Unusual self-assembly of a hydrophilic β-cyclodextrin inclusion complex into vesicles capable of drug encapsulation and release

Article abstract of DOI:10.1039/c4tb02114b

Formation of a hydrophilic β-cyclodextrin bis-inclusion complex and its self-assembly into supramolecular vesicles are described. The vesicles can be loaded with the anti-cancer drug doxorubicin and the loaded drug can be released upon addition of a competitive inclusion binder such as adamantane carboxylate.

Full text of DOI:10.1039/c4tb02114b

Journal of
Materials Chemistry B
View Article Online
View Journal | View Issue
COMMUNICATION
Unusual self-assembly of a hydrophilic
b-cyclodextrin inclusion complex into vesicles
capable of drug encapsulation and release†
Cite this: J. Mater. Chem. B, 2015,
, 3425
3
Received 22nd December 2014,
Accepted 28th March 2015
ab
ab
Nagaraj Nayak and Karical R. Gopidas*
DOI: 10.1039/c4tb02114b
www.rsc.org/MaterialsB
Formation of a hydrophilic b-cyclodextrin bis-inclusion complex hydrophilic, native b-CD bis-inclusion complex into vesicles.
and its self-assembly into supramolecular vesicles are described. We also show that these vesicles can be loaded with the anti-
The vesicles can be loaded with the anti-cancer drug doxorubicin cancer drug doxorubicin (DOX) and the loaded drug can be
and the loaded drug can be released upon addition of a competitive released upon addition of a competitive binding agent such as
inclusion binder such as adamantane carboxylate.
adamantane carboxylate (ADC).
Adamantane derivatives exhibit a very high tendency to form
9
Self-assembly of carefully designed cyclodextrin (CD) inclusion inclusion complexes with b-CD and hence the bis-adamantane
complexes into higher order structures is an important aspect derivative AD-AD studied here is expected to bind to two b-CD
of current CD research. For example, self-assemblies of CD molecules to give the bis-inclusion complex b-CDCAD-AD*
inclusion complexes into supramolecular polymers are well b-CD as shown in Scheme 1. We observed that the bis-inclusion
1
documented in the literature. Recently, several groups have complex undergoes spontaneous stacking, ultimately leading to
reported self-assembly of CD inclusion complexes into supra- the formation of vesicles as shown in Scheme 1. Data obtained
2
molecular vesicles. CDs are nontoxic and biocompatible and from isothermal titration calorimetry (ITC), NMR titration,
hence CD based vesicles would have potential uses as drug/ atomic force microscopy (AFM), transmission electron micro-
3
gene delivery agents. Vesicles intended for use as drug or gene scopy (TEM), scanning electron microscopy (SEM) and dynamic
delivery agents must undergo dispersion or dissociation into light scattering (DLS) experiments supported the self-assembly
molecular components in response to some external stimuli. It processes shown in Scheme 1.
has been shown that grafting of stimuli responsive sites onto
Synthesis and characterization of AD-AD are presented in the
the included guest or CD host can lead to CD based vesicles ESI.† Fig. S5 (ESI†) shows the ITC titration curve for the b-CD–AD-
2
b,4
5
capable of dispersion in response to chemical, electrochemical,
light, thermal or pH stimuli.
AD interaction. The experiment was carried out by taking b-CD
6
7
5c,8
In most of such studies
chemically modified CDs were used for the construction of
vesicles. Due to the large number of chemically equivalent
hydroxyl groups, controlled modification of CDs is challenging
and hence construction of vesicles using modified CDs is a
difficult task. There are only very few reports on self-assembly
2a,15
of host–guest complexes of native CDs into vesicles.
In this
paper we report the unusual and novel self-assembly of a
a
Photosciences and Photonics Section, Chemical Sciences and Technology Division,
CSIR-National Institute for Interdisciplinary Science and Technology (CSIR-NIIST),
Council of Scientific and Industrial Research (CSIR), Trivandrum 695019, India.
E-mail: gopidaskr@niist.res.in
b
Academy of Scientific and Innovative Research (AcSIR), New Delhi 110001, India
†
Electronic supplementary information (ESI) available: Synthesis and detailed
characterization of molecules, the ITC graph, additional AFM and TEM images,
1
the 2D-ROSEY NMR spectrum of AD-AD–b-CD interaction, the H NMR spectrum
showing vesicle disassembly, and additional CLSM images. See DOI: 10.1039/
c4tb02114b
Scheme 1 Graphical illustration of the chemical structure of AD-AD and
its host–guest complexation induced self-assembly into vesicles.
This journal is ©The Royal Society of Chemistry 2015
J. Mater. Chem. B, 2015, 3, 3425--3428 | 3425

View Article Online
Communication
2 mM, 200 mL) in the cell and AD-AD (10 mM, 40 mL) in the syringe
Journal of Materials Chemistry B
(
4
ꢁ1
4
ꢁ1
and gave values of K
a
= 6.4 ꢀ 10 M , DH = ꢁ8.19 ꢀ 10 J mol
,
ꢁ
1
ꢁ1
DS = ꢁ178 J mol deg and n = 0.45. When the titration was
carried out with AD-AD in the cell and b-CD in the syringe, the value
of n obtained was close to 2, suggesting that AD-AD interacts with
two b-CD molecules. In an earlier study we reported K
a
= 6.09 ꢀ
4
ꢁ1
4
ꢁ1
ꢁ1
ꢁ1
10 M , DH = ꢁ3.47 ꢀ 10 J mol , DS = ꢁ22.9 J mol deg and
10
n = 1.0 for a similar system with only one adamantane unit. The
fact that ꢁDH more than doubled and ꢁDS increased several folds
for AD-AD suggested that both adamantane units in AD-AD are
included in the b-CD cavity as shown in b-CDCAD-AD*b-CD
(
Scheme 1).
1
H NMR spectra of AD-AD in the presence of varying
amounts of b-CD, where the b-CD concentration changed from
–2 equivalents, are shown in Fig. 1, with the lower panel
0
showing the proton assignments of AD-AD. In Fig. 1 the dotted
vertical lines indicate the original peak positions and the
change in peak positions is indicated by arrows in the top
panel. Signals due to the adamantyl protons (H , H and H )
appear in the d 1.4–2.0 ppm range and all these peaks shifted
downfield in the presence of b-CD indicating encapsulation of
the adamantyl moieties into the CD cavity (the 2D-ROSEY
spectrum of the AD-AD–b-CD (1 : 2) system (Fig. S6, ESI†)
a
b
c
Fig. 2 (a) TEM image, (b) AFM image, (c) SEM image, (d) DLS profile and
ꢁ4
(
e) Tyndall effect of the b-CD–AD-AD system in water. [b-CD] = (1 ꢀ 10 M),
ꢁ5
[AD-AD] = 5 ꢀ 10 M for (a)–(e) (see the ESI† for additional TEM, AFM and
SEM images).
showed cross-peaks of adamantane H
a b c
, H and H protons with
the interior H-3 and H-5 protons of b-CD, which further TEM images of the particles obtained upon drop casting
ꢁ
5
confirmed inclusion of the adamantane moieties of AD-AD an aqueous solution containing AD-AD (5 ꢀ 10
M) and
ꢁ4
inside the b-CD cavity). A surprising observation in Fig. 1 is b-CD (1 ꢀ 10 M). Spherical structures ranging from 70–500 nm
the downfield shifting of almost all protons of AD-AD, which in diameter could be seen in the TEM images. Distinguishable
are not involved in the encapsulation process. We attributed features of each of these particles are a narrow dark particle
e h
the chemical shift changes observed for the H –H protons in skin and a central light region, which are typical for vesicle type
1
1
the b-CD–AD-AD system to secondary interactions involving structures. The dark particle skin, which most probably
b-CDCAD-AD*b-CD. In order to probe such secondary inter- corresponds to the vesicle membrane, has a thickness of
actions we have subjected the b-CD–AD-AD system to TEM, 10 nm for a 250 nm sized particle. The TEM images also
AFM, SEM and DLS studies.
showed that the interiors of some of these particles contain
In the b-CD–AD-AD system we observed the formation of dark objects which most probably are small aggregates of the
small particles even in very dilute solutions. Fig. 2a shows the inclusion complex which are captured inside the vesicles during
their formation. Formation of vesicles in the b-CD–AD-AD system
was confirmed by AFM studies as well (Fig. 2b). The AFM height
image and height profile showed particle heights of less than
1
0 nm and widths in the range 80–250 nm, suggesting that these
1
2
vesicles are sufficiently flattened on the mica surface. Fig. 2c
shows the SEM images of the vesicles. We were also successful in
obtaining SEM images of destroyed or opened particles (Fig. S8b,
ESI†) which confirmed that the particles are vesicles possessing
hollow interior cavities. Fig. 2d shows the DLS profile obtained
for the b-CD–AD-AD system in water. The study showed a narrow
size distribution of 150–400 nm for the particles with an average
hydrodynamic diameter of 256.7 nm. The presence of nano-
aggregates in the b-CD–AD-AD solution is further confirmed by
the observation of the Tyndall effect (Fig. 2e) upon passing a
laser beam through the above solution. Tyndall scattering will
occur only if the solution contains particles that can scatter laser
4b
light.
1
Formation of vesicles from native or functionalized CD
inclusion complexes has been reported by several groups. For
example, Hao and co-workers have recently reported inclusion
Fig. 1 H NMR titration spectra of AD-AD (10 mM) in the absence (A) and
presence (B–E) of b-CD (0.5–2 eq.) in D O. The inset shows the shifts of H
and H protons of AD-AD.
2
f
g
3
426 | J. Mater. Chem. B, 2015, 3, 3425--3428
This journal is ©The Royal Society of Chemistry 2015

View Article Online
Journal of Materials Chemistry B
complex formation between b-CD and curcumin as well as
Communication
2
m
0
2c
b-CD and N,N -bis(ferrocenylmethylene)diaminohexane and
self-assembly of the inclusion complexes into vesicles. Tao
2n
2l
et al. and Jing et al. also reported the formation of vesicles
from supramolecular complexes of cyclodextrins. In all these
cases one can identify the inclusion complex as a ‘supramolecular
amphiphile’ or a ‘supramolecular bola-amphiphile’, with the CD
acting as the hydrophilic head group and the alkyl or aryl residue
acting as the hydrophobic tail or the hydrophobic core. The AD-AD
molecule is water soluble and its hydrophilicity can be attributed to
the presence of two positive charges in the molecule. When the
adamantane units of AD-AD are included in the b-CD cavity the
bis-inclusion complex b-CDCAD-AD*b-CD is formed, which
has hydrophilic b-CD at both ends and hydrophilic, positively
charged pyridinium groups at the core. Thus b-CDCAD-AD*
b-CD is not a conventional bolaamphiphile and charge repulsion
may act against close packing. Hence we did not expect
b-CDCAD-AD*b-CD to self-assemble to give vesicles as shown
in Scheme 1. The fact that stacking occurs and vesicles are
formed suggests that the repulsive forces do not play a dominant
Fig. 3 CLSM (a) and transmission (b) images of DOX-loaded b-CD/AD-AD
role here and that the core unit may be sufficiently hydrophobic vesicles. Insets show enlarged images of a DOX loaded vesicle. CLSM
to drive the self-assembly process. The outer diameter of b-CD is (c) and transmission (d) images of the same after DOX release (see the ESI†
for additional images).
1.53 nm and geometrical requirements suggest that the core
units in successive layers (in Scheme 1) are also separated at
least by the same distance. Thus the distance between adjacent
pyridinium moieties is sufficiently large and hence the repulsive suggested that these vesicles may have potential applications as drug
1
3
forces could be very weak. A few water molecules can also be carriers. In order to see if the b-CD/AD-AD vesicles can actually carry
present near the charged sites leading to screening of the drug molecules, we have attempted to load the anti-cancer drug DOX
repulsive interactions. Alternatively, the positive pyridinium ions into these vesicles. In a typical experiment, DOX hydrochloride
ꢁ3
and the counter bromide ions may form tight ion pairs, which solution (1 ꢀ 10 M) was mixed with the vesicle solution
ꢁ
3
ꢁ4
can also eliminate repulsion between successive layers. The (1 ꢀ 10 M of b-CD and 5 ꢀ 10 M of AD-AD) and kept aside
molecular length of the bis-inclusion complex is B5 nm and overnight followed by dialysis to remove the uncomplexed DOX.
hence a vesicle membrane thickness of B10 nm (from TEM)
Confocal laser scanning microscopy (CLSM) images of the
suggested that the vesicles are formed by bilayer assemblies of DOX loaded vesicles are shown in Fig. 3a and b. The bright red
the inclusion complex. The two layers may be held together by luminescence due to DOX inside the vesicles can be clearly seen
hydrogen bonding interactions involving the hydroxyl groups at in Fig. 3, which confirms that these vesicles are loaded with
the narrow rims of the b-CD. There are seven hydroxyl groups at DOX. The DOX loading efficiency of the vesicles (see the ESI† for
the narrow rim of the b-CD and these can be engaged in the procedure) was found to be 7.06%. This value compares very
interlayer hydrogen bonding and stabilize the bilayer structure. well with the reported loading efficiency of 5.02% of pillar[6]arene
16
Formation of vesicles from the AD-AD–b-CD mixture has its based supramolecular vesicles. Both the empty and DOX-loaded
origin in the inclusion binding of adamantane moieties of vesicles were found to be stable for at least 3 weeks which was
AD-AD in the b-CD cavity. It is well known that guests included confirmed by SEM and DLS studies (Fig. S8c, ESI†).
in the CD cavity can be displaced using guest molecules with
We observed that the DOX loaded into these vesicles can be
higher binding constants. ADC is most commonly used for this released by disassembling the vesicles using ADC. For the
purpose as it exhibits a very high b-CD binding constant of release study, a solution of ADC (2.25 eq.) was added to the
5
ꢁ1 2e,14
2
.9 ꢀ 10 M
.
We observed that addition of 2.25 eq. of ADC DOX-loaded vesicle and the solution was kept aside for 2 h.
to the b-CDCAD-AD*b-CD vesicle led to disappearance of the CLSM images of the solution after vesicle disassembly are
vesicle structures in a few hours as evidenced from the absence shown in Fig. 3c and d. In Fig. 3c and d vesicles are not
1
of these structures in AFM images. The H NMR spectrum observed and the fluorescence of the dye is seen in most of
obtained after addition of ADC suggested that the adamantane the viewing area indicating that the vesicles are dispersed into
groups of AD-AD are pushed out of the CD cavity by ADC its constituents. This finding suggests that the supramolecular
(Fig. S9, ESI†).
vesicle formed in the b-CD–AD-AD system can have potential as
Vesicles can be used as carriers for controlled drug delivery a drug delivery agent.
and in such cases stimuli responsive, slow disruption of the
vesicle is an essential requirement for drug release.
In conclusion we have prepared supramolecular vesicles by the
spontaneous self-assembly of a bis-inclusion complex. Formation
3
a,b,d,4b,15
The fact that b-CD/AD-AD vesicles can be disrupted by ADC of the vesicular structures was proved by TEM, SEM, AFM, and DLS
This journal is ©The Royal Society of Chemistry 2015
J. Mater. Chem. B, 2015, 3, 3425--3428 | 3427

View Article Online
Communication
Journal of Materials Chemistry B
studies. These novel vesicles may have potential applications as
drug delivery systems.
3 (a) A. Pourjavadi, M. Adeli and M. Yazdi, New J. Chem., 2013,
37, 295–298; (b) C. Ortiz Mellet, J. M. Garcia Fernandez and
J. M. Benito, Chem. Soc. Rev., 2011, 40, 1586–1608;
(c) G. Tiwari, R. Tiwari and A. K. Rai, J. Pharm. BioAllied
Sci., 2010, 2, 72–79; (d) C. Alvarez-Lorenzo and A. Concheiro,
Chem. Commun., 2014, 50, 7743–7765.
4 (a) D. S. Guo, K. Wang, Y. X. Wang and Y. Liu, J. Am. Chem.
Soc., 2012, 134, 10244–10250; (b) K. Wang, D. S. Guo,
X. Wang and Y. Liu, ACS Nano, 2011, 5, 2880–2894.
We gratefully acknowledge the financial support by DAE-
BRNS (project no. 2007/37/37/BRNS) and CSIR, Government of
India. We are thankful to Mr Kiran Mohan and Mr Robert
Philip for TEM images and Mr J. S. Kiran for artwork. N.N.
thanks CSIR for a research fellowship. This article is contribu-
tion number NIIST-PPG 362.
5
(a) K. N. Power-Billard, R. J. Spontak and I. Manners, Angew.
Chem., Int. Ed., 2004, 43, 1260–1264; (b) H. Zhang, W. An,
Z. Liu, A. Hao, J. Hao, J. Shen, X. Zhao, H. Sun and L. Sun,
Carbohydr. Res., 2010, 345, 87–96; (c) H. Zhang, Z. Liu,
F. Xin, W. An, A. Hao, J. Li, Y. Li, L. Sun, T. Sun, W. Zhao,
Y. Li and L. Kong, Carbohydr. Res., 2011, 346, 294–304.
References
1
(a) M. Miyauchi and A. Harada, J. Am. Chem. Soc., 2004, 126,
1418–11419; (b) Y. Hasegawa, M. Miyauchi, Y. Takashima,
H. Yamaguchi and A. Harada, Macromolecules, 2005, 38,
724–3730; (c) A. Harada, Y. Takashima and H. Yamaguchi,
1
3
Chem. Soc. Rev., 2009, 38, 875–882; (d) X. Ma and H. Tian,
Acc. Chem. Res., 2014, 47, 1971–1981; (e) H. X. Zhao,
D. S. Guo, L. H. Wang, H. Qian and Y. Liu, Chem. Commun.,
6 (a) S. K. Nalluri, J. Voskuhl, J. B. Bultema, E. J. Boekema and
B. J. Ravoo, Angew. Chem., Int. Ed., 2011, 50, 9747–9751;
(b) Y. Wang, N. Ma, Z. Wang and X. Zhang, Angew. Chem.,
Int. Ed., 2007, 46, 2823–2826.
7 B.-W. Liu, H. Zhou, S.-T. Zhou, H.-J. Zhang, A.-C. Feng,
C.-M. Jian, J. Hu, W.-P. Gao and J.-Y. Yuan, Macromolecules,
2014, 47, 2938–2946.
2
012, 48, 11319–11321; ( f ) A. Harada, Y. Takashima and
M. Nakahata, Acc. Chem. Res., 2014, 47, 2128–2140;
g) X. Yan, F. Wang, B. Zheng and F. Huang, Chem. Soc.
(
Rev., 2012, 41, 6042–6065; (h) A. Tamura and N. Yui, Chem.
Commun., 2014, 50, 13433–13446.
(a) P. Xing, T. Sun and A. Hao, RSC Adv., 2013, 3,
8 T. Sun, Y. Li, H. Zhang, J. Li, F. Xin, L. Kong and A. Hao,
Colloids Surf., A, 2011, 375, 87–96.
2
2
4776–24793; (b) T. Sun, H. Yan, G. Liu, J. Hao, J. Su, S. Li,
9 J. Carrazana, A. Jover, F. Meijide, V. H. Soto and J. Vazquez
Tato, J. Phys. Chem. B, 2005, 109, 9719–9726.
P. Xing and A. Hao, J. Phys. Chem. B, 2012, 116, 14628–14636;
(
c) H. Zhang, J. Shen, Z. Liu, A. Hao, Y. Bai and W. An, 10 R. Krishnan and K. R. Gopidas, J. Phys. Chem. Lett., 2011, 2,
Supramol. Chem., 2010, 22, 297–310; (d) M. Felici, M. Marza- 2094–2098.
Perez, N. S. Hatzakis, R. J. Nolte and M. C. Feiters, Chem. – 11 A. Sorrenti, O. Illa and R. M. Ortuno, Chem. Soc. Rev., 2013,
Eur. J., 2008, 14, 9914–9920; (e) P. Falvey, C. W. Lim, R. Darcy, 42, 8200–8219.
T. Revermann, U. Karst, M. Giesbers, A. T. Marcelis, A. Lazar, 12 (a) S. V. Bhosale, C. H. Jani, C. H. Lalander, S. J. Langford,
A. W. Coleman, D. N. Reinhoudt and B. J. Ravoo, Chem. – Eur.
J., 2005, 11, 1171–1180; ( f ) Y. Kang, K. Guo, B. J. Li and
S. Zhang, Chem. Commun., 2014, 50, 11083–11092; (g) G. Chen
I. Nerush, J. G. Shapter, D. Villamaina and E. Vauthey, Chem.
Commun., 2011, 47, 8226–8228; (b) W. Jiang, Y. Zhou and
D. Yan, Chem. Soc. Rev., 2014, DOI: 10.1039/c4cs00274a.
and M. Jiang, Chem. Soc. Rev., 2011, 40, 2254–2266; (h) K. Wei, 13 H. Yang, Z. Ma, Z. Wang and X. Zhang, Polym. Chem., 2014,
J. Li, J. Liu, G. Chen and M. Jiang, Soft Matter, 2012, 8, 5, 1471–1476.
300–3303; (i) U. Kauscher, M. C. Stuart, P. Drucker, H. J. 14 M. V. Rekharsky and Y. Inoue, Chem. Rev., 1998, 98, 1875–1918.
Galla and B. J. Ravoo, Langmuir, 2013, 29, 7377–7383; 15 (a) J. Ding, L. Chen, C. Xiao, L. Chen, X. Zhuang and
3
(
4
j) Y. Chen, Y. M. Zhang and Y. Liu, Chem. Commun., 2010,
6, 5622–5633; (k) R. Donohue, A. Mazzaglia, B. J. Ravoo and
X. Chen, Chem. Commun., 2014, 50, 11274–11290;
(b) K. Wang, H. Q. Dong, H. Y. Wen, M. Xu, C. Li, Y. Y. Li,
H. N. Jones, D. L. Shi and X. Z. Zhang, Macromol. Biosci.,
2011, 11, 65–71; (c) R. Anand, F. Manoli, I. Manet, S. Daoud-
Mahammed, V. Agostoni, R. Gref and S. Monti, Photochem.
Photobiol. Sci., 2012, 11, 1285–1292.
R. Darcy, Chem. Commun., 2002, 2864–2865; (l) B. Jing,
X. Chen, X. Wang, C. Yang, Y. Xie and H. Qiu, Chem. – Eur.
J., 2007, 13, 9137–9142; (m) M. Ma, T. Sun, P. Xing, Z. Li, S. Li,
J. Su, X. Chu and A. Hao, Colloids Surf., A, 2014, 459, 157–165;
(n) W. Tao, Y. Liu, B. Jiang, S. Yu, W. Huang, Y. Zhou and 16 Y. Cao, X. Y. Hu, Y. Li, X. Zou, S. Xiong, C. Lin, Y. Z. Shen
D. Yan, J. Am. Chem. Soc., 2012, 134, 762–764.
and L. Wang, J. Am. Chem. Soc., 2014, 136, 10762–10769.
3
428 | J. Mater. Chem. B, 2015, 3, 3425--3428
This journal is ©The Royal Society of Chemistry 2015