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

Article abstract of DOI:10.1016/j.carbpol.2015.05.087

Abstract The objective of this study was the syntheses of well-defined glycodendrimer with entrapment efficiency by click reactions, with β-cyclodextrins (β-CDs) moiety to keep the biocompatibility properties, besides especially increase their capacity to

Full text of DOI:10.1016/j.carbpol.2015.05.087

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:
S0144-8617(15)00556-1
DOI:
Reference:
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
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Highlights:
 Novel glycodendrimer from β-CD in core and branches was synthesized.
 Supramolecular inclusion complex by dendrimer and MTX drug was prepared.
 MTT assay show that the resultant dendrimer is not cytotoxic to the cells.
 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
Applicable as Drug Nanocarrier
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Toomari Yousef, Namazi Hassan , Entezami Ali Akbar
Laboratory of Dendrimers and Nano-Biopolymers, Faculty of Chemistry, University of Tabriz,
Tabriz, Iran; Tel.: +98 413393121, Fax: +98 41 334 0191, E-mail address: namazi@tabrizu.ac.ir
Research Center for Pharmaceutical Nanotechnology (RCPN), Tabriz University of Medical
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
efficiency by click reactions, with β-cyclodextrins (β-CDs) moiety to keep the biocompatibility
properties, besides especially increase their capacity to load numerous appropriate sized guests.
The original dendrimer containing β-CD in both periphery and central was synthesized using
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 H NMR,
C
NMR and FTIR and its inclusion complex structure were investigated by SEM, DLS, DSC and
FTIR techniques. The cytotoxic effect of obtained compound and its inclusion complex with
MTX was analyzed using MTT test. The MTT test exhibited that the synthesized compound was
not cytotoxic to the cell line considered. The in vitro drug release study turned out that the
obtained β-CD dendrimer could be a suitable controlled drug delivery system for cancer
treatment.
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Keywords: β-cyclodextrin; dendrimer; nanocarrier; methotrexate; inclusion complex
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1. Introduction
Dendrimers are a class of polymeric materials having three-dimensional hyperbranched
macromolecules, monodispersed, defined spherical construction, nanoscopic objects with
amount of reactive periphery groups and host-guest entrapment properties (Shen, Li, Wu, Zhang
& Li, 2015; Tomalia et al., 1985). In this work we emphasis on dendrimers, these materials
firstly reported by Vögtle and co-workers as cascade molecules (Buhleier, Wehner & Vögtle,
1978) and followed by Tomalia et al. (Tomalia et al., 1985) named starburst dendrimers, and
Newkome et al. (Newkome, Yao, Baker & Gupta, 1985) as ‘arborols’. Since then, the study of
these materials, has been expands exponentially to all areas (Hawker & Fréchet, 1990; Namazi &
Adeli, 2003; Namazi & Adeli, 2005a; Namazi & Adeli, 2005b). Because of unique structures and
properties, dendrimers have concerned greatly attention for their uses in several fields. Among
them the usage of these compounds as drug delivery systems (DDS) has been of excessive
attention (Lai et al., 2007; Leng et al., 2013; Namazi & Toomari, 2011; Sun, Fan, Wang & Zhao,
2012; Tomalia & Fréchet, 2002).
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β-CD essentially biocompatible compound (Ortiz Mellet, Defaye & Garcia Fernandez, 2002),
and is one of the good candidates 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 been prepared (Hawker, Bosman & Harth, 2001). The β-CD
molecule is a torus-shaped oligosaccharide that consist of 7 glucose units connected through α-
1,4-glucosidic links (Namazi & Kanani, 2007, 2009; Saenger et al., 1998). The narrower and
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
hydrophilic. Because of these structures, β-CDs are known to form inclusion complexes with
hydrophobic molecules having the suitable dimension 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 of significance (Varghese, Al-
Busafi, Suliman & Al-Kindy, 2015).
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‘Glycodendrimer’ term is applied to define dendrimers that include carbohydrates in their
constructions (Chabre, Contino-Pepin, Placide, Shiao & Roy, 2008; Chabre & Roy, 2010).
Glycodendrimers having CD moiety in their assemblies are termed CD-dendrimers. Depending
on the core and branches groups, CD dendrimers can be separated into three chief categories.
The first categories (CD-based dendrimers), CDs are located on the periphery of dendrimers
(Adeli, Kalantari, Zarnega & Kabiri, 2012; Menuel, Duval, Cuc, Mutzenhardt & Marsura, 2007;
Menuel, Fontanay, Clarot, Duval, Diez & Marsura, 2008). In the second categories (CD-centered
dendrimers), a CD part as the central wherever all outlets (in primary face) are linked
(Newkome, Godinez & Moorefield, 1998). The third categories (CD-dendrimer conjugates),
dendrimers conjugated with CD moiety (Baussanne, Benito, Mellet, Garcia Fernandez, Law &
Defaye, 2000; Benito, Gomez-Garcia, Ortiz Mellet, Baussanne, Defaye & Garcia Fernandez,
2004; Wang, Shao, Qiao & Cheng, 2012). By the combination of CDs with dendrimer, the
positive properties of dendrimer and CDs improved (Menuel, Fontanay, Clarot, Duval, Diez &
Marsura, 2008).
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In this work, we report the preparation of a family of glycodendrimers having on β-CD scaffolds
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-
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
β-CD (98%, Merck) was achieved and used after desiccating under vacuum. p-Toluenesulfonyl
chloride (p-TsCl, Merck) was purified by chloroform and petroleum ether. Propargyl alcohol
(99%), methotrexate (MTX, 99%), MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium
4 3
bromide), copper sulfate (CuSO ; 99.99%), iodine (99.8%), triphenylphosphine (PPh ; 99%),
dimethyl sulfoxide (DMSO; 99.9%) and acetonitrile (99.8%) were purchased from Sigma
Aldrich and used as received. N,N-Dimethylformamide (DMF; 99.8%, Merck) was dried over
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1
1
CaH
2
and distilled under vacuum. Triethylamine (Et
3
N; Merck) was dried over CaH
2
and then
, 99%)
distilled. Acetone (99.8%), methanol (99.8%), diethyl ether (99.5%), sodium azide (NaN
3
and L-ascorbic acid sodium salt (99%) were obtained from Merck and used as received. Dialysis
tubing (benzoylated, molecular cut-off 2000 Da) was purchased from Sigma-Aldrich. T47D, a
human breast cancer cells were achieved from Pasteur Institute Cell Bank of Iran (Tehran, Iran)
and cultured in RPMI-1640 moderate complemented with 10% fetal bovine serum and 1%
o
penicillin/streptomycin solution at 37 C in a wetted incubator with 5% CO
2
. All the solvents
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)
To a solution of Ph P (2.07 g, 7.9 mmol) under stirring in dry DMF (9 mL), I
3
2
(2.09 g, 8.26
mmol) was added over 30 min. After the addition of I
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2
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
mmol) was then added, and the reaction mixture was stirred at 70 °C under argon atmosphere for
18 h. It was then concentrated through the elimination of DMF (approximately 5 mL) under
vacuum and the pH adjusted to 9-10 via the addition of NaOMe in MeOH (3 M, 3 mL) under an
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) (0.75
g, 89%) was obtained as a white powder after drying under high vacuum (Ashton, Königer,
Stoddart, Alker & Harding, 1996; Benkhaled, Cheradame, Fichet, Teyssié, Buchmann &
1
Guégan, 2008). M.p.: 222 °C (dec.). H NMR (400 MHz, DMSO-d
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; 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-(N
To a solution of β-CD-(I) (2) (0.75 g, 0.39 mmol) in anhydrous DMF (12 mL) at room
temperature were added NaN (0.25 g, 3.86 mmol). The reaction mixture was stirred at 80 °C
3 7
) ) (3)
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3
under argon atmosphere for 24 h. The suspension was concentrated under vacuum to 1/3 of the
starting volume before a large additional of water was added. After filtration, the residue was
washed with water and dried under high vacuum. β-CD-(N
3
)
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(3) (0.48 g, 94%) was obtained as
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a white powder. (Ashton, Königer, Stoddart, Alker & Harding, 1996). H NMR (400 MHz,
DMSO-d
3.35-3.32 (H-2, H-4, H-6a). FTIR (KBr, thin film; cm ): 3360 (str. OH), 2924 and 2856 (str.
CH), 2108 (str. N ), 1155–1049 (str. C–O).
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; 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),
-1
3
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2.4. Synthesis of 6-mono (p-toluenesulfonyl)-β-cyclodextrin (β-CD-OTs) (7)
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
water (165 mL) and sodium hydroxide (2.2 g, 55 mmol) in water (55 mL) were sequentially
added. After 10 min, p-toluenesulfonyl chloride (3.3 g, 17.4 mmol) in acetonitrile (22 mL) was
added drop by drop during 1 h. The reaction mixture was stirred for 4 h at ambient temperature,
and then neutralized (pH=12.5) with hydrochloric acid, the salts were eliminated by filtering and
the volume of solution was decreased to 2/3 of its initial volume by lyophilization. The β-CD-
OTs was crystallized (three times) by dissolving in boiling water and washed with acetone (2×7
mL), ether (2×9 mL) and dried. After recrystallizations, pure β-CD-OTs (1.34 g, 46%) was
achieved (Baussanne, Benito, Mellet, Garcia Fernandez, Law & Defaye, 2000).
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β-CD-OTs: M.p.: 182–183 C (dec.). H NMR (400 MHz, DMSO-d , δ, ppm): 7.75-7.73 (d, 2H,
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,
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)
−
1
ppm. FTIR (KBr, thin film; cm ): 3382(str. OH), 2927 (str. C–H), 1645 (str. C–C), 1601 (str.
C=C aromatic), 1414 (str. SO , asym.), 1158 (str. SO , sym.), 1027 (str. C–O).
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2.5. Synthesis of β-Alanine propargyl ester (6)
β-Alanine (2 g, 22.5 mmol) were stirred in freshly distilled trimethylsilyl chloride (4.89 mL, 45
mmol) in a flask followed by adding propargyl alcohol (20 mL) at room temperature for 15 h
under argon atmosphere. After the accomplishment of reaction (as checked by TLC), the reaction
solvent removed by rotary evaporator and the remaining material dissolved in a minimum
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
95% (3.49 g).
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H NMR (400 MHz, CDCl
-CO), 2.79-2.76 (CH -NH
77 (HC≡C), 54 (CH -O), 36 (CH
3
; δ, ppm): 4.7-4.65 (d, CH
2
-O), 3.23-3.2 (H-alkyne), 2.86-2.84 (t,
13
CH
2
2
2
). C NMR (100 MHz, CDCl3; δ, ppm): 173 (C=O), 78 (H C≡C),
-1
2
2
-C=O), 32 (CH
2
-NH
2
). FTIR (KBr, thin film; cm ): 3245 (str.
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)
In order to synthesize alkyne-terminated β-CD, β-Alanine propargyl ester hydrochloride (6)
(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 Et N (0.1 g, 0.36 mmol) were dissolved in dry DMF (5 mL) at 0 °C with stirring for 2 h
under argon atmosphere, then the reaction temperature increased from 0 °C to rt. After
maintaining the reaction temperature for 24 h in room temperature was increased to 35 °C for 12
h. The solvent was removed under vacuum and the precipitate was decanted into acetone (15
mL). The crude product after precipitating into acetone was prepared as a brown solid. For the
further purification, the product was purified by repeated recrystallization from water and
ethanol to give the final product as brown powders. The yield of β-Alanine propargyl ester
modified β-CD (8) was 65% (0.3 g).
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H NMR (400 MHz, DMSO-d
-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), 3.09-3.04 (m, β-CD-CH -NH), 2.96-2.93 (t, NH-CH -CH -C=O), 2.51-2.5 (DMSO-d ),
2.5-2.45 (NH-CH -CH -C=O), 2.21-2.2 (m, -NH). C NMR (100 MHz, CDCl3; δ, ppm): 170.4
(C=O of ester), 101.96 (β-CD C ), 82 (β-CD C ), 78 (O-CH -C≡C), 73.3-73 (β-CD C and C ),
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, δ, ppm): 5.76-5.66 (m, OH-2, OH-3), 4.88-4.82 (d, H-1 and H-
1′), 4.68 (O-CH
2
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2
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72 (β-CD C
2
and O-CH
2
-C≡C), 60.5 (β-CD C
6 2 2 2
), 53 (O-CH -C≡C), 51 (NH-CH -CH -C=O), 45
-1
(β-CD-CH -NH-) and 35 (NH-CH
2
2
-CH -C=O). FTIR (KBr, thin film; cm ): 3423 (str. OH),
2
3245 (str. H-C≡C), 2924 and 2856 (str. CH), 2126 (str. C≡C), 1740 (str. C=O), 1080–1052 (str.
C–O).
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2.7. Click reaction between β-CD-(N
β-CD dendrimer (9) was prepared by click reaction between (β-CD-(N
functionalized alkyne-terminated β-CD (8). The synthesis method is described as follows. (β-
CD-(N ) (3) (0.10 g, 0.076 mmol) was dissolved in t-butyl alcohol/H O mixture (12 mL, 1:1,
v/v), and mono alkyne-terminated β-CD (8) (1.33 g, 1.06 mmol, 2 equiv. per azide),
CuSO .5H O (2.66 mg, 0.01 mmol, 0.02 equiv. per azide groups), and sodium ascorbate (2.1 mg,
3 7
) and alkyne-terminated β-CD (9)
3 7
) ) (3) and mono
3
)
7
2
1
1
1
1
1
1
1
4
2
0.01 mmol, 0.02 equiv. per azide groups) were added into the solution. The brown solution was
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 NH OH was added until pH reached 7. After filtration, the
solvent was removed under reduced pressure, and the brown solid was obtained under vacuum.
The resulting crude product was purified by chromatography on a sephadex G-50 column using
deionized water as eluent. Finally, the yield of (9) was 53% (0.4 g).
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H NMR (400 MHz, DMSO-d
6
, δ, ppm): 7.95 (m, 7H, triazole), 5.72 (m, OH-2, OH-3), 4.89-4.8
-O), 4.5 (m, OH-6), 3.92-3.86 (m, β-CD-CH -triazole),
3.63-3.3 (m, H-3, H-4, H-5, H-2 and H-6), 2.99-2.89 (t, O=C-CH -CH -NH-), 2.78-2.68 (t, O=C-
CH -CH -NH-), 2.51-2.41 (m, NH-CH -C=O and DMSO-d ), 2.21-2 (m, NH). C NMR (100
MHz, CDCl3; δ, ppm): 170.47 (C=O of ester), 148 (CH of triazole ring), 124.31 (C of triazole
(m, H-1 and H-1′of β-CD and tiazole-CH
2
2
2
2
1
3
2
2
2
6
ring), 101.96 (β-CD C
1
), 82 (β-CD C
4
2 3 5
), 78 ((β-CD C ), 73-72 (β-CD C and C ), 59.99 (triazole-
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CH
2
2
-O-), 51 (β-CD-CH
2 2 2 2
-triazole), 45 (O=C-CH -CH -NH and β-CD-CH -NH-) and 39 (O=C-
−
1
CH
-CH -NH). FTIR (KBr, thin film; cm ): 3382(str. OH), 2926 (str. C–H), 1738 (str. C=O
2
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
The determination of content of β-CD in dendrimer (9) was similar to previous literature (Geok-
Lay & Tucker, 1986; Xu, Li & Sun, 2010). Briefly, 25 mg of dehydrated β-CD dendrimer was
refluxed in sulfuric acid (15 mL, 0.5 M) for 9 h at 100 °C, and then the solution was diluted to 50
mL with sulfuric acid (0.5 M). A portion (1.25 mL) of the diluted solution was mixed with 5
wt% phenol solution (0.75 mL), and then concentrated sulfuric acid (4.5 mL) was added. The
absorbance of the obtained solution was measured by using a UV-visible spectrophotometer at
490 nm (A490 nm). The glucose concentration (Cglucose) was calculated by a standard curve as
follows:
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Cglucose = 0.301A490nm + 0.014
−
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1
1
1
2
The curve is linear over Cglucose = 0.01–0.5 mg mL (R=0.99). The content of β-CD dendrimer in
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
(mg), Mβ-CD is the molecular weight of β-CD and 7 is the amount of glucose parts in the β-CD
fragment.
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2.9. Solid Samples Preparation
Solid inclusion complexes of MTX with β-CD-dendrimer (9) and β-CD were prepared by a
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 NH OH (25%) was added to help the dissolution of MTX) containing
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various amount of β-CD (25 mg) and β-CD-dendrimer (9) (100 mg). The obtained mixtures were
stirred at 37 °C for 24 h. After reaching equilibrium, the resultant solutions were centrifuged at
1300 rpm for 25 min and filtered. The measurements were accomplished in triplicate. Physical
mixtures of MTX and β-CD-dendrimer (9) were obtained by the same proportion with the
components by carefully mixing it in a ceramic mortar.
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2.10. Drug Loading and in Vitro Release
The encapsulation efficiency (EE) of the MTX-loaded β-CD and β-CD-dendrimer (9)
nanocarrier were calculated by following way: Briefly, solid inclusion complex (20 mg) was
taken into HCl (0.1 N, 5 mL) for 24 h, and the solid inclusion complex suspension was separated
by centrifugation at 13,000 rpm for 25 min under dark conditions. Supernatant of centrifugation
was analyzed for determine the loading content of MTX by UV spectrophotometer at 303 nm by
the following equation:
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1
1
1
1
3
4
Drug encapsulation efficiency % = (Mass of drug loaded (mg))/(Mass of total initial drug (mg) )
× 100
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1
5
6
Blank samples were prepared from β-CD and β-cyclodextrin dendrimer (9) without loaded MTX.
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
technique (Wu, Wang & Que, 2006). First, MTX-loaded β-CD and β-CD dendrimer (9) were
diluted by release moderate. Then, the resulting solutions (3 mL) were moved into a dialysis
bags (molecular cut-off 2000) and then the dialysis bags were placed in 30 ml of dissolution
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
of pH on drug release. At definite time intervals, external solution was taken and replenished
with equal volume of fresh buffer solution. The study of drug release was carried out for 72 h.
The amount of released MTX into buffer solution was analyzed by UV spectrophotometer at 307
(pH = 3) and 303 nm (pH = 7.4) respectively. The release experiments were performed in
triplicate.
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2.11. Characterization
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2
2.11.1. Nuclear magnetic resonance spectroscopy (NMR)
1
All NMR spectra were measured at 25 °C with a Bruker Avance spectrometer (400 MHz for H
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1
1
1
3 6 2
and 100 MHz for C) used in the Fourier transform mode. CDCl , DMSO-d and D O were used
as the solvent. The data are described as chemical shift (δ, ppm). HOD (4.79 ppm) or DMSO-d
6
(2.5 ppm) was used as interior references.
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15
2.11.2. Fourier transform infrared spectroscopy (FTIR)
The FTIR spectra of all samples were recorded on Bruker Tensor 27 Spectrophotometer by KBr
-1
disk method. The spectra were recorded over the range of 4000–400 cm .
16
17
18
19
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 (λ = 780 nm) at 25 °C and the scattering angle was kept at 170°. The
results were studied in CONTIN style. Measurements were recorded in triplicate.
14
Page 14 of 30

1
2
3
4
5
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.
-1
The heating of samples were performed at the rate of 10 °C min from 20 to 400 °C under argon
gas flow. A blank vacuum-packed pan was used as reference.
6
7
8
9
0
1
2.11.5. Scanning electron microscopy (SEM)
Surface morphology of β-CD-dendrimer, MTX, their complexes and physical mixtures were
studied by SEM (MIRA3 FEG-SEM, Tescan). The samples were attached onto carbon tabs
(double sided adhesive tape) stick to aluminum stumps. Specimens were covered with gold–
palladium (plasma deposition technique) with a BIO-RAD AC500. Pictures were achieved at an
excitation voltage of 15 kV.
1
1
1
1
1
2
3
4
2.11.6. UV-visible spectral analysis (UV-Vis)
UV-visible spectra were recorded using 1700 Shimadzu spectrophotometer with samples in a
quartz cell of path length 1 cm.
15
16
17
18
19
20
21
22
2.11.7. MTT assay and cell treatment
The in vitro cytotoxic effect of β-CD dendrimer, MTX, and their inclusion complex on human
breast cancer cells (T47D) was investigated by 72 h MTT tests. Concisely, T47D cultured in
RPMI-1640 moderate complemented with 10% fetal bovine serum and 1%
o
penicillin/streptomycin solution at 37 C in a wetted incubator with 5% CO
2
. Then 5000 cell/well
were cultured in a 96-well culture plate and incubated 24 h at 37 °C. The cells were incubated
with the MTX and inclusion complex at concentrations of 2-64 μM for 72 h. After incubation,
the moderate of all wells were removed, and MTT reagent (2 mg/mL in PBS) was added to all
15
Page 15 of 30

1
2
3
4
5
6
7
well. The cells were further incubated in dark for 4.5 h at 37 °C, washed with PBS (100 μL of
0.1 M, pH 7.4) and monitored by adding of 150 μL DMSO. Then, the absorbance was measured
at 570 nm during 15-30 minutes in the presence of Sorensen’s’ glycine buffer. All test condition
was completed in quadruple. Cell viability (%) was expressed through the living cells (%)
relative to controls. An IC50 (inhibitory concentration) value was distinct as the drug
concentration of compound at which 50% cell growing reserved. Then the IC50 value was
considered by the Prism 6.0 software (Graphpad, San Diego, USA).
8
9
0
1
2
3
4
5
3. Results and discussion
The detailed synthetic path for β-CD dendrimer (9) was shown in Scheme 1. As shown, β-CD
dendrimer (9) on the primary face having β-CD in both core and periphery was synthesized by a
click reaction, wherein β-CD is combined together via primary surface by the convergent
technique. For the first step, per azido-β-CD on the primary face as a core molecules (β-CD-
1
1
1
1
1
1
3 7
(N ) ) (3) was synthesized; at the second step, alkyne-terminated β-CD (8) precursors were
prepared. At the last step, azido groups were grafted to the junction points of alkyne-terminated
β-CD group via click reaction to produce β-CD dendrimer (9) on the primary face.
16
17
18
19
20
21
22
3.1. Preparation of per azide-functionalized β-CD precursor (3)
To prepare definite dendrimer, it is important to select appropriate multifunctional core that can
competently link with suitable moiety polymer precursors. β-CD-(I)
7
(2) was synthesized by
selective reaction of seven hydroxyl groups of β-CD on the primary face by iodine (Scheme 1).
The following treatment with NaN
3
3 7
in DMF directed to the preparation of β-CD-(N ) (3)
1
(Ashton, Königer, Stoddart, Alker & Harding, 1996). The study of β-CD-(N
3
)
7
by HNMR in
DMSO-d showed the thorough vanishing of hydroxyl proton (6-CH
6
2
OH) signal at 4.5 ppm,
16
Page 16 of 30

1
2
3
4
which is existent in β-CD (see supplementary data, Fig. S1). FTIR analysis (Fig. 1) of β-CD-
-1
3 7
(N ) showed the presence of new absorbance azide peak at 2108 cm . The obtained results
established that the hydroxyl groups on the primary face of β-CD were selectively substituted by
azide groups fully.
5
6
7
8
9
0
1
2
3
4
5
6
7
3.2. Preparation of mono-functionalized alkyne-terminated β-CD precursor (8)
The mono-functionalized alkyne-terminated β-CD precursor’s (8) was prepared by a multistep
reactions method, as shown in Scheme 1. The synthetic procedure to fabricate of mono-
functionalized alkyne-terminated β-CD (8) is as follows: β-alanine propargyl ester hydrochloride
(6) was obtained via the reaction of propargyl alcohol and β-alanine in the existence of
trimethylsilyl chloride. Then monofunctionalized β-CD derivative (7) (β-CD-OTs) was
synthesized and changed to (8) by replacement of the tosyl moiety with β-alanine propargyl ester
1
1
1
1
1
1
1
1
1
13
3
group by nucleophilic substitution (Scheme 1) in the presence of Et N. FTIR, H and C NMR
analysis were used to endorse the structure and purity of compound 8 (see supplementary data,
Fig. S2 and S3). Matching the spectra of 6 and 7, the vanishing of the peaks at 1601, 1414 and
−
1
1158 cm (consistent to the aromatic section and O=S=O), appearance of the peaks at 2128
-1
−1
cm (consistent to the alkyne section) and 1724 cm (consistent to the ester section) shows that
all these groups had reacted and were removed in workup.
18
19
20
21
22
3.3. Click reaction between β-CD-(N
3 7
) and alkyne-terminated β-CD (9)
β-CD dendrimer (9) on the primary face was prepared by Cu(I)-catalyzed click reaction between
1
13
3 7
β-CD-(N ) (3) and alkyne-terminated β-CD (8). H and C NMR study were used to approve
the structure and purity of (9). After the click reaction NMR analysis exhibited disappearance of
the terminal alkyne proton and corresponding appearance of a triazole proton at 7.8-8.1 ppm.
17
Page 17 of 30

1
1
2
3
4
5
6
7
8
9
0
1
2
3
The presence of the triazole proton around 8.0 ppm in the H NMR spectra was indicated the
formation of the β-CD dendrimer (9) completely (see supplementary data, Fig. S4). The integral
ratio of triazole proton to anomeric proton is about 1:7.9, which shows the successful “click”
1
3
3 7
reaction between β-CD-(N ) and alkyne-terminated β-CD (Scheme 1). C NMR analyses of (9)
showed the appearance of signals at 148, 124 ppm for carbon of triazole ring and 170.47 ppm,
which can be ascribed to carbonyl of ester (see supplementary data, Fig. S5). The synthetic
process of β-CD dendrimer (9) can be also monitored by FTIR spectra, as shown in Fig. 1. A
3 7
comparison to those of β-CD-(N ) and β-CD dendrimer (9) (Fig. 1) discovered the complete
-1
-1
disappearance of azide peak at 2108 cm and 2128 cm of alkyne peak for β-CD dendrimer (9).
Disappearance of azide and alkyne signals in the FTIR spectra of β-CD dendrimer (9), revealing
that the remaining end-capping substance has been removed completely from the last compound.
1
1
1
1
-1
Also, appearance of signals related to five-membered triazole ring at 1653 cm confirmed a
successful coupling reaction.
18
Page 18 of 30

1
2
Fig. 1.
3
3.4. Characterization of MTX:β-CD dendrimer inclusion complex
4
5
6
7
8
9
0
1
2
3
4
3.4.1. FTIR study of complex
Fig. 1 shows FTIR spectra of β-CD dendrimer (9), MTX, physical mixtures and respective
inclusion complexes. The inclusion complex formation of β-CD dendrimer (9) and a guest drug
-1
was confirmed by the FTIR spectra. β-CD dendrimer (9) showed 3412 cm (for OH stretching),
-1
-1
2927 cm (C–H stretching vibration), 1032 and 1135 cm (O–C–O stretching) and at 1658
correspond to H–O–H bending. The FTIR spectrum of MTX (Fig. 1) displayed distinctive peaks
-1
-1
1
1
1
1
1
at 1703 cm (for C=O stretching vibrations for carboxylic acid groups), 1655 cm
(corresponding to C=O stretching vibration of amide groups) and C–O stretch at 1097 and 1203
-1
-1
cm . The aromatic C=C stretch in 1455, 1500 and 1602 cm was observed. The physical
mixture spectrum showed approximate mixed spectra of MTX and β-CD dendrimer. All the
absorption peaks of MTX were enclosed by that β-CD dendrimer (9) in the FTIR spectrum of
19
Page 19 of 30

1
2
3
inclusion complex. The changes between the supramolecular inclusion complex and physical
mixture show that MTX was complexes into the cavity of β-CD dendrimer to form the inclusion
complex.
4
5
6
7
8
1.
3.4.2. Determination of β-CD in β-CD-dendrimer
The amount of β-CD in the β-CD-dendrimer (9) was calculated by the method of Koh and
Tucker (Geok-Lay & Tucker, 1986; Xu, Li & Sun, 2010) and the data disclose that CD
dendrimer contains 90.14 %β-CD in its construction, which is comparable to the real quantity
(88.64%).
9
0
1
2
3
4
5
6
7
8
9
0
1
2
.2
3.4.3. SEM analysis
1
1
1
1
1
1
1
1
1
1
2
2
2
The powdered forms of MTX, β-CD dendrimer (9), their physical mixture and inclusion
complexes were investigated by SEM analysis (Fig. 2). As shown in SEM images (i) β-CD
dendrimer is present in a globular morphology with cavity structures, (ii) MTX is present in
amorphous and crystalline states (iii) the structures of inclusion complexes appeared as
amorphous complexes and not crystals which is an evidence for the formation of inclusion
complexes. Otherwise, in SEM image the crystal photographs related to MTX and β-CD
dendrimer in the inclusion complex was not observed (iv) their physical mixtures presented
particles of MTX surrounded with β-CD dendrimer nanoparticles. The different constructions of
pure MTX, β-CD dendrimer (9) and their inclusion complex confirmed that MTX is comprised
into β-CD cavity of dendrimer (9) or dendritic network. The outcomes show that the successful
preparation of inclusion complex of β-CD-dendrimer (9) with MTX. From SEM observations it
seems that with formation of CD dendrimer 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
20
Page 20 of 30

1
2
aggregation or big clusters of mixture of many different shapes and size ranges of particles is
happened.
3
4
Fig. 2.
5
6
7
8
9
0
1
2
3.
3.4.4. DLS analysis
To evaluate the average hydrodynamic diameter of β-CD dendrimer (9) and its supramolecular
complex with MTX, DLS analyses were performed in water (Fig. 3). In relation to DLS results,
an average hydrodynamic diameter of 57 nm for pure β-CD dendrimer (9) and 78 nm for
supramolecular complex was found. The conversion in size is due to the formation inclusion
complex at the cavity of dendrimer and cyclodextrin moiety. The size obtained with SEM is
smaller than the results achieved from DLS. This is related to the SEM images measured the
particle size in solid state, whereas DLS in a solution.
1
1
1
21
Page 21 of 30

1
2
Fig. 3.
3
4
5
6
7
8
9
0
1
2
3
4
5
4.
3.4.5. DSC analysis
DSC analyses were carried out to study the physical state of the MTX in the inclusion complex.
DSC curves of β-CD dendrimer (9), MTX, physical mixture and their inclusion complex were
showed in Fig. 4. In DSC curves of MTX, the water loss happened in the 122.9 °C and the
melting peak at 180.9 °C. The curves of β-CD dendrimer (9) displayed endothermic peak at
138.8 °C and 319.6 °C matching to their loss of water and melting points, respectively. The DSC
curve of the physical mixture showed both typical signals of MTX at 185 °C with a slight shift
and β-CD dendrimer (9) at 326.4 °C. In the inclusion complex, the MTX melting point of curve
signal was fully vanished. The peaks at 235-258 °C have been cited by other authors in the range
of 210–240 °C for β-CD (Giordano, Novak & Moyano, 2001). The appearance of this peak
remains unclear, but seems to be related with crystal hydration through the preparation of β-CD
(Giordano, Novak & Moyano, 2001). This obviously recommended the actual formation of a
supramolecular inclusion complex between MTX and β-CD dendrimer (9).
1
1
1
1
1
1
22
Page 22 of 30

1
2
Fig. 4.
3
4
5
6
7
8
9
0
3.5. MTT assay
Fig. 5 shows the in vitro cytotoxic effect of β-CD dendrimer, MTX and their inclusion complex
with various concentrations of inclusion complex on T47D cells. Data analysis of MTT test
exhibited that IC50 of MTX and inclusion complex of β-CD dendrimer (9):MTX on T47D cells
was 7.4 and 4.9 µM for 72h MTT assays, respectively. These results display the cytotoxic effect
of inclusion complex of β-CD dendrimer (9):MTX on T47D cells was considerably greater than
that of free MTX. Also, β-CD dendrimer had a clearly low toxicity. The cell viability was higher
than 90% even if β-CD dendrimer concentration extended as high as 32 µM.
1
23
Page 23 of 30

1
2
Fig. 5.
3
4
5
6
7
8
9
0
1
2
3
4
5
3.6. Drug loading and in vitro drug release study
Drug encapsulation efficiency of β-CD dendrimer (9) and β-CD were estimated by UV
spectrophotometric technique and were found to be 79.8 and 52%, respectively. From the results
achieved by drug encapsulation efficiency of MTX with β-CD and β-CD dendrimer (9)
(comparison of β-CD with β-CD dendrimer (9)), it seemed that approximately 52% MTX may be
encapsulated in the cavity of β-CD and 27.8% MTX was entrapped in the dendrimer networks.
The EE of β-CD dendrimer (9) significantly improved compare with β-CD. High loading amount
of β-CD dendrimer (9) could be attributed to the inclusion of β-CD cavity to drug and
entrapment of MTX with dendritic network. Release profiles of the pure MTX (pH 7.4) and
MTX from the MTX/β-CD dendrimer and β-CD at pH 3 and pH 7.4 are shown in Fig. 6. The
results showed that the free MTX, complete diffusion was occurred within 8 h, but for β-CD and
β-CD dendrimer (9), a prolonged and slow release for 23 and 72 h were observed, respectively.
The in vitro cumulative release profiles of MTX from β-CD dendrimer (9) (Fig. 6) shows that
1
1
1
1
1
1
24
Page 24 of 30

1
2
3
4
5
6
7
8
9
0
1
2
3
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 was related to drugs diffusion and the dendrimer degradation.
The initial burst release may be perhaps caused by the drug attached on the dendrimer surface,
since the MTX might form hydrogen bonds with the hydroxyl groups of dendrimer. Sustained
release was achieved due to drug diffusion from the CD cavity and dendrimeric network. Also,
β-CD dendrimer (9) was showed to have a more controlled release behavior for the loaded MTX
when compared to β-CD. As shown in Fig. 6 the MTX release from nanocarrier was noticeably
pH dependent. The cumulative release profiles after initial burst show that 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 of the MTX/β-CD in pH 7.4 than in pH 3. It has been described that β-CD could
increase the release of hydrophobic drugs or dissolution profiles in acidic conditions (Stojanov &
Larsen, 2011). These finding showed that the β-CD dendrimer (9) nanocarrier could be a suitable
controlled drug delivery system for cancer treatment.
1
1
1
1
1
1
4
5
Fig. 6.
25
Page 25 of 30

1
2
3
4
5
6
7
8
9
0
1
2
4. Conclusion
The new glycodendrimer from β-CD in core and branches were synthesized through click
reaction with excellent coverage. The structure of β-CD dendrimer (9) was characterized and
confirmed with FTIR, NMR and DLS techniques. The obtained dendrimer showed the
remarkable encapsulation efficiency, 79.8% for MTX as model drug. The inclusion complex
structure of prepared dendrimer and MTX was determined by SEM, DLS, DSC and FTIR
techniques. The in vitro toxicity of the prepared compound and their inclusion complex with
MTX by the MTT test on T47D cells showed that the resultant dendrimer was not cytotoxic to
the cell line considered. The construction of the synthesized β-CD-dendrimer permitted two
kinds of potential entrapment places for the drug: in CD cavities and dendritic network. The in
vitro drug release results in buffer solution showed that the β-CD dendrimer nanocarrier could be
a suitable controlled drug delivery system for cancer treatment.
1
1
1
1
1
1
3
4
5
ACKNOWLEDGEMENTS
Authors gratefully acknowledge the financial support from the University of Tabriz and RCPN
of Tabriz University of Medical Science.
16
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