and 7 in D2O at different ionization states of
tertiary amines further support the hypothesis
(Figure 3b). The PEO segment did not change its
peak intensity and was used as an internal standard.
Throughout the ionization states, the proton reso-
nance peaks for the PR segment of 5 were easily
visualized, although the peak intensity decreased
and the width broadened at higher pH values,
reflecting the bulk aggregation of the copolymer.
For 7, the neutral state of the copolymer (i.e. 0%)
led to completely suppressed resonances in the PR
segment owing to the formation of highly compact
micelle cores. Transmission electron microscopy
(TEM) of 7 in aqueous solution demonstrated the
formation of micelles at pH 7.4 (above its pKa of
6.7) and complete micelle dissociation at pH 5.5
(Figure 3c). In comparison, no micelles were
formed from 5 at either of these pH values (data
not shown).
To investigate the intracellular activations of
pHAM particles, we examined nanoprobe 3 in
human H2009 lung cancer cells by confocal laser
scanning microscopy (Figure S3 in the Supporting
Information). It should be noted that nanoprobe 3
has an optimal pH transition at 6.3, which is ideally
suited to the study of nanoparticle activation in
early endosomes (pH 5.9–6.2).[13a,21] Because
pHAM nanoprobes are “silent” at neutral pH
values, we directly applied them in the culture
medium and monitored the kinetics of their uptake
and activation without the need to remove the
medium. Right after the nanoprobe addition,
neither the H2009 cells nor the medium showed
an observable fluorescence signal. At 15 min,
punctuate fluorescent dots appeared inside the cells. The
number of fluorescent dots increased over time. The signal-to-
noise ratio of the H2009 cells (SNRCell, using fluorescence
intensity at time t = 0 as the background noise) allowed
further quantification of the increased nanoprobe uptake and
activation over time. At 60 min, a 31-fold increase in SNRCell
((2.14 Æ 0.17) ꢁ 103) was observed over the medium
(SNRMed = 69.3 Æ 9.1, P < 0.001), where the majority of the
nanoprobes were still present. Then 0.1n HCl solution was
added to acidify the medium to pH 5.0, and considerable
increase in fluorescence intensity in the medium background
was found. A reverse trend of fluorescence contrast was
observed, where SNRCell was 74% of SNRMed (P < 0.05).
These data illustrate that pHAM nanoprobes can dramati-
cally increase the contrast sensitivity of cancer cells compared
to potentially always-ON nanoprobes.
Figure 3. Investigation of the ultra-pH-responsive properties of a representative
pHAM nanoprobe. a) pH titration curves of copolymers 5 and 7 and their
corresponding monomers. The added volumes of NaOH (VNaOH) were normalized
to the initial amount of amine residues ([R3N]0 in mmol). b) 1H NMR spectra (in
D2O) of 5 (top) and 7 (bottom) at different ionization states of the copolymers.
c) TEM image of 7 in pH 5.5 and 7.4 buffers at a polymer concentration of
2 mgmLÀ1
.
the micelles (pH 7.4) than the free dye (1.97 ns) at pH 7.4 or
the disassembled unimers at pH 5.5 (1.84 ns).
For the pH temporal response, stopped-flow experiments
showed that fluorescence activation was very fast, with most
nanoprobes fully activated within 5 ms at pH values lower
than their respective pKa values (e.g. t1/2 = 3.7 ms for 4,
Figure 2c). The ultrasensitive pH response was only observed
with 4, 3, 7, and 6. The fluorescence transition pH values (pHt,
the pH value at which F = 0.5(Fmax+Fmin)) were 5.4, 6.3, 6.8,
and 7.2 for nanoprobes 4, 3, 7, and 6, respectively (Figure 2b).
The other copolymers either did not show any pH response
(e.g. 1 in Figure 2a) or only broad pH responses (e.g. 2, 5, data
not shown). We hypothesize that hydrophobic micellization is
the driving force for the ultra-pH-responsive properties of
pHAM nanoparticles, and a critical threshold of hydropho-
bicity in the PR segment is necessary to achieve the
cooperative response. To test this hypothesis, we used
copolymers 5 and 7 as examples and compared their pH
titration curves and those of the corresponding monomers
(Figure 3a). Larger ring size (i.e. 7) resulted in higher
hydrophobicity in the PR segment owing to the extra
methylene groups. Copolymer 5 showed a broad pH response,
similar to both monomers over added volumes of NaOH. In
contrast, copolymer 7 had a much sharper pH transition, thus
demonstrating its better buffer capacity. 1H NMR spectra of 5
To further investigate whether different endocytic organ-
elles can selectively activate pHAM systems, we transfected
H2009 cells with green fluorescent protein (GFP)-fused
Rab5a and Lamp1 biomarkers in early endosomes and late
endosomes/lysosomes, respectively. Two pHAM nanoprobes
(3 and 4 with pHt of 6.3 and 5.4, respectively) were incubated
with H2009 cells, and confocal microscopy imaging was used
to examine the subcellular locations of pHAM nanoprobe
activation (Figure 4 and Figure S4 in the Supporting Infor-
Angew. Chem. Int. Ed. 2011, 50, 6109 –6114
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
6111