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Colloids and Surfaces B: Biointerfaces 79 (2010) 136–141

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

journa l homepage: www.e lsev ier .com/ locate /co lsur fb

wo-dimensional surface properties of an antimicrobial hydantoin at their–water interface: An experimental and theoretical study

bhishek Mandala,b, Ramya Santhana Gopala Krishnana, Sathiah Thennarasua,atya Panigrahib, Asit Baran Mandala,∗

Chemical Laboratory, Physical and Inorganic Chemistry Division, Central Leather Research Institute, Council of Scientific and Industrial Research (CSIR),dyar, Chennai 600020, IndiaDepartment of Biomedical Engineering, University of Saskatchewan, 57 Campus Drive, Saskatoon SK S7N 5A4, Canada

r t i c l e i n f o

rticle history:eceived 17 December 2009eceived in revised form 21 March 2010ccepted 27 March 2010vailable online 3 April 2010

eywords:

a b s t r a c t

5,5-Acetamidomethyl-5-methylimidazolidine-2,4-dione shows antimicrobial activity against bacteria atmillimolar concentrations well above its critical micellar concentration (cmc). Two-dimensional sur-face properties were investigated using Langmuir Film Balance to understand the membrane-activenature and nanomaterial behavior of this hydantoin derivative. Hydantoin forms an expanded nanofilmat air–water interface. The maximum limiting surface area (A0) and collapse pressure (�c) are dependenton hydantoin concentration. Hydantoin undergoes a change in orientation at the interface, in the pressureregion 2.5 and 7.5 mN m−1, corresponding to surface areas 51–15 and 41–12 Å2 molecule−1, respectively.

ntimicrobialydantoinelf-assembling characteristicsligomeric aggregates

A large collapse pressure (�c) in LB film indicates a role for hydrophobic interactions in the self-assemblyof hydantoin. Surface areas computed using Connolly method, are in good agreement with the experi-mental results. Monolayer studies suggest a dispersed state for hydantoin when its concentration is belowcmc, suggesting a mechanism for the observed bacteriostatic activity of hydantoin. In the present study,

first t

it has been found for thevery close to its cmc.

. Introduction

One of the problems associated with various industrial aqueousuid media such as metal working fluids used with metal workingquipment, arises from the susceptibility of the media to infes-ation and growth of microorganisms such as bacteria and fungi.ntimicrobial hydantoin compositions are generally added to such

ndustrial aqueous fluid media to reduce or inhibit the growth oficroorganisms. Efforts are being made in controlling this problem,hich continues to be a major annoyance to the metal work-

ng industry. Antimicrobacterial activity of aromatic derivativesf hydantoin has also been studied [1]. Recently, biocidal activityf hydantoin-containing polyurethane polymeric surface modifiersas been reported [2].

Although several therapeutic and pharmacological effects of

ydantoins have been studied recently [3–6], the self-assemblingature of hydantoin drugs has not been analyzed so far to theest of our knowledge. Knowledge of self-assembling properties ofydantoins will help in understanding their physicochemical and

∗ Corresponding author. Tel.: +91 44 24910846/24910897/24411630;ax: +91 44 24911589/24912150.

E-mail address: abmandal@hotmail.com (A.B. Mandal).

927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2010.03.042

ime that the minimum inhibitory concentration (MIC) of the hydantoin is

© 2010 Elsevier B.V. All rights reserved.

biological properties. Micellar characteristics of collagens, variouspolymers and peptides in aqueous and non-aqueous media havewidely been studied [7–19] in the recent past. Recently, we haveexplored the evidence of micelle formation [20] of 5-(�-acetamido-�-benzyl) methyl-5′-methyl imidazolidine-2,4-dione, a hydantoinderivative (Structure 1) in water medium. The evidence for an asso-ciation of high-mobility with two-dimensional charge transport inconjugated polymers has recently been reported by Sirringhaus etal. [21] in order to explore the supramolecular self-organization.Very recently, we have demonstrated [22,23] the 2D surface prop-erties of �-methoxy poly(ethylene glycol) macromonomer andhomopolymer at the air–water interface. In the present study,we (i) determined the minimal inhibitory concentration (MIC) ofthe hydantoin against Gram-positive and Gram-negative strainsin aqueous medium; (ii) measured surface pressure–area (�–A)isotherm as a function of concentration; and (iii) computed thesurface area of monomeric and oligomeric forms of the hydan-toin. In order to provide more insights on the genesis of theself-assembling characteristics of the hydantoin and its interac-

tion with bio-membranes, the information gathered in the presentstudy is coupled with the results obtained from previous investi-gations on the hydantoin [20,24]. In the present study, it has beenfound for the first time that the minimum inhibitory concentration(MIC) of the hydantoin is very close to its cmc.

A. Mandal et al. / Colloids and Surfaces B

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tructure 1. Molecular structure of 5,5-acetamidomethyl-5-methylimidazolidine-,4-dione, a hydantoin drug.

. Experimental details

.1. Materials and methods

The hydantoin drug was synthesized as described elsewhere25]. HPLC grade solvents such as chloroform and methanol weresed without further purification. Doubly distilled water (specificonductance 2–3 �S cm−1 at 22 ◦C) was used throughout the exper-ment. UV–vis and fluorescence spectral measurements were madet 22 ± 0.05 ◦C on Shimadzu UV-260 spectrometer and Hitachi 650-0 fluorimeter, respectively.

.2. Antibacterial assay

Bacteria were taken from glycerol stock solutions and inoculatednto 5 mL LB culture medium and incubated overnight at 37 ◦C. Theully grown cells were plated onto nutrient agar plates and incu-ated at 37 ◦C overnight or 36 h till single colonies were clearlyeen. Cells from single colonies were further inoculated into LBedium and incubated at 37 ◦C for 3–4 h, and the cell density was

iluted to 0.002 OD600. 1 mL of the diluted cell suspension contain-ng approximately 106 cells/mL, was added to autoclaved test tubesontaining serial concentrations of the hydantoin. The test tubesere incubated at 37 ◦C for 7–9 h. The concentration of hydantoin

n the test tube which showed no growth as measured by the OD600as taken as the MIC. 5 mL of LB medium was added to each of the

est tubes containing MIC and higher concentrations of hydantoin,nd incubated overnight at 37 ◦C in order to study the bacteriostaticr bacteriolytic nature of hydantoin. OD600 values ranging from 1.0o 1.6 were observed from overnight cultures.

.3. Measurement of �–A isotherms

In all the experiments that involved Langmuir film balance (LFB),urface pressure–molecular area (�–A) isotherms were measuredsing double distilled and deionized water obtained from a milli-Qystem (Millipore) as a sub phase. The purity of water was assessedrom the surface tension value (71.5–72 mN m−1 at 25 ◦C). A blankun on water using solvent alone was used to detect the presence ofny surface active impurity in the solvent. The �–A isotherms wereecorded on a single-barrier Langmuir-trough (NIMA Technologytd., UK, Model 611) at a continuous barrier speed of 150 cm2 min−1

sing thin films of hydantoin, which were obtained by spreading00 �L of the stock solutions in chloroform. The �–A isothermsere measured at 22 ◦C using triplicate samples containing 0.5, 0.7,

.9, 1.2, 1.5, 1.75, and 2 mM hydantoin. The LB films were depositedt a surface pressure � = 5 mN m−1 on freshly cleaned quartz platest a dipping speed of 2 mm min−1. The transfer ratios were 0.9–1.0or the upstroke and 0.7–0.8 for the down stroke. The observedransfer ratios are only an indication of homogeneous transfer

: Biointerfaces 79 (2010) 136–141 137

without having any stoichiometric meaning about the amount oftransferred material. After transferring the LB films onto the quartzslides, optical spectra were recorded on a Shimadzu UV-160A spec-trometer, with the LB films on the quartz slides located in thenormal sample position and the uncoated ones located in the ref-erence position. Regarding details of the LB techniques, we refer toour recent publications [22,23,26].

2.4. Computation of surface area

The computational details have been described in earlier pub-lications [27,28]. Briefly, the model of hydantoin molecule wascreated using a molecule builder program of the Cerius2 molecularsimulation package. The geometry of the molecule was subjected toenergy minimization using universal force field employing energyminimizer module of the Cerius2 package. The geometry corre-sponding to the minimum energy was used to calculate Connollysurface area of the molecule. Polar as well as the total Connollysurface areas were computed [27,28].

3. Results and discussion

3.1. Membrane-active mechanism

Two mechanisms have been proposed for antimicrobial activityof membrane-active agents. One involves a membrane mediatedtransient pore formation and the other is based on the interactionof membrane-active agents with the components of bacterial mem-brane, which is largely composed of a lipid bilayer. The hydantoinused in this study exhibited antimicrobial activity against microor-ganisms at relatively higher concentrations. The observed MICswere 650, 650, 700 and 750 �g against Escherichia coli, Staphylococ-cus sp., Pseudomonas sp., and Klebsiella sp., respectively. Althoughthe hydantoin inhibited the growth of the microorganisms at theindicated concentrations, further growth was observed when thesample cultures were supplemented with excess (fivefold) amountof growth medium. These data clearly suggest that the hydantoinacts as a bacteriostatic agent and not as a bactereolytic agent. As theobserved MIC values are closer to the CMC value of the hydantoin(1.5 mM [20]), we measured the surface properties to assess theaggregation states of the hydantoin. The concentration dependantaggregation behavior of hydantoin might provide clues about themembrane-active form (monomer or oligomeric aggregate) of thehydantoin.

3.2. Surface properties of the hydantoin film

Recently, we have determined the critical micellar concentra-tion of the hydantoin derivative in aqueous solution (cmc = 1.5 mMat 22 ◦C). The aggregation number for 4 mM hydantoin was foundto be 8 ± 1 [20]. Fig. 1 shows �–A isotherms recorded at 22 ◦C using0.5, 1.5, 1.75 and 2 mM solutions of hydantoin. This concentrationrange was chosen to study the film forming ability of hydantoinbelow cmc, at cmc and above cmc. The hydantoin forms a fairly sta-ble film at the air–water interface for a reasonable period of time(∼1 h) as observed from the reversible compression and expansionexperiments, which showed a very small hysteresis. The stabilityof the hydantoin film at higher concentration was also determinedby carrying out the monolayer experiment at constant pressure.The plot of surface area vs. time of films maintained at a con-stant pressure of 5 mN m−1 is shown in Fig. 2. The hydantoin films

were slightly contracted in the area by 3–4% with an exposure of500 s. The surface area dropped down slightly up to 250 s and after-ward was constant with time. From a close examination of the �–Aisotherm (Fig. 1, curve number 1), it is inferred that the hydantoinshows a gradual reduction in surface area with the slow increase

138 A. Mandal et al. / Colloids and Surfaces B: Biointerfaces 79 (2010) 136–141

Table 1Characteristics of the hydantoin film adsorbed at the air–water interface at 22 ◦C.

[Hydantoin] (mM) Surface area (A) (Å2 molecule−1) at a given surface pressure Collapse pressure(mN m−1)

Minimum surfacearea (Å2 molecule−1)

2.5 (mN m−1) 5.0 (mN m−1) 7.5 (mN m−1) 10.0 (mN m−1)

0.5 51.4 45.5 40.9 36.4 11.5 57.51.5 21.4 18.2 16.81.75 16.4 13.6 12.72.0 15.0 13.1 12.3

F2th

iiivtccgChifi

Fp

ig. 1. The �–A isotherms of various concentration of hydantoin in chloroform at2 ◦C. Curve nos. 1–4: 0.5, 1.5, 1.75, and 2 mM of hydantoin, respectively. Inset ishe plot of limiting surface area vs. concentration of hydantoin where the correctedydantoin concentrations are shown by open circle.

n pressure on compression. This is in contrast to the typical �–Asotherms of many amphipathic systems, which show a steepncrease in pressure with a small change in area [21]. Our obser-ations suggest that the hydantoin forms smooth expanded film athe air–water interface only at low concentrations well below theritical micelle concentration [24]. However, upon increasing theoncentration, the hydantoin exhibits more compact packing sug-esting an increase in packing density of hydantoin film [23,24,29].

omparing the collapse pressure (�c) at different concentrations ofydantoin, corresponding to the pressure of the deviation in linear-

ty of the �–A isotherms in the upper pressure region as calculatedrom the �–A isotherms (Fig. 1), it is observed that the �c valuencreases (from 11 to 16 mN m−1) with the increase in concentra-

ig. 2. Plot of surface area vs. time of films of hydantoin (3 mM) at a constantressure of 5 mN m−1 at 22 ◦C.

15.0 11.7 25.511.8 11.4 20.511.4 15.8 19.5

tion of the hydantoin (from 0.5 to 2.0 mM) (see Table 1). This isbecause of the rigidity arising from a close packing of the films likesolid condensed state. However, this is in contrast to the insolublemonolayer formed by the bis-amide molecule where the �c valuedecreases [24] (from 25 to 14 mN m−1) with the increase in con-centration (from 0.01 to 0.1 mM). Obviously, the pressure requiredfor the films to get collapsed increases as the stability of the films isincreased. The limiting surface areas were plotted against the con-centration of hydantoin (see inset in Fig. 1). We have for the firsttime demonstrated that the Langmuir film balance technique canbe used for the determination of cmc of the amphiphilic substancesin non-aqueous solvents, viz. determination of cmc of bis-amide inchloroform [24]. However, it was not reported so far for the deter-mination of cmc in aqueous solvent using Langmuir film balancetechnique. Therefore, in the present study, we have employed Lang-muir film balance technique for the first time to determine the cmcof hydantoin with modest hydrophobicity. Now, the question canbe raised as the hydantoin seems to be dissolved in water up to atleast 5 mM, and indicates the surface activity, in such case, the filmat the air/water interface is generally treated as the soluble (Gibbs)monolayer, rather than the insoluble (Langmuir) monolayer.

In Fig. 1, the concentration of hydantoin in chloroform is used.When the chloroform solution spreads on water, hydantoin issomewhat dissolved in water until the equilibrium is reached. It hasbeen found that very small amount of hydantoin viz. 0.05–0.02 mMdissolved in water sub phase from the hydantoin concentrationrange 0.5–2 mM. The dissolved concentrations of the hydantoinin water sub phase were determined by UV–vis spectroscopy (the�max value of hydantoin in water at 22 ◦C is 257 nm) [20]. There-fore, the molecular area obtained from the concentration of stocksolution in chloroform is not the same concentration. Accordingly,the limiting surface area of the hydantoin is plotted against the cor-rected hydantoin concentration, readily available at the air/waterinterface, is shown in Fig. 1 by open circle with dotted line. It isquite interesting to note that the cmc of the hydantoin obtainedfrom the plot of limiting surface area vs. concentration of hydan-toin is same (1.5 mM) for both corrected and uncorrected hydantoinconcentration (see inset of Fig. 1). Hence, these results suggest thatthe amount of hydantoin dissolved in bulk water is very insignif-icant in the present study. However, the hydantoin material(s)present at the air/water interface is more meaningful and accord-ingly, aggregation state and cmc would be reflected. Whether themonolayer formation is soluble or insoluble, it does not matter;the cmc obtained from the plot of limiting surface area vs. con-centration is analogous to surface area vs. concentration plot (boththe cases, the limiting surface area decreased with the increase ofamphiphilic concentration up to cmc) [24].

A clear break was obtained at the cmc (considered to be drymicelle formation at the interface, as suggested by Israelachvili etal. [30]), which is in good agreement with the results obtained from

other techniques: viz., surface tension measurements, UV–vis andfluorescence spectroscopy [20]. The limiting surface area decreaseswith the increase in concentration of hydantoin. The observation ofmicelle formation using monolayer technique is in good agreementwith our recent findings [24].

A. Mandal et al. / Colloids and Surfaces B: Biointerfaces 79 (2010) 136–141 139

FC

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otphTtthhf[cosicotTmts(weatac

ig. 3. Electronic spectra (of 20 deposited layers) of thin films of hydantoin at 22 ◦C.urve nos. 1–3: 0.5, 1.5, and 2 mM of hydantoin, respectively.

The plot of surface tension (�) vs. log[hydantoin] has beenhown in our recent publication [20] and the Gibbs molecular areaas determined using the equation

= −(

12.303 nRT

)(d�

d log C

)T

(1)

rea = (NA� )−1 (2)

here � = surface excess concentration, n = 1 in the present casehere the hydantoin molecules aggregated during the concen-

ration range (above cmc), R = 8.314 J mol−1 K−1, T = temperaturen Kelvin (here 295 K), � = surface tension in Newtons per meter,= concentration in moles per liter, and NA = 6.03 × 1023 (Avo-adro’s number). The Gibbs molecular area of the hydatoin wasound to be 25 ± 5 Å2 which is in good agreement with the limitingurface area obtained for the hydantoin molecule at high concen-ration (above cmc) during monolayer experiments (Langmuir filmalance measurements).

The change in slope of the linear portion of the �–A isothermsf the hydantoin suggests that the hydantoin undergoes a transi-ion causing a change of orientation at the interface in the surfaceressure region between 2.5 and 7.5 mN m−1 corresponding to theydantoin concentrations of 0.5, 1.5, 1.75 and 2.0 mM (see Table 1).he minimum limiting areas (A0) as determined from extrapola-ion of the linear portion at the most compressed state were foundo be 57.5, 25.5, 20.5 and 19.5 Å2 molecule−1 (cf. Table 1) for theydantoin concentrations 0.5, 1.5, 1.75 and 2 mM, respectively. Itas been reported that the area/molecule at the air–water inter-

ace is mainly dependent on the conformation at the interface21,22]. Thus, the smaller size observed for the molecule at higheroncentration (2.0 mM) must have stemmed from the preferredrientation or conformation at the air–water interface. This alsouggests a tendency towards a dehydration process or a reductionn hydration of the hydantoin film at concentrations higher thanmc. Similar observations in the reduction of hydration numberf the aqueous micellar assemblies at higher concentrations andemperatures have been observed from conductivity studies [17].he present monolayer studies also suggest that the hydantoin isore flexible and in highly dispersed state when the concentra-

ion of the hydantoin is sufficiently below the cmc. Three bluehifts and five red shifts were observed in the electronic spectraFig. 3) of thin films obtained from 0.5 mM solution, as comparedith the �max value of the corresponding solution spectra. How-

ver, only red shifts have been observed for the concentrations at

nd above the cmc of the hydantoin. Therefore, at higher concen-rations, hydantoin films behave like a condensed solid state ands stated earlier, packing density of the films increases at higheroncentrations thereby producing red shifts.

Fig. 4. Connolly surface of the hydantoin molecule with regard to (a) polar groups(S1), (b) polar groups (S2) and (c) total surface.

3.3. Solvent-accessible surfaces of the hydantoin

Connolly representation of solvent-accessible areas constitutesa continuous envelope of solvent molecules in contact with thesolute atoms that are accessible to the solvent [27,28]. This rep-resentation has found applications in enzymology, rational drugdesign, the elucidation of molecular diseases, and the recognitionof specific DNA sequences by proteins and drugs, and locating pos-sible antigenic determinants on viruses. A Connolly surface is thevan der Waals surface of the molecule that is accessible to a solventmolecule having a nonzero radius. The hydantoin is considered tobe a single-tail double-head (bipolar) molecule. Structure 1 showsthe polar surface areas S1 and S2 that are accessible to the sol-vent molecules. Connolly surface models of the hydantoin shownin Fig. 4 were used for computing various surface areas, total area

and volume. The results are presented in Table 2.

As the total surface areas of the hydantoin for contact and saddleconformations computed theoretically (cf. Table 2) with regard toboth S1 and S2 are quite larger compared to limiting surface areas

140 A. Mandal et al. / Colloids and Surfaces B: Biointerfaces 79 (2010) 136–141

Table 2The various surface areas, total areas and volume of the hydantoin drug moleculedetermined using Connolly method.

Area/volume Polar surface (S1)(area = 28.44 Å2)

Polar surface (S2)(area = 29.56 Å2)

Total surface

Contact (Å2) 58.089 43.898 115.658Saddle (Å2) 30.379 13.544 111.967Concave (Å2) 3.313 0.511 35.361

oigpeotpatstwau(tcoiwi1faaTtghcreaisF

3

ccimcaathMbma

Total areaa (Å2) 91.781 57.952 262.986Volume (Å3) 68.060 37.682 269.943

a Total area (Å2) = (contact + saddle + concave).

btained from monolayer experiments (cf. Table 1), the possibil-ties of contact and saddle conformations in terms of total polarroups are ruled out. However, contact areas in terms of individualolar groups are meaningful as they are in good agreement with thexperimental results. Also, these results depend upon the nature ofrientations. The results presented in Tables 1 and 2 suggest thathe contact surface area of S1 polar group is predominant over S2olar group when the concentration of the hydantoin is below cmc,nd the situation is reversed when the concentration of the hydan-oin is above cmc. It is interesting to note that when both the polarurface areas (S1 and S2) contributed significantly, their added con-ributions give rise to molecular surface area of ∼58 Å2 molecule−1,hich is in good agreement with the minimum limiting surface

rea of 57.5 Å2 molecule−1 obtained from monolayer experimentssing 0.5 mM hydantoin solution. The total concave surface area35.36 Å2) along with sufficient water molecules may contributeo the required limiting surface area of the hydantoin molecule atoncentrations below cmc. However, this possibility may be ruledut as the solubility of the hydantoin in water is poor. Accord-ng to Structure 1, the hydantoin has modest hydrophobicity and

hen it aggregates in aqueous solution to form micelles, the lim-ting areas obtained at the micellar level (Table 1) are in the range9.5–25.5 Å2 molecule−1, which is lower than the calculated sur-ace areas (see Table 2). Therefore, in order to get such low surfacereas at the micellar level, the hydantoin molecules must be foldedt the interface and some atoms may not be accessible to water.he red shift [26] observed with the thin film of hydantoin athe micellar level (Fig. 3) is in line with this proposition and sug-ests a compact structure for hydantoin assembly. However, theydantoin is more flexible and in highly dispersed state at belowmc. In a previous study involving both steady state and timeesolved fluorescence methods, we have demonstrated that aboutight molecules of hydantoin in aqueous solution aggregate to formmicelle [20]. Our results based on monolayer experiments are

n good agreement with the computational study suggesting thetabilization of an octameric aggregate of hydantoin as shown inig. 5.

.4. Bacteriostatic activity

The octameric aggregate formed at the air–water interfaceould be amphipathic, as it has been observed in several otherases. Under the antibacterial assay conditions used in this study,t is likely that a similar oligomeric aggregate is formed at the

embrane–water interface especially when the hydantoin con-entration (2.3–2.7 mM) is slightly above the cmc (1.5 mM). Suchn aggregate could be more amphipathic and is likely to exertprofound activity at the membrane–water interface leading to

he inhibition of growth of bacteria. When the concentration of

ydantoin is below cmc (i.e., when the test culture containingIC of hydantoin is diluted with LB medium), the equilibrium

etween the monomeric and oligomeric forms shifts towards theonomer state, allowing the growth of bacteria. This hypothesis

grees well with the observed bacteriostatic activity and two-

[

[

Fig. 5. The aggregation of hydantoin molecule determined by computer simulationtechnique.

dimensional surface properties, and is corroborated by Connollysimulation method.

4. Conclusions

Two-dimensional surface properties of the hydantoin, investi-gated by using Langmuir Film Balance and surface areas computedusing Connolly method including simulation, suggest the forma-tion of octameric aggregates at the air–water interface. The cmcobtained by Langmuir film balance technique is in good agreementwith the other techniques and therefore, Langmuir film balancetechnique may be considered as one of the complementary tech-niques for the determination of the cmc. The minimum inhibitoryconcentration (MIC) of the hydantoin against both Gram-positiveand Gram-negative bacteria (2.36–2.72 mM) is very much closeto the observed cmc of the hydantoin (1.5 mM). Considering theamphiphilic nature of the molecule, it appears that preformedoligomeric aggregates and not the monomer, which exert theobserved antimicrobial activity. This conclusion is consistent withthe cooperative binding of antimicrobial agents, which results inthe killing of the bacteria [31–33].

Acknowledgements

We thank Dr. V. Subramanian, Scientist, Chemical Laboratory,CLRI, for technical help in computational analysis and Dr. A.Gnanamani, Scientist, Bacteriology Laboratory, CLRI, for help inantimicrobial assay. The support of the Research Council is grate-fully acknowledged.

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