hydroxide salt in which zinc atoms are octahedrally coordinated with six hydroxide groups to give an empirical formula of Zn 5 (OH) 8 (NO 3 ) 2 ⋅ 2H 2 O [8].
Our previous work showed that hippuric acid intercalated into zinc layered hydroxide potentiated the cytotoxicity of tamoxifen against hepatocellular carcinoma HepG2 [9].e present study was conducted to investigate whether HAN could enhance the toxic effect for cytarabine, another chemotherapy used commonly to treat human promyelocytic leukemia.A parallel to that, antimicrobial activity of HAN nanocomposite against different micro-organisms was also determined.

Materials
Hippuric acid (C 9 H 9 NO 3 ) with 98% purity was purchased from Merck and was used as received.Cytarabine (C 9 H 13 N 3 O 5 ) with 99% purity was purchased from Bioscience (Tocris, Bristol, UK).Zinc oxide of the American chemical society reagent grade was purchased from Fisher Scienti�c, and dimethyl sulfoxide (DMSO) was purchased from Ajax Finechem and used without further puri�cation.Diphenylamine reagent containing 100 mL glacial acetic acid, 1.5 g diphenylamine, 1.5 mL concentrated sulfuric acid, and 0.5 mL 16 mg/mL acetaldehyde stock and trypan blue solution were purchased from Sigma-Aldich.e cell line HL-60 was purchased from the American type culture collection (ATCC number CCL-240).Deionized water was used in all the experiments.e microbial strains were provided by the UNiCC, Institute of Bioscience, Universiti Putra Malaysia.

Preparation of Hippurate Nanocomposite, HAN
Hippurate nanocomposite (HAN) was synthesized by direct reaction using zinc oxide (ZnO) as the starting material as reported previously [9][10][11].e concentrations of hippuric acid of 0.05, 0.2, and 0.4 molar were prepared using appropriate weights of active material in 20 mL of DMSO and adjusting the �nal volume to 50 mL by the addition of deionized water.A weight of 0.2 g of zinc oxide was suspended in 50 mL water.Hippurate solutions were added slowly dropwise to the suspended zinc oxide, with vigorous stirring until the addition was completed and the solution became clear; the pH was adjusted to 7.9 using an aqueous solution of NaOH (0.5 molar).e resulting precipitate was magnetically stirred for 18 hours at 70 ∘ C and washed with deionized water.Finally, the as-synthesized HAN was dried in an oven at 60 ∘ C overnight and kept in a sample bottle for further characterizations.

MTT Cytotoxicity Assay
Human promyelocytic leukemia (HL-60) cells were used in the present study.Cells were routinely cultured at 37 ∘ C a humidi�ed atmosphere with 5% CO 2 in 96-well plates for 24 hours.When cells were grown up to 80%-90% of cellular con�uence, the cells were treated with cytarabine alone and an equivalent cytostatic mixture of either hippuric acid or HAN.Aer 72 hours of incubation in a humidi�ed atmosphere with 5% CO 2 at 37 ∘ C, MTT assay with 3-(4,5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide was carried out with incubation times to determine the number of viable cells.Twenty microliters of MTT was added to all wells, and the cells were incubated for an additional 4 hours.Aer incubation, the upper part of the solution was removed to leave 30 L in all wells, and the formazan precipitate was dissolved in 10% sodium dodecyl sulfate in dimethyl sulfoxide containing 0.6% acetic acid.e microplates were then gently shaken in the dark for 30 minutes, and the absorbance at 570 nm and 630 nm (background) was measured spectrophotometrically using a microtiter plate reader.All experiments were carried out in triplicate.e IC 50 was generated from the dose-response curves for the cell line.MTT assy of HL-60 cells treated with a combination of doxorubicin with hippuric acid and HAN was used as positive control.

Antiproliferative Assay
Human promyelocytic leukemia (HL-60) cells were used; the cells were seeded at a density of 1 × 10 5 viable cells/mL in six-well plates.Aer incubation for 24 hours, the cells were treated with cytarabine (0.5 g/mL) alone and its combination with hippuric acid or HAN.e plates were incubated at 37 ∘ C in the presence of 5% CO 2 , for 24, 48, and 72 hours.Aer incubation, the media were aspirated off, and cells were washed with cold PBS buffer to get rid of dead cells and replaced with 1 mL of 0.05% trypsin-EDTA (2 mg/mL).e plates were incubated at 37 ∘ C for 10-15 minutes, until the majority of the cells were detached.e cells were harvested, and the cell suspension was centrifuged at 1000 rpm for 10 minutes, and the supernatant discarded.Twenty microliters of cell suspension was mixed with 20 L of 0.4% trypan blue solution.Cells were resuspended, and dyeexcluding viable cells were microscopically counted using a hemocytometer.

Examination of DNA Fragmentation
DNA fragmentation was quantitatively determined using diphenylamine reagent for cells treated with 1 g/mL cytarabine alone and in equal combination with either 1 g/mL hippuric acid or 1 g/mL HAN.A volume of 108 L of 5 molar perchloric acid was added, and samples were heated at 70 ∘ C for 15 min.Two volumes of a solution containing diphenylamine reagent were added, and the samples were stored at 4 ∘ C for 48 hours.e colorimetric reaction was determined spectrophotometrically at 575 nm using a UV spectrophotometer (UV-160A; Shimazu Co. Ltd., Tokyo, Japan).DNA aliquots from both pellet and supernatant were quanti�ed.e degree of DNA fragmentation referred to the percentage of DNA in the supernatant divided by the total DNA from the pellet and supernatant was determined.

Microbial Strains and Cultural Conditions
Four well-characterized species of bacteria including two Gram-positive micro-organisms: methicillin-resistant Staphylococcus aureus (MRSA) and Bacillus subtilis and two Gram-negative species: Salmonella choleraesuis and Pseudomonas aeruginosa, and yeast: Candida albicans were used in the screening of the synthesized HAN.Cultures were maintained on Luria-Bertani (LB) agar (1st Base, Singapore) or potato dextrose agar in case of yeast.Prior to incubation with nanoparticles, the bacteria were cultured overnight in 5 mL of LB broth (1st Base, Singapore) in a Certomat SII incubation shaker (Sartorius Stedim Biotech, Aubagne, France) at 37 ∘ C and 150 rpm until the culture reached an OD 600 of 1.0 (Ultrospec UV VIS 3000 pro, Amersham Pharmacia Biotech, Cambridge, England) corresponding to approximately 10 8 CFU mL −1 .e overnight cultures were diluted to approximately 10 7 CFU mL −1 using sterile LB broth.

Antimicrobial Activity Testing
e antimicrobial properties of the HAN nanocomposite were evaluated against the aforementioned micro-organisms using the disk diffusion method as described previously [12].Microorganisms were cultured in LB broth in case of bacteria (1st Base, Singapore) or potato dextrose broth (Fluka) in case of yeast to achieve a culture growth turbidity of 1 by measuring optical density (OD) spectrophotometrically at 600 nm.e suspension of each micro-organism was serially diluted in LB broth to a concentration of 1 ∼ 2 10 4 CFU/mL.e inhibitory activity was read aer 24 hours of incubation at 37 ∘ C. HAN-free discs (distilled water only) cultured under the same conditions were used as a control.

Characterization
Powder X-ray diffraction patterns were recorded with a Shimadzu XRD-6000 instrument (Shimadzu, Tokyo, Japan) using CuK radiation (  1418 Å) and a dwell time of 4 degrees per minute.Cell count was carried using Neubauer hemocytometer (Weber, England) using clear �eld microscopy (Nikon, Japan).e bacteria were cultured overnight in a Certomat SII incubation shaker (Sartorius Stedim Biotech, Aubagne, France).UV spectrophotometer (UV-160A; Shimadzu Co. Ltd., Tokyo, Japan) was used in the determination of DNA fragmentation.(ZLH) as the interlamella anion was 9.74 Å which appeared at 2 = 9.12 due to the 200 plane of the monoclinic structure [14].e PXRD pattern for HAN is shown in Figure 2(c) with basal spacing of 21.3 Å. e expansion of basal spacing from ZLH to HAN was due to the inclusion of hippuric acid anion, namely, hippurate into the ZLH lamella with a vertical monolayer orientation that warrants the expansion of the ZLH lamella [9].is can only be achieved when suitable concentration of hippuric acid is available in the mother liquor, under the experimental conditions as stated earlier.

Results and Discussion
At Figure 1(d) HAN prepared using 0.4 molar hippuric acid, a number of peaks are observed due to hippuric acid phase.is might be due to the non-intercalated hippuric acid, which was adsorbed onto the surface of ZLH.HAN prepared using 0.05 molar hippuric acid shows the presence of ZnO phase.HAN prepared using 0.2 molar hippuric acid produced sharp, symmetrical, and intense peaks, especially for the (003) peak, and the relatively pure intercalated compound was obtained at this concentration.As a result of relatively pure phase, HAN prepared from 0.2 molar hippuric acid was subsequently used for further characterizations.

Cytotoxicity of Combination of HAN with Cytarabine.
Figure 2 shows the cytotoxicity of hippuric acid, HAN nanocomposite, and their combinations with cytarabine against HL-60 cells.e cytotoxicity of cytarabine was investigated using human promyelocytic (HL-60) cells as described previosly [15].e hippuric acid and HAN T 1: Viability of HL-60 cells during incubation with cytarabine, combination of cytarabine with hippuric acid, and cytarabine with HAN nanocomposite.nanocomposite, up to a concentration of 50 g/mL did not show any inhibition in the cell growth of treated HL-60 cells (Figures 2(a) and 2(b)).In the absence of hippuric acid or HAN (Figure 2(c)), the cytarabine suppressed the growth of HL-60 cells with IC 50 values of 0.17 ± 0.09 g/mL.As shown in Figure 2(d) and Table 1, the combination of hippuric acid with cytarabine show inhibition in the cell growth, similarly with cytarabine action and with IC 50 0.17 ± 0.07 g/mL.e combination of cytarabine with HAN (Figure 2(e)) shows slightly higher tumor suppression as compared to cytarabine alone with IC 50 values of 0.16 ± 0.07 g/mL.is indicate that the suppression percentage using HAN reached 4.1%.A detailed look at Table 1, the results of MTT assay show that HAN exposure potentiates the toxic activity of cytarabine in HL-60 cells in a dose-dependent manner, and the lowest two combinations (0.62 g/mL and 0.31 g/mL) show the most notable effect.

Concentration of
Furthermore, compared with cytarabine, doxorubicin (Doxo) showed lower cytotoxicity toward the HL-60 cell line (Figures 3(a)-3(c)).Table 2 shows the IC 50 values for the combination of cytarabine and doxorubicin with hippuric acid and HAN nanocomposite.

Antiproliferative Effect of Cytarabine Alone and Combinations of Cytarabine with Hippuric Acid and HAN.
Figure 4(a) shows the effect of free cytarabine or in combination with hippuric acid and HAN on the proliferation of HL-60 cells at various incubation periods 24, 48, and 72 hours.Cytarabine and its combination with HAN suppressed the proliferation of HL-60 tumor cells.e combination of the drug with HAN was more efficient than the free drug in suppressing the tumor cells.
As shown in Table 3 at 24 hours, the combination of cytarabine with HAN showed 88.20% suppression of the proliferation as compared to the free cytarabine, which showed only 83.50% suppression.Additionally, at 72-hour treatment, the growth suppression for cytarabine with HAN showed 97.40%, compared to 95.50% for free cytarabine.e result in Figure 4(b) con�rmed that the combination of cytarabine with HAN can inhibit the cell proliferation more than doxorubicin with HAN.
In general, the current study showed that HAN did not potentiate the toxicity of cytarabine.However, these results are clearly distinct from our previous �ndings with tamoxifen, in which HAN showed a dramatic inhibition of HepG2 cell proliferation [9].  to HAN concentration.is result provides a rationale for the synergistic property of HAN when combined with cytarabine more than that was observed in MTT results.is indicates that drug delivery to the tumor cell was noticeably enhanced by nanocomposite with ZLH.In the nanocomposite system, however, drugs can reach the tumor cell membrane without any early decomposition, since the drug is stabilized and protected in the interlayer space of the ZLH layers.is result clearly con�rmed that the intercalation reaction not only resulted in avoidance of drug denaturation but also enhanced the permeability of the drug into the target cells without any noticeable side effects [17].

Antimicrobial Activity of Hippuric Acid and Its HAN
Nanocomposite. ere were many previous studies reported on the antimicrobial activity of hippuric acid [18][19][20][21][22]. e effect of HAN against different microorganisms is reported in the present study.e work principle depends on the measurement of the diameter of inhibition zones where the increase in the inhibition zone indicating the highest antimicrobial activity.HAN exhibited antimicrobial activity against the tested organisms by showing the clear inhibition zones using the disk diffusion method.e diameters of the inhibition zone are shown in Table 4. Similar to antibiotic sensitivity testing, the antimicrobial activity of the hippuric acid against different microorganisms was determined using the disk diffusion method, employing the nanoparticles impregnated disks.
In the present study, 100 g/mL suspension of hippuric acid and 25 g/mL suspension of HAN were used to impregnate 6 mm diameter sterilized �lter paper disks.e nanocomposite laden disks were placed on the surface of LB agar plates containting the tested microorganism.e microbial suspension (100 L of 10 4 CFU mL −1 ) was applied uniformly on the surface of the dried LB agar plates in case of bacteria or potato dextrose agar plates in case of yeast before placing the disks on the plate (3 plates per microorganism).e plates were incubated at 37 ∘ C for 24 hours.At the end of the incubation period, the average diameter (mm) of the inhibition zone surrounding the disk was measured using a ruler with up to 1 mm resolution.
e most susceptible organisms to the hippuric acid were Bacillus subtilis and Pseudomonas aeruginosa.In addition, Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus were more sensitive to HAN as compared to other micro-organisms, and this might be attributed to the smaller particle size of HAN as compared to hippuric acid alone.e small particle size of HAN allowed more drug uptake by Gram-negative bacteria.As shown in Table 4, hippuric acid and HAN show similar antimicrobial activity against all the tested organisms, although hippuric acid shows a slightly larger zone of inhibition than HAN.is might be attributed to that HAN is known of slow release of active hippuric acid.
It is worth mentioning that Pseudomonas aeruginosa and MRSA are major pathogens causing problematic infections in human and animals such as septicemia, wound, burns, and deadly infections [23,24].In addition, these two pathogens are known of multidrug resistance to several classes of currently used antibiotics making the treatment process problematic and concerning [25,26].Nanointercalation of 9.5 g hippuric acid (38% of the 25 g HAN) between two zinc-layered hydroxide (ZLH) increases its efficacy and exhibits the approximate reduction of 100 g/mL hippuric acid in bacterial growth aer 24 hours exposure as seen in Pseudomonas aeruginosa.e application of HAN would improve control of infections caused by these pathogens by its slow release of antibacterial activity.Our results were in accordance with the prospective importance of nanoparticles in the treatment of bacterial infections and nanomedicine [27,28].

Conclusion
A new nanocomposite compound in which hippuric acid is intercalated into the interlayer of zinc layered hydroxide was synthesized by direct reaction of zinc oxide aqueous suspension with anions of hippuric acid.e resulting phase pure nanocomposite was synthesized using 0.2 molar hippuric acid and had a basal spacing of 21.3 Å. e combination of cytarabine with HAN showed higher tumor suppression efficiency compared to cytarabine alone with IC 50 values 0.165 ± 0.070 g/mL.e exposure of HL-60 cancer cells to cytarabine alone resulted in 10.70 ± 0.96% DNA fragmentation compared to 18.90 ± 1.33% in the case of combination of cytarabine with HAN.Furthermore, the HAN nanocomposite showed antimicrobial activity against Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus.

Con�ict of �nterests
e authors declare that they have no con�ict of interests in this work.

F 2 :
MTT assays of HL-60 cell line treated with free hippuric acid, HAN and the combination of cytarabine with hippuric acid and HAN.

10. 4 .F 4 :T 4 :
Examination of DNA Fragmentation.DNA fragmentation is an important feature of nucleus that occurs in the apoptotic process.It is worth mentioning that Bouffard and Momparler have studied the effect of cytarabine versus DNA damages in leukemic cell HL60 line.Cytarabine at 5 m Antiproliferative assays of HL-60 cell line aer, 24 48, and 72 hours of treatment with free cytarabine and a combination of free cytarabine with hippuric acid and HAN (a) and positive control using doxorubicin (b).Antimicrobial activities of hippuric acid and HAN nanocomposite against different microorganisms aer 24 hours treatment

F 5 :
DNA fragmentation percentage of HL-60 cell line aer 72 hours by cytarabine and combinations of cytarabine with hippuric acid and HAN.
Antiproliferative assays of HL-60 cells aer 24, 48, and 72 hours treatment with cytarabine alone and combination of cytarabine with hippuric acid and HAN nanocomposite.