X. Zhao et al.
European Journal of Pharmaceutical Sciences 160 (2021) 105745
those delivery system, cyclodextrin (CD) complexation has become the
focus of interest for hydrophobic drug delivery due to its reliable safety
profile, simple preparation method and high drug loading capacity.
Cyclodextrins (CDs) are cyclic derivatives of starch that is obtained from
2. Materials and methods
2.1. Materials
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starch by enzymatic process (Hadaruga et al., 2019). They are torus
β-CD (99.0%, MW=1135.00) and p-toluenesulfonyl chloride were
purchased from Chengdu Kelong Chemical Co., Ltd.(Chengdu, China).
Biotin was obtained from Shanghai Duoduo Chemical Industries Co Ltd.
(Shanghai, China). Podophyllotoxin (98%) was obtained from Shanghai
Yuanye Bio-Technology Co., Ltd.(Shanghai, China). Triphenylphosphine
was obtained from Tianjin Kemiou Chemical Reagent Co., Ltd. (Tianjin,
China). All other chemical reagents were of analytical grade and all
materials were used according to the instructions.
shaped circular α-(1,4) linked oligosaccharides that have been exten-
sively used to improve the aqueous solubility, bioavailability and sta-
bility or decrease unfavorable side effects of drugs (Periasamya et al.,
2020; Carmen et al., 2005; Liu et al., 2003). A unique conical structure
with hydropbobic cavity is formed by the glucose chains in CDs, and
lipophilic compounds may enter and form water-soluble complexes that
alter the physical and chemical properties of drug (Gould and Scott,
2005).
α-, β- and γ-CD consist of six, seven and eight glucose units
respectively, are the most studied cyclodextrins. In particularly, β-CD is
more extensively used in drug delivery systems due to the appropriate
cavity size, good ability to combine aromatic units, ready availability,
easy production, and relatively economical price (Stappaerts et al.,
2018; Liao et al., 2015; Waleczek et al., 2003; Yao et al., 2014). How-
ever, the low water solubility (1.85g/100ml) of parent β-CD limits their
further application in pharmaceutical formulations (Periasamya et al.,
2020; Qiu et al., 2017). The relatively low water solubility of β-CD may
be owing to an internal hydrogen bond formed between the C-2-OH and
the C-3-OH of the neighboring glucose unit. The formation of the
hydrogen bond in the β-CD molecule result in a secondary belt, leading
to a rather rigid structure (Qiu et al., 2017). In addition, β-CD applica-
tion is also limited due to the lack of selectivity. The development of
site-specific delivery system with greater efficacy and lower toxicity is
recently an urgent need to overcome the limitation of conventional
therapy.
2.2. Chemical synthesis of mono-6-biotin-amino-6-deoxy-β-cyclodextrin
(B-CD)(Fig. 1)
Synthesis of mono-6-(p-toluenesulfony)-6-deoxy-β-cyclodextrin (β-CD-
OTs): β-CD-OTs was synthesized according to a previously reported
method (Abbas et al., 2017). Generally, β-CD (5g, 4.4mmol) was sus-
pended in 30 ml of ultra-pure water. Sodium hydroxide(8M, 1.75ml)
was added dropwise until the β-CD solution was clarified. A solution of
p-toluenesulfonyl chloride (1g, 5.28mmol) in 2.5 ml of acetonitrile was
added dropwise to the β-CD solution under vigorous stirring in an ice
water bath at 0-5◦C. Then hydrochloric acid(2M) was added to the
neutralize the solution. After stirring at 25◦C for 2h, a large amount of
white precipitate was formed. Then the resulting precipitate was
collected by vacuum filtration and recrystallized two or three times in
hot water. The obtained white product (β-CD-OTs) was dried at 40◦C
under vacuum and collected as a white solid(0.9563g,19.1%). 1H NMR
(400 MHz, DMSO-d6): δ 7.77-7.75(d, 2H), 7.45-7.43(d, 2H), 5.82-5.63
(m, 14H), 4.86-4.77(m, 7H), 4.52-4.43(m, 6H), 3.67-3.55(m, 28H),
3.40-3.28(overlap with HDO, m, 14H), 2.44-2.43(s, 3H). MS(ES),
m/z:1311.39[M+Na]+
Biotin, one of the B vitamins, also known as vitamin H, is a water-
soluble vitamin. As a cellular growth promoter, biotin and its de-
rivatives have already been used in the field of cancer studies and tissue
engineering (Na et al., 2003). Biotin was found in kidney, liver, pancreas
and milk (Park et al., 2006). Due to the rapid cell growth and enhanced
proliferation, cancer cells need more certain vitamins than normal cells.
Therefore, the receptors involved in the uptake of vitamins are usually
overexpressed on the surface of tumor cells and as a consequence these
surface receptors are useful as tumor-targeting biomarkers. It has been
reported that additional biotin is needed for the rapid growth and pro-
liferation of cancer cells (Bian et al., 2012). Specifically, biotin is present
in higher content in cancerous tissue than in normal tissue (Bagheri
et al., 2014). Coincidentally, biotin receptors have been reported to be
over-expressed on the surfaces of many types of tumor cells (Yan et al.,
2019). Highly proliferating cancer cells such as MDA-M231, MCF7,
A549, HeLa and HepG2 cells exhibit elevated biotin receptors in com-
parison with health cells. Therefore, biotin is a popular targeting agent
for drug delivery system. As a specific active targeting agent (Lammers
et al., 2008), biotin has been utilized in drug carriers to increase intra-
cellular uptake of drug and decrease toxicity in normal tissues (Bagheri
et al., 2014). When biotin conjugated with other drug via amide or ester
linkages, it spontaneously acts as a targeting moiety for specific inter-
action with tumor cells (Park et al., 2006). Previous report demonstrated
that a biotin and arginine modified hydroxypropyl-β-cyclodextrin could
improve the anticancer activity of paclitaxel(Yang et al., 2019). There-
fore, we hypothesized that biotin as a tumor specific ligand conjugated
with β-CD to improve its cancer selectivity is feasible.
Synthesis of mono-6-azide-6-deoxy-β-cyclodextrin(β-CD-N3): β-CD-OTs
(1g, 0.77mmol) and sodium azide (1.32g, 20.36mmol) were dissolved in
15ml of ultrapure water at 80◦C. The mixture solution was stirred at
80◦C for 24h. The mixture was cooled to room temperature and 70ml of
acetone was poured, immediately producing a white precipitate. The
resulting product (β-CD-N3) was collected with suction filtration and
vacuum dried overnight at 40◦C to obtain a white powder (yield:88%).
1H NMR(400 MHz, DMSO-d6): δ 5.76-5.61(m, 14H), 4.88-4.82(m, 7H),
4.51-4.42(m, 6H), 3.79-3.51(m, 28H), 3.42-3.31(overlap with HDO, m,
14H). MS(ES),m/z:1182.38[M+Na]+
Synthesis of mono-6-amino-deoxy-β-cyclodextrin(β-CD-NH2): β-CD-
NH2 was synthesized in a procedure described by Wei et al (Wei et al.,
2013). Briefly, β-CD-N3 (1g,0.86mmol) and triphenylphosphine(0.3g,
1.1mmol) were dissolved in 10 ml of N,N-dimethyl formamide and
stirred for 2h at 25◦C. Then 2ml distilled water was added, stirred at
90◦C for 2 hours, cooled to room temperature and 20ml of acetone was
poured, immediately producing a white precipitate. The white product
(β-CD-NH2) was collected by filtration and vacuum dried 60◦C to obtain
the desired product (yield:92%). 1H NMR(400 MHz, DMSO-d6): δ
5.78-5.58(m, 14H), 4.91-4.79(m,7H), 4.50-4.37(m, 6H), 3.76-3.48(m,
28H), 3.41-3.33(overlap with HDO, m, 14H). MS(ES),m/z:1134.39
[M+H]+
Synthesis of Biotin-N-hydroxysuccinimide ester(Biotin-NHS): Biotin
(97.6mg, 0.4mmol), N-hydroxysuccinimide (NHS, 48mg, 0.42mmol)
and N,N’-Dicyclohexyl- carbodiimide(130mg) were dissolved in 3 ml of
N,N-dimethylformamide. The reaction mixture was kept stirring in
condition of seal for 3h at 55◦C. The mixture was cooled to room tem-
perature and filtrated to remove the insoluble solid. The filtrate was
precipitated in diethyl ether under stirring in an ice water bath. The
precipitate was collected (Biotin-NHS) by filtration and dried in the air,
giving the desired product as white solid(yield: 92%). 1H NMR (400
MHz, DMSO-d6): δ 6.40-6.34(d, 2H), 4.32-4.29(m, 1H), 4.16-4.13(m,
1H), 3.13-3.08(m, 1H), 2.82-2.81(s, 4H), 1.69-1.38(m, 8H).
The purpose of this study is to improve the water solubility and
cancer selectivity of the PPT through the formation of PPT/B-CD in-
clusion complexes. The inclusion complexes of PPT/B-CD were prepared
and analyzed by water solubility, phase solubility, Job’s plot, 1H NMR
and 2D ROESY NMR, Powder X-ray diffraction(XRD), Fourier
transformation-infrared spectroscopy(FT-IR), Scanning electron micro-
scopy(SEM). In addition, the cell cytotoxicity experiment was conducted
to study the antitumor activity of the PPT/B-CD complexes. The cellular
uptake was carried out to investigate the targeting ability of B-CD with
rodamine B as a fluorescence probe.
2
Zhao, Xiu
