Increasing materials' response to two-photon NIR light via self-immolative dendritic scaffolds

Article abstract of DOI:10.1039/c2cc00072e

Photoactivation using two photons of NIR allows non-invasive biological manipulation. We applied the principle of dendritic amplification to improve the materials' sensitivity to NIR light. Light induced uncaging or release of l-glutamic acid was 2.8 fold

Full text of DOI:10.1039/c2cc00072e

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Showcasing research from Prof. Adah Almutairi’s
Laboratory for Bioresponsive Materials (part of the
KACST-UCSD Center of Excellence in Nanomedicine)
at UC San Diego.
As featured in:
Increasing materials’ response to two-photon NIR light via
self-immolative dendritic scaffolds
An alternative strategy to increasing efficiency of two-photon
release by using self-immolative dendritic architectures amplifies
release of caged molecules upon near infrared absorption.
See Adah Almutairi et al.,
Chem. Commun., 2012, 48, 9138.
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COMMUNICATION
Increasing materials’ response to two-photon NIR light via
self-immolative dendritic scaffoldsw
Nadezda Fomina,a Cathryn L. McFearina and Adah Almutairi*abcd
Received 5th January 2012, Accepted 16th March 2012
DOI: 10.1039/c2cc00072e
Photoactivation using two photons of NIR allows non-invasive
biological manipulation. We applied the principle of dendritic
amplification to improve the materials’ sensitivity to NIR light.
Light induced uncaging or release of L-glutamic acid was 2.8 fold
higher when incorporating 4-bromo-7-hydroxycoumarin (Bhc)
with self-immolative dendrimers compared with Bhc directly
conjugated to L-glutamic acid.
design a self-immolative5–7 dendrimer that translates a single
triggering event into a cascade of molecular rearrangements to
release multiple terminal groups. Self-immolative dendritic
structures have been applied to create stimuli-responsive
prodrugs8,9 and in analytical assays.10–12 Here we demonstrate
that the principle of dendritic amplification may be applied to
create materials with improved sensitivity to two-photon NIR
light. Conjugation of the known and well-studied two-photon
caging group, 4-bromo-7-hydroxycoumarin, to a self-immolative
quinone-methide based dendrimer results in a 3-fold higher
amount of uncaged L-glutamic acid (LGA) compared to Bhc
directly conjugated to LGA.
Photoactivation using near infrared (NIR) light has been
recognised as an attractive new avenue for non-invasive
delivery of bioactive molecules to target sites.1 Two-photon
excitation and uncaging provide excellent spatial and temporal
resolution for this method. However, the development of new
two-photon sensitive organic materials with sufficiently high
two-photon uncaging action cross-sections is still a challenge.
Despite a large amount of research dedicated to improving the
two-photon action cross section of organic protecting groups,
the progress has been rather slow due to the inability to predict
the cross-sections for two-photon excitation and the quantum
yields of uncaging based on the chromophore structure.2 The
first caging group generally applicable for two-photon uncaging
of biologically important molecules with an action cross section
of 1 GM at 740 nm was 4-bromo-7-hydroxycoumarin (Bhc).3
The largest two-photon uncaging action cross-section reported
to date is 10 GM,4 however, this caging group is obtained
through multi-step synthesis and its cytotoxicity has not been
evaluated. Therefore the field has achieved a 10 fold enhance-
ment in the action cross-section of biologically relevant two-
photon uncaging groups over a thirteen year period.
The structures of the zero (G0), first (G1), and second (G2)
generation quinone-methide based dendrimers incorporating
the Bhc photo triggering group and 1, 2, and 4 caged molecules
of LGA, respectively, are shown in Fig. 1. LGA was chosen as a
model small bioactive molecule commonly used in two-photon
uncaging studies.3,13–15 Compound G0 was used as a reference,
since it has been shown to release LGA upon two-photon
excitation.3 We anticipated that the branched structure of G1
and G2 allows for more incorporation of caged molecules and
should result in 2- and 4-fold enhanced release of LGA relative
to G0 upon a given dose of NIR stimulation.
The synthesis of the dendrimers is outlined in Scheme 1. The
zero generation dendrimer was synthesised by reacting compound
116 with LGA dimethyl ester hydrochloride, followed by
sequential removal of the methoxymethyl and methyl esters by
Although more efficient two-photon caging groups are being
developed, alternative strategies for increasing sensitivity to
two-photon NIR would be attractive. One way to increase
two photon sensitivity that has never been done before is to
a Skaggs School Pharmacy and Pharmaceutical Sciences, University of
California at San Diego, La Jolla, California 92093, USA.
E-mail: aalmutairi@ucsd.edu; Fax: +1 8 58 200 3815;
Tel: +1 8 58 822 2942
b Department of NanoEngineering, University of California at
San Diego, La Jolla, California 92093, USA
c Materials Science and Engineering, University of California at
San Diego, La Jolla, California 92093, USA
d Biomedical Sciences Programs, University of California at
San Diego, La Jolla, California 92093, USA
w Electronic supplementary information (ESI) available: Experimental
details. See DOI: 10.1039/c2cc00072e
Fig. 1 Structures of the dendrimers bearing a coumarin photo trigger
(blue) and caged LGA (red).
c
9138 Chem. Commun., 2012, 48, 9138–9140
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Scheme 1 Synthesis of G0, G1 and G2 dendrimers.
acid- and base-catalysed hydrolysis, respectively. The G1 and
G2 dendrimers were synthesised by a convergent route: compound
317 was reacted with LGA dimethyl ester hydrochloride to
afford intermediate 4. After removal of the BOC protecting
group, reacting 4 with 1 installed the triggering group. Finally,
sequential deprotection of 5 afforded G1. Similarly, G2 was
obtained by reacting 4 with 3 followed by installation of the
Bhc photo triggering group.
Cleavage of Bhc photo triggering groups by NIR light was
measured by HPLC. One mM solutions of the dendrimers in
PBS pH 7.4 were irradiated at 740 nm for 30 min. The
irradiated solutions were injected into HPLC with 4-hydroxy-
benzoic acid-n-hexyl ester as an internal standard and chromato-
grams were recorded at 280 nm. The fraction of the cleaved
triggering groups was calculated by integrating the peaks of
the dendrimers relative to the peak of the internal standard
before and after NIR exposure. As expected, photo triggered
removal of the Bhc group was of similar efficiency across the
dendrimer series and varied between 47 and 53% (Table 1).
The rearrangement of the quinone-methide dendrimers upon
this phototrigger was followed by HPLC-MS (Fig. 2).
Fig. 2 Disassembly of the intermediates generated by cleavage of Bhc
with NIR light monitored by HPLC-MS. (A) G1, (B and C) G2, (D)
structures of the intermediates, (E) schematic of dendrimer disassembly.
Irradiated solutions were incubated at 37 1C and aliquots
were removed periodically to determine the concentrations of
the intermediates (Fig. 2, IM 1 for G1 and IM 21 and IM 22
for G2) by integrating the peaks of the single ions, m/z = 628
(for IM 1 and IM 22, z = 1) and m/z = 795 (for IM 22, z = 2),
respectively. The signal from IM 1 gradually decreases within
96 hours, consistent with the previously published degradation
kinetics of quinone-methide dendrimers,18 while IM 21 remains
in solution for 120 hours (Fig. 2). The build-up of IM 22 in
solution over time and its subsequent disappearance after
196 h confirm the end-to-end degradation of G2. The total
efficiencies of G1 and G2 degradation were calculated to be
99% and 84.4% after 96 and 196 hours, respectively. The
increase in IM 1 and IM 22 over time in non-irradiated G1 and
G2 solutions results from partial degradation via dark hydro-
lysis and is taken into account when calculating the total
efficiencies of G1 and G2 degradation. Based on this result,
the expected amplification of the amount of uncaged LGA was
1.98 for G1 and 3.37 for G2.
Table 1 Exposure of G0, G1 and G2 to NIR light for 30 min
Expected Experimental
amplification amplification
%Photo
LGA
Generation triggereda releasedb/mg factor
factor
G0
G1
G2
47.6 (Æ6) 31.1 (Æ7)
52.9 (Æ2) 50.8 (Æ5)
46.7 (Æ3) 87.0 (Æ14)
1
1.98
3.37
1
1.63
2.80
a
b
Determined by HPLC relative to an internal standard. The values
obtained by Amplex Red assay after 72 h (G0), 96 h (G1) and 192 h
The release of caged LGA was quantified using an Amplex
Red enzymatic assay. After irradiation with NIR light for
30 min, the 1 mM solutions of G0, G1, and G2 were diluted
c
(G2) and corrected for controls. The numbers were calculated from
graphs in Fig. 2 and corrected for controls.
c
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Fig. 3 Release of LGA from G0, G1 and G2 after NIR exposure.
with PBS (pH 7.4) to 7, 3.5 and 1.75 mM, respectively, to
ensure that the final concentration of caged LGA was the same
across the series. The solutions were incubated at 37 1C and
aliquots were taken out at the specified time periods and
analysed for LGA (Fig. 3). G0 released 31 mg of LGA upon
irradiation and this served as the benchmark to determine the
amplification factors for G1 and G2. The kinetics of release by
G1 and G2 were consistent with the degradation profiles of the
intermediates (Fig. 2). Thus, G1 released 51 mg of the caged
acid over 96 h, while G2 showed release within 196 h (Table 1)
with an induction period of 48 h, yielding 88 mg of LGA. The
control solutions also showed some release of LGA after
prolonged incubation (over 100 h), pointing to non-specific
hydrolysis.
neurotransmitters, however, designing self-immolative dendri-
mers with faster kinetics of disassembly is of critical importance.
Our current efforts are focused on incorporating these dendritic
amplifiers into hydrogel nanocarriers that can be remotely
activated to release their cargo upon exposure to NIR light.
The authors thank the NIH Directors New Innovator
Award (1 DP2 OD006499-01) and King Abdul Aziz City of
Science and Technology (KACST) for financially supporting
this study.
Notes and references
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The results collected via the Amplex Red assay reveal that,
indeed, the amount of LGA released after 30 min of exposure
to NIR light is 1.63-fold higher for G1 and 2.80-fold higher for
G2, compared to G0, demonstrating that chemical amplification
through self-immolative constructs is a useful strategy to
increase the materials’ response to NIR light. However, the
amplification is slightly lower than the expected values of 1.98
and 3.37. We investigated whether the byproducts of the
dendrimers’ degradation, namely, 4-bromo-7-hydroxycoumarin,
2,6-bis-(hydroxymethyl)-p-cresol and 1,3-dimethyl-2-imidazol-
idinone,6,16 interfered with the enzymatic assay and ruled that
out as a cause (Fig. S1, ESIw). Because photobleaching is
enhanced in the two-photon regime compared to single photon
excitation,19 one possible explanation for the lower than
expected amplification is photochemical side reactions that occur
upon exposure to NIR light and consume the starting material
but do not lead to the desired liberation of the intermediates IM
1 and IM 21. Appearance of side products was observed by
HPLC, but their structures could not be deduced.
This work demonstrates that the principle of dendritic
amplification may be used to increase the responsiveness of
materials to two-photon excitation using well-studied triggering
groups. The results obtained with release of the small molecule
LGA should be translatable to uncaging of macromolecules or
even to the nano scale. It is important to note that for higher
generation dendrimers the overall degradation process is
slower because rearrangement reactions occur in series. This
is a useful strategy for increasing overall biological response to
light and achieving sustained release. For the uncaging of
19 G. H. Patterson and D. W. Piston, Biophys. J., 2000, 78,
2159–2162.
c
9140 Chem. Commun., 2012, 48, 9138–9140
This journal is The Royal Society of Chemistry 2012