Synthesis of the Dendritic Type β-Cyclodextrin on Primary Face via Click Reaction Applicable as Drug Nanocarrier

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Carbohydrate Polymers 132 (2015) 205–213

Contents lists available at ScienceDirect

Carbohydrate Polymers

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

ynthesis of the dendritic type �-cyclodextrin on primary face vialick reaction applicable as drug nanocarrier

oomari Yousefa, Namazi Hassana,b,∗, Entezami Ali Akbara

Laboratory of Dendrimers and Nano-Biopolymers, Faculty of Chemistry, University of Tabriz, Tabriz, IranResearch Center for Pharmaceutical Nanotechnology (RCPN), Tabriz University of Medical Science, Tabriz, Iran

r t i c l e i n f o

rticle history:eceived 10 January 2015eceived in revised form 29 April 2015ccepted 15 May 2015vailable online 23 June 2015

a b s t r a c t

The objective of this study was the syntheses of well-defined glycodendrimer with entrapment efficiencyby click reactions, with �-cyclodextrins (�-CDs) moiety to keep the biocompatibility properties, besidesespecially increase their capacity to load numerous appropriate sized guests. The original dendrimercontaining �-CD in both periphery and central was synthesized using click reaction. The entrapmentproperty of the �-CD-dendrimer was studied by methotrexate (MTX) drug. The chemical structure of

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eywords:-Cyclodextrinendrimeranocarrierethotrexate

nclusion complex

�-CD-dendrimer was characterized by H NMR, C NMR and FTIR and its inclusion complex structurewere investigated by SEM, DLS, DSC and FTIR techniques. The cytotoxic effect of obtained compound andits inclusion complex with MTX was analyzed using MTT test. The MTT test exhibited that the synthesizedcompound was not cytotoxic to the cell line considered. The in vitro drug release study turned out thatthe obtained �-CD dendrimer could be a suitable controlled drug delivery system for cancer treatment.

© 2015 Elsevier Ltd. All rights reserved.

. Introduction

Dendrimers are a class of polymeric materials having three-imensional hyperbranched macromolecules, monodispersed,efined spherical construction, nanoscopic objects with amountf reactive periphery groups and host-guest entrapment prop-rties (Shen, Li, Wu, Zhang, & Li, 2015; Tomalia et al., 1985).n this work we emphasis on dendrimers, these materials firstlyeported by Vögtle and co-workers as cascade molecules (Buhleier,

ehner, & Vögtle, 1978) and followed by Tomalia et al. (1985)amed starburst dendrimers, and Newkome, Yao, Baker, and Gupta1985) as ‘arborols’. Since then, the study of these materials, haseen expands exponentially to all areas (Hawker & Fréchet, 1990;amazi & Adeli, 2003; Namazi & Adeli, 2005a, 2005b). Becausef unique structures and properties, dendrimers have concernedreatly attention for their uses in several fields. Among them thesage of these compounds as drug delivery systems (DDS) has beenf excessive attention (Lai et al., 2007; Leng et al., 2013; Namazi &

oomari, 2011; Sun, Fan, Wang, & Zhao, 2012; Tomalia & Fréchet,002).

∗ Corresponding author at: Laboratory of Dendrimers and Nano-Biopolymers,aculty of Chemistry, University of Tabriz, Tabriz, Iran. Tel.: +98 41 339 3121;ax: +98 41 334 0191.

E-mail address: [email protected] (H. Namazi).

ttp://dx.doi.org/10.1016/j.carbpol.2015.05.087144-8617/© 2015 Elsevier Ltd. All rights reserved.

�-CD essentially biocompatible compound (Ortiz Mellet,Defaye, & Garcia Fernandez, 2002), and is one of the good candi-dates for the preparation of star polymers (Ritter & Tabatabai, 2002;Wen, Zhang, & Li, 2014; Zhang, Liu, & Li, 2013; Zhao, Yin, Zhang, &Li, 2013). Star polymers by using the core-first method have beenprepared (Hawker, Bosman, & Harth, 2001). The �-CD moleculeis a torus-shaped oligosaccharide that consist of 7 glucose unitsconnected through �-1,4-glucosidic links (Namazi & Kanani, 2007;Namazi & Kanani, 2009; Saenger et al., 1998). The narrower andwider ends of the CD, termed the primary and secondary face with7 and 14 hydroxyl groups, respectively. The cavity of �-CD is some-what hydrophobic and the external part of the CDs is hydrophilic.Because of these structures, �-CDs are known to form inclusioncomplexes with hydrophobic molecules having the suitable dimen-sion and form to promote their solubility (Namazi & Toomari, 2011;Uekama, Hirayama, & Irie, 1998; Zhang, Liu, & Li, 2011). Therefore,inclusion complexes of CDs with hydrophobic compounds are ofsignificance (Varghese, Al-Busafi, Suliman, & Al-Kindy, 2015).

‘Glycodendrimer’ term is applied to define dendrimers thatinclude carbohydrates in their constructions (Chabre, Contino-Pepin, Placide, Shiao, & Roy, 2008; Chabre & Roy, 2010).Glycodendrimers having CD moiety in their assemblies are termedCD-dendrimers. Depending on the core and branches groups, CD

dendrimers can be separated into three chief categories. The firstcategories (CD-based dendrimers), CDs are located on the peripheryof dendrimers (Adeli, Kalantari, Zarnega, & Kabiri, 2012; Menuel,Duval, Cuc, Mutzenhardt, & Marsura, 2007; Menuel et al., 2008). In

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he second categories (CD-centered dendrimers), a CD part as theentral wherever all outlets (in primary face) are linked (Newkome,odinez, & Moorefield, 1998). The third categories (CD-dendrimeronjugates), dendrimers conjugated with CD moiety (Baussannet al., 2000; Benito et al., 2004; Wang, Shao, Qiao, & Cheng, 2012).y the combination of CDs with dendrimer, the positive propertiesf dendrimer and CDs improved (Menuel et al., 2008).

In this work, we report the preparation of a family of glyco-endrimers having on �-CD scaffolds on the primary face in theirentral and periphery by using spacer arms, which was obtained bylick reaction (Scheme 1). Also, the encapsulation and drug deliveryroperty of obtained CD-dendrimers in buffer solutions using MTXs the guest molecule was studied.

. Materials and methods

.1. Materials

�-CD (98%, Merck) was achieved and used after desiccatingnder vacuum. p-Toluenesulfonyl chloride (p-TsCl, Merck) wasurified by chloroform and petroleum ether. Propargyl alcohol99%), methotrexate (MTX, 99%), MTT (3-[4,5-dimethylthiazol--yl]-2,5-diphenyl tetrazolium bromide), copper sulfate (CuSO4;9.99%), iodine (99.8%), triphenylphosphine (PPh3; 99%), dimethylulfoxide (DMSO; 99.9%) and acetonitrile (99.8%) were purchasedrom Sigma Aldrich and used as received. N,N-DimethylformamideDMF; 99.8%, Merck) was dried over CaH2 and distilled underacuum. Triethylamine (Et3N; Merck) was dried over CaH2 andhen distilled. Acetone (99.8%), methanol (99.8%), diethyl ether99.5%), sodium azide (NaN3, 99%) and l-ascorbic acid sodium salt99%) were obtained from Merck and used as received. Dialysisubing (benzoylated, molecular cut-off 2000 Da) was purchasedrom Sigma-Aldrich. T47D, a human breast cancer cells werechieved from Pasteur Institute Cell Bank of Iran (Tehran, Iran)nd cultured in RPMI-1640 moderate complemented with 10%etal bovine serum and 1% penicillin/streptomycin solution at7 ◦C in a wetted incubator with 5% CO2. All the solvents wereurified before use. Deionized water was used through the exper-

ments.

.2. Synthesis of heptakis (6-deoxy-6-iodo)-ˇ-cyclodextrinˇ-CD-(I)7) (2)

To a solution of Ph3P (2.07 g, 7.9 mmol) under stirring in dryMF (9 mL), I2 (2.09 g, 8.26 mmol) was added over 30 min. After theddition of I2 the solution temperature increased from room tem-erature (rt) to 50 ◦C. To this dark brown solution vacuum-dried-CD (1) (0.6 g, 0.53 mmol) was then added, and the reaction mix-

ure was stirred at 70 ◦C under argon atmosphere for 18 h. It washen concentrated through the elimination of DMF (approximatelymL) under vacuum and the pH adjusted to 9–10 via the additionf NaOMe in MeOH (3 M, 3 mL) under an argon atmosphere withffective cooling, for 30 min. The precipitate was formed by theddition of reaction mixture into MeOH (40 mL), which was washedith excess MeOH. �-CD-(I)7 (0.75 g, 89%) was obtained as a whiteowder after drying under high vacuum (Ashton, Königer, Stoddart,lker, & Harding, 1996; Benkhaled et al., 2008). M.p.: 222 ◦C (dec.).H NMR (400 MHz, DMSO-d6; ı, ppm): 6.05 (OH-2), 5.99 (OH-3),.98 (H-1), 3.81 (H-6b), 3.69–3.56 (H-3, H-5), 3.45–3.21 (H-2, H-4,-6a).

.3. Synthesis of heptakis (6-deoxy-6-azido)-ˇ-cyclodextrin

ˇ-CD-(N3)7) (3)

To a solution of �-CD-(I)7 (2) (0.75 g, 0.39 mmol) in anhy-rous DMF (12 mL) at room temperature were added NaN3 (0.25 g,

lymers 132 (2015) 205–213

3.86 mmol). The reaction mixture was stirred at 80 ◦C under argonatmosphere for 24 h. The suspension was concentrated under vac-uum to 1/3 of the starting volume before a large additional of waterwas added. After filtration, the residue was washed with waterand dried under high vacuum. �-CD-(N3)7 (3) (0.48 g, 94%) wasobtained as a white powder (Ashton et al., 1996). 1H NMR (400 MHz,DMSO-d6; ı, ppm): 5.93 (OH-2), 5.78 (OH-3), 4.9 (H-1), 3.79–3.7 (H-6b), 3.62–3.5 (H-3, H-5), 3.35–3.32 (H-2, H-4, H-6a). FTIR (KBr, thinfilm; cm−1): 3360 (str. OH), 2924 and 2856 (str. CH), 2108 (str. N3),1155–1049 (str. C–O).

2.4. Synthesis of 6-mono (p-toluenesulfonyl)-ˇ-cyclodextrin(ˇ-CD-OTs) (7)

To a solution of �-CD (2.5 g, 2.2 mmol) in water (110 mL), coppersulfate (1.65 g, 6.6 mmol) in water (165 mL) and sodium hydroxide(2.2 g, 55 mmol) in water (55 mL) were sequentially added. After10 min, p-toluenesulfonyl chloride (3.3 g, 17.4 mmol) in acetonitrile(22 mL) was added drop by drop during 1 h. The reaction mixturewas stirred for 4 h at ambient temperature, and then neutralized(pH = 12.5) with hydrochloric acid, the salts were eliminated by fil-tering and the volume of solution was decreased to 2/3 of its initialvolume by lyophilization. The �-CD-OTs was crystallized (threetimes) by dissolving in boiling water and washed with acetone(2× 7 mL), ether (2× 9 mL) and dried. After recrystallizations, pure�-CD-OTs (1.34 g, 46%) was achieved (Baussanne et al., 2000).

�-CD-OTs: M.p.: 182–183◦C (dec.). 1H NMR (400 MHz, DMSO-

d6, ı, ppm): 7.75–7.73 (d, 2H, Ar), 7.43–7.41(d, 2H, Ar), 5.72 (m, OH-2, OH-3), 4.83 (7H, H-1), 4.76 (H-1′), 4.51 (m, 14H, OH-6), 4.33–4.3(m, H-6-OTs), 3.64–3.2 (m, 28 H, H-3, H-4, H-5, H-2), 2.4 (s, 3 H,Me-OTs) ppm. FTIR (KBr, thin film; cm−1): 3382 (str. OH), 2927(str. C–H), 1645 (str. C–C), 1601 (str. C C aromatic), 1414 (str. SO2,asym.), 1158 (str. SO2, sym.), 1027 (str. C–O).

2.5. Synthesis of ˇ-Aalanine propargyl ester (6)

�-Alanine (2 g, 22.5 mmol) were stirred in freshly distilledtrimethylsilyl chloride (4.89 mL, 45 mmol) in a flask followed byadding propargyl alcohol (20 mL) at room temperature for 15 hunder argon atmosphere. After the accomplishment of reaction (aschecked by TLC), the reaction solvent removed by rotary evapora-tor and the remaining material dissolved in a minimum amount ofmethanol and so add ether to precipitate appears. After filtrationand washing with ether residues the final (�-alanine propar-gyl ester hydrochloride) product obtained. The yield was 95%(3.49 g).

1H NMR (400 MHz, CDCl3; ı, ppm): 4.7–4.65 (d, CH2–O),3.23–3.2 (H-alkyne), 2.86–2.84 (t, CH2–CO), 2.79–2.76 (CH2–NH2).13C NMR (100 MHz, CDCl3; ı, ppm): 173 (C O), 78 (H C C), 77(HC C), 54 (CH2–O), 36 (CH2–C O), 32 (CH2–NH2). FTIR (KBr, thinfilm; cm−1): 3245 (str. CH-alkyne), 2980 and 2826 (str. CH), 2128(str. (C C)), 1742 (str. C O), 1012 (C–O).

2.6. Synthesis of mono alkyne-terminated ˇ-CD on the primaryface (8)

In order to synthesize alkyne-terminated �-CD, �-Alaninepropargyl ester hydrochloride (6) (0.176 g, 1.08 mmol), mono [6-O-(p-toluenesulfonyl)]-�-cyclodextrin (7) (0.47 g, 0.36 mmol) andEt3N (0.1 g, 0.36 mmol) were dissolved in dry DMF (5 mL) at 0 ◦Cwith stirring for 2 h under argon atmosphere, then the reactiontemperature increased from 0 ◦C to rt. After maintaining the reac-

tion temperature for 24 h in room temperature was increased to35 ◦C for 12 h. The solvent was removed under vacuum and the pre-cipitate was decanted into acetone (15 mL). The crude product afterprecipitating into acetone was prepared as a brown solid. For the

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thesis

flpw

O(32–1C(33(

Scheme 1. Synthetic routes employed for the syn

urther purification, the product was purified by repeated recrystal-ization from water and ethanol to give the final product as brownowders. The yield of �-Alanine propargyl ester modified �-CD (8)as 65% (0.3 g).

1H NMR (400 MHz, DMSO-d6, ı, ppm): 5.76–5.66 (m, OH-2,H-3), 4.88–4.82 (d, H-1 and H-1′), 4.68 (O–CH2–C C), 4.50–4.49

m, OH-6), 3.61–3.2 (m, H-6a, H-6b, H-3, H-4, H-5, H-2, C CH),.09–3.04 (m, �-CD–CH2–NH), 2.96–2.93 (t, NH–CH2–CH2–C O),.51–2.5 (DMSO-d6), 2.5–2.45 (NH–CH2–CH2–C O), 2.21–2.2 (m,NH). 13C NMR (100 MHz, CDCl3; ı, ppm): 170.4 (C = O of ester),01.96 (�-CD C1), 82 (�-CD C4), 78 (O–CH2–C C), 73.3–73 (�-D C3 and C5), 72 (�-CD C2 and O–CH2–C C), 60.5 (�-CD C6), 53

O–CH2–C C), 51 (NH–CH2–CH2–C O), 45 (�-CD–CH2–NH–) and5 (NH–CH2–CH2–C O). FTIR (KBr, thin film; cm−1): 3423 (str. OH),245 (str. H–C C), 2924 and 2856 (str. CH), 2126 (str. C C), 1740str. C O), 1080–1052 (str. C–O).

of �-CD-based dendrimer (9) via “click” reaction.

2.7. Click reaction between ˇ-CD-(N3)7 and alkyne-terminatedˇ-CD (9)

�-CD dendrimer (9) was prepared by click reaction between(�-CD-(N3)7) (3) and mono functionalized alkyne-terminated �-CD (8). The synthesis method is described as follows. (�-CD-(N3)7)(3) (0.10 g, 0.076 mmol) was dissolved in t-butyl alcohol/H2Omixture (12 mL, 1:1, v/v), and mono alkyne-terminated �-CD(8) (1.33 g, 1.06 mmol, 2 equiv. per azide), CuSO4·5H2O (2.66 mg,0.01 mmol, 0.02 equiv. per azide groups), and sodium ascorbate(2.1 mg, 0.01 mmol, 0.02 equiv. per azide groups) were added intothe solution. The brown solution was stirred for 24 h at 70 ◦C. The

solvent of reaction mixture was evaporated by vacuum. The crudeproduct was liquefied in water and NH4OH was added until pHreached 7. After filtration, the solvent was removed under reducedpressure, and the brown solid was obtained under vacuum. The

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esulting crude product was purified by chromatography on aephadex G-50 column using deionized water as eluent. Finally,he yield of (9) was 53% (0.4 g).

1H NMR (400 MHz, DMSO-d6, ı, ppm): 7.95 (m, 7H, triaz-le), 5.72 (m, OH-2, OH-3), 4.89–4.8 (m, H-1 and H-1′ of �-CDnd tiazole-CH2-O), 4.5 (m, OH-6), 3.92–3.86 (m, �-CD-CH2-riazole), 3.63–3.3 (m, H-3, H-4, H-5, H-2 and H-6), 2.99–2.89t, O C–CH2–CH2–NH–), 2.78–2.68 (t, O C–CH2–CH2–NH–),.51–2.41 (m, NH–CH2–C O and DMSO-d6), 2.21–2 (m, NH). 13CMR (100 MHz, CDCl3; ı, ppm): 170.47 (C O of ester), 148 (CH of

riazole ring), 124.31 (C of triazole ring), 101.96 (�-CD C1), 82 (�-CD4), 78 (�-CD C2), 73–72 (�-CD C3 and C5), 59.99 (triazole–CH2–O–),1 (�-CD–CH2–triazole), 45 (O C–CH2–CH2–NH and �-D–CH2–NH–) and 39 (O C–CH2–CH2–NH). FTIR (KBr, thinlm; cm−1): 3382 (str. OH), 2926 (str. C–H), 1738 (str. C O ester),655 (str. triazole ring), 1036 (str. C–O).

.8. Determination of ˇ-CD in dendrimer

The determination of content of �-CD in dendrimer (9) wasimilar to previous literature (Geok-Lay & Tucker, 1986; Xu, Li,

Sun, 2010). Briefly, 25 mg of dehydrated �-CD dendrimer wasefluxed in sulfuric acid (15 mL, 0.5 M) for 9 h at 100 ◦C, and then theolution was diluted to 50 mL with sulfuric acid (0.5 M). A portion1.25 mL) of the diluted solution was mixed with 5 wt% phenol solu-ion (0.75 mL), and then concentrated sulfuric acid (4.5 mL) wasdded. The absorbance of the obtained solution was measured bysing a UV-visible spectrophotometer at 490 nm (A490 nm). The glu-ose concentration (Cglucose) was calculated by a standard curve asollows:

glucose = 0.301A490nm + 0.014

The curve is linear over Cglucose = 0.01–0.5 mg mL−1 (R = 0.99).he content of �-CD dendrimer in dry state was calculated as fol-ows:

-CD (%) = Cglucose(mg mL−1) × 50 mL × M�-CD

7 × Mglucose × m�-CD dendrimer

here Mglucose is the molecular weight of glucose, m�-CD-dendrimer ishe weight of �-CD dendrimer (mg), M�-CD is the molecular weightf �-CD and 7 is the amount of glucose parts in the �-CD fragment.

.9. Solid samples preparation

Solid inclusion complexes of MTX with �-CD-dendrimer (9)nd �-CD were prepared by a common co-precipitation technique.riefly, excess of MTX (10 mg) was added to 40 mL pure water (amall quantity of NH4OH (25%) was added to help the dissolutionf MTX) containing various amount of �-CD (25 mg) and �-CD-endrimer (9) (100 mg). The obtained mixtures were stirred at7 ◦C for 24 h. After reaching equilibrium, the resultant solutionsere centrifuged at 1300 rpm for 25 min and filtered. The measure-ents were accomplished in triplicate. Physical mixtures of MTX

nd �-CD-dendrimer (9) were obtained by the same proportionith the components by carefully mixing it in a ceramic mortar.

.10. Drug loading and in vitro release

The encapsulation efficiency (EE) of the MTX-loaded �-CD and-CD-dendrimer (9) nanocarrier were calculated by following way:riefly, solid inclusion complex (20 mg) was taken into HCl (0.1N,

mL) for 24 h, and the solid inclusion complex suspension was

eparated by centrifugation at 13,000 rpm for 25 min under darkonditions. Supernatant of centrifugation was analyzed for deter-ine the loading content of MTX by UV spectrophotometer at

lymers 132 (2015) 205–213

303 nm by the following equation:

Drug encapsulation efficiency (%)

=(

Mass of drug loaded (mg)Mass of total initial drug (mg)

)× 100

Blank samples were prepared from �-CD and �-cyclodextrindendrimer (9) without loaded MTX. All set samples were analyzedin triplicate.

In vitro release of MTX-loaded �-CD and �-CD dendrimer (9)were evaluated by a dialysis technique (Wu, Wang, & Que, 2006).First, MTX-loaded �-CD and �-CD dendrimer (9) were diluted byrelease moderate. Then, the resulting solutions (3 mL) were movedinto a dialysis bags (molecular cut-off 2000) and then the dialy-sis bags were placed in 30 mL of dissolution medium with stirringat 100 rpm at 37 ± 0.5 ◦C. Phosphate-buffered saline (PBS) solutionpH = 7.4 and sodium acetate buffer solution pH = 3 as release mediawere used to study the influence of pH on drug release. At definitetime intervals, external solution was taken and replenished withequal volume of fresh buffer solution. The study of drug releasewas carried out for 72 h. The amount of released MTX into buffersolution was analyzed by UV spectrophotometer at 307 (pH = 3)and 303 nm (pH = 7.4) respectively. The release experiments wereperformed in triplicate.

2.11. Characterization

2.11.1. Nuclear magnetic resonance spectroscopy (NMR)All NMR spectra were measured at 25 ◦C with a Bruker Avance

spectrometer (400 MHz for 1H and 100 MHz for 13C) used in theFourier transform mode. CDCl3, DMSO-d6 and D2O were used asthe solvent. The data are described as chemical shift (ı, ppm). HOD(4.79 ppm) or DMSO-d6 (2.5 ppm) was used as interior references.

2.11.2. Fourier transform infrared spectroscopy (FTIR)The FTIR spectra of all samples were recorded on Bruker Ten-

sor 27 Spectrophotometer by KBr disk method. The spectra wererecorded over the range of 4000–400 cm−1.

2.11.3. Dynamic light scattering (DLS)DLS measurements were performed on a Nanotrac Wave

from Microtrac instruments equipped with a 10 mW He–Ne laser(� = 780 nm) at 25 ◦C and the scattering angle was kept at 170◦. Theresults were studied in CONTIN style. Measurements were recordedin triplicate.

2.11.4. Differential scanning calorimetry (DSC)DSC analyses of the different samples were recorded on a

Linseis-L 63/45 DSC instrument. Thermal behaviors of the samples(5 ± 0.05 mg) were investigated in aluminum crimped pans. Theheating of samples were performed at the rate of 10 ◦C min−1 from20 to 400 ◦C under argon gas flow. A blank vacuum-packed pan wasused as reference.

2.11.5. Scanning electron microscopy (SEM)Surface morphology of �-CD-dendrimer, MTX, their complexes

and physical mixtures were studied by SEM (MIRA3 FEG-SEM, Tes-can). The samples were attached onto carbon tabs (double sidedadhesive tape) stick to aluminum stumps. Specimens were cov-ered with gold–palladium (plasma deposition technique) with aBIO-RAD AC500. Pictures were achieved at an excitation voltage of15 kV.

2.11.6. UV–visible spectral analysis (UV–vis)UV–visible spectra were recorded using 1700 Shimadzu spec-

trophotometer with samples in a quartz cell of path length 1 cm.

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.11.7. MTT assay and cell treatmentThe in vitro cytotoxic effect of �-CD dendrimer, MTX, and their

nclusion complex on human breast cancer cells (T47D) was inves-igated by 72 h MTT tests. Concisely, T47D cultured in RPMI-1640

oderate complemented with 10% fetal bovine serum and 1% peni-illin/streptomycin solution at 37 ◦C in a wetted incubator with 5%O2. Then 5000 cell/well were cultured in a 96-well culture platend incubated 24 h at 37 ◦C. The cells were incubated with theTX and inclusion complex at concentrations of 2–64 �M for 72 h.

fter incubation, the moderate of all wells were removed, and MTTeagent (2 mg mL−1 in PBS) was added to all well. The cells were fur-her incubated in dark for 4.5 h at 37 ◦C, washed with PBS (100 �L of.1 M, pH 7.4) and monitored by adding of 150 �L DMSO. Then, thebsorbance was measured at 570 nm during 15–30 min in the pres-nce of Sorensen’s’ glycine buffer. All test condition was completedn quadruple. Cell viability (%) was expressed through the livingells (%) relative to controls. An IC50 (inhibitory concentration)alue was distinct as the drug concentration of compound at which0% cell growing reserved. Then the IC50 value was considered byhe Prism 6.0 software (Graphpad, San Diego, USA).

. Results and discussion

The detailed synthetic path for �-CD dendrimer (9) was shownn Scheme 1. As shown, �-CD dendrimer (9) on the primary faceaving �-CD in both core and periphery was synthesized by a clickeaction, wherein �-CD is combined together via primary surfacey the convergent technique. For the first step, per azido-�-CD onhe primary face as a core molecules (�-CD-(N3)7) (3) was synthe-ized; at the second step, alkyne-terminated �-CD (8) precursorsere prepared. At the last step, azido groups were grafted to the

unction points of alkyne-terminated �-CD group via click reactiono produce �-CD dendrimer (9) on the primary face.

.1. Preparation of per azide-functionalized ˇ-CD precursor (3)

To prepare definite dendrimer, it is important to select appro-riate multifunctional core that can competently link with suitableoiety polymer precursors. �-CD-(I)7 (2) was synthesized by selec-

ive reaction of seven hydroxyl groups of �-CD on the primaryace by iodine (Scheme 1). The following treatment with NaN3 inMF directed to the preparation of �-CD-(N3)7 (3) (Ashton et al.,996). The study of �-CD-(N3)7 by 1HNMR in DMSO-d6 showedhe thorough vanishing of hydroxyl proton (6-CH2OH) signal at.5 ppm, which is existent in �-CD (see supplementary data, Fig.1). FTIR analysis (Fig. 1) of �-CD-(N3)7 showed the presence of newbsorbance azide peak at 2108 cm−1. The obtained results estab-ished that the hydroxyl groups on the primary face of �-CD wereelectively substituted by azide groups fully.

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.carbpol.2015.05.087

.2. Preparation of mono-functionalized alkyne-terminated ˇ-CDrecursor (8)

The mono-functionalized alkyne-terminated �-CD precursor’s8) was prepared by a multistep reactions method, as shownn Scheme 1. The synthetic procedure to fabricate of mono-unctionalized alkyne-terminated �-CD (8) is as follows: �-alanineropargyl ester hydrochloride (6) was obtained via the reaction ofropargyl alcohol and �-alanine in the existence of trimethylsilyl

hloride. Then monofunctionalized �-CD derivative (7) (�-CD-OTs)as synthesized and changed to (8) by replacement of the tosyloiety with �-alanine propargyl ester group by nucleophilic sub-

titution (Scheme 1) in the presence of Et3N. FTIR, 1H and 13C NMR

lymers 132 (2015) 205–213 209

analysis were used to endorse the structure and purity of com-pound 8 (see supplementary data, Figs. S2 and S3). Matching thespectra of 6 and 7, the vanishing of the peaks at 1601, 1414 and1158 cm−1 (consistent to the aromatic section and O S O), appear-ance of the peaks at 2128 cm−1 (consistent to the alkyne section)and 1724 cm−1 (consistent to the ester section) shows that all thesegroups had reacted and were removed in workup.

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.carbpol.2015.05.087

3.3. Click reaction between ˇ-CD-(N3)7 and alkyne-terminatedˇ-CD (9)

�-CD dendrimer (9) on the primary face was prepared byCu(I)-catalyzed click reaction between �-CD-(N3)7 (3) and alkyne-terminated �-CD (8). 1H and 13C NMR study were used to approvethe structure and purity of (9). After the click reaction NMR analysisexhibited disappearance of the terminal alkyne proton and cor-responding appearance of a triazole proton at 7.8–8.1 ppm. Thepresence of the triazole proton around 8.0 ppm in the 1H NMRspectra was indicated the formation of the �-CD dendrimer (9)completely (see supplementary data, Fig. S4). The integral ratio oftriazole proton to anomeric proton is about 1:7.9, which showsthe successful ‘click’ reaction between �-CD-(N3)7 and alkyne-terminated �-CD (Scheme 1). 13C NMR analyses of (9) showed theappearance of signals at 148, 124 ppm for carbon of triazole ringand 170.47 ppm, which can be ascribed to carbonyl of ester (seesupplementary data, Fig. S5). The synthetic process of �-CD den-drimer (9) can be also monitored by FTIR spectra, as shown inFig. 1. A comparison to those of �-CD-(N3)7 and �-CD dendrimer(9) (Fig. 1) discovered the complete disappearance of azide peakat 2108 cm−1 and 2128 cm−1 of alkyne peak for �-CD dendrimer(9). Disappearance of azide and alkyne signals in the FTIR spectraof �-CD dendrimer (9), revealing that the remaining end-cappingsubstance has been removed completely from the last compound.Also, appearance of signals related to five-membered triazole ringat 1653 cm−1 confirmed a successful coupling reaction.

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.carbpol.2015.05.087

3.4. Characterization of MTX:ˇ-CD dendrimer inclusion complex.

3.4.1. FTIR study of complexFig. 1 shows FTIR spectra of �-CD dendrimer (9), MTX, phys-

ical mixtures and respective inclusion complexes. The inclusioncomplex formation of �-CD dendrimer (9) and a guest drugwas confirmed by the FTIR spectra. �-CD dendrimer (9) showed3412 cm−1 (for OH stretching), 2927 cm−1 (C–H stretching vibra-tion), 1032 and 1135 cm−1 (O–C–O stretching) and at 1658correspond to H–O–H bending. The FTIR spectrum of MTX (Fig. 1)displayed distinctive peaks at 1703 cm−1 (for C O stretching vibra-tions for carboxylic acid groups), 1655 cm−1 (corresponding to C Ostretching vibration of amide groups) and C–O stretch at 1097 and1203 cm−1. The aromatic C C stretch in 1455, 1500 and 1602 cm−1

was observed. The physical mixture spectrum showed approxi-mate mixed spectra of MTX and �-CD dendrimer. All the absorptionpeaks of MTX were enclosed by that �-CD dendrimer (9) in theFTIR spectrum of inclusion complex. The changes between thesupramolecular inclusion complex and physical mixture show thatMTX was complexes into the cavity of �-CD dendrimer to form theinclusion complex.

3.4.2. Determination of ˇ-CD in ˇ-CD-dendrimerThe amount of �-CD in the �-CD-dendrimer (9) was calculated

by the method of Koh and Tucker (Geok-Lay & Tucker, 1986; Xuet al., 2010) and the data disclose that CD dendrimer contains

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Fig. 1. FTIR spectra of (a) �-CD-(N3)7 (3), (b) alkyne-terminated �-CD (8), (c) �-CD dendrimer (9), (d) pure MTX, (e) inclusion complexes and (f) respective physical mixtures.

Fig. 2. SEM photographs of (a) �-CD dendrimer (9), (b) MTX, (c) MTX and �-CD dendrimer physical mixture (1:1 molar ratio) and (d) MTX/�-CD dendrimer inclusion complex.

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Y. Toomari et al. / Carbohydrate Polymers 132 (2015) 205–213 211

FM

9q

3

msaiswOa(woc(

ig. 3. DLS measurement of �-CD dendrimer (9) and their inclusion complex withTX in aqueous solution.

0.14%�-CD in its construction, which is comparable to the realuantity (88.64%).

.4.3. SEM analysisThe powdered forms of MTX, �-CD dendrimer (9), their physical

ixture and inclusion complexes were investigated by SEM analy-is (Fig. 2). As shown in SEM images (i) �-CD dendrimer is present inglobular morphology with cavity structures, (ii) MTX is present

n amorphous and crystalline states (iii) the structures of inclu-ion complexes appeared as amorphous complexes and not crystalshich is an evidence for the formation of inclusion complexes.therwise, in SEM image the crystal photographs related to MTXnd �-CD dendrimer in the inclusion complex was not observediv) their physical mixtures presented particles of MTX surrounded

ith �-CD dendrimer nanoparticles. The different constructions

f pure MTX, �-CD dendrimer (9) and their inclusion complexonfirmed that MTX is comprised into �-CD cavity of dendrimer9) or dendritic network. The outcomes show that the successful

Fig. 4. DSC curves of (a) MTX; (b) physical mixture; (c) �-CD dend

Fig. 5. In vitro cytotoxicity of �-CD dendrimer (9), MTX and inclusion complex of�-CD dendrimer:MTX measured by the MTT test against T47D cells. (Mean ± SD;n = 3).

preparation of inclusion complex of �-CD-dendrimer (9) with MTX.From SEM observations it seems that with formation of CD den-drimer a new cavity with average size diameter of 70 nm is formed.In the case of SEM photograph for dendrimers with larger cavity≥100 nm probably the aggregation or big clusters of mixture ofmany different shapes and size ranges of particles is happened.

3.4.4. DLS analysisTo evaluate the average hydrodynamic diameter of �-CD den-

drimer (9) and its supramolecular complex with MTX, DLS analyseswere performed in water (Fig. 3). In relation to DLS results, an aver-age hydrodynamic diameter of 57 nm for pure �-CD dendrimer (9)and 78 nm for supramolecular complex was found. The conversionin size is due to the formation inclusion complex at the cavity of

dendrimer and cyclodextrin moiety. The size obtained with SEM issmaller than the results achieved from DLS. This is related to theSEM images measured the particle size in solid state, whereas DLSin a solution.

rimer (9) and (d) MTX/�-CD dendrimer inclusion complex.

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F opartn

3

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3

asITT�tic

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ig. 6. Cumulative drug release (%) of MTX from �-CD and MTX:�-CD dendrimer nan= 3).

.4.5. DSC analysisDSC analyses were carried out to study the physical state of the

TX in the inclusion complex. DSC curves of �-CD dendrimer (9),TX, physical mixture and their inclusion complex were showed in

ig. 4. In DSC curves of MTX, the water loss happened in the 122.9 ◦Cnd the melting peak at 180.9 ◦C. The curves of �-CD dendrimer9) displayed endothermic peak at 138.8 ◦C and 319.6 ◦C match-ng to their loss of water and melting points, respectively. The DSCurve of the physical mixture showed both typical signals of MTXt 185 ◦C with a slight shift and �-CD dendrimer (9) at 326.4 ◦C. Inhe inclusion complex, the MTX melting point of curve signal wasully vanished. The peaks at 235–258 ◦C have been cited by otheruthors in the range of 210–240 ◦C for �-CD (Giordano, Novak, &oyano, 2001). The appearance of this peak remains unclear, but

eems to be related with crystal hydration through the preparationf �-CD (Giordano et al., 2001). This obviously recommended thectual formation of a supramolecular inclusion complex betweenTX and �-CD dendrimer (9).

.5. MTT assay

Fig. 5 shows the in vitro cytotoxic effect of �-CD dendrimer, MTXnd their inclusion complex with various concentrations of inclu-ion complex on T47D cells. Data analysis of MTT test exhibited thatC50 of MTX and inclusion complex of �-CD dendrimer (9):MTX on47D cells was 7.4 and 4.9 �M for 72 h MTT assays, respectively.hese results display the cytotoxic effect of inclusion complex of-CD dendrimer (9):MTX on T47D cells was considerably greater

han that of free MTX. Also, �-CD dendrimer had a clearly low tox-city. The cell viability was higher than 90% even if �-CD dendrimeroncentration extended as high as 32 �M.

.6. Drug loading and in vitro drug release study

Drug encapsulation efficiency of �-CD dendrimer (9) and �-D were estimated by UV spectrophotometric technique and were

ound to be 79.8 and 52%, respectively. From the results achieved

y drug encapsulation efficiency of MTX with �-CD and �-CDendrimer (9) (comparison of �-CD with �-CD dendrimer (9)), iteemed that approximately 52% MTX may be encapsulated in theavity of �-CD and 27.8% MTX was entrapped in the dendrimer

icles in buffer solution (pH 3 and 7.4) and pure MTX in pH = 7.4 at 37 ◦C. (Mean ± SD;

networks. The EE of �-CD dendrimer (9) significantly improvedcompare with �-CD. High loading amount of �-CD dendrimer (9)could be attributed to the inclusion of �-CD cavity to drug andentrapment of MTX with dendritic network. Release profiles of thepure MTX (pH 7.4) and MTX from the MTX/�-CD dendrimer and�-CD at pH 3 and pH 7.4 are shown in Fig. 6. The results showedthat the free MTX, complete diffusion was occurred within 8 h, butfor �-CD and �-CD dendrimer (9), a prolonged and slow releasefor 23 and 72 h were observed, respectively. The in vitro cumula-tive release profiles of MTX from �-CD dendrimer (9) (Fig. 6) showsthat about 22% of the drug was released in the burst release phase(after 4 h). After the primary burst, sustained release up to 72 h wasrelated to drugs diffusion and the dendrimer degradation. The ini-tial burst release may be perhaps caused by the drug attached on thedendrimer surface, since the MTX might form hydrogen bonds withthe hydroxyl groups of dendrimer. Sustained release was achieveddue to drug diffusion from the CD cavity and dendrimeric network.Also, �-CD dendrimer (9) was showed to have a more controlledrelease behavior for the loaded MTX when compared to �-CD. Asshown in Fig. 6 the MTX release from nanocarrier was noticeably pHdependent. The cumulative release profiles after initial burst showthat almost 68 and 57% of MTX was released at pH 3 and pH 7.4,respectively. These results can be because of the lower solubility ofthe MTX/�-CD in pH 7.4 than in pH 3. It has been described that�-CD could increase the release of hydrophobic drugs or dissolu-tion profiles in acidic conditions (Stojanov & Larsen, 2011). Thesefinding showed that the �-CD dendrimer (9) nanocarrier could bea suitable controlled drug delivery system for cancer treatment.

4. Conclusion

The new glycodendrimer from �-CD in core and branches weresynthesized through click reaction with excellent coverage. Thestructure of �-CD dendrimer (9) was characterized and confirmedwith FTIR, NMR and DLS techniques. The obtained dendrimershowed the remarkable encapsulation efficiency, 79.8% for MTXas model drug. The inclusion complex structure of prepared den-

drimer and MTX was determined by SEM, DLS, DSC and FTIRtechniques. The in vitro toxicity of the prepared compound andtheir inclusion complex with MTX by the MTT test on T47D cellsshowed that the resultant dendrimer was not cytotoxic to the

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ell line considered. The construction of the synthesized �-CD-endrimer permitted two kinds of potential entrapment places forhe drug: in CD cavities and dendritic network. The in vitro drugelease results in buffer solution showed that the �-CD dendrimeranocarrier could be a suitable controlled drug delivery system forancer treatment.

cknowledgements

Authors gratefully acknowledge the financial support from theniversity of Tabriz and RCPN of Tabriz University of Medical Sci-nce.

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