ARTICLES

PUBLISHED ONLINE: 15 APRIL 2012 | DOI: 10.1038/NCHEM.1326

Acyclic cucurbit[n]uril molecular containers

enhance the solubility and bioactivity of poorly
soluble pharmaceuticals
Da Ma1, Gaya Hettiarachchi2, Duc Nguyen2, Ben Zhang1, James B. Wittenberg1, Peter Y. Zavalij1,
Volker Briken2 * and Lyle Isaacs1 *

The solubility characteristics of 4070% of new drug candidates are so poor that they cannot be formulated on their own,
so new methods for increasing drug solubility are highly prized. Here, we describe a new class of general-purpose
solubilizing agentsacyclic cucurbituril-type containerswhich increase the solubility of ten insoluble drugs by a factor of
between 23 and 2,750 by forming containerdrug complexes. The containers exhibit low in vitro toxicity in human liver,
kidney and monocyte cell lines, and outbred Swiss Webster mice tolerate high doses of the container without sickness or
weight loss. Paclitaxel solubilized by the acyclic cucurbituril-type containers kills cervical and ovarian cancer cells more
efciently than paclitaxel alone. The acyclic cucurbituril-type containers preferentially bind cationic and aromatic drugs,
but also solubilize neutral drugs such as paclitaxel, and represent an attractive extension of cyclodextrin-based technology
for drug solubilization and delivery.

A
barrier facing the pharmaceutical industry is that an esti- are prepared by expensive multi-step syntheses; (iv) some are
mated 4070% of new chemical entities belong to unstable or insoluble in water; (v) some contain undesirable
Biopharmaceutics Classication System (BCS) Class II, transition-metal ions.
which comprises compounds with low solubility but high intestinal Recently, we and others2729 have been exploring a new class of
permeability13. There is therefore an urgent requirement for molecular containers known as cucurbit[n]urils (CB[n], n 5, 6,
methods that improve the solubility of poorly soluble drug candi-
dates. Accordingly, methods that trap high-energy forms of active
pharmaceutical ingredients (APIs) and thereby enhance their rate O O O OO O
and extent of dissolution have been developed, including the cre- N N N N NN N N N N
ation of nanocrystalline solid forms or solid dispersions of the N N
Cucurbit[n]uril
API4,5. Other established methods to improve solubility include (CB[n])
salt formation, and the use of pro-drugs, pharmaceutical co-crystals, N N n = 5, 6, 7, 8, 10
NN N N N N
drugdendrimer constructs and complexation within a molecular N N N N
container610. In spite of their relatively poor recognition properties O
O OO O
(low Ka and poor selectivity), a-, b- and g-cyclodextrin11 derivatives O n-5
are the molecular containers of choice for drug solubilization and
delivery10,12,13, because they are generally regarded as being safe, RO O OR
are inexpensive and are available on an industrial scale. Examples O
OR O
of the b-cyclodextrin derivatives include hydroxypropyl b-CD RO
(HP-b-CD) and Captisol, the latter having been used to formulate O RO RO
O
a number of marketed APIs14. O OR
RO OR
As well as cyclodextrins, supramolecular chemists have studied RO
other classes of molecular container compounds, including calixar- O
enes15, cyclophanes16 and self-assembled cages and capsules1719. OR -CD R = H
RO
These synthetic molecular containers have been used to control O O
OR
chemical reactions1922, to study non-covalent interactions OR
between species held in intimate contact23, and to prepare molecular O
OR
machines2426. However, there are several reasons why none of these OR ORO OR
O RO O
synthetic molecular containers has been commercialized as a RO
general-purpose solubilizing agent for insoluble APIs: (i) their O OR
rigid macrocyclic structures allow complexation with only a small
subset of insoluble APIs; (ii) the containerguest complexes HP--CD: R16.1 = H; R4.9 = CH2CH(OH)CH3
often exhibit inappropriate kinetics of dissociation; (iii) some Captisol: R14 = H; R7 = (CH2)4SO3Na

1
Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA, 2 Department of Cell Biology and Molecular
Genetics, University of Maryland, College Park, Maryland 20742, USA; These authors contributed equally to this work. *e-mail: vbriken@umd.edu;
lisaacs@umd.edu

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ARTICLES NATURE CHEMISTRY DOI: 10.1038/NCHEM.1326

O O O O O O O O
(CH2O)n, 8 M HCl MeSO3H
HN NH 50 C HN N N NH N N 50 C N N N N N N N N
H H H HH H + O O O H HH H O
HN NH 62% HN N N NH N N 36% N N N N N N N N

O O 3 O O 4 O O 5 O O

O O O O
OH O(CH2)3SO3Na OR OR
O NaOH 5, TFA N N N N N N N N
O dioxane, RT 70 C, 3 h
S H HH H
+
O 81% 40% N N N N N N N N
6 10 8
OH O(CH2)3SO3Na OR OR
O O O O

1 R = (CH2)3SO3Na

O O O O
OH OR OR OR
10, NaOH 5, TFA
dioxane, RT N N N N N N N N
70 C, 3 h
H HH H
27% 30% N N N N N N N N
7 OH 9 OR OR OR
O O O O

2 R = (CH2)3SO3Na

Figure 1 | Synthesis of acyclic CB[n] containers 1 and 2. The synthesis proceeds via key building blocks 3 and 4, which are prepared on a large (.300 g)
scale. Condensation of glycoluril dimer 3 and cyclic ether 4 yields glycoluril tetramer 5. Aromatic wall building blocks 8 and 9 are obtained by reaction of 6
or 7 with propanesultone (10). Glycoluril tetramer building block 5 undergoes a double electrophilic aromatic substitution reaction with aromatic wall building
blocks 8 or 9 to deliver 1 or 2 on a multigram scale.

7, 8, 10). Of the known examples of CB[n], only CB[7] features good
water solubility (2030 mM)30 in combination with hostguest rec-
ognition properties. Accordingly, several groups have performed
toxicology studies to demonstrate the biocompatibility of
CB[7]31,32, have explored the ability of CB[7] to form complexes
with pharmaceutical agents (which in some cases increases their
solubility)3339, and studied the bioactivity of the CB[7]drug com-
plexes4042. All carriers for drug delivery have distinct advantages
and limitations, and an important goal of the eld is to enlarge
the toolbox available to formulate the diverse range of structures
of insoluble pharmaceuticals. For macrocyclic CB[n], two of the
potential limitations are their structural rigidity (which limits the
size of drugs that can be encapsulated) and their often slow
CB[7]guest dissociation kinetics43 (which may not be ideal for
some applications). We envisioned that certain acyclic CB[n]-type
receptors would be pre-organized but sufciently exible to recog-
nize a broad range of hydrophobic pharmaceuticals for drug solubil-
ization and delivery applications. Here, we report the synthesis of
acyclic CB[n]-type receptors 1 and 2, their ability to enhance the
solubility of ten poorly soluble pharmaceuticals, the low in vitro
and in vivo cytotoxicity of 1, and nally that aqueous solutions of
1paclitaxel have higher bioactivity than paclitaxel alone.
Results and discussion
Design of acyclic CB[n]-type containers. Our requirements for a
new class of general-purpose solubilizing agents for hydrophobic
pharmaceuticals include (i) excellent aqueous solubility, (ii) the
ability to solubilize a wide array of insoluble pharmaceuticals, and
(iii) the ability to release them rapidly and completely. The
chemical structures of the prototypical members of this new
family of molecular containers (1 and 2) are shown in Fig. 1.
Compounds 1 and 2 comprise a central C-shaped glycoluril
tetramer unit that provides the nucleus of a hydrophobic
cavity29,44,45, which endows 1 and 2 with the ability to bind to
hydrophobic species, together with two ureidyl CO portals,
which promote cation binding. Compounds 1 and 2 also contain
two terminal substituted aromatic rings, which were envisioned to
enable 1 and 2 to interact with the wide variety of poorly soluble
and aromatic pharmaceuticals by p2p interactions. Finally, 1 and
2 feature four sodium sulfonate groups that greatly enhance their
aqueous solubility.
Synthesis and properties of containers 1 and 2. The synthesis of
1 and 2 proceeds by a building-block approach involving several
known key intermediates that are available from inexpensive
starting materials on a large scale (Fig. 1). For example, glycoluril
dimer 3 is available on a .300 g scale by the condensation of
glycoluril and paraformaldehyde in concentrated HCl. Similarly,
we have developed a 700 g synthesis of previously known cyclic
ether 446 by means of a simple two-step sequence. The synthesis
of C-shaped glycoluril tetramer building block 5 by the
condensation of 3 and 4 has been described previously45
(the synthetic procedures and characterization data section of the
Supplementary Information describes the synthesis of 5 on a 76 g
scale). Reaction of 6 with 1,3-propanesultone (10) under basic
conditions gave aromatic wall building block 8 in 81% yield, while
reaction of 7 with 1,3-propanesultone (10) under basic conditions
gave 9 in 27% yield. Heating a mixture of 5 and 8 in TFA at
70 8C gave 1 by double electrophilic aromatic substitution
reactions in 40% yield, while the same reaction of 5 and 9 gave 2
in 30% yield. There are several noteworthy aspects regarding the
synthesis of 1 and 2: (i) the building block approach renders each
condensation step selective, delivering a single major product; (ii)
all of the intermediates are puried by scalable washing or
recrystallization procedures; (iii) the starting materials are
inexpensive. We have scaled up the synthesis of 1 to deliver a 60 g
batch and believe it can be adapted to deliver industrial-scale
quantities of 1. We have not yet optimized the synthesis of 2.

504 NATURE CHEMISTRY | VOL 4 | JUNE 2012 | www.nature.com/naturechemistry

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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1326
We rst assessed the intrinsic solubility of 1 and 2. For this
purpose, we stirred an excess of solid 1 with water or buffer until
equilibrium was established. After removal of insoluble 1 or 2 by
centrifugation, we determined the concentration of 1 in solution
by 1H NMR integration versus 1,3,5-benzene tricarboxylic acid as
internal standard (Supplementary Figs S7S9). In H2O the solubility
of 1 is 346 mM, and in sodium phosphate buffer it is 105 mM.
Although it is important for carriers for drug delivery to have
high aqueous solubility, it is equally or even more important that
the containerdrug complex has good solubility, as this is the
form that is delivered. Our design of 1 and 2 incorporate four sul-
fonate groups to enhance the solubility of both the containers and
the containerguest complexes. Compound 1 is signicantly more
soluble than CB[7] (2030 mM) and anomalously soluble com-
pared to a related tetra-carboxylic acid container (12 mM) pre-
pared previously45. Compound 2 has more modest solubility in
H2O (18 mM) and sodium phosphate buffer (14 mM), but its
larger aromatic rings allow it to enhance the solubility of insoluble
drugs at lower concentrations. The solubility and recognition prop-
erties (vide infra) of 1 and 2 enable their use as general-purpose
solubilizing agents.
X-ray crystal structures of containers 1 and 2. We obtained single
crystals of 1 and 2 as their CF3CO2H solvates (Fig. 2). As expected
(based on a literature precedent45), 1 and 2 display a C-shaped
conformation with glycolurils and aromatic rings dening a
hydrophobic cavity. The (CH2)3SO3Na solubilizing groups are
oriented away from the hydrophobic cavity, which leaves the
cavity free for complexation. In Fig. 2, one molecule of CF3CO2H
occupies the hydrophobic cavity and forms a hydrogen bond to a
ureidyl CO group of 1 and 2. These X-ray crystal structures of 1
and 2 present a static picture of their structures. In solution, the
conformational behaviours of acyclic 1 and 2 are more adaptable
than those of macrocyclic cyclodextrins or CB[n] because the
fused polycyclic backbones of 1 and 2 are able to ex like a
hand44,45 at the bridging CH2 groups to accommodate larger
guests. The ability of the cavities in 1 and 2 to expand promises
to greatly increase the range of poorly soluble pharmaceuticals
that can be formulated. Furthermore, in contrast to the relatively
rigid CB[n] hosts, which undergo constrictive binding processes
(slow association and slow dissociation kinetics)4749, the exible
acyclic CB[n]-type receptors release their guests rapidly under
suitable conditions.
Containers 1 and 2 self-associate weakly. A prerequisite for 1 and
2 to function as solubilizing agents for hydrophobic pharmaceuticals
is that they are not strongly self-associated in water. When we diluted
a sample of 1 from 64 mM to 10 mM, the 1H NMR resonance for the
ArH protons on the tips of the substituted o-xylylene walls of 1
underwent only small changes in chemical shift (,0.1 ppm),
suggesting a low propensity for self-association. Fitting the change
in chemical shift versus [1] to a twofold self-association model50
yielded Ks 47 M21. Similar experiments using 2 gave Ks
624 M21. Accordingly, self-association of 1 and 2 will not impinge
signicantly on their ability to act as general-purpose solubilizing
agents for insoluble pharmaceuticals.
Containers 1 and 2 enhance the solubility of poorly soluble
pharmaceuticals. We next tested the ability of 1 and 2 to enhance
the solubility of ten poorly soluble but clinically important
pharmaceutical agents (Fig. 3a). Four of the ten compounds
are anticancer agents (paclitaxel, tamoxifen, melphalan and
camptothecin) and the remaining six (albendazole, cinnarizine,
clopidogrel, amiodarone, indomethacin and tolfenamic acid) fall
into the anthelmintic, antihistamine, anticlotting, anti-arrhythmic
and non-steroidal anti-inammatory classes. This test set of
NATURE CHEMISTRY | VOL 4 | JUNE 2012 | www.nature.com/naturechemistry
2012 Macmillan Publishers Limited. All rights reserved.
ARTICLES
a
b
Figure 2 | X-ray crystal structures of 1 and 2. a, Cross-eyed stereoview of
the structure of 1 in the crystal. b, Cross-eyed stereoview of the structure of
2 in the crystal. The glycoluril tetramer units of 1 and 2 result in their overall
C shape, and the aromatic walls help to dene the hydrophobic cavity. The
crystal structures of both 1 and 2 feature a molecule of CF3CO2H held inside
the cavity by OH...O hydrogen bonding. C, grey; H, white; N, blue; O, red;
F, green; hydrogen bond, redyellow striped.
pharmaceutical agents spans a range of sizes (small, albendazole;
large, paclitaxel). All ten drugs have aromatic rings, and eight (the
exceptions being paclitaxel and indomethacin) have one or more
basic nitrogen atoms, which are expected to be protonated under
the experimental conditions because of the well-known ability of
CB[n]-type receptors to shift the pKa values of their guests29. Both
structural elements promote binding to the cavity of 1 and 2
dened by p-systems and ureidyl CO portals.
Solubility enhancement studies were carried out using the phase
solubility method50. Experimentally, we stirred solutions containing
known concentrations of 1 or 2 with an excess of solid drug until
equilibrium was achieved. Excess insoluble drug was removed by cen-
trifugation and the concentration of soluble drug in the supernatant
was determined by 1H NMR integration of drug resonances versus
1,3,5-benzene tricarboxylic acid, subsequently added as an internal
standard. Using these data we created phase solubility diagrams
(plots of [drug] versus [1] or [2]) for each of the ten drugs. All ten
pharmaceutical agents demonstrated substantial increases in solubi-
lity (by factors of 232,750; Fig. 3a and Supplementary Information)
at high [1] or [2] relative to the intrinsic solubility of the drug. Of the
10 drugs studied, only albendazole and camptothecin have been solu-
bilized previously using macrocyclic CB[n]. For albendazole, the
highest solubilities obtained with CB[6] (5.8 mM), CB[7] (7.1 mM)
and CB[8] (2.7 mM)37,40,41 are comparable to those reported here
for 1 (6.8 mM) and 2 (4.5 mM). For camptothecin, the solubilities
reported with CB[7] (0.4 mM) and CB[8] (47 mM)36 are signicantly
lower than achieved with 2 (11.6 mM). Figure 3b,c shows the phase
solubility diagrams obtained for two anticancer agents (paclitaxel
and tamoxifen) using containers 1, 2 or HP-b-CD. Container 1 exhi-
bits far superior solubilization ability towards paclitaxel than HP-b-
CD, whereas the solubilization of tamoxifen achieved with 2 is
approximately threefold better than with HP-b-CD. Overall, the
excellent solubility enhancements achieved with 1 and 2 towards a
range of drugs and the favourable comparison relative to known con-
tainers CB[n] and HP-b-CD suggests a wide applicability of 1 and 2
in drug solubilization and delivery.
The phase solubility diagram for paclitaxel (Fig. 3b) is linear at
low [1] and curves upwards at higher [1]. This shape of phase
505
ARTICLES NATURE CHEMISTRY DOI: 10.1038/NCHEM.1326

a Et b 12
O HO NH2 Cl
O Ph 1
O OH Ph
Ph 10
O N Cl

[Paclitaxel] (mM)
O NH O Melphalan 8
1: 655-fold
Ph O H O
6
HO O O H O O(CH2)2NMe2
OH S N O
Ph NH 4
O O Tamoxifen
Paclitaxel N 1: 23-fold; 2: 118-fold
1: 2,750-fold 2
Albendazole HP--CD
1: 226-fold; 2: 149-fold 0

Bu O 0 20 40 60 80 100
O
CO2Me Cl [Container] (mM)
I
O N
N c 1.5
O
I Cl S O 2
OH

[Tamoxifen] (mM)
N(CH2CH2CH3)2 O 1
Amiodarone Clopidogrel Indomethacin
1: 267-fold; 2: 340-fold 1: 1,220-fold 2: 56-fold

HO2C 0.5 HP--CD

H O
Ph N Cl N
N N N
Ph
O 0
Ph
Cinnarizine Tolfenamic acid Camptothecin 0 2 4 6 8 10
OH O
1: 354-fold 2: 37-fold 2: 580-fold [Container] (mM)

Figure 3 | Phase solubility diagrams allow a determination of the enhancement in solubility for poorly soluble drugs in the presence of molecular
containers 1, 2 or HP-b-CD. Such phase solubility diagrams are constructed by stirring a known concentration of container with an excess of solid drug until
equilibrium is reached, followed by centrifugation and determination of the concentration of drug in the supernatant by 1H NMR spectroscopy relative to an
internal standard of known concentration. a, Structures of the ten drugs used in this study and the solubility enhancements achieved with 1 and 2. b, Phase
solubility diagram for paclitaxel in the presence of molecular containers 1 (lled circles) and HP-b-CD (open circles). c, Phase solubility diagram measured for
tamoxifen in the presence of molecular containers 2 (lled circles) and HP-b-CD (open circles).

solubility diagram is known as an AP-type plot and indicates the
presence of a higher-order complex (for example 12drug)50.
Because paclitaxel has such poor intrinsic solubility characteristics
we were not able to measure a Ka value for 1paclitaxel by
traditional titration methods. Fortunately, the linear region of
Fig. 3b (up to 33 mM) can be subjected to a linear t with Ka
slope/(S0(1 slope)), where S0 is the intrinsic solubility of paclitaxel.
In this way, we determined a Ka value for the 1paclitaxel complex of
1.9 104 M21 (Supplementary Fig. S38). Values of Ka in this range
are desirable for drug delivery applications because drug release
occurs upon dilution of the containerdrug complex within the bio-
logical environment51. We were surprised that paclitaxelthe largest
and only neutral druggave the largest solubility enhancement
(2,750-fold) and displayed evidence of higher-order complexes.
These results indicate that (i) 1 is able to expand its cavity by
exing the bridging CH2 groups of the glycoluril oligomer backbone
and (ii) 1 is able to bind to neutral guests via p2p interactions,
the hydrophobic effect, and CONH...OC hydrogen bonds in the
absence of CO...cation (ion-dipole) interactions. These results
are signicant because they suggest that 1 may serve as a solubilizing
agent for drugs that contain aromatic, hydrophobic or cationic
substructures, which, of course, encompasses a signicant swathe
of the structure space of pharmaceutical agents. The phase solubility
diagrams for 2 and tamoxifen (Fig. 3c) or camptothecin
(Supplementary Fig. S17) are linear up to [2] 10 mM, which rep-
resents ideal 1:1 binding behaviour. Other drugs (Supplementary
Figs S11S14, S20) display relatively linear regions at low container
concentrations followed by plateau regions at higher concentrations.
Phase solubility diagrams of this shape are referred to as AN-type
plots and generally signify the presence of solution non-ideality or
self-association of the container, or both.
In vitro and in vivo toxicity studies. Biocompatibility must be
established before compounds can be used in therapeutic
applications. We used cell viability (MTS) and cell lysis
(Adenylate Kinase (AK) release) assays to evaluate the toxicity of
uncomplexed 1 in three cell lines: human kidney cells (HEK 293,
Fig. 4a,b), human liver cells (HepG2, Fig. 4c,d) and human
monocyte cells (THP-1, Fig. 4e,f ). HEK 293 and HepG2 cells were
used because the kidney and liver are organs where substantial
amounts of drugs accumulate for processing and clearance by the
body, hence making these key locations at which high toxicity
could occur. The THP-1 cell line was used to investigate any
detrimental effects of 1 towards immune cells. In these
experiments, antibiotics erythromycin and erythromycin estolate
were used as points of comparison to assess the toxicity of 1.
Distilled water was used as a positive control (assigned 100% AK
release) and untreated cells (UT) were used as a reference in the
MTS assay (set to 100% cell viability). Finally, a haemolysis assay
(Fig. 4g) was used to determine whether 1 induces red blood cell
lysis following intravenous injections.
HEK 293 cells treated with concentrations of 1 up to 10 mM
showed only a minor reduction in cell viability (20%) relative to
the untreated samples (100%) in the MTS assay (Fig. 4a). The comp-
lementary AK release assay (Fig. 4b) did not show signicant cell
lysis even at [1] 10 mM. In contrast, erythromycin reduced the
cell viability by 50% and induced 25% of maximal cell lysis at
1 mM concentration (Fig. 4a,b). These results conrm that
container 1 demonstrates low cytotoxicity in the human HEK 293
cell line. The human liver cells (HepG2) responded similarly to
treatment with 1 (Fig. 4c,d). Indeed, a reduction of 20% in cell
viability and no increase in cell lysis were observed at the highest
dose ([1] 10 mM). For comparison purposes, we performed a

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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1326 ARTICLES
a b 120 ***
120
100
** 100
Cell viability (%)

AK release (%)
80 ***
80
*** 60
60
40 *** UT = untreated
40 ***
20 *
D = distilled water
** ***
20 0 E = Erythromycin
0
***
20 EE = Erythromycin estolate
UT D E EE 1 UT D E EE 1 Stx = Staurosporine
c = 0.01 mM
d = 0.1 mM
120 140 ***
* *** = 1 mM
Cell viability (%)

100 *** *** 120 = 10 mM

AK release (%)
80 *** 100
80
60 ** * ***
60
40 *** 40
***
20 *** 20
0 0
UT D E EE 1 UT D E EE 1
e f g
200 120 *** 120
***
** **
100 100
Cell viability (%)

Haemolysis (%)
150
AK release (%)

80 80

100 60 60

40 40
50
20 20
*** ***
*** ***
0 0 0
UT Stx 1 UT Stx 1 PBS D 1

Figure 4 | In vitro cell viability, cell death and haemolysis assays performed with container 1. af, Plots of cell viability (MTS assay; a,c,e) and cell death
(AK release assay; b,d,f) obtained for 1 (0.01 mM, 0.1 mM, 1 mM, 10 mM) after 48 h incubation with three cell lines: HEK 293 cells (a,b), HepG2 cells (c,d)
and THP-1 cells (e,f). Data presented in af are the average values obtained from triplicate experiments and the corresponding standard deviation values.
g, Haemolysis assay conducted using puried human red blood cells diluted in phosphate buffered saline and then incubated with 1 for 3 h. Data represent the
average and standard deviation values from three replicate experiments with four donors. For all panels, unpaired t-test analysis was used (*P 0.010.05;
**P 0.0010.01; ***P , 0.001).

similar assay with HP-b-CD (Supplementary Fig. S48) in HepG2 that the containerdrug complexes would be an attractive alterna-
cells, and this also showed low toxicity up to a concentration tive to currently used technologies that rely on cyclodextrin deriva-
of 10 mM. In contrast, under similar conditions Cremophor tives or surfactants such as Cremophor EL.
EL shows high non-specic toxicity (Supplementary Fig. S48). In
human monocytes (THP-1 cell line), cell viability was even slightly Bioactivity of solubilized paclitaxel. We next investigated whether
increased relative to untreated cells (111%) upon treatment with 1 the increased solubility of paclitaxel achievable in the presence of
(10 mM); the percent cell lysis was also lower (2%) than in untreated
cells (20%) (Fig. 4e,f ). Finally, we performed a haemolysis assay with 1.4
primary human red blood cells (Fig. 4g), and did not observe a
signicant increase in cell lysis (4% for [1] 10 mM) compared
to the untreated samples (3%).
Weight dayn / weight day0

1.2
To assess the in vivo toxicity of container 1 we performed a
maximal tolerated dose (MTD) study using outbred Swiss
Webster mice. Mice were dosed via bolus injection of 1 into the
tail vein three times over 8 days and monitored for an additional 1.0
two weeks for signs of sickness and weight changes (Fig. 5). We
PBS 616 mg kg1
were pleased to observe that even those mice receiving the
maximal dose ([1] 1,230 mg kg21) appeared healthy and contin- 0.8 154 mg kg1 924 mg kg1
ued to increase in weight throughout the study at a rate that was not 308 mg kg1 1,230 mg kg1
signicantly different from the control group of mice treated with * Dosing day
phosphate buffered saline. 0.6
Together, the in vitro and in vivo results demonstrate that com- 0* 2 4* 6 8* 10 12 14 16 18 20 22
pound 1 is well tolerated by human cells and in the mouse animal Day no.
model after intravenous injection. In contrast, one of the commonly
used drug formulation agents, Cremophor EL, has shown cytotoxic Figure 5 | MTD study performed for container 1. Female Swiss Webster
and immunohypersensitivity side effects52. A large number of low- mice (n 5 per group) were dosed via the tail vein on days 0, 4 and 8
molecular-weight drugs exhibit relatively low solubility and thus (*, dosing day) with different concentrations of 1 or phosphate buffered
need a vehicle to increase bioavailability. Given the ability of 1 saline. The total amount of 1 per kg of body weight is indicated. The
and 2 to increase the solubility of clinically important anticancer normalized average weight change per study group (n 5) is indicated.
agents such as paclitaxel, tamoxifen and camptothecin, we envision Error bars represent standard error of the mean.

NATURE CHEMISTRY | VOL 4 | JUNE 2012 | www.nature.com/naturechemistry 507

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ARTICLES NATURE CHEMISTRY DOI: 10.1038/NCHEM.1326

a b c

d e f

120
***

Fragmented nuclei (%)

***
100
80
60
40
20
0
UT Stx 1 1P P
Compounds

g 150 h 100
Apoptosis (% control)

Apoptosis (% control)

100

50

50

0 0
5 4 3 2 1 0 5 4 3 2 1 0
Log [paclitaxel] (mM) Log [paclitaxel] (mM)

Figure 6 | Cell death induction assays performed on cancer cells treated with staurosporine (Stx), 1, paclitaxel solubilized with 1 (1P) and paclitaxel (P)
alone. ae, Micrographs of HeLa cells that were incubated for 24 h with cell culture medium alone (a), staurosporine (1 mM) (b), 1 (5 mM) (c), paclitaxel
(2 mM) (d), a solution containing 1 (5 mM) and paclitaxel (0.6 mM) (e). Nuclei are stained in green and actin in red. f, Percent of cells with fragmented
nuclei in ae. Scale bar for all panels, 25 mm. g,h, EC50 determination for 1paclitaxel on HeLa (g) and SK-OV-3 (h) cancer cells. Cells were incubated with
solutions containing 1 (5 mM) and the indicated concentrations of paclitaxel for 24 h. The average and standard deviation of cell apoptosis induction was
determined for six replicates and normalized to the amount of apoptosis detected in cells killed with the apoptosis inducer staurosporine (1 mM). The best-t,
nonlinear regression curve is indicated by the broken line.

1 translates into improved in vitro biological activity. Accordingly,
we incubated HeLa cells with a saturated solution of paclitaxel
(2 mM) or with a solution containing paclitaxel (0.6 mM)
solubilized with 1 (5 mM) (Fig. 6d,e). Cell death was detected by
morphological changes, which were highlighted by staining the
actin skeleton, and nuclear fragmentation. Quantication of the
percentage of cells with fragmented nuclei demonstrated that after
24 h less than 5% of paclitaxel-treated cells were dead, whereas
close to 90% of cells treated with 1paclitaxel died (Fig. 6f ). To
further characterize the killing of cancer cells by 1paclitaxel,
doseresponse experiments using SK-OV-3 and HeLa cells were
performed (Fig. 6g,h). From these experiments, we determined
the EC50 values for 1paclitaxel towards HeLa cells (0.7 mM)
and SK-OV-3 cells (0.8 mM). Unfortunately, we were unable to
measure the EC50 for paclitaxel solubilized with Cremophor EL
for comparison, because Cremophor EL exhibits high non-specic
toxicity on its own towards HeLa cells (Supplementary Fig. S49).
These results demonstrate that binding of paclitaxel inside
container 1 does not interfere with the bioactivity of the drug, but
instead leads to increased bioavailability and hence increased
cancer killing activity relative to the unbound drug. Our results
establish 1 and 2 as promising new tools with which to formulate
a distinct subset of poorly soluble drug candidates relative to
other molecular containers53,54.
In summary, we have prepared acyclic CB[n]-type molecular
containers 1 and 2 that have excellent aqueous solubility.
Containers 1 and 2 increase the solubility of insoluble drugs by a
factor of up to 2,750 by forming soluble containerdrug complexes.
Toxicity studies using assays for metabolic activity and cell death
demonstrate the low in vitro toxicity of 1 in HEK 293, HepG2
and THP-1 cells. MTD studies performed using 1 in outbred
Swiss Webster mice demonstrate that even extremely high doses
of 1 (1,230 mg kg21) do not result in weight loss or sickness.
Finally, we showed that the increased concentrations of paclitaxel

508 NATURE CHEMISTRY | VOL 4 | JUNE 2012 | www.nature.com/naturechemistry

2012 Macmillan Publishers Limited. All rights reserved.

NATURE CHEMISTRY DOI: 10.1038/NCHEM.1326
enabled by complexation inside 1 resulted in the more efcient
killing of HeLa and SK-OV-3 cancer cells. A key aspect of the mol-
ecular design of 1 and 2 is the partial preorganization of the acyclic
CB[n]-type compounds, which allows the containers to expand
their cavities to accommodate larger drugs. Finally, the demon-
stration that paclitaxel complexed to 1 retains its in vitro anticancer
activity establishes that drugs contained inside acyclic CB[n]-type
containers are released and remain active. Accordingly, it is straight-
forward to imagine that access to a family of acyclic CB[n]-type con-
tainers that enhance the solubility of a broad range of poorly soluble
pharmaceuticals hold promise in treating numerous diseases.
Methods
The materials used for the chemical and biological experiments described in this
Article were obtained from commercial sources. Standard methods were used for
chemical synthesis, self-association studies and to construct the phase solubility
diagrams as described in detail in the Supplementary Information. The X-ray crystal
structures of 1 and 2 have been deposited with the Cambridge Crystallographic Data
Centre (nos CCDC-851130 and CCDC-851131). The cell culture medium consisted
of Eagles minimum essential medium (ATCC, 30-2003) containing 10% fetal calf
serum (ThermoScientic) and 1% penicillin/streptomycin (Mediatech). The in vitro
toxicology studies (MTS assay, Promega; AK assay, Lonza; cell death ELISA, Roche)
were performed according to standard methodology provided by the vendors. The
animal study was performed at the University of Maryland Greenbaum Cancer
Center Translation Laboratories under the supervision of R. Lapidus. The animal
procedures were in accordance with the NIH Guide for the Care and Use of
Laboratory Animals and were approved by the Institutional Animal Care and Use
Committee of the University of Maryland (protocol no. 0405001). Detailed
procedures can be found in the Supplementary Information (section on materials
and methods for the in vitro and in vivo biological experiments).
Received 21 November 2011; accepted 5 March 2012;
published online 15 April 2012
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1326
Acknowledgements
The authors acknowledge the Maryland Department of Business and Economic
Development (Nano-Bio Initiative), the Maryland Technology Development Corporation
and the National Science Foundation (CHE-1110911) for funding.
Author contributions
D.M., G.H., V.B. and L.I. designed the research. D.M., G.H., D.N., B.Z., J.B.W. and P.Y.Z.
performed the research. D.M., G.H., D.N., B.Z., J.B.W., P.Y.Z., V.B. and L.I. analysed data.
D.M., G.H., V.B. and L.I. wrote the paper.
Additional information
The authors declare competing nancial interests: details accompany the full-text HTML
version of the paper at www.nature.com/naturechemistry. Supplementary information
and chemical compound information accompany this paper at www.nature.com/
naturechemistry. Reprints and permission information is available online at http://www.
nature.com/reprints. Correspondence and requests for materials should be addressed to
V.B. and L.I.
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