A pH-sensitive near-infrared fluorescent probe with alkaline p: K a for chronic wound monitoring in diabetic mice

Article abstract of DOI:10.1039/c9cc02289a

A pH-sensitive near-infrared fluorescent probe with alkaline pK_a, AlkaP-1, was developed by incorporating a benzoyl hydrazine group into a cyanine dye. The significant fluorescence changes in the alkaline regions enable the probe to monitor the alkalization process from acute wounds to chronic wounds in diabetic mice.

Full text of DOI:10.1039/c9cc02289a

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A pH-sensitive near-infrared fluorescent probe with alkaline pKa
for chronic wounds monitoring in diabetic mice
Received 00th January 20xx,
Accepted 00th January 20xx
a
b
a
a
a
a
a
b
Hengtang Mai, Yu Wang , Shuang Li, Ruizhen Jia, Sixian Li, Qian Peng, Yan Xie, Xiang Hu and
a
Song Wu*
DOI: 10.1039/x0xx00000x
www.rsc.org/
A pH-sensitive near-infrared fluorescent probe with alkaline pKa, Include glass electrode, electrochemical method, and
AlkaP-1, was developed by incorporating a benzoyl hydrazine luminescent probes, suffering from either only single-spot
group into a cyanine dye. The significant fluorescence changes in value detectable, or on-spot, and real time detection
8
the alkaline regions enable the probe to monitor the alkalization inapplicable. There is a great demand for the development of
process from acute wounds to chronic wounds in diabetic mice.
straightforward tools with high spatio-temporal resolutions.
Near-infrared (NIR) fluorescence imaging of biological
parameters is a burgeoning area in biomedical sciences. It is
well-known that NIR emission from 650 to 900 nm has the
biological merits of less photodamage, deep tissue
penetration, and less autofluorescence interference created by
In complex bioorganisms like human being, pH
environments vary vastly from strong acidic gastric fluid to
basic pancreatic secretion, which are vital to perform normal
1
physiological functions. Abnormalities of pH environments in
biosystems are usually indicators of occurrence of diseases,
such as in the case of development of chronic wounds.
9
biomolecules in the living system. Many types of pH-sensitive
2
NIR fluorescence probes have been developed, for the various
Chronic wounds become caught in a state of inflammation,
making higher levels of degrading proteases, which alter
components of the extracellular matrix that are vital to the
wound healing process. pH plays a significant role in affecting
cellular processes in the wound, which in turn affects the
10
purposes from diagnosis of tumors to measurements of small
11
changes in living cells and organells. However, most of these
probes function in acidic regions with pKa less than 7.5, which
are unsuitable for monitoring pH changes in alkaline chronic
wounds. Therefore, it is very important to develop NIR
fluorescent probes with an appropriate alkaline pKa for
practical bioimaging applications on the chronic wounds.
Herein we report our efforts on the development of AlkaP-1,
a new pH-sensitive NIR fluorescence probe, which is obtained
by rational design (Scheme 1). For the first time, AlkaP-1 is
successfully employed to visualize alkalization in the course of
chronic wound development from acute wound in a
transgenetic diabetic mouse model due to its unique optical
characteristics toward pH.
3
healing process. Skin surface on a health human is slight acidic
4
(
4.2-5.9), but neutralized by body fluid leakage when the
underlying tissue wounded (acute wound), and more alkaline
(
7.14-8.9) at the stage of chronic wound if delayed healing
5
happened under certain pathological circumstances. Many
types of chronic wounds, including venous leg ulcer, diabetic
foot ulcer and bedsore, not only seriously affect the quality of
life of the patients, but also bring about a heavy financial
6
burden. It is reported that the cost of the treatment of
chronic wounds accounts for one-third of the dermatological
health budget in the United States. Monitoring the wound pH
may provide a tool to determine the condition of the wound
7
bed and help to optimize the clinical therapeutic schedule. So
far the reported tools for pH measurement for chronic wounds
a. Hubei Province Engineering and Technology Research Center for Fluorinated
Pharmaceuticals, School of Pharmaceutical Sciences, Wuhan University, Wuhan,
Hubei, 430072, P. R. China.
b.
Department of Orthopaedic Trauma and Microsurgy, Zhongnan Hospital of
Wuhan University, Wuhan, Hubei, 430071, China.
Electronic Supplementary Information (ESI) available: Experimental section,
HRMS and NMR, optical spectra, determination of pKa, stability and selectivity
experiment, cytotoxicity assay, live cell imaging, and animal experiments. See
DOI: 10.1039/x0xx00000x
Scheme 1 Chemical structures of the NIR fluorescent
compounds (1, 2 and 3) and their structural responses to pH
changes.
†
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Given the pH environments in chronic wounds, we consider observed, which is not good enough for the chrVoienwicArtwicloe uOnnldines
pH monitoring.
DOI: 10.1039/C9CC02289A
that an ideal pH-sensitive NIR fluorescence probe for these
diseases requires showing significant fluorescent changes
among narrow pH ranges (ca. 7.0-9.0). The key point to design
Next, we sought to tune the optical performance of the
fluorescent compounds containing acylhydrazine by structural
modification, using 1 as a lead compound. Introduction of an
electron-withdrawing group to benzene ring may reduce the
electron density of the terminal amino group and make the
amino group less alkaline, by which pKa may be reduced.
Based on the above analysis, we designed compound 2 by
introduction of bromine atom adjacent to the acylhydrazine
group. Meanwhile, to prove the effect of different substituents
on optical performance, compound 3, an analogue of
compound 1, with an electron-donating methoxy group, was
also designed. Compound 2 and 3 were synthesized via a
similar synthetic route to compound 1 and characterized
a
pH-sensitive
probe
relies
on
the
protonization/deprotonization of a reactive moiety attached to
a NIR fluorophore based on the photoinduced electron
transfer (PET) mechanism. The reported reactive moieties,
1
2
13
including morpholinyl group , piperazinyl group , indolyl
group , anilino group
1
4
10a,10b,11a
11b,11c
, , etc,
have been utilized to
construct probes responsive to acidic pH due to their relative
low pKa values. We postulated that acylhydrazine compounds,
1
5
which are reported to possess higher pKa, may act as
reactive moieties of the probes that are responsive to alkaline
pH conditions for chronic wounds. For the proof-of-concept
test, we initially designed compound 1 containing a benzoyl
hydrazine cationic group, which can enhance the water-
solubility of the probe, attached to a widely used NIR
(Scheme S1, ESI † ). Their optical performances to pH were
evaluated (Fig. S1, ESI † ). The overall absorbance and
fluorescence responsive tendencies of the both compounds
toward pH are similar to that of compound 1. But at alkaline
regions from 7.0-9.0, the fluorescence change ratios for
compound 2 and 3 are 10.0 and 0.6-fold, respectively. The
pkas of compound 2 and 3, were calculated to be 8.01 and
1
6
fluorophore Cy7 via an ether linker. Using 3-hydroxybenzoate
as the starting material, we synthesized the intermediate tert-
butyl 2-(3-hydroxybenzoyl) hydrazine carboxylate (1b) via a
two-step reaction including hydrazinolysis by hydrazine and
8
.89, respectively (Fig. S2b-c, ESI†). Apparently, substituents
protection
of
acylhydarzine
group
by
N-tert-
with different electronic effects can alter the pKa values,
leading to the different optical performances to pH. Among
the pH range from 7.0 to 9.0, the higher pka of the compound
corresponds to the weaker fluorescence changes. Considering
that compound 2 has largest fluorescence changes among 7.0-
butoxycarbonylation (Boc). The next condensation between
1
1
b and CyCl (IR-780) under basic conditions gave the precursor
c, followed by the deprotection of Boc group to offer the
desired green compound 1 (Scheme S1, ESI†). Compound 1
was fully characterized by NMR and HRMS.
9
.0, it was selected for the further bioimaging studies and
The optical response of compound 1 toward pH was then
studied in citrate-phosphate and carbonate-bicarbonate
buffers, respectively. As shown in Fig. 1a, the absorption
peaks around 770 nm gradually decreased with the increasing
pH among 4.5-10.5. Interestingly, the fluorescence spectra as
shown in Fig. 1b exhibited a quite different variation tendency.
At acidic regions among 4.5 to 7.0, the fluorescence displayed
weak changes, while sharply dropped among the alkaline pH
region. The decreasing of fluorescence may ascribed to the
PET effect, namely, the cationic compound 1 turns to its
nonfluorescent non-protonated form with pH increasing. The
pka of compound 1, was calculated to be 8.48 (Fig. S2a, ESI†).
The above consequences proved our hypothesis that
introduction of acylhydrazine to the fluorophore makes major
fluorescence changes in alkaline regions. But unfortunately, at
the pH region of 7.0-9.0 that we are interested, only an
intermediate fluorescence intensity change about 2.6-fold was
named as AlkaP-1. The quantum yield of AlkaP-1, was
measured to be 4.2% at pH 4.5 and 0.6% at pH 9.5, respective
ly, suggesting the great environmental impact on the optical
properties of the probe (Fig. S3, ESI†).
We investigated the photostability of AlkaP-1 at acidic and
alkaline conditions, respectively, by irradiating them at 715 nm
and measuring fluorescence intensity at different time points
(Fig. S4, ESI†). At pH 4.5, the fluorescence intensity of AlkaP-1
showed only a maximum decrease by 14.5% after 1 h
irradiation, while an even lower decrease ratio of 7.0% could
be observed at pH 9.5, indicating the good photostability of
the probe under the both conditions. Meanwhile, the
selectivity of AlkaP-1 to pH in the presence of a variety of
metal ions, anions, biothiols, and amino acids was investigated
by fluorescence spectra at pH 4.5, 7.5, and 10.0, respectively
(Fig. S5, ESI † ). All the biologically relevant species exhibited
little influences on fluorescence intensities, suggesting the
good selectivity of the probe.
A standard cell viability protocol (MTT assay) was employed
to study the cytotoxicity of the probe on Ewing’s Sarcoma cells
A673 by treating cells with various concentrations of AlkaP-1
(5, 10, 20 µM) for 24 h (Fig. S6, ESI † ). AlkaP-1 showed low
cytotoxicity and good compatibility with over 80% cell viability
at the highest concentration of 20 µM. We further determined
the optimal intracellular working concentration of AlkaP-1.
The fluorescence imaging in living cells was conducted by
Fig. 1 (a) Absorbance spectra of 5 µM compound 1 with
different pH buffers containing 10 % DMSO. (b)Fluorescence
spectra of 10 µM compound 1 with different pH buffers incubating Ewing's Sarcoma cells A673 with 5, 10, 15, and 20
containing 10 % DMSO at excitation of 715 nm.
µM AlkaP-1 in PBS, respectively. The fluorescence intensity in
2
| J. Name., 2012, 00, 1-3
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coincident with that observed in vitro. ApparenVitelwy,ArtAicllekOanPli-n1e
could indeed detect the pH changes partic in
regions in living cells.
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The development of chronic wounds, especially in diabetic
ulcers, usually accompanies with an increase of the alkalinity
due to wound infections, which therefore result in delayed
1
7
wound healing. A transgenetic diabetes-impaired db/db
mouse model has been used for studies of chronic wound
1
8
healing in diabetic foot ulcers. To study the pH changes
during chronic wounds development, we created six excisional
wounds, roughly in two parallel lines on the back of one db/db
mouse. These excisional wounds are reliable to form chronic
wounds that are hard to heal. AlkaP-1 was then sprayed into
the each wound bed on the right side at 1, 4, and 7 days,
respectively, after the excision. Meanwhile, the wounds on the
left side keep intact for comparison. As shown in Fig. 4, the
bright fluorescence could be observed at the stage of acute
Fig. 2 Fluorescence images of Ewing's Sarcoma cells incubated
with 5, 10, 15, and 20 µM AlkaP-1 in PBS, respectively. Images
were taken using the confocal fluorescence microscope at 60 wound (day 1), but gradually decreased as the time went on,
×
magnification, scale bars = 50 µm.
reflecting that pH of the wound bed is increasing.
Semiquantitative analysis to fluorescence intensity of each
cells increased with concentration of the probe, and reached wound by employing imaging system analysis software showed
its maximum at 15 µM (Fig. 2), while the higher concentration about 45% of decreased fluorescence from day 1 to day 7 (Fig
at 20 µM did not offer significant fluorescence increasing. We S7a, ESI †). Meanwhile, the average surface pH changes of the
1
9
thus chose the concentration of 15 µM for the next studies wound bed measured by a flat glass pH electrode showed
based on the optical performance and cytotocixity evaluation increase from 6.97 to 8.06 within 7 days (Fig S7b, ESI †). Both
in cells.
approaches reflect the alkalization processes during the
In order to investigate whether the AlkaP-1 could be used to development of chronic wounds. In addition, we also noticed
reflect pH changes in living cells, the fluorescence imaging of that the pH distribution of every wound is uneven (Fig. 4a-c),
live cells with different intracellular pH values was conducted which may be useful to visualize the heterogeneity of pH
by incubating Ewing’s Sarcoma cells A673 with 15 µM AlkaP-1, within the wound bed during the different developmental
+
+
and then with nigericin (15 µg/mL) (H /K ionophore) in buffers stages of chronic wounds. The above observations, not only
with different pH values at 4.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, prove the alkalization processes occur during the course of the
respectively, where nigericin was used to equilibrate the delayed healing of chronic wounds, but also provide the
1
3
intracellular and extracellular pHs. As shown in Fig. 3, the detailed information of the pH distributions in the whole
cellular fluorescence irregularly decreased with the increasing wound area.
pH. In the acidic region from 4.5 to 7.0, only a low fluorescence
change about 8.4% was observed, while it dropped about probe, AlkaP-1, based on benzoyl hydrazine structure by
6.9% in the alkaline regions from 7.0 to 9.0. This tendency is
rational design. The characteristic responses to pH in the
In summary, we developed a pH-sensitive NIR fluorescent
7
Fig. 3 Fluorescence images of Ewing 's Sarcoma cells incubated with 15 µM AlkaP-1 for 30 min in buffers with different pH values
of 4.5, 7.0, 7.5, 8.0, 8.5, 9.0 and 9.5 containing 15 µg/mL nigericin. Images were collected using a confocal fluorescence
microscope magnification, scale bars = 25 µM. Quantified relative fluorescence intensity of a certain pH was obtained employing
Image-Pro Plus software and presented as mean ± SD, with n = 3.
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alkaline regions render AlkaP-1 ability to visualize the pH
changes in the course of chronic wounds development in living
system. In addition, the uneven pH distribution of wound
disclosed by the probe, suggests that we may correlate the pH
with the heterogeneous morphology of wounds. Our probe
provides a potential tool to evaluate the condition of the
wound bed, which will benefit the treatment of the related
diseases.
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This research reported in this publication was supported by
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An alkaline pH-sensitive near-infrared fluorescence probe can monitor pH changes in the course
of chronic wounds developments in mice.