.
Angewandte
Communications
tantly, in protein-rich environments such as the bloodstream,
non-PEGylated dendrimers have been shown to interact
extensively with endogenous proteins; this interaction can
decrease the rate of oxygen quenching by an order of
magnitude or more.[6] We have observed that fourth gener-
ation dye-tagged poly(amido) amine (G4 PAMAM) “star-
burst” dendrimers were taken up by monolayer OvCa
cultures after 40 min (Figure S2); the same molecules pene-
trated throughout OvCa 3D spheroids of greater than 250 mm
diameter after 3.5 h at low concentrations (500 nm) with no
observable toxicity (Figure S3, Movie S1 in the Supporting
Information). PAMAM dendrimers are known to form
complexes with proteins such as bovine serum albumin
(BSA) and are readily endocytosed.[10,11] We therefore
reasoned that a phosphorescent porphyrin coupled to one
or more higher-generation (ꢀ G2) PAMAM dendrons would
benefit from the cell- and spheroid-penetrating properties of
PAMAM while also interacting with cellular proteins, thereby
creating an oxygen sensor with a useful dynamic range for
biological experiments.[10,11] For this work, we selected
palladium tetracarboxytetrabenzoporphyrin (PdTCTBP) as
our phosphorescent porphyrin core because its lifetime
properties have been characterized in the literature, its
synthesis is well-known, and its NIR emission (810 nm) lies
in the “optical window” ideal for tissue imaging. Additionally,
the spectral profile of PdTCTBP minimizes overlap with
known regions of intense autofluorescence and it can be
excited by readily available laser lines (445 nm and 635 nm).[6]
Prior synthetic schemes involving synthetic modification
of commercially available PAMAM dendrons followed by
convergent assembly around an oxygen-sensitive porphyrin
resulted in low yields. Therefore, to produce the spheroid-
permeable oxygen sensor described herein, we developed
a click-based strategy to rapidly assemble PAMAM-like
subunits and couple them to an oxygen-sensitive porphyrin
core. PAMAM dendrons with azide/alkyne functionality have
been reported in the literature but they problematically make
use of extremely slow reactions and require months to
synthesize.[12] In contrast, our scheme enables the rapid
assembly of PAMAM-like dendrons from three branched
subunits (Figure 1a and Figure S5). Briefly, the synthetic
scheme relies on the differing labilities of triethylsilyl (TES)
and triisopropylsilyl (TIPS) alkyne-protecting groups, which
allows sequential subunit ligations while avoiding polymeri-
zation (Figure 1b, Figure S6). First, the TIPS-protected
“starting” subunit 1d is reacted with 2 equivalents of the
“intermediate” subunit 2c and catalytic quantities of sodium
ascorbate and CuSO4. Following isolation of the reaction
product 4a, the TES protecting groups are removed by using
mild conditions that leave the focal-point TIPS protecting
group intact, thereby yielding 4b and preparing the growing
dendron for another round of ligation. Primary amine
functionality can then be added by ligation of the “final”
subunit 3b to create 4c, the focal-point alkyne of which is
then deprotected by using tetrabutylammonium fluoride to
yield 4d.[13] Each dendron growth phase can be conducted
overnight, and the product can be isolated without the use of
chromatography. This allows higher-generation triazole-
bridged PAMAM-like dendrons to be synthesized in
a matter of days as opposed to months. Once complete, the
dendrons can be conjugated to any central moiety in a single
convergent step. In this study, we coupled the dendrons to
palladium tetraazidotetrabenzoporphyrin (5b), an azido-
functionalized PdTCTBP derivative, to give the CAOS
nanoconjugate 6a, which was then deprotected to give 6b
(Figure 1c).
By using this sequential click method, we synthesized
third-generation (G3) CAOS nanoconjugates from G2
PAMAM-like dendrons. The palladium tetraazidotetraben-
zoporphyrin core is capable of reacting with up to four
equivalents of dendrons. In practice, a distribution of adducts
was observed. The G3 CAOS nanoconjugates were charac-
terized by NMR and MALDI-TOF MS as being composed of
mono-, di- and tri-dendron adducts; NMR integration sug-
gests that, on average, between two and three dendrons are
attached to each nanoconjugate (Figures S8, S29–31).
The G3 CAOS nanoconjugates were studied in a dense,
protein-rich environment and were found to exhibit uniform
oxygen sensitivity. To evaluate their photophysical properties,
we dissolved the nanoconjugates in 2% bovine serum
albumin (BSA) and monitored their phosphorescence life-
time as a function of pO2.[6] Oxygen quenching of phosphor-
escence is traditionally described using the Stern–Volmer
relationship [Eq. (1)]:
1
t
1
t0
ð1Þ
¼ kq½pO2ꢁ þ
Where t is the exponential lifetime of the phosphor, kq is
the quenching coefficient, [pO2] is the partial pressure of
dissolved oxygen, and t0 is the exponential lifetime of the
phosphor in the absence of oxygen.[6] kq is a function of the
chemical environment of the phosphor and reflects the
sensorꢀs dynamic range; in general, a lower kq value is
desirable. We compared the kq and t0 values of the G3 CAOS
nanoconjugates to those of Oxyphor G2 (Figure 2, Table 1),
which possesses a PdTCTBP core surrounded by second
generation glutamate dendrimers.[6,7] The order of magnitude
of the kq value observed for the G3 CAOS nanoconjugates is
consistent with that of Oxyphor G2, the calibration is linear,
and the dynamic range is sufficient for oxygen measurement
across the physiological range.[6]
Table 1: Calibration constants for the G3 CAOS probe and Oxyphor G2.
t0 [ms][a]
kq [mmHgꢂ1 sꢂ1 [b]
]
G3 CAOS probe
Oxyphor G2
173
178
377
283
[a] t0 =phosphorescence lifetime in zero oxygen.
[b] kq =phosphorescence quenching constant.
Having verified that the G3 CAOS nanoconjugates
exhibit the desired photophysical properties, we next tested
their uptake in cells. Given the low quantum yield (< 1%) of
PdTCTBPs in ambient oxygen conditions and the low NIR
throughput and sensitivity of commercially available micro-
3672
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 3671 –3674
Nichols, Alexander J.
