Deep cavitand vesicles - Multicompartmental hosts

Article abstract of DOI:10.1039/c2cc34065h

The synthesis and characterization of vesicles assembled from deep cavitands in water is reported. These vesicles act as hosts for three different types of guests: the cavitands bind small guest molecules, the bilayer attracts larger hydrophobic guests an

Full text of DOI:10.1039/c2cc34065h

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Deep Cavitand Vesicles – Multicompartmental Hosts
Jens Kubitschke, Sacha Javor, and Julius Rebek, Jr.*
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
the vesicles decreased in size to a range of 100 - 300 nm (Figure
2b).
5
The synthesis and characterization of vesicles assembled from
deep cavitands in water is reported. These vesicles act as
hosts for three different types of guests: the cavitands bind
small guest molecules, the bilayer attracts larger hydrophobic
guests and the inner aqueous compartment contains
55
10 hydrophilic molecules.
Deep cavitands are vase-like molecules that fold around small
guest molecules and isolate them from the medium.[1,2] This can
lead to several encapsulation phenomena like stabilization of
reactive species,[3] contortion of guest conformations,[4] sensing
15 of biomessengers[5] and catalysis of bimolecular reactions.[6]
A
few examples exist that show these intriguing properties can be
maintained in water.[7,8] The same applies to some deep cavitands
incorporated into micelles[9-11] and bilayer structures,[12,13]
mimicking biological membranes. However, this approach of
20 embedding cavitands into phospholipids is limited, especially in
terms of cavitand concentration. One-component vesicles with a
surface consisting entirely of receptor-like cavities are an
alternative. The molecular host-guest interactions of vesicles are
mainly carried out using cyclodextrin derivatives.[14] Ravoo et al.
25 have reported examples including light-induced release of
DNA[15] and adhesion of vesicles triggered by light[16] or metal
ions.[17] Additional vesicles consisting of curcubiturils[18] and
calixarenes[19] have been reported, as has an example of a simple
resorcinarene.[20] Here we report deep cavitands that form
30 vesicles and maintain their ability to host small guest molecules
through hydrophobic interactions in water. Larger hydrophobic
and hydrophilic guests can be bound in the hydrophobic bilayer
structure and the inner aqueous compartment of the vesicles,
respectively (Figure 1). The ability to sequester three different
35 types of guest molecules offers new departures in the use of the
deep cavitand vesicles as carriers for drug delivery applications.
Figure 1. Schematic representation of an idealized deep cavitand vesicle,
assuming a bilayer structure with interdigitated packing of alkyl chains
and TEG groups facing water on the inside and outside of the vesicle. The
60 small colored balls represent three different types of guest molecules.
Scheme 1. Synthesis of amphiphilic cavitand 8.
The synthesis of water-soluble, neutral cavitands relied on
tetraethylene glycol (TEG) groups attached at the upper rim.
(Scheme 1). The TEG monomethylether 1 was extended with
40 ethyl acrylate 2 then converted to acid chloride 5 using a
sequence described elsewhere.[21] The acid chloride 5 was used to
acylate the previously described octaamino cavitand 6[22] and
afforded the octaamide 7. Finally, addition of 1-decanethiol[23] to
the alkene feet of 7 yielded the amphiphilic thioether cavitand 8.
45 Cavitand 8 exhibited an unexpectedly high water-solubility (39
mM!) apparently due to the formation of vesicles. On simple
dissolution in water, the compound formed regularly shaped
bilayer structures as revealed by TEM measurements. Vesicles
with a distribution of sizes ranging from 100 nm to 5 ꢀm were
50 observed (Figure 2a). Some of the vesicles (< 10%) featured a
multilamellar structure and a few larger structures suggested that
the vesicles undergo fusion or fission (see ESI). Upon sonication,
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walls. Interestingly, the removal of most of the excess of the
55 guest seemed also to restore the vesicle sizes to the range
observed for the cavitands alone (Figure 2a and 5c) suggesting
that the geometry of the vesicles can be reversibly controlled by
introduction of a suitable guest.
Figure 2. TEM images of vesicles of cavitand
8 using sodium
silicotungstate (pH 8) as stain. a) obtained simply upon dissolving
compound 8 in water (4.7 mM). b) obtained after sonication of the
solution for 3 h using a benchtop 100 W and 42 kHz ultrasound bath.
5
Cavitand 7 was water-soluble as well, but as indicated by the
progressive darkening of the aqueous solutions, it was only
moderately stable under these conditions. This was presumably
due to reactions involving the alkene feet. In contrast, no
10 degradation was observed for cavitand 8. The thioether feet are
well-known to interact with gold surfaces,[23,24] which might be
an potentially useful feature to disrupt the vesicles. Alternatively,
in the context of in vivo drug delivery, these functional groups
can be readily oxidized by reactive oxygen species found in the
15 vicinity of inflamed or otherwise damaged tissues, a possible
means of triggering release.[25]
1
60 Figure 3. Upfield regions of the H NMR (600 MHz, D2O) spectra for a
variety of guest molecules and their association constants Ka. [8]:[guest] =
1.6:1.5, 0.54:1.8, 1.2:2.6, 0.48:2.3, 0.53:6.4, 0.25:16 mM. Whenever
permitted by sufficient guest solubility, the values were obtained by
titration and the given numbers are the average of 3 - 4 data points. All
65 concentrations were determined by integration relative to the external
standard signal at 0.04 ppm (see ESI for details).
However, the thioether feet were not essential to the formation of
vesicles.[20] For instance, a TEG substituted cavitand with short
ethyl feet instead of the long thioether feet of 8 also formed
20 vesicles (size range: 100 - 1000 nm; see ESI). Nonetheless, its
water-solubility was significantly lower (11 mM) and likely
reflects less stable vesicles originating from the loss of
intramolecular hydrophobic interactions of the aliphatic feet.
As vesicle components, the cavitands fully maintained their host
25 properties. When suitable guest molecules 9-13 were added to a
1
solution of cavitand 8 in D2O, the H NMR spectra featured the
characteristic far upfield shifts of guest signals caused by the
aromatic panels that surround the guest in kinetically stable
caviplexes (Figure 3). The guest complexation was in accordance
30 with that observed for other water-soluble cavitands and is
essentially driven by the hydrophobic effect. It also depends on
the size and shape of guests as well as electrostatic, CH-π, cation-
π and van der Waals interactions.[26,27] The range of values
determined (up to Ka = 1800 for 2-adamantanone 9) was higher
35 than the typical values observed in water with other structurally
related but non vesicle-forming cavitands.[11,27,28] Instead,
consistent with the TEM data, the measured values were similar
to those reported for cavitands incorporated into micelles or lipid
membranes.[9-12] Acetylcholine 14 did not show any complexation
40 as is the usual case of other octaamide cavitands in water.[11,28,29]
Figure 4. a) Structure of synthetic fluorescent guest 15 and corresponding
70 guest signals in the upfield region of 1H NMR (600 MHz, D2O) spectrum
of cavitand 8 (Ka = 430 at pD10). b) TEM image of vesicles of cavitand 8
after addition of 15 (4.7 mM, 1:1 mixture). c) Fluorescent confocal
microscopy image of the mixture of 8 and 15 after Sephadex® G-50
column chromatography.
A fluorescent guest with an adamantane anchor 15 was
synthesized using fluorescein isothiocyanate and
1-adamantanemethylamine 12. After addition to an aqueous
1
solution of cavitand 8, the H NMR spectrum showed the typical
75 Pyrene fluorescence was used to investigate the encapsulation of
large hydrophobic guests inside the bilayer structure of the
vesicles and assess the propensity for aggregation of 8. The
fluorescence emission spectrum of pyrene is highly sensitive to
its local microenvironment and is often used to determine the
80 critical micelle concentration (CMC).[30] Pyrene as a guest is too
big to fit in the cavity of cavitand 8 (no guest signals in the NMR
spectrum). Nonetheless, fluorescence spectroscopy showed that
pyrene was embedded in a hydrophobic domain - apparently the
bilayer – that was already present at cavitand concentrations as
45 guest signals, while TEM measurements showed that the vesicles
were still intact (Figure 4), even though the observed size range
was smaller (100 - 1300 nm).
After separating most of the excess of fluorescent guest 15 in
solution from the vesicles by using a Sephadex® G-50 column,
50 fluorescence spectroscopy indicated an accumulation of
fluorescent guests on the surface of the vesicles (Figure 5). In
combination with the NMR evidence, the fluorescence suggests
the position of the actively binding cavities of 8 inside the vesicle
2
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[1] D. M. Rudkevich, G. Hilmersson, J. Rebek Jr., J. Am. Chem. Soc.
low as 1 ꢀM (see ESI), indicating very stable assemblies. For
comparison, classical surfactants such as alkyl dimethyl-
ammonium propane sulfonates or alkyl trimethylammonium
bromides have CMC’s in the low millimolar range.[30]
1998, 120, 12216-12225.
[2] A. R. Renslo, J. Rebek Jr., Angew. Chem. 2000, 112, 3419-3421;
Angew. Chem. Int. Ed. 2000, 39, 3281-3283.
5
Currently, several anti-cancer agents are administered as
liposomal formulations in order to reduce toxic side effects of the
compounds.[31,32] However, active methods to control the release
of the encapsulated drug are still desirable. Doxorubicin
hydrochloride was chosen as a well-known and fluorescent anti-
55 [3] T. Iwasawa, E. Mann, J. Rebek Jr., J. Am. Chem. Soc. 2006, 128,
9308-9309.
[4] A. Scarso, L. Trembleau, J. Rebek Jr., J. Am. Chem. Soc. 2004, 126,
13512-13518.
10 cancer agent to investigate the ability of our vesicles to
encapsulate large hydrophilic drug molecules in their inner
aqueous compartment. Accordingly, the amphiphilic 8 was
dissolved in an aqueous solution of doxorubicin hydrochloride,
and the excess drug was separated from the vesicles by dialysis.
15 The presence of the drug in the inner aqueous compartment of the
vesicles was demonstrated by comparison of differential
interference contrast and fluorescence microscopy measurements
(Figure 6). Preliminary measurements showed a release of
doxorubicin hydrochloride upon addition of the surfactant Triton
20 X-165 (see ESI). Alternative methods of drug release, especially
those that exploit the specific recognition capabilities of the
cavities on the vesicle’s surface would also be useful.
[5] E. Biavardi, C. Tudisco, F. Maffei, A. Motta, C. Massera, G. G.
60
Condorelli, E. Dalcanale, Proc. Natl. Acad. Sci. 2012, 109, 2263-
2268.
[6] J. Kang, J. Rebek Jr., Nature 1997, 385, 50-52.
[7] L. Trembleau, J. Rebek Jr., Science 2003, 301, 1219-1220.
[8] R. J. Hooley, S. M. Biros, J. Rebek Jr., Angew. Chem. 2006, 118,
3597-3599; Angew. Chem. Int. Ed. 2006, 45, 3517-3519.
[9] M. P. Schramm, R. J. Hooley, J. Rebek, J. Am. Chem. Soc. 2007, 129,
9773-9779.
65
[10] Y. J. Kim, M. T. Lek, M. P. Schramm, Chem. Commun. 2011, 47,
9636-9638.
70 [11] S. Javor, J. Rebek Jr., J. Am. Chem. Soc. 2011, 133, 17473-17478.
[12] Y. Liu, P. Liao, Q. Cheng, R. J. Hooley, J. Am. Chem. Soc. 2010,
132, 10383-10390.
[13] K. M. Feher, H. Hoang, M. P. Schramm, New J. Chem. 2012, 36,
874-876.
75 [14] J. Voskuhl, B. J. Ravoo, Chem. Soc. Rev. 2009, 38, 495-505.
[15] S. K. M. Nalluri, J. Voskuhl, J. B. Bultema, E. J. Boekema, B. J.
Ravoo, Angew. Chem. Int. Ed. 2011, 50, 9747-9751.
[16] S. K. M. Nalluri, B. J. Ravoo, Angew. Chem. Int. Ed. 2010, 49, 5371-
5374.
80 [17] S. K. M. Nalluri, J. B. Bultema, E. J. Boekema, B. J. Ravoo, Chem.
Sci. 2011, 2, 2383-2391.
Figure 5. Confocal microscopy images of vesicles of cavitand 8 after
25 encapsulation of doxorubicin hydrochloride and dialysis. a) differential
interference contrast image; b) fluorescence microscopy image.
[18] H.-K. Lee, K. M. Park, Y. J. Jeon, D. Kim, D. H. Oh, H. S. Kim, C.
K. Park, K. Kim, J. Am. Chem. Soc. 2005, 127, 5006-5007.
[19] K. Helttunen, P. Shahgaldian, New J. Chem. 2010, 34, 2704-2714.
85 [20] Y. Tanaka, M. Miyachi, Y. Kobuke, Angew. Chem. 1999, 111, 565-
567; Angew. Chem. Int. Ed. 1999, 38, 504-506.
In conclusion, we have described an amphiphilic deep cavitand
that forms vesicles in water. These vesicles are capable of
encapsulating three different types of guests: small guest
30 molecules inside the cavities on the surface of the vesicles, larger
hydrophobic guests inside the bilayer structure and hydrophilic
guests in the inner aqueous compartment of the vesicles. The
[21] F. Bellouard, F. Chuburu, J.-J. Yaouanc, H. Handel, Y. Le Mest,
Eur. J. Org. Chem. 1999, 12, 3257-3261.
[22] S. Saito, D. M. Rudkevich, J. Rebek, Org. Lett. 1999, 1, 1241-1244.
90 [23] E. U. Thoden van Velzen, J. F. J. Engbersen, D. N. Reinhoudt, J.
Am. Chem. Soc. 1994, 116, 3597-3598.
combination offers
a promising carrier for drug delivery
applications. New methods to control the release of drug
35 molecules from the inside of the vesicles are under study,
especially those making use of the cavities on the vesicle surface.
[24] Y. Liu, T. Taira, M. C. Young, D. Ajami, J. Rebek, Q. Cheng, R. J.
Hooley, Langmuir 2012, 28, 1391-1398.
[25] W. Vogt, Free Radic. Biol. Med. 1995, 18, 93-105.
95 [26] S. M. Biros, E. C. Ullrich, F. Hof, L. Trembleau, J. Rebek, J. Am.
Chem. Soc. 2004, 126, 2870-2876.
We are grateful to the Skaggs Institute for financial support and
the Alexander von Humboldt foundation for a Feodor Lynen
40 fellowship for J. K. and the Swiss National Science Foundation
for a fellowship for S. J. We especially thank M. Wood and W. B.
Kiosses for performing the TEM and fluorescence microscopy
measurements.
[27] A. Lledó, J. Rebek Jr, Chem. Commun. 2010, 46, 8630-8632.
[28] T. Haino, D. M. Rudkevich, A. Shivanyuk, K. Rissanen, J. J. Rebek,
Chem. Eur. J. 2000, 6, 3797-3805.
100 [29] D. A. Ryan, J. Rebek, J. Am. Chem. Soc. 2011, 133, 19653-19655.
[30] D. López-Díaz, M. M. Velázquez, Chem. Educator 2007, 12, 327-
330.
Notes and references
45 Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, CA 92037
[31] E. Soussan, S. Cassel, M. Blanzat, I. Rico-Lattes, Angew. Chem. Int.
Ed. 2009, 48, 274-288.
† Electronic Supplementary Information (ESI) available: experimental
procedures, characterization of new compounds, 1H NMR spectra,
105 [32] T. M. Allen, P. R. Cullis, Science 2004, 303, 1818-1822.
additional TEM
50 DOI: 10.1039/b000000x/
images,
fluorescence
spectra.
See
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