UV and near-IR triggered release from polymeric nanoparticles

Article abstract of DOI:10.1021/ja102595j

A new light-sensitive polymer containing multiple light-sensitive triggering groups along the backbone and incorporating a quinone-methide self-immolative moiety was developed and formulated into nanoparticles encapsulating a model pharmaceutical Nile Red. Triggered burst release of the payload upon irradiation and subsequent degradation of the nanoparticles were observed. This system is designed to be versatile where the triggering group can be sensitive to a number of wavelengths.

Full text of DOI:10.1021/ja102595j

Published on Web 06/22/2010
UV and Near-IR Triggered Release from Polymeric Nanoparticles
Nadezda Fomina, Cathryn McFearin, Marleen Sermsakdi, Osayimwense Edigin, and Adah Almutairi*
School of Pharmacy and Pharmaceutical Sciences, Departments of NanoEngineering and of Materials Science and
Engineering, UniVersity of California at San Diego, La Jolla, California 92093
Received March 29, 2010; E-mail: aalmutairi@ucsd.edu
Scheme 1. Synthesis of Light-Responsive Polymer 2
Abstract: A new light-sensitive polymer containing multiple light-
sensitive triggering groups along the backbone and incorporating
a quinone-methide self-immolative moiety was developed and
formulated into nanoparticles encapsulating a model pharma-
ceutical Nile Red. Triggered burst release of the payload upon
irradiation and subsequent degradation of the nanoparticles were
observed. This system is designed to be versatile where the
triggering group can be sensitive to a number of wavelengths.
With the rapid progress of nanotechnology over the past decade,
there is growing interest in polymeric biomaterials that can be
remotely disassembled in a controlled fashion upon an external
stimulus but otherwise are stable under physiological conditions.1
molecule payload upon irradiation. Its design allows for adaptation
Various internal and external stimuli, such as pH,2 specific
to a variety of stimuli responsive triggering groups.
enzymes,3 temperature,4-6 ultrasound,7,8 and light,9 are being
The monomer design is based on the self-immolative quinone-
explored. Optical stimulus is especially attractive as it can be
methide system.11-14 The cleavage of the triggering group by light
remotely applied for a short period of time with high spatial and
induces cyclization of the diamine spacer, which in turn unmasks
temporal precision. Near-infrared (NIR) light can penetrate deeper
an unstable quinone-methide moiety. Incorporation of this moiety
into a polymer chain causes degradation of the polymer backbone
upon irradiation with light.
Monomer 1 was synthesized according to a previously published
into tissue and has many in ViVo applications.10 Despite these
advantages, there is a dearth of biomaterials that can efficiently
respond to light, especially NIR light.
To increase the efficiency of response we designed a polymer
procedure11 with slight modifications (see Supporting Information).
using self-immolative monomers.11-14 In such systems the sensitiv-
4,5-Dimethoxy-2-nitrobenzyl alcohol15 was chosen despite its low
ity to a stimulus is increased due to a domino effect, where a single
two-photon uncaging cross section (0.01 GM)16 compared to
triggering event leads to multiple output reactions. However, if the
6-bromo-coumarins (1 GM)17 or fluorene-based systems (5 GM),18
triggering is not very efficient, the polymer scission will be
because it is well-studied and readily available, making it a good
incomplete. Another way to increase the sensitivity and to increase
proof-of-concept photolabile group.
the number of fragmented chains is by introducing multiple light-
Monomer 1 was copolymerized with adipoyl chloride to yield a
regular copolymer. The low molecular weight oligomers were
sensitive triggering groups per polymer chain (Figure 1).
removed by repeated precipitation of the crude polymer with cold
ethanol, yielding the final product with a molecular weight of 65 000
Da and PDI of 1.54 (characterized by GPC relative to polystyrene
standards) with 44% yield.
Cleavage of the triggering groups by irradiation at 350 and 750
nm, via one- and two-photon processes respectively, was monitored
by observing the changes in the absorbance spectrum of polymer
2 in acetonitrile/H2O (9/1). Upon light exposure, the peak at 346
nm, corresponding to 4,5-dimethoxy-2-nitrobenzyl carbamate de-
creased, while a new peak at 400 nm appeared, corresponding to
the cleaved 4,5-dimethoxy-2-nitrosobenzaldehyde16 (Figures S1,
S2). The absorption spectrum remained unchanged after 15 min of
irradiation with 350 nm light, indicating complete deprotection,
while it was necessary to irradiate the system for 5 h at 750 nm to
observe changes in the absorption spectrum, consistent with the
Figure 1. Degradation of light-sensitive polymer 2 upon irradiation.
Herein, we describe a new light-sensitive degradable polymer
containing a quinone-methide self-immolative moiety, which can
be triggered to degrade through multiple light-sensitive groups along
the backbone (Scheme 1). We also demonstrate that nanoparticles
formulated from this polymer are capable of releasing their small
low two-photon uncaging cross section of the 4,5-dimethoxy-2-
nitrobenzyl group. However, more efficient two-photon uncaging
coumarin based groups may be used in place of the nitrobenzyl
group and are currently under investigation to make the system
more sensitive to NIR light and biologically innocuous.
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C O M M U N I C A T I O N S
Figure 2. (A) Degradation of polymer 2 upon exposure to 350 nm light
for defined time periods in acetonitrile/H2O (9/1) and (B) GPC traces of
polymer 2 before irradiation (red), upon irradiation for 15 min in
acetronitrile/H20/Et3N (9/0.5/0.5) (green), and after 24 h of incubation at
37 °C after 15 min of irradiation (blue).
Figure 4. Fluorescence intensity of Nile Red encapsulated within polymeric
nanoparticles and upon irradiation with 350 nm light, monitored at 630 nm
with excitation at 540 nm.
Figure 3. An illustration of the formulation of nanoparticles, their
degradation, and light-triggered release of their encapsulated Nile Red
payload.
The degradation of polymer 2 was studied by GPC and proton
NMR in acetonitrile/water solutions. The polymer solutions were
exposed to UV light (350 nm) for various periods of time and
incubated at 37 °C. Samples were removed and analyzed. The
degree of polymer degradation showed strong dependence on the
irradiation time (Figure 2). The initial drop in molecular weight in
the first few minutes after UV irradiation is likely to be mostly
due to the loss of the triggering groups, while further reduction in
molecular weight is due to the cleavage of the polymer backbone
as a result of cyclization and elimination reactions within the self-
immolative monomer unit. The difference in the degradation degree
is especially evident in the samples irradiated for 5 and 15 min:
more triggering groups are cleaved. Consequently, the polymer
chains degrade into smaller fragments. Although the estimated
molecular weights of the fragments level off at 20 000 Da, the
molecular weight of monomer 1 (m/z ) 544.19) was estimated by
GPC to be 3500 Da; therefore these fragments may be oligomers.
Notably, only a small portion of all the triggering groups needs to
be cleaved to induce a reduction in the molecular weight of the
polymer.
The proton NMR of polymer 2 before irradiation in CDCl3
showed all the characteristic peaks and splittings (Figure S6). As
expected,14 in CD3CN/D2O the NMR peaks broadened (Figure S7).
Upon irradiation and incubation at 37 °C for 18 h the NMR peaks
corresponding to the benzylic protons of the 4,5-dimethoxy-2-
nitrobenzyl group disappeared and the remaining peaks shifted
accordingly (Figure S8). Sharper monomer peaks, indicating the
presence of urea and cresol emerged, overlapping with broader
polymer and oligomer peaks.
Figure 5. Fluorescence intensity of Nile Red encapsulated within polymeric
nanoparticles and after 20 min irradiation increments with 750 nm light,
monitored at 630 nm with excitation at 540 nm.
Two-photon irradiation of polymer 2 for 5 h showed a similar
degree of degradation as that after 5 min of one-photon irradiation
(Figure S16).
To evaluate the properties of the new polymer for controlled
light-triggered release, nanoparticles were formulated by the single
emulsion method (Figure 3), encapsulating the small hydrophobic
molecule dye Nile Red. This small molecule was chosen because
of its excellent photostability. The Z-average diameter of the
nanoparticles was 170 nm and PDI ) 0.191, as determined by
dynamic light scattering (DLS).
The release of the Nile Red payload upon irradiation was
observed by fluorescence spectroscopy. Nanoparticles were redis-
persed in PBS pH 7.4, and the fluorescence intensity of the
suspension was recorded. After irradiation with 350 nm light for 1
min, the fluorescence intensity dropped by 67%, indicating burst
release of the dye from the nanoparticles into a more polar medium
(Figures 3 and 4).19,20 On the other hand, a suspension of
nanoparticles that was not irradiated exhibited unchanged fluores-
cence intensity over several days. Interestingly, prolonged irradiation
of nanoparticles did not result in a further drop of fluorescence
signal.
Further degradation of nanoparticles of 2 after UV irradiation
was observed by DLS at 37 °C in PBS buffer at pH 7.4 and pH
10. No particles were detected after 4 days of incubation at pH 10
while in pH 7.4 the particles degraded within 10 days.
The cyclization of the diamine linker has been shown to be the
rate-determining step of the self-immolation within the quinone-
methide unit, and it has been shown to accelerate in the presence
of triethylamine.11 Thus, we measured polymer degradation in the
presence of triethylamine (Figure S15) and observed an increase
in the rate of the polymer degradation (Figure S14).
We also explored the possibility of triggering the release of Nile
Red by NIR light through two-photon absorption. The suspension
of nanoparticles in PBS pH 7.4 was irradiated at 750 nm for 20
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min intervals followed by 10 min of incubation at 37 °C. A gradual
decrease in the fluorescence intensity of Nile Red was observed
during the 4 h of irradiation (Figure 5).
spectra of polymer 2 upon photolysis, fluorescence intensity of Nile
Red encapsulated in PLGA particles, H NMR spectra of polymer 2
before and after irradiation, and GPC traces of the degrading polymer
2. This material is available free of charge via the Internet at http://
pubs.acs.org.
1
The observation of burst release of Nile Red upon UV irradiation
while the polymer degradation is slower suggests the possibility
of a secondary mechanism of release. A change in hydrophobicity
of the particles upon cleavage of the triggering group may be
involved.9 The rapid and efficient unmasking of a large number of
the secondary amino groups may make the particles rapidly more
permeable to water. This may explain the rapid release of Nile Red
upon UV irradiation. However, the two-photon unmasking process
is much less efficient which could explain the slower Nile Red
release in the NIR two-photon regime. Notably, the final degradation
of the nanoparticles is an important property for in ViVo biological
applications that require materials to degrade into easily excretable
fragments.
To rule out the possibility of spontaneous release caused by
simple cavitation, poly(lactic-co-glycolic acid) (PLGA) nanopar-
ticles encapsulating Nile Red were formulated by the same method
and exposed to UV and NIR light in the same fashion. As expected,
no release of Nile Red was observed in this case (Figures S3 and
S4).
References
(1) Wang, W.; Alexander, C. Angew. Chem., Int. Ed. 2008, 47, 7804–7806.
(2) Murthy, N.; Xu, M.; Schuck, S.; Kunisawa, J.; Shastri, N.; Frechet, J. M. J.
Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 4995–5000.
(3) Veronese, F. M. S., O.; Pasut, G.; Mendichi, R.; Andersson, L.; Tsirk, A.;
Ford, J.; Wu, G.; Kneller, S.; Davies, J.; Duncan, R. Bioconjugate Chem.
2005, 16, 775–784.
(4) Chung, J. E. Y., M.; Yamato, M.; Aoyagi, T.; Sakurai, Y.; Okano, T. J. J.
Controlled Release 1999, 62, 115–127.
(5) Liu, S. Q.; Tong, Y. W.; Yang, Y. Y. Biomaterials 2005, 26, 5064–5074.
(6) Na, K.; Lee, K. H.; Lee, D. H.; Bae, Y. H. Eur. J. Pharm. Sci. 2006, 27,
115–122.
(7) Gao, Z. G.; Fain, H. D.; Rapoport, N. J. Controlled Release 2005, 102,
203–222.
(8) Nelson, J. L.; Roeder, B. L.; Carmen, J. C.; Roloff, F.; Pitt, W. G. Cancer
Res. 2002, 62, 7280–7283.
(9) Goodwin, A. P.; Mynar, J. L.; Ma, Y. Z.; Fleming, G. R.; Frechet, J. M. J.
J. Am. Chem. Soc. 2005, 127, 9952–9953.
(10) Near-Infrared Applications in Biotechnology; Raghavachari, R., Ed.;Prac-
tical Spectroscopy Series 25; Marcel Dekker: New York, 2001.
(11) Amir, R. J.; Pessah, N.; Shamis, M.; Shabat, D. Angew. Chem., Int. Ed.
2003, 42, 4494–4499.
(12) Sagi, A.; Weinstain, R.; Karton, N.; Shabat, D. J. Am. Chem. Soc. 2008,
130, 5434–5435.
(13) Weinstain, R.; Sagi, A.; Karton, N.; Shabat, D. Chem. Eur. J. 2008, 14,
6857–6961.
(14) DeWit, M. A.; Gillies, E. R. J. Am. Chem. Soc. 2009, 131, 18327–18334.
(15) Patchornik, A; Amit, B.; Woodward, R. B. J. Am. Chem. Soc. 1970, 92,
6333–6335.
(16) Aujard, I.; Benbrahim, C.; Gouget, M.; Ruel, O.; Baudin, J. B.; Neveu, P.;
Jullien, L. Chem. Eur. J. 2006, 12, 6865–6879.
(17) Furuta, T.; Wang, S. S. H.; Dantzker, J. L.; Dore, T. M.; Bybee, W. J.;
Callaway, E. M.; Denk, W.; Tsien, R. Y. Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 1193–1200.
(18) Gug, S.; Bolze, F.; Specht, A.; Bourgogne, C.; Goeldner, M.; Nicoud, J.-
F. Angew. Chem., Int. Ed. 2008, 47, 9525–9529.
(19) Sackett, D. L.; Wolff, J. Anal. Biochem. 1987, 167, 228–234.
(20) Dutta, A. K.; Kamada, K.; Ohta, K. J. Photochem. Photobiol. A 1996, 93,
57–64.
In conclusion, we have described a novel light-sensitive nano-
particle capable of controlled triggered burst release of small
hydrophobic molecules. The versatile design of this system allows
the triggering group to be sensitive to internal or remote stimuli.
Current efforts are exploring a number of more efficient photo-
triggers sensitive to a range of wavelengths.
Acknowledgment. We thank the NIH New Innovator Award
(DP20D006499) and the Skaggs School of Pharmacy and Phar-
maceutical Sciences for their generous funding of this study.
Supporting Information Available: Experimental details for the
synthesis of polymer 2 and formulation of the nanoparticles, absorption
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