Synthesis of the dendritic type β-cyclodextrin on primary face via click reaction applicable as drug nanocarrier

Accepted Manuscript Title: Synthesis of the Dendritic Type ␤-Cyclodextrin on Primary Face via Click Reaction Applicable as Drug Nanocarrier Author: Toomari Yousef Namazi Hassan Entezami Ali Akbar PII: DOI: Reference:

S0144-8617(15)00556-1 http://dx.doi.org/doi:10.1016/j.carbpol.2015.05.087 CARP 10038

To appear in: Received date: Revised date: Accepted date:

10-1-2015 29-4-2015 15-5-2015

Please cite this article as: Yousef, T., Hassan, N., and Akbar, E. A.,Synthesis of the Dendritic Type rmbeta-Cyclodextrin on Primary Face via Click Reaction Applicable as Drug Nanocarrier, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.05.087 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Highlights:  Novel glycodendrimer from β-CD in core and branches was synthesized.

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 Supramolecular inclusion complex by dendrimer and MTX drug was prepared.

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 MTT assay show that the resultant dendrimer is not cytotoxic to the cells.

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 Results showed that the developed dendrimer could offer a controlled delivery of MTX.

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Synthesis of the Dendritic Type β-Cyclodextrin on Primary Face via Click Reaction

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Applicable as Drug Nanocarrier

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Toomari Yousef, Namazi Hassan*, Entezami Ali Akbar

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Laboratory of Dendrimers and Nano-Biopolymers, Faculty of Chemistry, University of Tabriz,

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Tabriz, Iran; Tel.: +98 413393121, Fax: +98 41 334 0191, E-mail address: [email protected]

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Research Center for Pharmaceutical Nanotechnology (RCPN), Tabriz University of Medical

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Science, Tabriz, Iran

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Abstract

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The objective of this study was the syntheses of well-defined glycodendrimer with entrapment

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efficiency by click reactions, with β-cyclodextrins (β-CDs) moiety to keep the biocompatibility

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properties, besides especially increase their capacity to load numerous appropriate sized guests.

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The original dendrimer containing β-CD in both periphery and central was synthesized using

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click reaction. The entrapment property of the β-CD-dendrimer was studied by methotrexate

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(MTX) drug. The chemical structure of β-CD-dendrimer was characterized by 1H NMR,

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NMR and FTIR and its inclusion complex structure were investigated by SEM, DLS, DSC and

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FTIR techniques. The cytotoxic effect of obtained compound and its inclusion complex with

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MTX was analyzed using MTT test. The MTT test exhibited that the synthesized compound was

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not cytotoxic to the cell line considered. The in vitro drug release study turned out that the

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obtained β-CD dendrimer could be a suitable controlled drug delivery system for cancer

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treatment.

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Keywords: β-cyclodextrin; dendrimer; nanocarrier; methotrexate; inclusion complex

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1. Introduction

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Dendrimers are a class of polymeric materials having three-dimensional hyperbranched

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macromolecules, monodispersed, defined spherical construction, nanoscopic objects with

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amount of reactive periphery groups and host-guest entrapment properties (Shen, Li, Wu, Zhang

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& Li, 2015; Tomalia et al., 1985). In this work we emphasis on dendrimers, these materials

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firstly reported by Vögtle and co-workers as cascade molecules (Buhleier, Wehner & Vögtle,

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1978) and followed by Tomalia et al. (Tomalia et al., 1985) named starburst dendrimers, and

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Newkome et al. (Newkome, Yao, Baker & Gupta, 1985) as ‘arborols’. Since then, the study of

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these materials, has been expands exponentially to all areas (Hawker & Fréchet, 1990; Namazi &

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Adeli, 2003; Namazi & Adeli, 2005a; Namazi & Adeli, 2005b). Because of unique structures and

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properties, dendrimers have concerned greatly attention for their uses in several fields. Among

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them the usage of these compounds as drug delivery systems (DDS) has been of excessive

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attention (Lai et al., 2007; Leng et al., 2013; Namazi & Toomari, 2011; Sun, Fan, Wang & Zhao,

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2012; Tomalia & Fréchet, 2002).

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β-CD essentially biocompatible compound (Ortiz Mellet, Defaye & Garcia Fernandez, 2002),

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and is one of the good candidates for the preparation of star polymers (Ritter & Tabatabai, 2002;

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Wen, Zhang & Li, 2014; Zhang, Liu & Li, 2013; Zhao, Yin, Zhang & Li, 2013). Star polymers

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by using the core-first method have been prepared (Hawker, Bosman & Harth, 2001). The β-CD

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molecule is a torus-shaped oligosaccharide that consist of 7 glucose units connected through α-

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1,4-glucosidic links (Namazi & Kanani, 2007, 2009; Saenger et al., 1998). The narrower and

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wider ends of the CD, termed the primary and secondary face with 7 and 14 hydroxyl groups,

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respectively. The cavity of β-CD is somewhat hydrophobic and the external part of the CDs is

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hydrophilic. Because of these structures, β-CDs are known to form inclusion complexes with

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hydrophobic molecules having the suitable dimension and form to promote their solubility

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(Namazi & Toomari, 2011; Uekama, Hirayama & Irie, 1998; Zhang, Liu & Li, 2011). Therefore,

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inclusion complexes of CDs with hydrophobic compounds are of significance (Varghese, Al-

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Busafi, Suliman & Al-Kindy, 2015).

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‘Glycodendrimer’ term is applied to define dendrimers that include carbohydrates in their

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constructions (Chabre, Contino-Pepin, Placide, Shiao & Roy, 2008; Chabre & Roy, 2010).

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Glycodendrimers having CD moiety in their assemblies are termed CD-dendrimers. Depending

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on the core and branches groups, CD dendrimers can be separated into three chief categories.

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The first categories (CD-based dendrimers), CDs are located on the periphery of dendrimers

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(Adeli, Kalantari, Zarnega & Kabiri, 2012; Menuel, Duval, Cuc, Mutzenhardt & Marsura, 2007;

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Menuel, Fontanay, Clarot, Duval, Diez & Marsura, 2008). In the second categories (CD-centered

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dendrimers), a CD part as the central wherever all outlets (in primary face) are linked

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(Newkome, Godinez & Moorefield, 1998). The third categories (CD-dendrimer conjugates),

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dendrimers conjugated with CD moiety (Baussanne, Benito, Mellet, Garcia Fernandez, Law &

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Defaye, 2000; Benito, Gomez-Garcia, Ortiz Mellet, Baussanne, Defaye & Garcia Fernandez,

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2004; Wang, Shao, Qiao & Cheng, 2012). By the combination of CDs with dendrimer, the

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positive properties of dendrimer and CDs improved (Menuel, Fontanay, Clarot, Duval, Diez &

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Marsura, 2008).

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In this work, we report the preparation of a family of glycodendrimers having on β-CD scaffolds

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on the primary face in their central and periphery by using spacer arms, which was obtained by

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click reaction (Scheme 1). Also, the encapsulation and drug delivery property of obtained CD-

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dendrimers in buffer solutions using MTX as the guest molecule was studied.

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2. Materials and methods

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2.1. Materials

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β-CD (98%, Merck) was achieved and used after desiccating under vacuum. p-Toluenesulfonyl

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chloride (p-TsCl, Merck) was purified by chloroform and petroleum ether. Propargyl alcohol

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(99%), methotrexate (MTX, 99%), MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium

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bromide), copper sulfate (CuSO4; 99.99%), iodine (99.8%), triphenylphosphine (PPh3; 99%),

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dimethyl sulfoxide (DMSO; 99.9%) and acetonitrile (99.8%) were purchased from Sigma

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Aldrich and used as received. N,N-Dimethylformamide (DMF; 99.8%, Merck) was dried over

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CaH2 and distilled under vacuum. Triethylamine (Et3N; Merck) was dried over CaH2 and then

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distilled. Acetone (99.8%), methanol (99.8%), diethyl ether (99.5%), sodium azide (NaN3, 99%)

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and L-ascorbic acid sodium salt (99%) were obtained from Merck and used as received. Dialysis

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tubing (benzoylated, molecular cut-off 2000 Da) was purchased from Sigma-Aldrich. T47D, a

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human breast cancer cells were achieved from Pasteur Institute Cell Bank of Iran (Tehran, Iran)

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and cultured in RPMI-1640 moderate complemented with 10% fetal bovine serum and 1%

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penicillin/streptomycin solution at 37 oC in a wetted incubator with 5% CO2. All the solvents

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were purified before use. Deionized water was used through the experiments.

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2.2. Synthesis of Heptakis (6-Deoxy-6-iodo)-β-cyclodextrin (β-CD-(I)7) (2)

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To a solution of Ph3P (2.07 g, 7.9 mmol) under stirring in dry DMF (9 mL), I2 (2.09 g, 8.26

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mmol) was added over 30 min. After the addition of I2 the solution temperature increased from

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room temperature (rt) to 50 °C. To this dark brown solution vacuum-dried β-CD (1) (0.6 g, 0.53

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mmol) was then added, and the reaction mixture was stirred at 70 °C under argon atmosphere for

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18 h. It was then concentrated through the elimination of DMF (approximately 5 mL) under

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vacuum and the pH adjusted to 9-10 via the addition of NaOMe in MeOH (3 M, 3 mL) under an

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argon atmosphere with effective cooling, for 30 min. The precipitate was formed by the addition

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of reaction mixture into MeOH (40 mL), which was washed with excess MeOH. β-CD-(I)7 (0.75

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g, 89%) was obtained as a white powder after drying under high vacuum (Ashton, Königer,

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Stoddart, Alker & Harding, 1996; Benkhaled, Cheradame, Fichet, Teyssié, Buchmann &

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Guégan, 2008). M.p.: 222 °C (dec.).1H NMR (400 MHz, DMSO-d6; δ, ppm): 6.05 (OH-2), 5.99

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(OH-3), 4.98 (H-1), 3.81 (H-6b), 3.69–3.56 (H-3, H-5), 3.45–3.21 (H-2, H-4, H-6a).

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2.3. Synthesis of Heptakis (6-Deoxy-6-azido)-β-cyclodextrin (β-CD-(N3)7) (3)

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To a solution of β-CD-(I)7 (2) (0.75 g, 0.39 mmol) in anhydrous DMF (12 mL) at room

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temperature were added NaN3 (0.25 g, 3.86 mmol). The reaction mixture was stirred at 80 °C

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under argon atmosphere for 24 h. The suspension was concentrated under vacuum to 1/3 of the

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starting volume before a large additional of water was added. After filtration, the residue was

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washed with water and dried under high vacuum. β-CD-(N3)7 (3) (0.48 g, 94%) was obtained as

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a white powder. (Ashton, Königer, Stoddart, Alker & Harding, 1996). 1H NMR (400 MHz,

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DMSO-d6; δ, 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),

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3.35-3.32 (H-2, H-4, H-6a). FTIR (KBr, thin film; cm-1): 3360 (str. OH), 2924 and 2856 (str.

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CH), 2108 (str. N3), 1155–1049 (str. C–O).

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

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To a solution of β-CD (2.5 g, 2.2 mmol) in water (110 mL), copper sulfate (1.65 g, 6.6 mmol) in

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water (165 mL) and sodium hydroxide (2.2 g, 55 mmol) in water (55 mL) were sequentially

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added. After 10 min, p-toluenesulfonyl chloride (3.3 g, 17.4 mmol) in acetonitrile (22 mL) was

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added drop by drop during 1 h. The reaction mixture was stirred for 4 h at ambient temperature,

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and then neutralized (pH=12.5) with hydrochloric acid, the salts were eliminated by filtering and

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the volume of solution was decreased to 2/3 of its initial volume by lyophilization. The β-CD-

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OTs was crystallized (three times) by dissolving in boiling water and washed with acetone (2×7

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mL), ether (2×9 mL) and dried. After recrystallizations, pure β-CD-OTs (1.34 g, 46%) was

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achieved (Baussanne, Benito, Mellet, Garcia Fernandez, Law & Defaye, 2000).

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β-CD-OTs: M.p.: 182–183 °C (dec.). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 7.75-7.73 (d, 2H,

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Ar), 7.43-7.41(d, 2H, Ar), 5.72 (m, OH-2, OH-3), 4.83 (7 H, H-1), 4.76 (H-1′), 4.51 (m, 14 H,

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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)

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ppm. FTIR (KBr, thin film; cm−1): 3382(str. OH), 2927 (str. C–H), 1645 (str. C–C), 1601 (str.

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C=C aromatic), 1414 (str. SO2, asym.), 1158 (str. SO2, sym.), 1027 (str. C–O).

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2.5. Synthesis of β-Alanine propargyl ester (6)

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β-Alanine (2 g, 22.5 mmol) were stirred in freshly distilled trimethylsilyl chloride (4.89 mL, 45

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mmol) in a flask followed by adding propargyl alcohol (20 mL) at room temperature for 15 h

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under argon atmosphere. After the accomplishment of reaction (as checked by TLC), the reaction

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solvent removed by rotary evaporator and the remaining material dissolved in a minimum

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amount of methanol and so add ether to precipitate appears. After filtration and washing with

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ether residues the final (β-alanine propargyl ester hydrochloride) product obtained. The yield was

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95% (3.49 g).

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CH2-CO), 2.79-2.76 (CH2-NH2). 13C NMR (100 MHz, CDCl3; δ, ppm): 173 (C=O), 78 (H C≡C),

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77 (HC≡C), 54 (CH2-O), 36 (CH2-C=O), 32 (CH2-NH2). FTIR (KBr, thin film; cm-1): 3245 (str.

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CH-alkyne), 2980 and 2826 (str. CH), 2128 (str. (C≡C)), 1742 (str. C=O), 1012 (C-O).

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2.6. Synthesis of mono alkyne-terminated β-CD on the primary face (8)

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In order to synthesize alkyne-terminated β-CD, β-Alanine propargyl ester hydrochloride (6)

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(0.176 g, 1.08 mmol), mono [6-O-(p-toluenesulfonyl)]-β-cyclodextrin (7) (0.47 g, 0.36 mmol)

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and Et3N (0.1 g, 0.36 mmol) were dissolved in dry DMF (5 mL) at 0 °C with stirring for 2 h

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under argon atmosphere, then the reaction temperature increased from 0 °C to rt. After

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maintaining the reaction temperature for 24 h in room temperature was increased to 35 °C for 12

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h. The solvent was removed under vacuum and the precipitate was decanted into acetone (15

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mL). The crude product after precipitating into acetone was prepared as a brown solid. For the

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further purification, the product was purified by repeated recrystallization from water and

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ethanol to give the final product as brown powders. The yield of β-Alanine propargyl ester

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modified β-CD (8) was 65% (0.3 g).

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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,

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C≡CH), 3.09-3.04 (m, β-CD-CH2-NH), 2.96-2.93 (t, NH-CH2-CH2-C=O), 2.51-2.5 (DMSO-d6),

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2.5-2.45 (NH-CH2-CH2-C=O), 2.21-2.2 (m, -NH). 13C NMR (100 MHz, CDCl3; δ, ppm): 170.4

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(C=O of ester), 101.96 (β-CD C1), 82 (β-CD C4), 78 (O-CH2-C≡C), 73.3-73 (β-CD C3 and C5),

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H NMR (400 MHz, CDCl3; δ, ppm): 4.7-4.65 (d, CH2-O), 3.23-3.2 (H-alkyne), 2.86-2.84 (t,

H NMR (400 MHz, DMSO-d6, δ, ppm): 5.76-5.66 (m, OH-2, OH-3), 4.88-4.82 (d, H-1 and H-

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72 (β-CD C2 and O-CH2-C≡C), 60.5 (β-CD C6), 53 (O-CH2-C≡C), 51 (NH-CH2-CH2-C=O), 45

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(β-CD-CH2-NH-) and 35 (NH-CH2-CH2-C=O). FTIR (KBr, thin film; cm-1): 3423 (str. OH),

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3245 (str. H-C≡C), 2924 and 2856 (str. CH), 2126 (str. C≡C), 1740 (str. C=O), 1080–1052 (str.

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C–O).

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2.7. Click reaction between β-CD-(N3)7 and alkyne-terminated β-CD (9)

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β-CD dendrimer (9) was prepared by click reaction between (β-CD-(N3)7) (3) and mono

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functionalized alkyne-terminated β-CD (8). The synthesis method is described as follows. (β-

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CD-(N3)7) (3) (0.10 g, 0.076 mmol) was dissolved in t-butyl alcohol/H2O mixture (12 mL, 1:1,

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v/v), and mono alkyne-terminated β-CD (8) (1.33 g, 1.06 mmol, 2 equiv. per azide),

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CuSO4.5H2O (2.66 mg, 0.01 mmol, 0.02 equiv. per azide groups), and sodium ascorbate (2.1 mg,

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0.01 mmol, 0.02 equiv. per azide groups) were added into the solution. The brown solution was

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stirred for 24 h at 70 °C. The solvent of reaction mixture was evaporated by vacuum. The crude

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product was liquefied in water and NH4OH was added until pH reached 7. After filtration, the

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solvent was removed under reduced pressure, and the brown solid was obtained under vacuum.

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The resulting crude product was purified by chromatography on a sephadex G-50 column using

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deionized water as eluent. Finally, the yield of (9) was 53% (0.4 g).

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(m, H-1 and H-1′of β-CD and tiazole-CH2-O), 4.5 (m, OH-6), 3.92-3.86 (m, β-CD-CH2-triazole),

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3.63-3.3 (m, H-3, H-4, H-5, H-2 and H-6), 2.99-2.89 (t, O=C-CH2-CH2-NH-), 2.78-2.68 (t, O=C-

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CH2-CH2-NH-), 2.51-2.41 (m, NH-CH2-C=O and DMSO-d6), 2.21-2 (m, NH).

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MHz, CDCl3; δ, ppm): 170.47 (C=O of ester), 148 (CH of triazole ring), 124.31 (C of triazole

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ring), 101.96 (β-CD C1), 82 (β-CD C4), 78 ((β-CD C2), 73-72 (β-CD C3 and C5), 59.99 (triazole-

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H NMR (400 MHz, DMSO-d6, δ, ppm): 7.95 (m, 7H, triazole), 5.72 (m, OH-2, OH-3), 4.89-4.8

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C NMR (100

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CH2-O-), 51 (β-CD-CH2-triazole), 45 (O=C-CH2-CH2-NH and β-CD-CH2-NH-) and 39 (O=C-

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CH2-CH2-NH). FTIR (KBr, thin film; cm−1): 3382(str. OH), 2926 (str. C–H), 1738 (str. C=O

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ester), 1655 (str. triazole ring), 1036 (str. C–O).

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Scheme 1.

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2.8. Determination of β-CD in dendrimer

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The determination of content of β-CD in dendrimer (9) was similar to previous literature (Geok-

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Lay & Tucker, 1986; Xu, Li & Sun, 2010). Briefly, 25 mg of dehydrated β-CD dendrimer was

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refluxed in sulfuric acid (15 mL, 0.5 M) for 9 h at 100 °C, and then the solution was diluted to 50

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mL with sulfuric acid (0.5 M). A portion (1.25 mL) of the diluted solution was mixed with 5

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wt% phenol solution (0.75 mL), and then concentrated sulfuric acid (4.5 mL) was added. The

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absorbance of the obtained solution was measured by using a UV-visible spectrophotometer at

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490 nm (A490 nm). The glucose concentration (Cglucose) was calculated by a standard curve as

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follows:

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Cglucose = 0.301A490nm + 0.014

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The curve is linear over Cglucose = 0.01–0.5 mg mL−1(R=0.99). The content of β-CD dendrimer in

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dry state was calculated as follows:

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Where Mglucose is the molecular weight of glucose, mβ-CD-dendrimer is the weight of β-CD dendrimer

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(mg), Mβ-CD is the molecular weight of β-CD and 7 is the amount of glucose parts in the β-CD

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fragment.

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2.9. Solid Samples Preparation

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Solid inclusion complexes of MTX with β-CD-dendrimer (9) and β-CD were prepared by a

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common co-precipitation technique. Briefly, excess of MTX (10 mg) was added to 40 mL pure

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water (a small quantity of NH4OH (25%) was added to help the dissolution of MTX) containing 12

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various amount of β-CD (25 mg) and β-CD-dendrimer (9) (100 mg). The obtained mixtures were

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stirred at 37 °C for 24 h. After reaching equilibrium, the resultant solutions were centrifuged at

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1300 rpm for 25 min and filtered. The measurements were accomplished in triplicate. Physical

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mixtures of MTX and β-CD-dendrimer (9) were obtained by the same proportion with the

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components by carefully mixing it in a ceramic mortar.

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2.10. Drug Loading and in Vitro Release

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The encapsulation efficiency (EE) of the MTX-loaded β-CD and β-CD-dendrimer (9)

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nanocarrier were calculated by following way: Briefly, solid inclusion complex (20 mg) was

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taken into HCl (0.1 N, 5 mL) for 24 h, and the solid inclusion complex suspension was separated

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by centrifugation at 13,000 rpm for 25 min under dark conditions. Supernatant of centrifugation

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was analyzed for determine the loading content of MTX by UV spectrophotometer at 303 nm by

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the following equation:

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Drug encapsulation efficiency % = (Mass of drug loaded (mg))/(Mass of total initial drug (mg) )

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× 100

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Blank samples were prepared from β-CD and β-cyclodextrin dendrimer (9) without loaded MTX.

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All set samples were analyzed in triplicate.

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In vitro release of MTX-loaded β-CD and β-CD dendrimer (9) were evaluated by a dialysis

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technique (Wu, Wang & Que, 2006). First, MTX-loaded β-CD and β-CD dendrimer (9) were

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diluted by release moderate. Then, the resulting solutions (3 mL) were moved into a dialysis

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bags (molecular cut-off 2000) and then the dialysis bags were placed in 30 ml of dissolution

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medium with stirring at 100 rpm at 37±0.5 °C. Phosphate-buffered saline (PBS) solution pH =

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7.4 and sodium acetate buffer solution pH = 3 as release media were used to study the influence

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of pH on drug release. At definite time intervals, external solution was taken and replenished

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with equal volume of fresh buffer solution. The study of drug release was carried out for 72 h.

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The amount of released MTX into buffer solution was analyzed by UV spectrophotometer at 307

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(pH = 3) and 303 nm (pH = 7.4) respectively. The release experiments were performed in

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triplicate.

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2.11. Characterization

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2.11.1. Nuclear magnetic resonance spectroscopy (NMR)

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All NMR spectra were measured at 25 °C with a Bruker Avance spectrometer (400 MHz for 1H

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and 100 MHz for 13C) used in the Fourier transform mode. CDCl3, DMSO-d6 and D2O were used

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as the solvent. The data are described as chemical shift (δ, ppm). HOD (4.79 ppm) or DMSO-d6

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(2.5 ppm) was used as interior references.

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2.11.2. Fourier transform infrared spectroscopy (FTIR)

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The FTIR spectra of all samples were recorded on Bruker Tensor 27 Spectrophotometer by KBr

15

disk method. The spectra were recorded over the range of 4000–400 cm-1.

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2.11.3. Dynamic light scattering (DLS)

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DLS measurements were performed on a Nanotrac Wave from Microtrac instruments equipped

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with a 10 mW He-Ne laser (λ = 780 nm) at 25 °C and the scattering angle was kept at 170°. The

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results were studied in CONTIN style. Measurements were recorded in triplicate.

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2.11.4. Differential scanning calorimetry (DSC)

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DSC analyses of the different samples were recorded on a Linseis-L 63/45 DSC instrument.

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Thermal behaviors of the samples (5±0.05 mg) were investigated in aluminum crimped pans.

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The heating of samples were performed at the rate of 10 °C min-1 from 20 to 400 °C under argon

5

gas flow. A blank vacuum-packed pan was used as reference.

6

2.11.5. Scanning electron microscopy (SEM)

7

Surface morphology of β-CD-dendrimer, MTX, their complexes and physical mixtures were

8

studied by SEM (MIRA3 FEG-SEM, Tescan). The samples were attached onto carbon tabs

9

(double sided adhesive tape) stick to aluminum stumps. Specimens were covered with gold–

10

palladium (plasma deposition technique) with a BIO-RAD AC500. Pictures were achieved at an

11

excitation voltage of 15 kV.

12

2.11.6. UV-visible spectral analysis (UV-Vis)

13

UV-visible spectra were recorded using 1700 Shimadzu spectrophotometer with samples in a

14

quartz cell of path length 1 cm.

15

2.11.7. MTT assay and cell treatment

16

The in vitro cytotoxic effect of β-CD dendrimer, MTX, and their inclusion complex on human

17

breast cancer cells (T47D) was investigated by 72 h MTT tests. Concisely, T47D cultured in

18

RPMI-1640

19

penicillin/streptomycin solution at 37 oC in a wetted incubator with 5% CO2. Then 5000 cell/well

20

were cultured in a 96-well culture plate and incubated 24 h at 37 °C. The cells were incubated

21

with the MTX and inclusion complex at concentrations of 2-64 μM for 72 h. After incubation,

22

the moderate of all wells were removed, and MTT reagent (2 mg/mL in PBS) was added to all

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moderate

complemented

with

10%

fetal

bovine

serum

and

1%

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Page 15 of 30

well. The cells were further incubated in dark for 4.5 h at 37 °C, washed with PBS (100 μL of

2

0.1 M, pH 7.4) and monitored by adding of 150 μL DMSO. Then, the absorbance was measured

3

at 570 nm during 15-30 minutes in the presence of Sorensen’s’ glycine buffer. All test condition

4

was completed in quadruple. Cell viability (%) was expressed through the living cells (%)

5

relative to controls. An IC50 (inhibitory concentration) value was distinct as the drug

6

concentration of compound at which 50% cell growing reserved. Then the IC50 value was

7

considered by the Prism 6.0 software (Graphpad, San Diego, USA).

8

3. Results and discussion

9

The detailed synthetic path for β-CD dendrimer (9) was shown in Scheme 1. As shown, β-CD

10

dendrimer (9) on the primary face having β-CD in both core and periphery was synthesized by a

11

click reaction, wherein β-CD is combined together via primary surface by the convergent

12

technique. For the first step, per azido-β-CD on the primary face as a core molecules (β-CD-

13

(N3)7) (3) was synthesized; at the second step, alkyne-terminated β-CD (8) precursors were

14

prepared. At the last step, azido groups were grafted to the junction points of alkyne-terminated

15

β-CD group via click reaction to produce β-CD dendrimer (9) on the primary face.

16

3.1. Preparation of per azide-functionalized β-CD precursor (3)

17

To prepare definite dendrimer, it is important to select appropriate multifunctional core that can

18

competently link with suitable moiety polymer precursors. β-CD-(I)7 (2) was synthesized by

19

selective reaction of seven hydroxyl groups of β-CD on the primary face by iodine (Scheme 1).

20

The following treatment with NaN3 in DMF directed to the preparation of β-CD-(N3)7 (3)

21

(Ashton, Königer, Stoddart, Alker & Harding, 1996). The study of β-CD-(N3)7 by 1HNMR in

22

DMSO-d6 showed the thorough vanishing of hydroxyl proton (6-CH2OH) signal at 4.5 ppm,

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Page 16 of 30

which is existent in β-CD (see supplementary data, Fig. S1). FTIR analysis (Fig. 1) of β-CD-

2

(N3)7 showed the presence of new absorbance azide peak at 2108 cm-1. The obtained results

3

established that the hydroxyl groups on the primary face of β-CD were selectively substituted by

4

azide groups fully.

5

3.2. Preparation of mono-functionalized alkyne-terminated β-CD precursor (8)

6

The mono-functionalized alkyne-terminated β-CD precursor’s (8) was prepared by a multistep

7

reactions method, as shown in Scheme 1. The synthetic procedure to fabricate of mono-

8

functionalized alkyne-terminated β-CD (8) is as follows: β-alanine propargyl ester hydrochloride

9

(6) was obtained via the reaction of propargyl alcohol and β-alanine in the existence of

10

trimethylsilyl chloride. Then monofunctionalized β-CD derivative (7) (β-CD-OTs) was

11

synthesized and changed to (8) by replacement of the tosyl moiety with β-alanine propargyl ester

12

group by nucleophilic substitution (Scheme 1) in the presence of Et3N. FTIR, 1H and

13

analysis were used to endorse the structure and purity of compound 8 (see supplementary data,

14

Fig. S2 and S3). Matching the spectra of 6 and 7, the vanishing of the peaks at 1601, 1414 and

15

1158 cm−1 (consistent to the aromatic section and O=S=O), appearance of the peaks at 2128

16

cm-1(consistent to the alkyne section) and 1724 cm−1 (consistent to the ester section) shows that

17

all these groups had reacted and were removed in workup.

18

3.3. Click reaction between β-CD-(N3)7 and alkyne-terminated β-CD (9)

19

β-CD dendrimer (9) on the primary face was prepared by Cu(I)-catalyzed click reaction between

20

β-CD-(N3)7 (3) and alkyne-terminated β-CD (8). 1H and

21

the structure and purity of (9). After the click reaction NMR analysis exhibited disappearance of

22

the terminal alkyne proton and corresponding appearance of a triazole proton at 7.8-8.1 ppm.

13

C NMR

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C NMR study were used to approve

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Page 17 of 30

The presence of the triazole proton around 8.0 ppm in the 1H NMR spectra was indicated the

2

formation of the β-CD dendrimer (9) completely (see supplementary data, Fig. S4). The integral

3

ratio of triazole proton to anomeric proton is about 1:7.9, which shows the successful “click”

4

reaction between β-CD-(N3)7 and alkyne-terminated β-CD (Scheme 1). 13C NMR analyses of (9)

5

showed the appearance of signals at 148, 124 ppm for carbon of triazole ring and 170.47 ppm,

6

which can be ascribed to carbonyl of ester (see supplementary data, Fig. S5). The synthetic

7

process of β-CD dendrimer (9) can be also monitored by FTIR spectra, as shown in Fig. 1. A

8

comparison to those of β-CD-(N3)7 and β-CD dendrimer (9) (Fig. 1) discovered the complete

9

disappearance of azide peak at 2108 cm-1 and 2128 cm-1 of alkyne peak for β-CD dendrimer (9).

10

Disappearance of azide and alkyne signals in the FTIR spectra of β-CD dendrimer (9), revealing

11

that the remaining end-capping substance has been removed completely from the last compound.

12

Also, appearance of signals related to five-membered triazole ring at 1653 cm-1 confirmed a

13

successful coupling reaction.

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Page 18 of 30

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Fig. 1.

M

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

4

3.4.1. FTIR study of complex

5

Fig. 1 shows FTIR spectra of β-CD dendrimer (9), MTX, physical mixtures and respective

6

inclusion complexes. The inclusion complex formation of β-CD dendrimer (9) and a guest drug

7

was confirmed by the FTIR spectra. β-CD dendrimer (9) showed 3412 cm-1 (for OH stretching),

8

2927 cm-1 (C–H stretching vibration), 1032 and 1135 cm-1 (O–C–O stretching) and at 1658

9

correspond to H–O–H bending. The FTIR spectrum of MTX (Fig. 1) displayed distinctive peaks

10

at 1703 cm-1 (for C=O stretching vibrations for carboxylic acid groups), 1655 cm-1

11

(corresponding to C=O stretching vibration of amide groups) and C–O stretch at 1097 and 1203

12

cm-1. The aromatic C=C stretch in 1455, 1500 and 1602 cm-1 was observed. The physical

13

mixture spectrum showed approximate mixed spectra of MTX and β-CD dendrimer. All the

14

absorption peaks of MTX were enclosed by that β-CD dendrimer (9) in the FTIR spectrum of 19

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Page 19 of 30

1

inclusion complex. The changes between the supramolecular inclusion complex and physical

2

mixture show that MTX was complexes into the cavity of β-CD dendrimer to form the inclusion

3

complex.

4

1.

5

The amount of β-CD in the β-CD-dendrimer (9) was calculated by the method of Koh and

6

Tucker (Geok-Lay & Tucker, 1986; Xu, Li & Sun, 2010) and the data disclose that CD

7

dendrimer contains 90.14 %β-CD in its construction, which is comparable to the real quantity

8

(88.64%).

9

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3.4.2. Determination of β-CD in β-CD-dendrimer

3.4.3. SEM analysis

The powdered forms of MTX, β-CD dendrimer (9), their physical mixture and inclusion

11

complexes were investigated by SEM analysis (Fig. 2). As shown in SEM images (i) β-CD

12

dendrimer is present in a globular morphology with cavity structures, (ii) MTX is present in

13

amorphous and crystalline states (iii) the structures of inclusion complexes appeared as

14

amorphous complexes and not crystals which is an evidence for the formation of inclusion

15

complexes. Otherwise, in SEM image the crystal photographs related to MTX and β-CD

16

dendrimer in the inclusion complex was not observed (iv) their physical mixtures presented

17

particles of MTX surrounded with β-CD dendrimer nanoparticles. The different constructions of

18

pure MTX, β-CD dendrimer (9) and their inclusion complex confirmed that MTX is comprised

19

into β-CD cavity of dendrimer (9) or dendritic network. The outcomes show that the successful

20

preparation of inclusion complex of β-CD-dendrimer (9) with MTX. From SEM observations it

21

seems that with formation of CD dendrimer a new cavity with average size diameter of 70 nm is

22

formed. In the case of SEM photograph for dendrimers with larger cavity ≥100 nm probably the

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Page 20 of 30

aggregation or big clusters of mixture of many different shapes and size ranges of particles is

2

happened.

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3

Fig. 2.

pt

4 3.

3.4.4. DLS analysis

6

To evaluate the average hydrodynamic diameter of β-CD dendrimer (9) and its supramolecular

7

complex with MTX, DLS analyses were performed in water (Fig. 3). In relation to DLS results,

8

an average hydrodynamic diameter of 57 nm for pure β-CD dendrimer (9) and 78 nm for

9

supramolecular complex was found. The conversion in size is due to the formation inclusion

10

complex at the cavity of dendrimer and cyclodextrin moiety. The size obtained with SEM is

11

smaller than the results achieved from DLS. This is related to the SEM images measured the

12

particle size in solid state, whereas DLS in a solution.

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1 2

Fig. 3. 4.

3.4.5. DSC analysis

4

DSC analyses were carried out to study the physical state of the MTX in the inclusion complex.

5

DSC curves of β-CD dendrimer (9), MTX, physical mixture and their inclusion complex were

6

showed in Fig. 4. In DSC curves of MTX, the water loss happened in the 122.9 °C and the

7

melting peak at 180.9 °C. The curves of β-CD dendrimer (9) displayed endothermic peak at

8

138.8 °C and 319.6 °C matching to their loss of water and melting points, respectively. The DSC

9

curve of the physical mixture showed both typical signals of MTX at 185 °C with a slight shift

10

and β-CD dendrimer (9) at 326.4 °C. In the inclusion complex, the MTX melting point of curve

11

signal was fully vanished. The peaks at 235-258 °C have been cited by other authors in the range

12

of 210–240 °C for β-CD (Giordano, Novak & Moyano, 2001). The appearance of this peak

13

remains unclear, but seems to be related with crystal hydration through the preparation of β-CD

14

(Giordano, Novak & Moyano, 2001). This obviously recommended the actual formation of a

15

supramolecular inclusion complex between MTX and β-CD dendrimer (9).

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Page 22 of 30

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1

Fig. 4.

M

2 3.5. MTT assay

4

Fig. 5 shows the in vitro cytotoxic effect of β-CD dendrimer, MTX and their inclusion complex

5

with various concentrations of inclusion complex on T47D cells. Data analysis of MTT test

6

exhibited that IC50 of MTX and inclusion complex of β-CD dendrimer (9):MTX on T47D cells

7

was 7.4 and 4.9 µM for 72h MTT assays, respectively. These results display the cytotoxic effect

8

of inclusion complex of β-CD dendrimer (9):MTX on T47D cells was considerably greater than

9

that of free MTX. Also, β-CD dendrimer had a clearly low toxicity. The cell viability was higher

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than 90% even if β-CD dendrimer concentration extended as high as 32 µM.

23

Page 23 of 30

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1 Fig. 5.

3

3.6. Drug loading and in vitro drug release study

4

Drug encapsulation efficiency of β-CD dendrimer (9) and β-CD were estimated by UV

5

spectrophotometric technique and were found to be 79.8 and 52%, respectively. From the results

6

achieved by drug encapsulation efficiency of MTX with β-CD and β-CD dendrimer (9)

7

(comparison of β-CD with β-CD dendrimer (9)), it seemed that approximately 52% MTX may be

8

encapsulated in the cavity of β-CD and 27.8% MTX was entrapped in the dendrimer networks.

9

The EE of β-CD dendrimer (9) significantly improved compare with β-CD. High loading amount

10

of β-CD dendrimer (9) could be attributed to the inclusion of β-CD cavity to drug and

11

entrapment of MTX with dendritic network. Release profiles of the pure MTX (pH 7.4) and

12

MTX from the MTX/β-CD dendrimer and β-CD at pH 3 and pH 7.4 are shown in Fig. 6. The

13

results showed that the free MTX, complete diffusion was occurred within 8 h, but for β-CD and

14

β-CD dendrimer (9), a prolonged and slow release for 23 and 72 h were observed, respectively.

15

The in vitro cumulative release profiles of MTX from β-CD dendrimer (9) (Fig. 6) shows that

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Page 24 of 30

about 22 % of the drug was released in the burst release phase (after 4 h). After the primary

2

burst, sustained release up to 72 h was related to drugs diffusion and the dendrimer degradation.

3

The initial burst release may be perhaps caused by the drug attached on the dendrimer surface,

4

since the MTX might form hydrogen bonds with the hydroxyl groups of dendrimer. Sustained

5

release was achieved due to drug diffusion from the CD cavity and dendrimeric network. Also,

6

β-CD dendrimer (9) was showed to have a more controlled release behavior for the loaded MTX

7

when compared to β-CD. As shown in Fig. 6 the MTX release from nanocarrier was noticeably

8

pH dependent. The cumulative release profiles after initial burst show that almost 68 and 57% of

9

MTX was released at pH 3 and pH 7.4, respectively. These results can be because of the lower

10

solubility of the MTX/β-CD in pH 7.4 than in pH 3. It has been described that β-CD could

11

increase the release of hydrophobic drugs or dissolution profiles in acidic conditions (Stojanov &

12

Larsen, 2011). These finding showed that the β-CD dendrimer (9) nanocarrier could be a suitable

13

controlled drug delivery system for cancer treatment.

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Fig. 6. 25

Page 25 of 30

4. Conclusion

2

The new glycodendrimer from β-CD in core and branches were synthesized through click

3

reaction with excellent coverage. The structure of β-CD dendrimer (9) was characterized and

4

confirmed with FTIR, NMR and DLS techniques. The obtained dendrimer showed the

5

remarkable encapsulation efficiency, 79.8% for MTX as model drug. The inclusion complex

6

structure of prepared dendrimer and MTX was determined by SEM, DLS, DSC and FTIR

7

techniques. The in vitro toxicity of the prepared compound and their inclusion complex with

8

MTX by the MTT test on T47D cells showed that the resultant dendrimer was not cytotoxic to

9

the cell line considered. The construction of the synthesized β-CD-dendrimer permitted two

10

kinds of potential entrapment places for the drug: in CD cavities and dendritic network. The in

11

vitro drug release results in buffer solution showed that the β-CD dendrimer nanocarrier could be

12

a suitable controlled drug delivery system for cancer treatment.

13

ACKNOWLEDGEMENTS

14

Authors gratefully acknowledge the financial support from the University of Tabriz and RCPN

15

of Tabriz University of Medical Science.

16

References

17 18 19 20 21 22 23 24 25

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Wu, W., Wang, Y., & Que, L. (2006). Enhanced bioavailability of silymarin by selfmicroemulsifying drug delivery system. Eur J Pharm Biopharm, 63(3), 288-294. Xu, J., Li, X., & Sun, F. (2010). Cyclodextrin-containing hydrogels for contact lenses as a platform for drug incorporation and release. Acta Biomaterialia, 6(2), 486-493. Zhang, Z.-X., Liu, K. L., & Li, J. (2011). Self-Assembly and Micellization of a Dual Thermoresponsive Supramolecular Pseudo-Block Copolymer. Macromolecules, 44(5), 11821193. Zhang, Z.-X., Liu, K. L., & Li, J. (2013). A Thermoresponsive Hydrogel Formed from a Star– Star Supramolecular Architecture. Angewandte Chemie International Edition, 52(24), 61806184. Zhao, F., Yin, H., Zhang, Z., & Li, J. (2013). Folic acid modified cationic gamma-cyclodextrinoligoethylenimine star polymer with bioreducible disulfide linker for efficient targeted gene delivery. Biomacromolecules, 14(2), 476-484.

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Captions of Figures and Scheme

2

Scheme 1.Synthetic routes employed for the synthesis of β-CD-based dendrimer (9) via ‘‘click’’

3

reaction.

4 5

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

6 7

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

8 9

Fig. 3. DLS measurement of β-CD dendrimer (9) and their inclusion complex with MTX in aqueous solution.

10 11

Fig. 4. DSC curves of (a) MTX; (b) physical mixture; (c) β-CD dendrimer (9) and (d) MTX/βCD dendrimer inclusion complex.

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Fig. 5. In vitro cytotoxicity of β-CD dendrimer (9), MTX and inclusion complex of β-CD

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dendrimer:MTX measured by the MTT test against T47D cells. (Mean ± SD; n = 3)

14 15 16

Fig. 6. Cumulative drug release (%) of MTX from β-CD and MTX:β-CD dendrimer nanoparticles in buffer solution (pH 3 and 7.4) and pure MTX in pH = 7.4 at 37 °C. (Mean ± SD; n=3)

19 20 21 22 23 24

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Ac ce

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