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Materials Chemistry and Physics 124 (2010) 623–627

Contents lists available at ScienceDirect

Materials Chemistry and Physics

journa l homepage: www.e lsev ier .com/ locate /matchemphys

ccumulation of harmful compounds by the composite of DNA and cyclodextrin:ffect on intramolecular cavity of cyclodextrin

asanori Yamada ∗, Jun Shikano, Yoshimi Haradaepartment of Chemistry, Faculty of Science, Okayama University of Science, Ridaicho, Kita-Ku, Okayama 700-0005, Japan

r t i c l e i n f o

rticle history:eceived 4 April 2010eceived in revised form 24 June 2010ccepted 10 July 2010

eywords:iomaterialolymers

a b s t r a c t

Alpha-cyclodextrin (˛-CD), ˇ-CD, and �-CD are composed of six, seven, and eight d-glucopyranoseresidues, respectively, and have a hydrophobic cavity in their molecules. In this study, the water-solublecyclodextrin-immobilized poly(allylamine) (PCD) with various cavity sizes was synthesized. These ˛-PCD, ˇ-PCD, and �-PCD showed stability constants of 9.9 × 102 M−1, 4.5 × 103 M−1, and 6.2 × 103 M−1 tothe 1,8-ANS molecule, respectively. Additionally, these PCD polymers formed water-insoluble compositematerials by mixing with the double-stranded DNA, which can accumulate planar structure-containingharmful compounds, such as dioxin and polychlorobiphenyl (PCB). Therefore, we demonstrated the

omposite materialrganic compounds

accumulation of various harmful compounds by the DNA–PCD composite materials having various cav-ity sizes. As a result, although the DNA–ˇ-PCD composite material could encapsulate the non-planarstructure-containing harmful compounds, such as bisphenol A and diethylstilbestrol, the DNA–˛-PCDcomposite material did not interact with these molecules. These phenomena were due to the effect onthe intramolecular cavity of the CD in the polymer. In contrast, all of the DNA–PCDs could accumulate

ainin

the planar structure-contin the CD.

. Introduction

ˇ-Cyclodextrin (ˇ-CD), a cyclic oligosaccharide composed ofeven d-glucopyranose residues, has an intramolecular cavityf 0.78 nm and can encapsulate various non-planar structure-ontaining harmful compounds, such as bisphenol A andiethylstilbestrol, into its cavity [1–3]. Therefore, ˇ-CD and itserivatives have been used as absorbents of harmful compoundsnd as a sensor material for monitoring bisphenol A [4–6]. In con-rast, ˛-cyclodextrin (˛-CD) and �-cyclodextrin (�-CD) comprisedf six and eightd-glucopyranose residues have intramolecular cavi-ies of 0.57 nm and 0.95 nm, respectively [1–3]. Therefore, the ˛-, ˇ-nd �-CDs can encapsulate different organic molecules in their cav-ties. However, the relations between the cavity size of the CD andhe accumulation of the non-planar structure-containing harmfulompounds, such as bisphenol A and diethylstilbestrol, have notet been reported to the best of our knowledge.

Double-stranded DNA, one of the most important biopolymers

n living systems, is a known functional material [7–10]. Addition-lly, the double-stranded DNA has the property for the selectiveccumulation of harmful compounds with planar structures, suchs dioxin-derivatives, polychlorobiphenyl (PCB)-derivatives, and

∗ Corresponding author. Tel.: +81 86 256 9550; fax: +81 86 256 9757.E-mail address: myamada@chem.ous.ac.jp (M. Yamada).

254-0584/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.matchemphys.2010.07.024

g compounds by an intercalation mechanism regardless of the cavity size

© 2010 Elsevier B.V. All rights reserved.

benzo[a]pyrene [11–13]. In this case, although the DNA materi-als could accumulate planar structure-containing compounds byintercalation, they cannot accumulate the non-planar structure-containing compounds, such as bisphenol A and diethylstilbestrol.Therefore, we recently reported the DNA–ˇ-CD composite mate-rial by mixing the double-stranded DNA and ˇ-CD-immobilizedpolymer [14]. This composite material had properties of both thedouble-stranded DNA, such as intercalation, and the ˇ-CD, such asencapsulation of an organic molecule into the intramolecular cav-ity. Therefore, these materials could accumulate not only harmfulcompounds with a planar structure, but also non-planar molecules,such as bisphenol A and diethylstilbestrol. However, these studieshave been demonstrated using only the ˇ-CD-immobilized poly-mer and could not show the relation between the CD cavity sizeand the harmful compounds.

In this study, we synthesized the water-soluble CD-immobilizedpolymer with various intramolecular cavity sizes and preparedthe water-insoluble DNA–CD composite material by mixing withthe double-stranded DNA. As a result, not only the DNA–ˇ-CDcomposite material, but also the DNA–�-CD composite materialaccumulated the non-planar structure-containing harmful com-

pounds, such as diethylstilbestrol. However, the affinity of harmfulcompounds for the DNA–�-CD material was lower than that ofthe DNA–ˇ-CD material. Surprisingly, the DNA–˛-CD compositematerial with the smallest intramolecular cavity could not accu-mulate these harmful compounds. These results suggested that the

624 M. Yamada et al. / Materials Chemistry and Physics 124 (2010) 623–627

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Scheme 1. Molecular structures of cyclodextr

NA–ˇ-CD composite was an effective material for the accumula-ion of harmful compounds from an aqueous solution.

. Experimental

.1. Materials

Double-stranded DNA (sodium salt from salmon milt, molecular weight;5 × 106) and poly(allylamine hydrochloride) (PAA·HCl, molecular weight; 1 × 105)ere obtained from Yuki Fine Chemical Co., Ltd., Tokyo, Japan, and Nittooseki Co., Ltd., Tokyo, Japan, respectively. ˛-CD, ˇ-CD, and �-CD were pur-hased from Hayashibara Biochemical Labs., Inc., Okayama, Japan, Wako Purehemical Industries, Osaka, Japan, and Kanto Chemical Co., Inc., Tokyo, Japan,espectively. The molecular structures of CD are shown in Scheme 1. The-toluenesulfonyl chloride, 2,4,6-triisopropylbenzensulfonyl chloride, ethidiumromide, dibenzo-p-dioxin, dibenzofuran, biphenyl, benzene, bisphenol A, diethyl-tilbestrol, 1-anilinonaphthalene-8-sulfonate (1,8-ANS) were obtained from Wakoure Chemical Industries, Acros Organics, Morris Plains, NJ, or Tokyo Kasei Industriestd., Tokyo, Japan.

.2. Synthesis of CD-immobilized poly(allylamine)

The monotosylations of ˛-CD (˛-CDOTs) and ˇ-CD (ˇ-CDOTs) at the 6-positionere carried out by a reported procedure [15–17]. Additionally, mono-6-(2,4,6-

riisopropylbenzenesulfonyl)-�-CD (�-CDOTri) was synthesized by the reportedethods [18].

The ˛-CD-, ˇ-CD-, and �-CD-immobilized poly(allylamine) (˛-PCD, ˇ-PCD, and-PCD, respectively) were synthesized by the partially modified procedure [19,20].he molecular structure of PCD is shown in Scheme 1. To a solution of potassiumydroxide (2.4 g, 36 mmol) in methanol (135 ml), poly(allylamine hydrochloride)3.45 g, 36 mmol) was added in small portions, the solution was stirred for 1 h andhen left at 5 ◦C for 12 h. The potassium chloride was filtered off and the filtrateas evaporated to dryness. The resulting free poly(allylamine) (1 g, 17.5 mmol) wasissolved in 45 ml of water, and the ˛-CDOTs (0.99 g, 0.88 mmol), ˇ-CDOTs (1.1 g,.88 mmol), or �-CDOTri (8.2 g, 5.3 mmol) were added while stirring at 75 ◦C. Thisolution was stirred for 12–72 h at 75 ◦C or 95 ◦C. Finally, the reaction mixture was

ltered and the filtrate was transferred to a dialysis membrane (Wako Pure Chemical

ndustries) and then dialyzed against water for 6 days. After freeze-drying, a whiteowder was obtained.

The identification of the synthetic polymer ˛-PCD was based on 1H NMR spec-rum using a LNM-LA300 (JEOL Ltd., Tokyo, Japan). 1H NMR (300 MHz, D2O): ı 1.202H, CH2-a); 1.57 (1H, CH-b); 2.38 (3H, CH3-TsO); 2.62 (2H, CH2-c); 3.58–3.87 (36H,

cyclodextrin-immobilized poly(allylamine).

2-H, 3-H, 4-H, 5-H, and 6a,6b-H); 4.96 (6H, 1-H); 7.33 and 7.63 (4H, ar H). The inte-gral ratio data in the 1H NMR spectrum showed a 1.5% degree of ˛-CD substitutionin ˛-PCD.

The identification of the synthetic polymer ˇ-PCD was based on 1H NMR. 1HNMR (300 MHz, D2O): ı 1.21 (2H, CH2-a); 1.56 (1H, CH-b); 2.39 (3H, CH3-TsO); 2.62(2H, CH2-c); 3.63–3.85 (42H, 2-H, 3-H, 4-H, 5-H, and 6a,6b-H); 5.02 (7H, 1-H); 7.33and 7.67 (4H, ar H). The integral ratio data in the 1H NMR spectrum showed a 1.3%degree of ˇ-CD substitution in ˇ-PCD.

The identification of the synthetic polymer �-PCD was based on 1H NMR. 1HNMR (300 MHz, D2O): ı 1.40 (2H, CH2-a); 1.86 (1H, CH-b); 2.90 (2H, CH2-c); 3.61–3.91(48H, 2-H, 3-H, 4-H, 5-H, and 6a,6b-H); 5.32 (8H, 1-H); 8.07 (2H, ar H). The integralratio data in the 1H NMR spectrum showed a 1.9% degree of �-CD substitution in�-PCD.

2.3. Fluorescence spectra of 1,8-ANS with PCD

1,8-ANS was dissolved in 20 mM Tris–HCl (pH 7.4, containing 100 mM NaCl).The concentration of 1,8-ANS was 1 �M. This solution of 2 ml was added in quartzcuvette and mixed with the ˛-, ˇ-, or �-PCD solution (0–200 �l, 1 mM PCD (concen-tration of [CD] in ˛-, ˇ-, or �-PCD) in 20 mM Tris–HCl (pH 7.4, containing 100 mMNaCl)). Fluorometric measurements were carried out using a F-2500 fluorescencespectrophotometer (Hitachi Co., Ltd., Tokyo, Japan) at 25 ◦C. The fluorescence spectrawere measured at the excitation wavelength of 350 nm [16].

2.4. Preparation of the DNA–PCD composite material

The DNA–PCD composite materials were prepared by mixing of aqueous DNA(200 �l, 10 mg ml−1) and PCD (50 �l, 50 mg/ml−1) solutions on a glass plate (Mat-sunami Glass Ind., Ltd., Osaka, Japan). These DNA–PCD composite materials wererinsed with pure water (5 ml × 5 times) to remove the DNA and PCD, which had notgelled, and then stored in pure water for more than 2 days. On the other hand, theUV-irradiated double-stranded DNA films were prepared by a reported procedure[11].

2.5. IR spectra of the DNA–PCD composite material

DNA, DNA–PCD composite, and PCD materials were dried at room temperatureovernight. The infrared (IR) absorption spectra of these materials were measuredby the attenuated total reflection (ATR) method using a FT-IR 8400 Fourier trans-form infrared spectrometer (Shimadzu Corp., Kyoto, Japan). The IR spectrum wasmeasured with a resolution of 4 cm−1.

M. Yamada et al. / Materials Chemistry

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ig. 1. Fluorescence spectra of 1,8-ANS at various ˇ-PCD concentrations at 25 ◦C.1,8-ANS] = 1.0 �M; [ˇ-CD in ˇ-PCD] = 0–100 �M; �ex = 250 nm; *H2O Raman.

.6. Accumulation of harmful compounds by the DNA–PCD composite material

Dibenzo-p-dioxin, dibenzofuran, biphenyl, benzene, bisphenol A, and diethyl-tilbestrol were used as the model harmful compounds, such as endocrineisruptors. The aqueous harmful compound solutions were prepared by previ-usly reported methods [12]. The concentrations of the aqueous dibenzo-p-dioxin,ibenzofuran, biphenyl, benzene, bisphenol A, and diethylstilbestrol solutions were.1, 1.4, 1.3, 13, 4.2, and 1.3 �M, respectively. The DNA–PCD composite materialas incubated in each of the aqueous harmful compound solutions (3 ml) for 24 h

t room temperature. The composite materials were separated from the aque-us solutions. The amounts of accumulated compounds were determined fromhe absorption spectra of the aqueous solutions in the absence or presence of theNA–PCD composite material. The absorption spectra were recorded by a U-2010V-Vis spectrophotometer (Hitachi Co., Ltd.).

. Results and discussion

.1. Encapsulation property of ˛-, ˇ-, and �-PCD polymer

The 1,8-ANS molecule, one of the famous fluorescence reagents,s encapsulated into the CD’s cavity and can indicate fluorescence16]. Fig. 1 shows the fluorescence spectra of 1,8-ANS in an aque-us ˇ-PCD solution. The fluorescence intensity of 1,8-ANS increasedith the amount of ˇ-PCD and a slight blue shift in �max was

bserved. The blue shift and the increase in the fluorescence inten-ity by the encapsulation of organic molecules into the CD’s cavityave been reported [16]. Additionally, 1,8-ANS in an aqueous PAAolution, which does not immobilize the CD, did not show the bluehift and increased fluorescence intensity. Therefore, these results

uggested that the ˇ-PCD polymer can encapsulate the 1,8-ANSnto its cavity. Similar results, such as the increased fluorescence,

ere also obtained in the aqueous ˛-PCD and �-PCD solutions (dataot shown). These results suggested that CD in the PCD polymeras maintained the encapsulation property of organic molecules,

ig. 2. The plot of �I−1 vs. [CD in PCD]. (a) ˇ-PCD polymer; (b) ˛-PCD polymer; (c) �-PChe solid line is the results of the least-squares fit.

and Physics 124 (2010) 623–627 625

such as 1,8-ANS, into its intramolecular cavity. If the observed flu-orescence change (�I) is proportional to the concentration of theformed inclusion complex, then the relationship shown in Eq. (1)is derived under the condition of [CD in PCD]total � [1,8-ANS]total,

�I−1 = K−1

�I∞1:1[CD in PCD]+ 1

�I∞1:1(1)

where [CD in PCD]total, K, and �I∞1:1 are the total concentra-tion of CD in PCD, the stability constant of the 1:1 encapsulatedcomplex, and the fluorescence intensity at infinity [CD in PCD],respectively, when all the 1,8-ANS is bound to the CD in the PCDpolymer. A similar equation has been reported [16]. The plot of�I−1 vs. [ˇ-CD in ˇ-PCD] is a linear function (R2 = 0.999) as shownin Fig. 2(a). The value of Kˇ-CD was estimated from the slope tobe 4.5 × 103 M−1. Similarly, the values of K˛-CD (9.9 × 102 M−1) andK�-CD (6.2 × 103 M−1) were estimated from the slopes in Fig. 2(b)and (c), respectively. The stability constant of 1,8-ANS in an aqueous˛-PCD solution was lower than that of the ˇ- and �-PCDs. Generally,the cavity size relation in the cyclodextrin is ˛-CD < ˇ-CD < �-CD,and ˛-CD cannot stably encapsulate large organic molecules into itsintramolecular cavity. Therefore, the ˛-PCD polymer, which immo-bilized the ˛-CD, showed the lower stability constant for 1,8-ANS.

3.2. Preparation of the DNA–PCD composite material

The DNA–PCD composite materials were prepared by the mix-ing of the aqueous DNA and PCD solutions on a glass plate. Thecomposite material could form at all the PCD polymers, such as the˛-, ˇ-, and �-PCD polymers. These composite materials were elasticmaterials. The elasticity of the composite material decreased withthe decrease in the concentration, and the composite material wasnot formed at the low concentration (<5 mg ml−1). The DNA–PCDcomposite materials were stored in ultrapure water for more than2 days to remove the small amount of the remaining water-solubleDNA and PCD polymer and then used for further experiments.

Next, we examined the properties of the double-strandedDNA in the DNA–PCD composite material. Generally, the double-stranded DNA interacts with ethidium bromide, one of the famousintercalating reagents of the double-stranded DNA, and indicates ared fluorescence during UV irradiation (254 nm) [9–11]. When thiscomposite material was incubated in an aqueous ethidium bro-mide solution, the white material was dyed red (data not shown).Additionally, this composite material with the ethidium bromideshowed a red fluorescence during UV irradiation (254 nm). Thesephenomena were obtained for all of the composite materials,

such as the DNA–˛-PCD, DNA–ˇ-PCD, and DNA–�-PCD compos-ite materials. These results suggested that the DNA structure inthe composite material has maintained a double-stranded struc-ture and the intercalative function of planar structure-containingcompounds.

D polymer. The �I−1 values were determined by the fluorescence measurements.

626 M. Yamada et al. / Materials Chemistry and Physics 124 (2010) 623–627

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ig. 3. IR spectra of DNA–PCD composite materials. (a) DNA–˛-PCD composite; (b)NA–ˇ-PCD composite; (c) DNA–�-PCD composite; (d) pure DNA materials. The IR

pectrum was measured at the resolution of 4 cm−1.

.3. Molecular structure of the DNA–PCD composite material

The molecular structure of the DNA–PCD composite materialas confirmed by IR spectrometry. Fig. 3 shows the IR spectra of (a)

he DNA–˛-PCD composite, (b) the DNA–ˇ-PCD composite, (c) theNA–�-PCD composite, and (d) the DNA materials. The absorptionand at 1240 cm−1 in the pure DNA material, related to the antisym-etric vibration of the phosphate group [13,14,21,22], was shifted

o a lower wavenumber, 5–15 cm−1, by the composite with PCDsee the dashed line in Fig. 3). This shift in the phosphate group isue to the electrostatic interaction between the phosphate groupf DNA and the positively charged amino group of the PCD poly-er, and similar phenomena have been reported for other polyion

omplexes [13,14]. These results suggest that the amino group ofCD in the composite material binds to the phosphate group of DNAy an electrostatic interaction and forms the acid–base complex. Inontrast, the absorption band at 1300–1600 cm−1, related to thetretching vibration of the nucleic acid base [21], did not change.his result suggests that the double-strand conformation of DNA inhe DNA–PCD composite material has been maintained.

.4. Accumulation of harmful compounds by the DNA–PCDomposite material

Model endocrine disruptors and harmful compounds, such asisphenol A, diethylstilbestrol, benzene, dibenzo-p-dioxin, diben-ofuran, and biphenyl, were dissolved in water. The DNA–PCDomposite material was incubated for 24 h in an aqueous solu-ion of each harmful compound, and then the amounts of theompounds were determined by measuring the absorption spectraf the solutions. Previously, we reported the selective accumula-ion of the planar structure-containing harmful compounds, suchs dioxin- and PCD-derivatives, by an intercalative mechanismnto the double-stranded DNA [12,13]. In this case, pure DNA

aterial cannot accumulate the non-planar structure-containingompounds, such as diethylstilbestrol [12,13]. Fig. 4(a) shows the

bsorption spectra of diethylstilbestrol in the absence (solid line)nd presence (dashed line) of the DNA–ˇ-PCD composite mate-ial. When the DNA–ˇ-PCD composite material was added to anqueous diethylstilbestrol solution, the absorbance of the solutionecreased; that is, ca. 35% of diethylstilbestrol was accumulated

Fig. 4. Absorption spectra of diethylstilbestrol in the absence (solid line) and pres-ence (dashed line) of the DNA–PCD composite material. (a) DNA–ˇ-PCD composite;(b) DNA–˛-PCD composite; (c) DNA–�-PCD composite materials.

by the DNA–ˇ-PCD composite material. Therefore, we demon-strated the accumulation of diethylstilbestrol by the DNA–˛-PCDand DNA–�-PCD composite materials. Fig. 4(b) and (c) showsthe absorption spectra of diethylstilbestrol by the DNA–˛-PCDand DNA–�-PCD composite materials, respectively. Surprisingly,when the DNA–˛-PCD composite material was added to the aque-ous diethylstilbestrol solution, no decrease in absorbance wasobserved. In contrast, although a decrease in absorbance wasobserved by the addition of the DNA–�-PCD composite material,the accumulated amount of diethylstilbestrol was lower than thatof the DNA–ˇ-PCD composite material. These results suggestedthat the accumulated amount of diethylstilbestrol was relatedto the cavity size of the cyclodextrin, and the diethylstilbestrolwas effectively accumulated by the DNA–ˇ-PCD composite mate-rial.

Fig. 5 shows the molecular structures and the accumulatedamounts of various harmful compounds by the DNA–PCD com-

posite material. The non-planar structure-containing compound,such as bisphenol A, was accumulated by the DNA–ˇ-PCD compos-ite material. The DNA–˛-PCD and DNA–�-PCD composite materialscould not accumulate the bisphenol A. The differences in the accu-mulated amounts are due to the effect of the cavity size in the CD.

M. Yamada et al. / Materials Chemistry

Fig. 5. Accumulated amounts of various harmful compounds by DNA–˛-PCD com-pmtv

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osite (open bar), DNA–ˇ-PCD composite (closed bar), and DNA–�-PCD compositeaterials (diagonal bar). The accumulated amount was determined by the absorp-

ion spectra in the absence and presence of the DNA–PCD composite material. Eachalue represents the mean of three separation determinations.

imilar results were also obtained during the fluorescence mea-urements of bisphenol A and the PCD polymers with various cavityizes (data not shown). In contrast, the DNA–PCD composite mate-ial could accumulate the planar structure-containing compounds,uch as dioxin- and PCD-derivatives. The accumulated amounts ofhese compounds with the planar structure were higher than thatf the UV-irradiated DNA film, which consisted of pure DNA [12].herefore, these accumulations were due not only to the interca-ation of harmful compounds into the double-stranded DNA, butlso their encapsulation into the hydrophobic cavity of the CD. Inact, these phenomena, such as the encapsulation of biphenyl oribenzo-p-dioxin into the hydrophobic cavity of the CD, have beeneported [23,24]. These results suggested that the DNA–PCD com-osite material can accumulate the model endocrine disruptors andarmful compounds. Especially, the DNA–ˇ-PCD composite mate-

ial with the immobilization of ˇ-CD strongly interacted with thearmful compounds, such as dibenzo-p-dioxin and biphenyl. Addi-ionally, since the ˛-PCD has the smallest cavity in polymer, theNA–˛-PCD composite material did not show an effective accu-ulation of the harmful compounds.

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and Physics 124 (2010) 623–627 627

4. Conclusion

We synthesized the cyclodextrin-immobilized poly(allylamine)(PCD) with the various cavity sizes. The ˛-PCD, ˇ-PCD, and �-PCD had stability constants of 9.9 × 102 M−1, 4.5 × 103 M−1, and6.2 × 103 M−1 to the 1,8-ANS molecule, respectively. These PCDpolymers formed a composite material by mixing in an aque-ous DNA solution. Therefore, we demonstrated the accumulationof harmful compounds using the DNA–PCD composite materialwith various cavity sizes. As a result, the DNA–PCD compositematerial can accumulate not only non-planar structure-containingcompounds, such as bisphenol A and diethylstilbestrol, but alsoplanar structure-containing compounds, such as dioxin- andPCB-derivatives. Especially, the accumulated amount of dibenzo-p-dioxin in the DNA–ˇ-PCD composite material was greater than75%. In contrast, the DNA–˛-PCD composite material, which hasthe smallest intramolecular cavity, did not show any accumulationof bisphenol A and diethylstilbestrol.

Acknowledgements

This work was supported by matching fund subsidy for privateuniversities from MEXT (Ministry of Education, Culture, Sports, Sci-ence and Technology of Japan). Additionally, the part of this workwas supported by Wesco Scientific Promotion Foundation.

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