Mitochondria-localized: In situ generation of rhodamine photocage with fluorescence turn-on enabling cancer cell-specific drug delivery triggered by green light

Article abstract of DOI:10.1039/d0cc03524f

In this work, we have developed a rhodamine dye based water-soluble, mitochondria-indicating photocage, which gets activated in the mitochondria producing a fluorescent signal and on-demand releases the caged anticancer drug, chlorambucil, in the cancer cells selectively upon irradiation of green light.

Full text of DOI:10.1039/d0cc03524f

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Mitochondria-localized In Situ Generation of Rhodamine
Photocage with Fluorescence Turn-On Enabling Cancer Cell-
Specific Drug Delivery Triggered by Green Light
Received 00th January 20xx,
Accepted 00th January 20xx
Amrita Paul,a Rakesh Mengji,b,c Manoranjan Bera,a Mamata Ojha,a Avijit Jana*b,c,d and N. D.
DOI: 10.1039/x0xx00000x
Pradeep Singh*a
In this work, we have developed a rhodamine dye based water- readily oxidized back to the parent fluorescent dye by the
soluble, mitochondria-indicating photocage, which gets activated reactive oxygen species (ROS) or by cellular redox systems that
in the mitochondria producing fluorescent signal and on-demand accumulate in mitochondrial membranes.5 Therefore, the
release the caged anticancer drug, chlorambucil in the cancer cells dihydrorhodamine derivatives, like dihydrorhodamine 123 and
selectively upon irradiation of green light.
dihydrorhodamine 6G are commonly used as fluorescent
mitochondrial dyes.6 In addition, rhodamine dyes have been
widely used as laser dyes and fluorescent markers for labeling
biomolecules.7 However, to date, rhodamine based dyes have
not been developed as a photocage. Therefore, we intend to
develop a new photocage based on rhodamine dye, which will
imbibe all the qualities of rhodamine, for the release of the
carboxylate based anticancer drug via C-O bond cleavage by
green light (λ ~ 546 nm) (Fig. 1).
The effectiveness of cancer diagnosis and therapy can be
improved, avoiding severe side effects of the cancer
treatment, by developing anticancer drug delivery systems
(DDSs) capable of selectively and efficiently killing tumor cells
without affecting normal tissues.1,2 Light triggered activation of
the prodrugs in the biological environment, allows localized
and non-invasive treatment, as light is a mild reagent capable
of providing high spatio-temporal control. Therefore,
photocages, the chemical moieties caging active molecules
through covalent linkage and uncaging them upon irradiation,
are increasingly gaining popularity for therapeutic applications,
like drug delivery.3 However, the photocages that are highly
competent to detect the target site, get localized at the target
site and selectively deliver the drug, in order to have reduced
side effects along with improved therapeutic efficiency, are
still desirable.
Rhodamine is a class of triarylmethane dyes that contain
xanthene core. Rhodamine dyes possess several advantageous
properties like, (i) well compatibility to the biological system,
(ii) high photostability, (iii) good aqueous solubility, (iv) high
molar extinction coefficient above 500 nm, (v) high fluorescent
quantum yield, and (vi) low degree of triplet formation.4
Moreover, the fascinating property of rhodamine dyes is that
their fluorescence get quenched in chemically reduced form
and is regained upon oxidation. For example, “dihydro”
derivatives of rhodamine are non-fluorescent and they are
Mitochondria are vital subcellular organelle that play vital
roles in regulating cell metabolism like, energy production,
cellular signaling, regulating apoptosis, and most importantly
they are the major sources of endogenous ROS.8 In addition, it
has been observed that most of the cancer cells overproduce
(~10-fold) ROS constantly compared to the normal cells owing
to the oncogenic stimulation, mitochondrial malfunction and
increased metabolic activity of cancers.9 Our strategy is to
design a DDS, which can sense the tumor microenvironment
and get activated only in the cancer cells in order to have
highly efficient cancer therapy avoiding the severe side effects.
With this idea, we developed a DDS, based on the newly
designed rhodamine photocage. The photocage in the reduced
form (HRhod-cbl) is inactive and does not fluoresce until it
enters the actively respiring cells, where it is oxidized by the
intracellular ROS generated in the mitochondria to form bright
red fluorescent active rhodamine photocage (Rhod-cbl), in
situ. The active photocage (Rhod-cbl) produced, thus gets
localized in the mitochondria generating a fluorescent signal,
which in turn enables us to locate the exact site for the
irradiation to uncage the drug. Herein, we have developed a
rhodamine based water-soluble photocage which is generated
in situ localized at mitochondria, to report the in vitro
mitochondria-specific, cancer cell-selective anticancer drug
(chlorambucil) delivery triggered by green light, for the
efficient cancer therapy (Fig. 1).
a. Department of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur-
721302, West Bengal, India. E-mail: ndpradeep@chem.iitkgp.ac.in
b. Department of Applied Biology, CSIR-Indian Institute of Chemical Technology
Hyderabad, Uppal Road, Hyderabad 500007.
c.
Department of Organic Synthesis and Process Chemistry, CSIR-Indian Institute of
Chemical Technology Hyderabad Uppal Road, Hyderabad 500007.
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India.
d.
†
Electronic
Supplementary
Information
(ESI)
available:
See
DOI: 10.1039/x0xx00000x
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in the fluorescence intensity (λmax ~ 585 nm) weVrieewwAritticnleeOsnsleinde
†
DOI: 10.1039/D0CC03524F
(Fig. S8, ESI ), when we treated HRhod-cbl with 100 µM of
other ROS like potassium superoxide (KO2), hydroxyl radical
(∙OH) (generated by typical Fenton reaction between Fe2+ and
H2O2),11 hypochlorous acid (HOCl) and peroxynitrite (ONOO−)
(prepared according to the reported method).12 Further from
the RP-HPLC analysis, it was observed that treating HRhod-cbl
with 100 µM of five different ROS separately, for 30 min
caused the complete conversion of HRhod-cbl to Rhod-cbl (Fig.
S9, ESI†), whereas no significant aerial oxidation of HRhod-cbl
in the neat state (without solvent) and aqueous phosphate
buffer (0.1 M, pH = 7.0) solution was observed (Fig. S10, ESI†)
in absence of light.
Fig. 1. Schematic presentation of the mitochondria-localized in situ generation of
rhodamine-based photocage with fluorescent turn-on for the photoinduced anticancer
drug delivery selectively in the cancer cells.
The rhodamine based photocage (4) was synthesized
according to the procedure depicted in Scheme 1.
Commercially available rhodamine B dye was reduced with
NaBH4 and I2 in THF producing compound 2. Esterification
reaction between compound 2 and the anticancer drug,
chlorambucil (cbl) in the presence of 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide
(EDC)
and
4-
dimethylaminopyridine (DMAP) yielded compound 3. Finally,
oxidation of 3 with chloranil produced the photocage (Rhod-
cbl) (4). The products were characterized by 1H NMR, 13C NMR
and HRMS spectroscopy (Fig. S1–S6, ESI†).
Rhod-cbl exhibited only 6% decomposition in aqueous solution Fig. 2. a) Normalized absorption and emission spectra of Rhod-cbl (4) in water.
The excitation wavelength for the emission spectrum, λex = 560 nm. (C = 10-5 M).
b) Fluorescence spectra of HRhod-cbl (10 µM) with different concentrations of
H2O2 (0-100 µM) in acetonitrile/HEPES buffer (1:19). Inset images of vials
containing HRhod-cbl solution under UV light: before (left) and after (right)
(pH = 7) in dark after the period of 21 days, exhibiting its
hydrolytic stability. The solubility of 4 in aqueous phosphate
buffer (0.1 M, pH = 7.4) was found to be 10 mg/ mL at 30 °C.
addition of H2O2.
The photorelease of the anticancer drug (cbl) from Rhod-cbl
(4) was studied by irradiating 4 (1×10-4 M) in aqueous
phosphate buffer (0.1 M, pH = 7.0), with green light (λ ~ 546
nm) isolated from a medium-pressure mercury lamp (125 W).
The percentage of drug released from the Rhod-cbl (4) after 1
h of irradiation was observed to be 89 % (Fig. S11, ESI†). The
photochemical quantum yield (Фp) of decomposition of Rhod-
cbl upon irradiation was calculated as 0.026, using potassium
ferrioxalate as an actinometer.13
Scheme 1. Synthesis of rhodamine based photocage, Rhod-cbl (4).
The UV/Vis absorption spectrum of Rhod-cbl (4) exhibited a
strong band with absorption maximum (λmax) at 563 nm (Fig.
2a) and molar absorptivity (Ɛmax) of 11.6 × 104 M-1 cm-1. On the
other hand, the emission maximum was observed at 590 nm
with fluorescence quantum yield (Фf) to be 0.33
using
rhodamine B (Φf = 0.31 in water) as the standard, with solvent
refractive index correction.10 The absorption spectra of HRhod-
OH (2) and HRhod-cbl (3) was also recorded (Fig. S7, ESI†).
To better understand the fate of the HRhod-cbl in presence of
ROS, the solution of HRhod-cbl (10 µM) in acetonitrile/HEPES
buffer (1:19) was treated with different concentration of H2O2
(0-100 µM) and the reaction was monitored by the
fluorescence spectroscopy (Fig. 2b). It was observed that there
was a gradual increase in fluorescence intensity at λmax = 585
nm, upon gradual addition of H2O2 (0-100 µM) and the solution
became non-fluorescent to red fluorescent. A similar increase
Fig. 3. RP-HPLC chromatograms of Rhod-cbl (4) at regular time intervals of
irradiation with light (λ ~ 546 nm). The y-axes are offset by 50 mAU and the x-
axes are offset by 30 s to facilitate better visualization. AU = arbitrary units.
The photolysis of Rhod-cbl (4) was monitored by reversed
phase (RP)-HPLC (Fig. 3) with acetonitrile/water (1:1) as the
mobile phase at a constant flow rate (1 mL min-1). The peak at
retention time (tR) 14.73 min in the RP-HPLC chromatogram at
the 0 min represented the Rhod-cbl (4). With the increase in
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irradiation time, a gradual depletion of the peak at tR 14.73 visualization of the cytoplasmic distribution of Rhod-cbl. Then
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DOI: 10.1039/D0CC03524F
a confocal laser-scanning
was observed, indicating the photodegradation of Rhod-cbl images were recorded using
(4). There was the concomitant appearance and increase in the microscope (CLSM). It was observed that Rhod-cbl colocalized
intensity of two new peaks at tR 12.64 min and 11.15 min, with MTG (Fig. S22 ia-via, ESI†), but not with LTG (Fig. S22ib-
corresponding to the photoproduct (5) and the released cbl vib, ESI†). Further, the quantitative colocalization of Rhod-cbl
(determined by the coinjection of standard cbl and Rhod-OH with MTG was studied by colocalization pixel map analysis and
(5)). The formation of Rhod-OH (5) (Fig. S12, ESI†) was scatter plot, which clearly demonstrated an extensive
confirmed from 1H NMR (Fig. S13, ESI†) and HRMS analysis colocalization of the Rhod-cbl in the mitochondria (Fig. S22va
(Fig. S14, ESI†). We also investigated the photorelease ability of and via, ESI†) over lysosome (Fig. S22vb and vib, ESI†).
the rhodamine based photocage using rhodamine- In order to follow the kinetics of the intracellular oxidation of
phenylacetic acid conjugate (Rhod-phac) by the 1H NMR HRhod-cbl to Rhod-cbl in real-time in the mitochondria, the
spectroscopy (see Fig. S15, ESI†).
live B16F10 melanoma cancer cells were pretreated with
Based on the literature14 precedence we proposed that the HRhod-cbl and were counterstained with MTG. From the
photorelease of caged cbl from Rhod-cbl (4) occurs following confocal images, a gradual increase in the fluorescence (red)
the steps (Scheme 2), (i) 4 gets excited to its singlet excited intensity was observed with increasing incubation time (Fig. 4),
state (4*) upon irradiation. The fluorescent lifetime (singlet also preferential colocalization of red pixels with green pixels
state lifetime) of 4* was 2.01 ns, obtained from a time- of MTG (yellow fluorescence in Fig. 4 iii(a-e)) were noted. The
correlated single photon counting (TCSPC) experiment (Fig. time-dependent enhancement of fluorescence intensity in the
S16, ESI†).15 (ii) In the singlet excited state it undergoes B16F10 cells without any external inducer indicated the
heterolytic C − O bond cleavage forming the ion pair oxidation of HRhod-cbl to generate Rhod-cbl in situ by the
intermediate (6). It was observed that the presence of singlet endogenous ROS produced in mitochondria, causing the
state quencher azulene (1 mM) quenched the localization of the DDS (Rhod-cbl) in the mitochondria. In order
photodegradation of Rhod-cbl (Fig. S16, ESI†), whereas the to have a better visualization of the intracellular distribution of
photodegradation was not quenched by triplet state quencher, the DDS, 3D Z-stacked confocal images of B16F10 cells treated
potassium sorbate (0-1 mM) (Fig. S18, ESI†), indicating the with Rhod-cbl and MTG were recorded (Fig. S23, ESI†)
uncaging occurs from the singlet state. (iii) The cationic part demonstrating profound colocalization of Rhod-cbl and MTG.
reacts with water to yield the photoproduct, Rhod-OH (5) and
the anionic part undergoes protonation to form the free cbl.
Rhod-OH (5) (open form) is fluorescent and remains in
equilibrium with the non-fluorescent spirocyclic form (7). The
pKcycl of 5 is 9.2, hence at the neutral and physiological pH (7.4)
the photoproduct exists in the fluorescent open form.16 We
observed that the photouncaging followed first order reaction
kinetics (Fig. S19, ESI†) with rate constant (kp) 5.16 × 10-4 s-1.
Fig. 4. CLSM images of live B16F10 cell lines incubated with HRhod-cbl and MTG:
(i) red channel, (ii) green channel, (iii) merged image of (i and ii), (iv)
Scheme 2. Possible mechanism of photorelease of cbl from Rhod-cbl.
colocalization pixel map of the corresponding red and green channel, (v)
corresponding scatter plot: concentrated pixels along the diagonals indicate a
high degree of colocalization of Rhod-cbl (produced after endogenous oxidation
of HRhod-cbl) and MTG. Scale bar: 25 µm.
Further, the temporal control over the photorelease of drug
was demonstrated by exposing Rhod-cbl periodically to light
and dark, the release was observed only during irradiation (Fig.
S20, ESI†).
After observing the light triggered drug release ability of Rhod-
cbl, in vitro cellular internalization and distribution of Rhod-cbl
in B16F10 (cancerous) cell line and CHO (non-cancerous) cell
line (Fig. S21, ESI†) was investigated. The B16F10 and CHO cells
were treated with Rhod-cbl for 4h followed by counterstaining
with commercially available lysotracker green (LTG) and
The extent of in vitro cellular uptake of HRhod-cbl in B16F10
and CHO cell were quantified by flow cytometry analysis,
tracking the mitochondrial oxidation of HRhod-cbl to form
Rhod-cbl in real-time (Fig. S24, ESI†). Time-dependent
fluorescence enhancement owing to the oxidation of HRhod-
cbl was significantly higher in cancerous B16F10 cells (Fig.
S24a, ESI†) compared to non-cancerous CHO cells (Fig. S24b,
ESI†). The notable higher emission intensity (~ 10 folds) in the
mitotracker green (MTG) separately to have
a better
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B16F10 cells compared to the CHO cells can be directly situ formation of active red fluorescent photocage in the
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DOI: 10.1039/D0CC03524F
correlated to the fact that the cancer cells have elevated level presence of ROS generated in the mitochondria. This enabled
of ROS compared to the normal cells.17 Therefore, HRhod-cbl the localization of the DDS in the mitochondria of the cancer
can be considered as an ideal tool for the specific sensing of cells having over expressed ROS, causing more efficient and
cancer cells.
target-specific cancer treatment with lower side effects,
Light triggered anticancer activity of HRhod-cbl (3) selectively demonstrated by in vitro studies. Therefore, the developed
to the cancer cells was investigated by measuring the rhodamine based photocage itself serves the key roles of
cytotoxicity of 3 in both B16F10 and CHO cells by MTT assay18 sensing, targeting, releasing, imaging, and treatment, thereby
[MTT
=
3-(4,5-dimethylthiazol-2-yl)-2,5- providing a promising platform for efficient cancer diagnosis
diphenyltetrazoliumbromide, a yellow tetrazole] before and and therapy.
after irradiation. It was observed that both B16F10 (Fig. 5a) We thank DST SERB (Grant No. DIA/2018/000019) for financial
and CHO (Fig. 5c) cells showed cell viability of about 80 % after support and DST-FIST for 600 and 400 MHz NMR. A. Paul is
incubation with HRhod-cbl (5-40 μM) in dark for 72 h. thankful to IIT Kharagpur for fellowship. AJ and RM thanks to
However, the cell viability of cancerous B16F10 cells incubated DST, for the INSPIRE Faculty Grant (IFA15, CH171, GAP0546).
with HRhod-cbl (5-40 μM) decreased to 20 % after irradiation The CSIR-IICT Communication number for this manuscript is
of green light (Fig. 5b). On the contrary, the cell viability of IICT/Pubs./2020/113.
non-cancerous CHO cells remained above 70 % even after light
exposure (Fig. 5d). This clearly indicated that HRhod-cbl got
oxidized and generated the active DDS (Rhod-cbl) in situ more
Conflicts of interest
efficiently in the cancer cells (B16F10), which in presence of
light released the anticancer drug (cbl), thereby causing the
enhanced cytotoxicity after irradiation. Moreover, HRhod-cbl
exhibited enhanced cytotoxicity in cancer cells over
commercial anticancer drug chlorambucil in the presence of
light (Fig. 5b). The cytotoxicity of the photoproduct (Rhod-OH)
was also assessed. It exhibited lower cytotoxicity (cell viability
of more than 80 %) in dark as well as after light irradiation on
both B16F10 and CHO cells (Fig. 5a-d). This indicated that the
rhodamine photoproduct has good biocompatibility.
Therefore, the results of MTT assay collectively validated that
the precursor of our rhodamine based DDS (HRhod-cbl)
exhibited an efficient anticancer activity selectively to the
cancer cells in the presence of light.
There are no conflicts to declare.
Notes and references
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Fig. 5. Cell viability assay of HRhod-cbl, Rhod-OH, and free drug (cbl) in in (a)
B16F10 cell line in dark, (b) B16F10 cell line after light irradiation, (c) CHO cell
line in the dark, and (d) CHO cell line after light irradiation. Values are presented
as mean ± SD.
In conclusion, we have utilized the well-known dye, rhodamine
to develop a water-soluble photocage that can be activated by
green light (546 nm) for the release of anticancer drug. The
photocage exhibited efficient uncaging ability in the water and
physiological medium with good chemical and photochemical
quantum yields. The rhodamine based photoresponsive drug
delivery system showed the inherent property of locating the
mitochondria by fluorescence turn-on phenomenon, due to in
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Mitochondria-localized In Situ Generation of Rhodamine Photocage with
DOI: 10.1039/D0CC03524F
Fluorescence Turn-On Enabling Cancer Cell-Specific Drug Delivery Triggered
by Green Light
Amrita Paul,a Rakesh Mengji,b,c Manoranjan Bera,a Mamata Ojha,a Avijit Jana*b,c,d and N. D. Pradeep Singh*a
a.Department of Chemistry, Indian Institute of Technology Kharagpur 721302, West Bengal, India E-mail:
ndpradeep@chem.iitkgp.ernet.in
b. Department of Applied Biology, CSIR-Indian Institute of Chemical Technology Hyderabad, Uppal Road, Hyderabad 500007.
c.
Department of Organic Synthesis and Process Chemistry, CSIR-Indian Institute of Chemical Technology Hyderabad Uppal Road,
Hyderabad 500007.
d. Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India.
In this work, we have developed a new multi-tasking water-soluble photocage based on well-known
rhodamine dye for the cancer cells selective anticancer drug delivery triggered by green light. The
active fluorescent photocage is generated in situ, localized in the mitochondria in the presence of
reactive oxygen species (ROS). This helps to detect the cancer microenvironment, having elevated ROS
by fluorescence reporting and deliver the drug on demand by irradiating the indicated target sites.