Synthetic micelle sensitive to IR light via a two-photon process

Article abstract of DOI:10.1021/ja0523035

A micellar assembly of molecules constituted of poly(ethylene glycol) as the hydrophilic component and 2-diazo-1,2-naphthoquinone as the hydrophobic component was shown to be destroyed in a two-photon photoreaction triggered by infrared light with release of an encapsulated fluorescent probe molecule. Copyright

Full text of DOI:10.1021/ja0523035

Published on Web 06/25/2005
Synthetic Micelle Sensitive to IR Light via a Two-Photon Process
Andrew P. Goodwin, Justin L. Mynar, Yingzhong Ma, Graham R. Fleming, and Jean M. J. Fre´chet*
Department of Chemistry, UniVersity of California, MS 1460, Berkeley, California 94720-1460
Received April 9, 2005; E-mail: frechet@berkeley.edu
Much exciting work has been done to encapsulate drug molecules
in macromolecular structures, such as micelles1 and liposomes,2
that can accumulate in a tumor and release their payload. Despite
significant advances in tumor targeting,3 however, these therapeutics
will spread throughout the body, as well.4 If the stability of the
drug complex could be controlled by a spatially directed external
agent, such as light, the drug might be released at the tumor site
exclusively. While skin absorbs UV and visible light quite readily,
light between 750 and 1000 nm has been shown to penetrate the
skin with fewer risks of damage to the irradiated area.5a Infrared
light has been used for local generation of singlet oxidation for
photodynamic therapy5 and liposome degradation,2f but to our
knowledge, no one has synthesized a micellar system capable of
encapsulating a hydrophobic molecule and releasing it under
infrared light.
Figure 1. Solubility change in 2-diazo-1,2-naphthoquinone derivatives after
Wolff rearrangement in buffered water.
Scheme 1. Synthesis of Functional Amphiphilea
Functional micelles have found extensive use in drug delivery
for their ability to encapsulate and retain hydrophobic drugs in their
self-assembled interior. When exposed to a specific stimulus, such
as pH change,6 the hydrophobic tail of molecules constituting the
micelle becomes hydrophilic and the micelle is destroyed. 2-Diazo-
1,2-naphthoquinones (DNQ) constitute an attractive trigger group
because their UV-induced Wolff rearrangement7 can result in a
drastic change in water solubility, a property that has led to the
widespread incorporation of DNQ in industrial UV photoresists.
The photoproduct, a 3-indenecarboxylic acid of pKa ) 4.5,8 should
be ionized in aqueous buffer and would, therefore, be strongly
hydrophilic (Figure 1). Moreover, Urdabayev and Popik recently
discovered that DNQ undergoes this same reaction via a two-photon
process under 800 nm laser light,9 a useful wavelength for external
targeting.
Functional amphiphile 4 was designed as a PEG-lipid with DNQ
on the end of a hydrophobic tail. Its synthesis (Scheme 1) involved
treatment of 12-aminododecanoic acid with di-tert-butyldicarbonate
to protect the amine as the tert-butyl carbamate followed by
esterification of the free acid with poly(ethylene glycol) monomethyl
ether (Aldrich, Mn ∼ 750) using DCC coupling to install the
hydrophilic segment of the amphiphile. The amine was then
deprotected with trifluoroacetic acid and coupled to 2-diazo-1,2-
naphthoquinone-5-sulfonyl chloride to afford the final product in
86% yield over four steps.
To determine if this molecule indeed formed micelles in water,
its ability to encapsulate Nile Red, a fluorescence probe, was
examined.10 Excitation of the hydrophobic Nile Red with 550 nm
light in an aqueous medium results in a relatively low fluorescence
with a λmax of 660 nm. However, if the dye resides in a hydrophobic
environment, such as the interior of a micelle, its fluorescence
emission intensity increases dramatically and experiences a blue
shift. Solutions with varying concentrations of 4 in 10 mM
phosphate buffer at pH 7.4 were treated with Nile Red and
equilibrated 2 h to allow for encapsulation. The fluorescence
emission spectra for these solutions excited at 550 nm show a
pronounced increase in emission intensity with increasing concen-
a Reagents and conditions: (a) Boc2O, Et3N, CH2Cl2, 99%; (b) poly-
(ethylene glycol) monomethyl ether, DCC, DMAP, DPTS, CH2Cl2, 90%;
(c) TFA, CH2Cl2; (d) 2-diazo-1,2-naphthoquinone-5-sulfonyl chloride, Et3N,
CH2Cl2, 97% from 2.
Figure 2. (a) Emission spectra of Nile Red (λexc ) 550 nm) in solutions
of varying concentrations of 4 in 10 mM phosphate buffer at pH 7.4. (b)
Emission intensity at 642 nm versus the log of concentration (mg/mL) of
4.
tration of 4 (Figure 2a). A plot of the relative fluorescence intensity
at 642 nm (λmax) versus the log of concentration shows a nonlinear
relationship, suggesting formation of multimolecular micelle (Figure
2b). At low concentrations (e.g., 0.05 mg/mL), the weak emission
indicates that the Nile Red is in water and, thus, few micelles are
present. The steady increase in emission with increasing concentra-
tion shows the formation of micelles. The observed inflection point
corresponds to a critical micelle concentration (CMC) of 0.15 mg/
mL, or 0.13 mM. Further evidence of micelle formation was
provided by dynamic light scattering (DLS), which at 1.0 mg/mL,
showed particles of approximately 7 nm in diameter (see Supporting
Information). Below the CMC, the scattering intensity fell below
the detection limit of the instrument.
To demonstrate the sensitivity of the micelle to UV light, the
change in Nile Red emission was examined as a function of the
photoreaction of 4 at concentrations above and below the CMC.
Solutions of 4 at concentrations of 0.05 and 1.0 mg/mL in 10 mM
phosphate buffer at pH 7.4 were equilibrated with Nile Red as
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10.1021/ja0523035 CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Figure 3. (a) UV-vis spectra of 4 at 0.05 mg/mL in 10 mM phosphate
buffer at pH 7.4 at various timepoints during irradiation at 350 nm. (b)
Fluorescence measurements of Nile Red encapsulated in 1.0 mg/mL of 4
in 10 mM phosphate buffer at pH 7.4 at same timepoints.
Figure 4. (a) Emission spectra of Nile Red (λexc ) 550 nm) encapsulated
in 1.0 mg/mL of 4 in 10 mM phosphate buffer at pH 7.4 after irradiation
at 795 nm. (b) Fluorescence emission intensity of Nile Red at 640 nm versus
time of irradiation. The relative intensities of Nile Red in solutions
containing 4 are comparable, but these are not comparable with the data
obtained from Nile Red in SDS.
before. The solutions were irradiated at 350 nm, and both the UV
absorbance and fluorescence emission spectra were measured at
various times. With increasing irradiation time, the UV absorption
maxima at 330 and 399 nm decreased steadily, showing conversion
of DNQ to the 3-indenecarboxylate (Figure 3a). Also observed was
a slight shoulder at approximately 255 nm, which supports
formation of the deprotonated carboxylate.9a This is expected to
change the amphiphilic properties of the molecule and destroy any
micellar formation.
Acknowledgment. We thank NIH (GM 65361 and EB 002047),
NSF-DMR, and the U.S. Department of Energy (DE-AC03-
765F00098) for support of this research.
Supporting Information Available: Experimental details, light
scattering experiments, and additional photophysical data. This material
is available free of charge via the Internet at http://pubs.acs.org.
During this same experiment, the emission from the Nile Red
decreased sharply and shifted from λmax 642 to 660 nm, indicating
that the Nile Red was being released into water (Figure 3b). Also
observed were a sharp decrease in light scattering intensity and an
increase in surface tension consistent with that seen in other
photosensitive micelles11 (Supporting Information). When the same
experiment was performed on the sample with a concentration of
4 below the CMC, the UV absorbance changed as before, but the
fluorescence of the Nile Red was low and changed very little. Thus,
the change in fluorescence is caused by the destruction of a micelle,
a result of the photoreaction of 4.
Analogous studies were performed to show the sensitivity of 4
to infrared light. Two solutions of 4 with Nile Red were prepared
as in the above experiment but instead irradiated with a Ti-sapphire
pulse laser at 795 nm. The fluorescence emission of Nile Red was
monitored again through fluorescence at various times (Figure 4a).
After only 15 min of irradiation, the fluorescence emission
decreased by about 60%, while after 30 min, the emission fell to
25% of its original intensity. Again, a system with 4 at a
concentration of 0.05 mg/mL with Nile Red showed little to no
fluorescence change, while a solution of Nile Red in 20 mM sodium
dodecyl sulfate showed no decrease in emission following irradia-
tion (Figure 4b). Thus, it appears that the Nile Red has been released
due to the destruction of an IR-sensitive micelle.
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