Selective tracking of lysosomal cu2+ ions using simultaneous target- and location-activated fluorescent nanoprobes

Article abstract of DOI:10.1021/ac503240x

Levels of lysosomal copper are tightly regulated in the human body. However, few methods for monitoring dynamic changes in copper pools are available, thus limiting the ability to diagnostically assess the influence of copper accumulation on health status. We herein report the development of a dual target and location-activated rhodamine-spiropyran probe, termed Rhod-SP, activated by the presence of lysosomal Cu²⁺. Rhod-SP contains a proton recognition unit of spiropyran, which provides molecular switching capability, and a latent rhodamine fluorophore for signal transduction. Upon activation by lysosomal acidic pH, Rhod-SP binds with Cu²⁺ by spiropyran-based proton activation, promoting, in turn, rhodamine ring opening, which shows a "switched on" fluorescence signal. However, to protect Rhod-SP from degradation and interference by the physiological environment, it is engineered on mesoporous silica nanoparticles (MSNs), and the surface of Rhod-SP@MSNs is further anchored with β-cyclodextrin (β-CD) to enhance the solubility and bioavailability of Rhod-SP@MSN-CD. Next, to enhance cell specificity, a guiding unit of c(RGDyK) peptide conjugated adamantane (Ad-RGD) as prototypical system, is incorporated on the surface of Rhod-SP@MSN-CD to target integrin α_vβ₃ and α_vβ₅ overexpressed on cancer cells. Fluorescence imaging showed that both Rhod-SP@MSN-CD and Rhod-SP@MSN-CD-RGD were suitable for visualizing exogenous and endogenous Cu²⁺ in lysosomes of living cells. This strategy addresses some common challenges of chemical probes in biosensing, such as spatial resolution in cell imaging, the solubility and stability in biological system, and the interference from intracellular species. The newly designed nanoprobe, which allows one to track, on a location-specific basis, and visualize lysosomal Cu²⁺, offers a potentially rich opportunity to examine copper physiology in both healthy and diseased states.

Full text of DOI:10.1021/ac503240x

Article
pubs.acs.org/ac
2+
Selective Tracking of Lysosomal Cu Ions Using Simultaneous
Target- and Location-Activated Fluorescent Nanoprobes
†
,§
†,§
‡
†
‡
†
†
Yinhui Li, Yirong Zhao, Winghong Chan, Yijun Wang, Qihua You, Changhui Liu, Jing Zheng,
†
†
,†
Jishan Li, Sheng Yang, and Ronghua Yang*
†State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Collaborative
Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha, 410082, China
‡
Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Kowloon, Hong Kong, China
*
S Supporting Information
ABSTRACT: Levels of lysosomal copper are tightly regulated
in the human body. However, few methods for monitoring
dynamic changes in copper pools are available, thus limiting
the ability to diagnostically assess the influence of copper
accumulation on health status. We herein report the
development of a dual target and location-activated rhod-
amine−spiropyran probe, termed Rhod-SP, activated by the
2
+
presence of lysosomal Cu . Rhod-SP contains a proton
recognition unit of spiropyran, which provides molecular switching capability, and a latent rhodamine fluorophore for signal
transduction. Upon activation by lysosomal acidic pH, Rhod-SP binds with Cu2+ by spiropyran-based proton activation,
promoting, in turn, rhodamine ring opening, which shows a “switched on” fluorescence signal. However, to protect Rhod-SP
from degradation and interference by the physiological environment, it is engineered on mesoporous silica nanoparticles
(
MSNs), and the surface of Rhod-SP@MSNs is further anchored with β-cyclodextrin (β-CD) to enhance the solubility and
bioavailability of Rhod-SP@MSN-CD. Next, to enhance cell specificity, a guiding unit of c(RGDyK) peptide conjugated
adamantane (Ad-RGD) as prototypical system, is incorporated on the surface of Rhod-SP@MSN-CD to target integrin α β and
v
3
α β overexpressed on cancer cells. Fluorescence imaging showed that both Rhod-SP@MSN-CD and Rhod-SP@MSN-CD-RGD
v
5
2+
were suitable for visualizing exogenous and endogenous Cu in lysosomes of living cells. This strategy addresses some common
challenges of chemical probes in biosensing, such as spatial resolution in cell imaging, the solubility and stability in biological
system, and the interference from intracellular species. The newly designed nanoprobe, which allows one to track, on a location-
2
+
specific basis, and visualize lysosomal Cu , offers a potentially rich opportunity to examine copper physiology in both healthy
and diseased states.
opper plays a crucial role in many cellular processes, and
its concentration at the cellular level is related to severe
fluorescent platform with a mitochondrial-targeting triphenyl-
C
phosphonium moiety for localizing the probe to the
1
−3
19,20
physiological and pathological events.
Because of its
organelle.
However, the imperfect targeting ability of
essential, yet toxic, nature, intracellular copper distribution is
strictly controlled by cells. It has been demonstrated that some
human copper transporters localize late in endosomes and
these guidance systems, along with the nonspecific distribution
of the analyte throughout the cell, resulted in considerable
background signal from cytosol and other organelles, making it
impossible to achieve high resolution at the suborganelle level.
In addition, the poor solubility of the probe molecules,
combined with the degradation of the molecules by lysosomal
4
−6
lysosomes to facilitate cellular copper uptake.
However,
excess copper excreted from cytoplasm can accumulate in
lysosomes, causing a highly toxic state and resulting in serious
21,22
7
,8
acid and hydrolase,
as well as interference from other
infant liver damage, such as Wilson’s disease. On the other
hand, the loss of copper homeostasis can result in Alzheimer’s
intracellular species, have remained challenging. Nonetheless,
we have been committed to solving these problems within the
context of our ongoing interest in developing highly sensitive
9
,10
disease and Menkes disease.
Therefore, it is desirable to
develop new molecular tools to monitor lysosomal copper
concentrations.
23−25
and selective spiropyran-based molecular probes.
As a
While numerous molecular tools for intracellular Cu2+ have
consequence of our efforts, we proposed herein a new strategy
2
+
11−15
for real-time targeting and selective tracking of lysosomal Cu
been developed and applied to intracellular imaging,
of
ions. The new designed fluorescent spiropyran-based probe,
which capability to selective track the metal in an organelle have
16−18
been rarely reported.
In the seminal work of Chang et al., a
targetable fluorescent probe for imaging exchangeable
mitochondrial Cu pools was recently proposed. The probe
was chemically designed by combining a Cu+ responsive
Received: August 26, 2014
Accepted: December 1, 2014
+
16
©
XXXX American Chemical Society
A
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Analytical Chemistry
Article
termed Rhod-SP, which contains an proton recognition unit of
Scheme 1. (A) Synthetic Route for Rhod-SP. (B) Proposed
Scheme of Fluorescence Response of Rhod-SP by Binding of
H and Cu
2
+
spiropyran and a latent Cu -complexed rhodamine fluoro-
phore, allows tracking on a location-specific basis and
+
2+
2
+
visualizing lysosomal Cu .
To ensure that Rhod-SP can most accumulate in lysosomes
2
+
and efficiently track Cu without deleterious interference from
biological systems, we chose mesoporous silica nanoparticles
(
MSNs) as the probe reservoir, a familiar nanocarrier, and
26−30
lysosomal marker.
The surface of Rhod-SP@MSNs was
then anchored with β-cyclodextrin (β-CD) as a gatekeeper, to
eliminate interference from intracellular species, but allow Cu2+
ions to enter the nanopores and react with Rhod-SP. To
enhance cell specificity, a c(RGDyK) peptide conjugated with
adamantane (Ad-RGD) was incorporated on the surface of
Rhod-SP@MSN-CD to target integrin α β and α β overex-
v
3
v 5
31
pressed on cancer cells, resulting in Rhod-SP@MSN-CD-
RGD that targets lysosomal Cu via the specific recognition of
integrin α β and α β overexpressed in cancer cells. On the
2
+
v
3
v 5
basis of these strategies, the ability to monitor the dynamics of
lysosomal copper at high spatial resolution offers a potentially
rich opportunity to examine copper physiology in both healthy
and diseased states.
EXPERIMENTAL SECTION
■
Preparation of Rhod-SP-Based Nanoprobes. Synthesis
of Rhod-SP. The synthesis route of Rhod-SP is shown in
Scheme 1A (for details, see Supporting Information).
Compound 1 was obtained via the Duff reaction by using
anhydrous trifluoroacetic acid, and the reaction of Rhodamine
B with hydrazine hydrate afforded compound 2. Then,
compounds 1 and 2 were mixed at room temperature and
stirred for 12 h, giving compound 3. Finally, Rhod-SP was
obtained via Knoevenagel condensation reaction of compound
3
and 1,2,3,3-tetramethyl-3H-indolium in 75% yield.
Preparation of Rhod-SP@MSN-CD and Rhod-SP@MSN-
CD-RGD. MSN-NH was prepared according to a protocol
2
32
given in the literature. Briefly, purified MSN-NH (10 mg)
2
was added to an acetonitrile solution (5 mL) of Rhod-SP (20
mM). The solution mixture was stirred at room temperature for
2
4 h, to allow encapsulation of Rhod-SP by MSN-NH . The
2
resulting particles were then separated by centrifugation and
dispersed in a mixture solution (dimethyl sulfoxide/water
(
DMSO/H O) = 1:1, v/v) with mono-6-deoxy-6-(p-tolylsul-
2
of 550 nm with an emission wavelength range from 540 nm to
fonyl)-β-cyclodextrin (6-OTs-β-CD) (90 mg). The mixture was
allowed to react for 24 h at 45 °C under nitrogen atmosphere
to yield Rhod-SP@MSN-CD. The resulting material was
filtered and extensively washed with ultrapure water and
ethanol and dried under high vacuum at 60 °C. To modify the
RGD to Rhod-SP@MSN-CD, Rhod-SP@MSN-CD (1.0 mg)
and adamantine-functionalized RGD (0.2 mg) were added to
7
00 nm.
To measure leakage of Rhod-SP from MSNs, Rhod-SP@
MSN and Rhod-SP@MSN-CD were suspended in 300 μL of
buffer solution, respectively, sealed in a homemade dialysis tube
with a molecular weight cutoff of 8000−14 000 Da, and then
2
+
immersed in 1.7 mL of buffer solution containing 10.0 μM Cu
at 37 °C. The system was moderately shaken. The fluorescence
intensity of dialysis fluid was measured at certain time points
with an excitation wavelength of 550 nm.
1.0 mL of ultrapure water. The mixture reacted for 0.5 h at
room temperature. After the reaction, Rhod-SP@MSN-CD-
RGD complexes were washed three times by centrifugation at
Cell Imaging. HeLa cells were incubated with 50 μg/mL
14 000 rpm with ultrapure water.
Rhod-SP@MSN-CD for 3.0 h in the culture medium, followed
by supplementation with 50 μM CuCl for 12.0 h. Cells were
Spectrophotometric Measurements. Both fluorescence
2
and UV-vis absorption measurements of Rhod-SP@MSN-CD
were conducted in sodium acetate/acetic acid buffer solution.
For Cu2 assay, 1.0 mL sodium acetate/acetic acid buffer (20
mM, pH 5.0) containing 1.0 μM Rhod-SP or 50 μg/mL Rhod-
SP@MSN-CD was first introduced to a quartz cell. Following
also treated with EDTA before and after incubation with Rhod-
SP@MSN-CD for 2.0 h, respectively. In addition, after cells
+
were supplemented with CuCl and incubated with Rhod-SP@
2
MSN-CD, 1,8-bis(dimethylamino)naphthalene (DMAN), a
common proton sponge, was further incubated with cells for
2.0 h to promote lysosomal damage. Cells were washed three
times with PBS and then analyzed by confocal fluorescence
2
+
the additions of different concentration of Cu solution, the
fluorescence intensities were recorded at excitation wavelength
B
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Analytical Chemistry
Article
microscopy. To monitor exogenous Cu2+ in cells, the cells were
internalize into cells by endocytosis and mainly accumulate in
2
6,29,30
pretreated with different concentrations of CuCl solution for
lysosomes.
Finally, to enhance cell specificity, β-CD was
2
1
2.0 h and then incubated with 50 μg/mL Rhod-SP@MSN-CD
for 3.0 h.
To characterize Rhod-SP@MSN-CD for labeling lysosomes,
modified with a targeting vector peptide cyclo(Arg-Gly-Asp-
31
Phe-Lys(mpa)) conjugated with adamantane (denoted as Ad-
RGD) on β-CD via host−guest interactions of β-CD and Ad
(see Scheme 2A). This Ad-RGD, when incorporated as a
a lysosome tracker, LysoTracker Green, was used as the
standard lysosome marker. HeLa cells were incubated with 50
μg/mL Rhod-SP@MSN-CD in culture medium for 3.0 h. To
stain lysosomes, the cells were incubated with the indicated
concentrations of LysoTracker Green 1.0 μM for 30 min before
Scheme 2. (A) Synthetic Route of Rhod-SP@MSN-CD-
RGD. (B) Schematic Illustration of the Nanoprobe Rhod-
2
+
SP@MSN-CD-RGD for Cu Imaging in Lysosomes
imaging. (Rhod-SP@MSN-CD channel: λex = 559 nm, λem
=
5
70−600 nm bandpass. LysoTracker Green channel: λ = 488
ex
nm, λ = 505−530 nm bandpass. Yellow: co-localization of red
and green fluorescence).
em
Transmission Electron Microscopy Analysis. HeLa cells
were treated with Rhod-SP@MSN-CD in media for 3.0 h at 37
°C. After washing with PBS, the cells were detached and
centrifuged. The cell pellets were fixed with 2.5% glutaralde-
hyde in 0.1 M phosphate buffer for 2.5 h, dehydrated using an
ascending alcohol series (20%, 40%, 60%, 80%, and 100%
twice) for 20 min for each change, and then embedded in
Araldite resin at 65 °C overnight. A 70 nm section was placed
on a TEM grid and stained with saturated uranyl acetate and
0
.2% Reynolds lead citrate before TEM imaging. Images were
collected using a JEOL 1200 EXII scanning and transmission
electron microscope (Japan Electron Optics Laboratory,
Peabody, MA).
The Binding Specificity of Rhod-SP@MSN-CD-RGD.
The binding specificity of Rhod-SP@MSN-CD-RGD to HeLa
cells was performed using confocal laser scanning microscopy.
MCF-7 cells were used as a negative control. HeLa cells and
MCF-7 cells were seeded in cell culture dishes at a density of 4
component with Rhod-SP@MSN-CD, exhibited specific cell
binding to α β and α β integrin receptors overexpressed on
4
×
10 cells/dish in RPMI 1640 medium with 15% fetal calf
v
3
v 5
serum and allowed to grow for 2 days. The HeLa and MCF-7
cells were respectively incubated with Rhod-SP@MSN-CD-
RGD (50 μg/mL) in culture medium with different incubation
times.
the membrane of most cancer cells, thereby improving
internalization efficiency of the nanoprobe through receptor-
mediated endocytosis (Scheme 2B). The newly designed
nanoprobe not only makes full use of the acidic pH
environment to activate the probe, but also inhibits the
macromolecules of proteins and enzymes from diffusing into
the nanopores to degrade the chemical probe, which allows one
to track in a location-specific manner and visualize lysosomal
RESULTS AND DISCUSSION
■
2
+
To reliably acquire signals activated by Cu under acidic
lysosomal environment (pH = 4.0−6.0), a rhodamine-−
piropyran fluorescent probe, termed Rhod-SP, that recognizes
2+
Cu with high spatial resolution.
2
+
the presence of lysosomal Cu and then binds with it through
spiropyran-based proton activation was designed and synthe-
sized (see Scheme 1A, as well as Figures S1−S4 in the
Supporting Information). Rhod-SP possessed a spiropyran
recognition unit, which is a well-known metastable system
Figure S5 and Figure 1A show the absorption and
fluorescence spectra of Rhod-SP. At pH 8.0, Rhod-SP exhibited
no absorption in the visible region and was essentially
nonfluorescent, because of the dual closure of rhodamine and
2+
spiropyran moieties. Upon the addition of Cu , no significant
change in absorption or fluorescence was noted, indicating that
the spirolactam moiety of Rhod-SP remained intact. In an
acidic medium (pH 5.0), however, the absorption spectra of
Rhod-SP showed a significant band at 400−460 nm, which
could be ascribed to the protonated merocyanine form of
spiropyran, but, still, no characteristic absorption of the
rhodamine moiety was observed. However, upon the addition
3
3,34
susceptible to the activation of UV light or protonation,
and a latent fluorescent signal transduction unit of rhodamine.
Rhod-SP can be activated by acidic pH to release the ligating
2
+
phenolic group required for binding Cu via collaborative
coordination with CO and CN that promotes rhodamine
ring-opening and shows a “switched on” fluorescence signal
(
see Scheme 1B). However, the fluorescent signal was silent
2+
when Rhod-SP was allowed into an alkaline environment. Next,
to confer the probe with the ability to accumulate in lysosomes
and, at the same time, bypass interference and degradation, we
used mesoporous silica nanoparticles (MSNs) as a protective
nanocoating, entrapping the probe in MSN channels. Mean-
while, MSNs were conjugated with β-cyclodextrin (β-CD) as a
of Cu , an absorption at 550 nm emerged concomitant with a
2
+
fluorescent emission at 590 nm, indicating Cu -induced
rhodamine ring-opening via metal-mediated coordination
37
reaction in an acidic medium. The inset of Figure 1A, and
Figure S6 in the Supporting Information, further confirmed the
pH dependence of Rhod-SP for Cu2+ sensing. Furthermore, an
3
5,36
2+
gatekeeper,
to eliminate interference from intracellular
increase of fluorescence with increasing Cu concentration was
2
+
species, but allow Cu ions to enter the nanopores and react
with Rhod-SP. By their nanosize, MSNs are expected to
observed over the pH range of 4.0−6.0, which corresponds to
22
the acidic pH microenvironment in lysosomes.
C
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Article
Supporting Information), primarily because of the strong
2
+
binding between the sulfhydryl function and Cu . These
results suggested that free Rhod-SP for Cu2+ detection would
be severely thwarted by interference from complex biological
systems.
Based on the above results, it can be inferred that Rhod-SP
would be a good lysosome-activatable Cu2+ probe. However, to
protect Rhod-SP from degradation and interference by the
physiological environment, we engineered Rhod-SP on
mesoporous silica nanoparticles (MSNs) as a protective
nanocoat entrapping Rhod-SP molecules in MSNs channels
Figure 1. (A) Fluorescence emission spectra (λ = 550 nm) of Rhod-
ex
(
Rhod-SP@MSN). The surface of MSNs was then function-
SP under different conditions: (a), 1.0 μM Rhod-SP in 50% buffer−
alized with a β-cyclodextrin (β-CD) as a gatekeeper to cap the
CH CN (v/v) solution (pH 7.0); (b) the solution described for that in
3
2+
pores and enhance the dispersibility and inertness of MSNs in
panel a, + 10.0 μM Cu ; (c) 1.0 μM Rhod-SP in 50% buffer-CH CN
3
aqueous media (see Scheme 2A). MSN-NH was prepared
(
1
v/v) solution (pH 5.0); and (d) the soluton described for panel c +
0.0 μM Cu . Inset shows the effect of pH on the fluorescence
2
34
2+
according to protocols described in the literature. Trans-
mission electron microscopy (TEM) and powder X-ray
diffraction (PXRD) confirmed the well-ordered structures
with typical MCM-41 hexagonal arrangements, having an
approximate diameter of 80 nm (Figure 2). Using a diffusion
emission of Rhod-SP in the absence (●) and presence (▲) of 10.0
2+
μM Cu . (B) Real-time fluorescence records (λ /λ = 550 nm/590
ex ex
nm) of Rhod-SP upon addition of Cu2 in 50% buffer−CH CN
+
3
solution (pH 7.0), following several repeated concentration step
changes between 200 μM HCl solution and then 200 μM NaOH
solution.
Next, the binding mode between Rhod-SP and Cu2 was
+
1
verified by H NMR analysis (see Figure S7 in the Supporting
Information). The peaks at δ 12.60 and 9.18 ppm for the two
2+
protons (OH, NCH) disappeared after the addition of Cu ,
implicating that nitrogen and oxygen take part in Cu2+ binding.
In addition, other peaks for the active protons broadened by
the increase of proton exchange, and both the peaks shifted
upfield because of the electron-deshielding effect of Cu2+ on
them. From the ESI-MS spectrum (Figure S8 in the Supporting
Information), a new m/z peak was found to be 863.82 in its
2
+
ESI-MS upon addition of Cu , which was assigned to [Rhod-
2
+ +
SP + Cu ] . The (A − A )/A value displayed a maximum at a
0
0
molar fraction of ∼0.508 by Job’s method in Figure S9 in the
Supporting Information, indicating a 1:1 stoichiometry of Cu2+
Figure 2. (A) TEM images and (B) small-angle powder XRD patterns
to Rhod-SP in the complex, where A and A represent the
absorbance at 550 nm of Rhod-SP without or with Cu ,
respectively.
of MSN-NH , MSN-CD, and Rhod-SP@MSN-CD. (C) The
0
2
2+
cumulative release profiles of Rhod-SP from Rhod-SP@MSN and
Rhod-SP@MSN-CD in buffer solution (20 mM, pH 5.0) containing
+
1
0.0 μM Cu2 at 37 °C.
As shown in Figure 1B, further investigation revealed rapid
and complete reversibility of the response of Rhod-SP to Cu2+
with pH changes. A slight increase in fluorescence intensity at
protocol, Rhod-SP molecules were entrapped in MSN-NH . β-
2
5
90 nm could be observed upon the addition of Cu2+ in neutral
CD was installed on the Rhod-SP-loaded MSN surface via the
amidation reaction between MSN-NH2 and mono(6-(p-
toluenesulfonyl))permethylated β-cyclodextrin (β-CD) (de-
noted as Rhod-SP@MSN-CD). The loading efficiency of
Rhod-SP in MSN-CD was estimated to be ∼12 wt %. The
treatment processes of the MSNs were characterized using
TEM, PXRD, the zeta potential, and nitrogen adsorption−
desorption isotherms. From Figure 2A, the average diameter of
MSN-CD and Rhod-SP@MSN-CD showed no significant
solution, but a strong fluorescence was realized at acidic pH.
Importantly, fluorescence could be “switched off” at alkaline
pH, indicating that Rhod-SP could be exploited for the real-
2
+
time monitoring of Cu in a complex intracellular environ-
ment. Collectively, these in vitro results demonstrated that the
Rhod-SP fluorescence signal could reversibly become quenched
in cytoplasm, but, conversely, it would be illuminated in acidic
subcellular organelles, such as endosome or lysosome.
Selectivity is another important parameter by which to assess
the performance of a fluorescent probe in a complex cell
environment. At pH 5.0 (see Figure S10 in the Supporting
Information), the fluorescence intensity of Rhod-SP displayed
differences from that of MSN-NH . However, while an obvious
2
border was observed around the treated MSN-CD, XRD
patterns showed that the low-angle reflections indexed as (110)
and (200) disappeared after loading with Rhod-SP, because of
an effect from the filled pores (Figure 2B). In addition, nitrogen
adsorption−desorption isotherms showed the typical Type IV
+
+
2+
2+
negligible variation in the presence of K , Na , Mg , Ca ,
2+
2+
3+
2+
3+
2+
2+
−
2−
Zn , Fe , Fe , Hg , Cr , Cd , Pb , and Cl , SO , as well
4
2
−1
as aspartic acid (Asp), glutamic acid (Glu), ATP, glucose, and
β-glucosidase (β-GCD). However, the strong fluorescence
caused by Cu2 weakened rapidly upon addition of biological
thiols, such as cysteine (Cys), glutathione (GSH), bovine
serum albumin (BSA), and Caspase-1 (see Figure S11 in the
curves with a surface area of 693 m g and an average pore
size of 2.8 nm of MSN-NH , but the surface area and pore size
2
+
decreased with increasing surface density and infilling (Figure
S12 in the Supporting Information). The zeta potential values
of MSN-NH , MSN-CD, and Rhod-SP@MSN-CD were +37.5,
2
D
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Analytical Chemistry
14.2, and +27.8 mV, respectively (see Figure S13 in the
Article
+
enhancement of the emission intensity that covered 0 to 10.0
2
+
Supporting Information). Furthermore, Fourier transform
infrared (FTIR) spectroscopy (see Figure S14 in the
μM Cu , and a signal-to-background ratio (S/B) of 16.8 was
achieved in the presence of 5.0 μM Cu2+ (see Figure 3B); here,
S/B is defined as
1
3
Supporting Information) and C CP-MAS solid-state NMR
spectroscopy provided solid evidence for the successful
functionalization of MSNs with β-CD rings (Figure S15 in
the Supporting Information). Therefore, Rhod-SP molecules
were effectively entrapped in MSNs without being released for
F − Fbuffer
S/B =
F0 − Fbuffer
2
+
more than 48 h (Figure 2C). Nonetheless, Cu could freely
diffuse into the inner mesopores through the cavities of the β-
CD rings to react with Rhod-SP (see Figure S16 in the
Supporting Information). Figure S17 in the Supporting
Information shows that the uniformly distributed MSN-CD
exhibited an improved long-term stability in cell culture
medium, which was mainly attributed to the enhanced water-
solubility after conjugating with β-CD. It was worth noting that
the wavelength of maximum emission showed a slight blue shift
from 590 nm to 575 nm, but no observable change in
absorption spectra upon loading Rhod-SP into the hydrophobic
inner mesopores (see Figure S18 in the Supporting
Information).
where F is the fluorescence intensity at 575 nm of Rhod-SP@
MSN-CD with Cu , F the fluorescence intensity at 575 nm of
2+
0
2+
Rhod-SP@MSN-CD without Cu , and Fbuffer the fluorescence
intensity at 575 nm of Rhod-SP@MSN-CD with a buffer.
There was good linearity between S/B and the concentrations
of Cu2+ in the range of 0.1−4.0 μM. The detection limit based
on 3σ/slope was estimated to be 1.26 nM, which exhibited
higher sensitivity toward Cu2+ than that of free Rhod-SP (62.5
nM), as shown in Figure 3B (representative fluorescence
spectra are shown in Figure S19 in the Supporting
Information). This was most likely because the good
hydrophobic environment offered by the nanopores could
strengthen the interaction between Rhod-SP molecules and
Cu . Moreover, because of the protective effect of MSNs
capped with β-CD, Rhod-SP@MSN-CD exhibited high stability
and excellent anti-interference ability in complex biological
samples (see Figure 3C, as well as Figure S20 in the Supporting
Information).
2+
Figure 3A shows fluorescence spectra changes of Rhod-SP@
2+
MSN-CD in response to different concentrations of Cu . The
2
+
addition of increasing concentrations of Cu to the aqueous
solution of Rhod-SP@MSN-CD at pH 5.0 elicited a dramatic
Following characterization of the spectroscopic properties of
Rhod-SP@MSN-CD, we sought to apply Rhod-SP@MSN-CD
to image fluctuations in exchangeable copper pools in living
cells. The cytotoxicity of both Rhod-SP and Rhod-SP@MSN-
CD on living cells was first evaluated by employing standard
cell viability protocols (MTT assay) (see Figure S21 in the
Supporting Information). After culturing HeLa cells with Rhod-
SP and Rhod-SP@MSN-CD for 24 h, their cellular viability was
over 95%, and no significant difference in morphology was
observed, even when the concentration was increased up to
1
0.0 μM Rhod-SP or 100 μg/mL Rhod-SP@MSN-CD. Next,
Rhod-SP@MSN-CD was used for confocal imaging experi-
ments to further verify its ability to detect intracellular
exchangeable copper using HeLa cells as a model cell line. As
shown in Figure 4, and in Figure S22 in the Supporting
Information, HeLa cells showed no detectable fluorescence
signals without the addition of CuCl (Figure 4a), but strong
2
fluorescence could be visualized when they were pretreated
with exogenous CuCl (Figure 4b). Moreover, we observed a
2
tendency toward increased fluorescence with increasing
concentrations of CuCl (see Figure S23 in the Supporting
2
Information). However, when CuCl -supplemented cells
2
(
Figure 4b) were further treated with the reducing reagent
2
+
ascorbate to globally deplete intracellular Cu stores, the
fluorescence completely disappeared, essentially because
ascorbate is known to change the redox equilibrium to promote
the reduction of Cu2+ to Cu+ (Figure 4c). We further
Figure 3. (A) Fluorescence emission spectra of Rhod-SP@MSN-CD
+
(
λex = 550 nm) in the presence of different concentrations of Cu2 in
38
buffer solution (20 mM, pH 5.0). The arrows indicate the change in
signal observed as the Cu2 concentration increases (0.05, 0.1, 0.2, 0.4,
+
2+
investigated fluorescence change caused by endogeneous Cu
0
.6, 0.8, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 8.0, and 10.0 μM).
by treating HeLa cells with pyrrolidine dithiocarbamate
Dotted line represents the fluorescence spectrum of buffer solution.
(
PDTC), the precursor for promoting oxidation of glutathione
(
(
B) S/B of Rhod-SP@MSN-CD, as a function of Cu2+ concentration.
C) S/B of Rhod-SP (1.0 μM, red bars) and Rhod-SP@MSN-CD (50
2+
39
to increase the intracellular level of Cu ions. Compared with
the results shown in Figure 4a, it can be seen in Figure 4d that a
weak fluorescence signal is emitted from PDTC-treated cells.
These results can be interpreted to mean that changes in both
exogenous and endogeneous Cu2+ can be monitored by Rhod-
SP@MSN-CD. Since Rhod-SP selectively responded to the
2+
μg/mL, blue bars) to Cu (50.0 μM) in buffer solution, in 50% FBS,
and in the presence of possible competitors (K , Mg , Zn , Ca /
+
2+
2+
2+
−
2−
50.0 μM, Cl , SO4 /100.0 μM, Asp, Glu, ATP, Cys, GSH, glucose/
5.0 mM, BSA/0.1 mg/mL, and β-glucosidase (β-GCD), Caspase-1/
5U, and a mixture containing all of the tested substances) in buffer
+
2+
solution.
coexistence of H and Cu in vitro, it could be hypothesized
E
dx.doi.org/10.1021/ac503240x | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry
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Figure 4. Confocal microscopy images of HeLa cells using Rhod-SP@
MSN-CD under different conditions: (a) cells incubated with Rhod-
SP@MSN-CD (50 μg/mL) for 3.0 h; (b) cells supplemented with 50
μM CuCl for 12.0 h and then incubated with Rhod-SP@MSN-CD
2
(50 μg/mL) for 3.0 h; (c) cells from panel (b), treated with 1.0 mM
ascorbate for 2.0 h; (d) cells from panel (a), treated with 100 μM
PDTC for 2.0 h; and (e) cells from panel (b), treated with a proton
sponge of DMAN for 2.0 h (λex = 559 nm; λem = 570−600 nm). (f)
Graph showing quantification of mean fluorescence intensity of each
condition (a−e), normalized to the control condition. Statistical
analyses were performed with a two-tailed Student’s t-test. Asterisk (*)
indicates P < 0.05 (n = 3), and error bars are ±SD (SD = standard
deviation).
Figure 5. (A) Rhod-SP@MSN-CD colocalizes to the lysosome in
HeLa cells. Cells were stained with (a) 1.0 μM LysoTracker Green
(Channel 1, λ = 488 nm; λ = 505−530 nm) for 0.5 h at 37 °C, (b)
ex
em
50 μg/mL Rhod-SP@MSN-CD (Channel 2, λex = 559 nm; λ
=
em
570−600 nm) with CuCl (50.0 μM) for 3.0 h at 37 °C, (c) overlay of
2
panels a and b, (d) intensity profile of region of interest (ROI) across
the HeLa cell, (e) intensity scatter plot of LysoTracker Green and
Rhod-SP@MSN-CD, and (f) simulated image (the white pixels, as
represented by the scatter plot shown in panel e, were highlighted by
selecting the points within the noted red rectangular region). (B)
TEM micrographs of HeLa cells incubated with (a) MSNs and (b)
Rhod-SP@MSN-CD for 3.0 h.
that the intracellular response behavior of Rhod-SP@MSN-CD
happened in the acidic lysosomal microenvironment rather
than the cytoplasm or other organelles with neutral environ-
ments. To demonstrate this proposition, HeLa cells were
treated with 1,8-bis(dimethylamino)naphthalene (DMAN), a
40
common proton sponge, to promote lysosomal damage.
From Figure 4e, an obvious decrease in fluorescence was
observed in these cells from the proton-sponge effect, leading
to the successful escape of Rhod-SP@MSN-CD into the
cytoplasm, preliminarily proving that the fluorescence enhance-
ment of Rhod-SP@MSN-CD was dependent on lysosomal
acidity. Fluorescence imaging analysis convincingly proved that
the cells could only be illuminated by Rhod-SP@MSN-CD in
internalized by cells and trapped inside lysosomes. The superior
colocalization results can be attributed to two factors. First, the
MSNs could forwardly accumulate into lysosomes after
internalizing into cells by endocytosis. Second, the acidic
lysosomal environment could effectively activate Rhod-SP to
2+
function a good response to Cu . Thus, Rhod-SP@MSN-CD
exhibits excellent spatiotemporal resolution capability for
2
+
2+
the simultaneous presence of Cu and lysosomal protons.
As expected, Rhod-SP@MSN-CD displayed well-localized
fluorescence within lysosomes. HeLa cells were co-stained with
Rhod-SP@MSN-CD for 3.0 h in the presence of exogenous
specifically sensing lysosomal Cu .
Since β-CD on the surface of Rhod-SP-loaded MSNs confers
the capability of nanoparticles to further conjugate with various
functional receptors, a guiding unit of c(RGDyK) peptide
conjugated adamantane (Ad-RGD) as prototypical system was
incorporated on the surface of Rhod-SP@MSN-CD by host−
guest interactions of β-CD and Ad to enhance cell specificity
(see Scheme 2). The characteristics confirmed that c(RGDyK)
peptide was well-modified on the surface of MSNs. After
conjugation of Rhod-SP@MSN-CD with Ad-RGD peptide, the
zeta potential of the nanoprobes changed from +27.8 mV of
Rhod-SP@MSN-CD to +6.6 mV of Rhod-SP@MSN-CD-RGD
(Figure S24 in the Supporting Information). The dynamic light
scattering (DLS) analysis also indicated that diameter of the
obtained nanoprobes changed from ∼80 nm of Rhod-SP@
MSN-CD to ∼98 nm of Rhod-SP@MSN-CD-RGD with
excellent dispersion (Figure S25 in the Supporting Informa-
tion). Moreover, FTIR spectroscopy gave direct evidence,
because of the emerging absorption bands of acylamide
vibration and carboxyl stretching vibration at 1541 and 1670
2+
Cu and LysoTracker Green for 5 min at 37 °C. As shown in
Figure 5A (panels (a)−(c)), the fluorescent images between
Rhod-SP@MSN-CD and LysoTracker Green overlapped very
well. The intensity profiles of the linear regions of interest
across HeLa cells stained with Rhod-SP@MSN-CD and
LysoTracker also tended toward synchronization (panel (d)
in Figure 5A). From the intensity correlation plots (panel (e) in
Figure 5A), a high Pearson’s colocalization coefficient of 0.942
and overlap coefficient of 1.626 were obtained, respectively, and
the white pixels (Figure 5Af) identified those lysosomes with
high-intensity distribution of the two dyes. The results
indicated that Rhod-SP@MSN-CD was specifically accumu-
lated in lysosomes and activated by the coexistence of acidic pH
2
+
and Cu . To provide additional physical evidence for
subcelluar residence of nanoprobe, TEM analysis was applied
to image the cell uptake of MSNs and Rhod-SP@MSN-CD.
From Figure 5B, it can be clearly seen that both were
−1
cm , respectively (Figure S26 in the Supporting Information).
F
dx.doi.org/10.1021/ac503240x | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry
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Then, the cell specificity of Rhod-SP@MSN-CD-RGD was
verified using flow cytometry and confocal imaging experiments
by incubating the probe with HeLa cells and MCF-7 cells,
respectively. Flow cytometry experiments (Figure S27 in the
Supporting Information) in HeLa cells revealed a marked shift
in population distribution from low fluorescence intensity to
high fluorescence intensity when the cells were supplemented
targeted chemical probes by simply changing the target
recognition element, hence making it applicable to other
analytes of interest.
ASSOCIATED CONTENT
■
*
S
Supporting Information
More experimental details and additional spectroscopic data as
noted in text. This material is available free of charge via the
Internet at http://pubs.acs.org.
2+
with Cu . This response can be ascribed to the high affinity
between the cyclic RGD peptide and both α β and α β
v 5
v
3
4
1
integrins, which are expressed at high levels on HeLa cells,
but at lower levels on MCF-7 cells, in turn, leading to enhanced
endocytosis of the nanoprobe. Subsequent analysis using
confocal imaging further confirmed that receptor mediation
conferred efficient endocytosis. Significant fluorescence was
clearly visualized in HeLa cells after being incubated with Rhod-
SP@MSN-CD-RGD for 30 min (see Figures S28A and S28B in
the Supporting Information), and high colocalization between
Rhod-SP@MSN-CD-RGD and LysoTracker was seen in
lysosomes, but only a weak fluorescent signal was observed in
MCF-7 cells, even if the incubation time reached 3.0 h (see
Figures S28C and S28D in the Supporting Information).
Moreover, TEM confirmed that Rhod-SP@MSN-CD-RGD
exhibited a higher uptake efficiency into HeLa cells and faster
lysosomal accumulation (30 min) than that of MSNs and
Rhod-SP@MSN-CD (see Figure S29 in the Supporting
Information). Clearly, the presence of RGD peptides on the
surface of nanoprobes conferred both targeting and internal-
ization properties to specific cell populations.
AUTHOR INFORMATION
■
Corresponding Author
*Fax: +86-731-8882 2523. E-mail: Yangrh@hnu.edu.cn.
Author Contributions
These authors contributed equally to this work.
§
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors acknowledge financial support through the
National Natural Science Foundation of China (Nos.
21305036, 21135001, and J1103312), the Foundation for
Innovative Research Groups of NSFC (No. 21221003), the
“973” National Key Basic Research Program (No.
2011CB91100-0), Hunan Provincial Natural Science Founda-
tion of China (No. 2015JJ3035), and the Fundamental
Research Funds for the Central Universities.
Finally, we evaluated the potential application of Rhod-SP@
2+
MSN-CD-RGD in the diagnosis and evaluation of Cu -related
diseases by incubating Rhod-SP@MSN-CD-RGD with live
human normal liver cells (HL-7702) and human hepatoma cells
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