A two-photon responsive naphthyl tagged: P -hydroxyphenacyl based drug delivery system: Uncaging of anti-cancer drug in the phototherapeutic window with real-time monitoring

Article abstract of DOI:10.1039/d0cc01903h

We report a two-photon responsive drug delivery system (DDS), namely, p-hydroxyphenacyl-naphthalene-chlorambucil (pHP-Naph-Cbl), having a two-photon absorption (TPA) cross-section of ≥20 GM in the phototherapeutic window (700 nm). Our DDS exhibited both AIE and ESIPT phenomena, which were utilized for the real-time monitoring of anti-cancer drug release.

Full text of DOI:10.1039/d0cc01903h

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DOI: 10.1039/D0CC01903H.
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Two-photon responsive Napthyl tagged p-hydroxyphenacyl based
drug delivery system: uncaging of anti-cancer drug in the
phototherapeutic window with real-time monitoring
Received 00th January
20xx,
Accepted 00th January
20xx
Amit Kumar Singh,a Moumita Kundu,b Samrat Roy,c Biswajit Roy,a Sk. Sheriff Shah,a Asha V Nair,a
Bipul Pal,c Mahitosh Mondal,b and N. D. Pradeep Singh *a
DOI:
10.1039/x0xx00000x
We report a two-photon responsive drug delivery system (DDS) tetraphenylene-p-hydroxyphenacyl-chlorambucil11 conjugate
namely, p-hydroxyphenacyl-naphthalene-chlorambucil (pHP- which released the anti-cancer drug only in the aggregated
Naph-Cbl) having two-photon absorption (TPA) cross-section of ≥ state on exposure to visible light (λ ≥410 nm) with real time
20 GM in the phototherapeutic window (700 nm). Our DDS monitoring ability. Though, several visible light activated p-
exhibited both AIE and ESIPT phenomena, which were utilized for hydroxyphenacyl based DDSs have been reported. To the best
the real-time monitoring of anti-cancer drug release.
of our knowledge, p-hydroxyphenacyl based DDS with
Light-induced drug delivery systems (DDSs) have recently uncaging ability in the phototherapeutic window (650-950 nm)
gained significant importance in the area of biological sciences is not still known, which hinders their practical usage in the
because of their ability to provide spatial and temporal control biological applications.12 To overcome the above-mentioned
over the drug release. To date, different types of light-induced limitation, two-photon responsive p-hydroxyphenacyl based
DDSs have been developed using the well-known DDS has been targeted. Elles et al. reported two-photon
phototriggers like o-nitrobenzyl,1 coumarinyl,2 quinolinyl,3 p- activated p-hydroxyphenacyl based DDS for the release of ATP
hydroxyphenacyl,4 anthracenyl5, and o-hydroxy cinnamate6. or DEP on exposure to light of wavelength 570 nm.13 But the
Among them, photoresponsive DDSs constructed based on p- above designed two-photon responsive DDS didn’t exhibit
hydroxyphenacyl phototrigger have captured great attention promising uncaging ability in the region of 650-950 nm.14
due to its unique properties which include: (i) large Stokes shift Hence, there is a need to develop two-photon responsive p-
(ii) fast release (iii) high photochemical quantum yield and (iv) hydroxyphenacyl based DDS having large two-photon
formation of biologically benign photoproduct.
absorption (TPA) cross-section in the phototherapeutic
Because of the aforementioned unique properties of p- window.15-19
hydroxyphenacyl phototrigger, several phenacyl based DDSs
Recent studies show that enhancing the internal charge
have been developed for the release of various important transfer (ICT) in a  conjugated donor (D) and acceptor (A) (D-
biomolecules like neurotransmitters, enzyme switches, -A) systems leads to large TPA cross-section.20-22 Based on the
ATP/DEP etc.7-9 In 2016, Singh group developed p- above interesting idea, we intend to construct p-
hydroxyphenacyl–benzothiazole as a DDS for the release of hydroxyphenacyl based DDS with good uncaging ability in the
anti-cancer drug chlorambucil in the visible wavelength region phototherapeutic window. For this purpose, we have
(λ ≥410 nm) using excited-state intramolecular proton transfer incorporated Naphthalene moiety to the p-hydroxyphenacyl
(ESIPT) phenomenon.10 In continuation, the same group also group so that a strong ICT can occur, thereby resulting in red-
developed another DDS based on p-hydroxyphenacyl namely, shifted absorption and enhanced two-photon uncaging cross-
section (Fig. 1). Our newly designed photoresponsive DDS,
pHP-Naph-Cbl provided the following advantages (i) two-
photon absorption in the phototherapeutic window (700 nm),
(ii) exhibits AIE phenomenon, thus significantly releases the
drug in the aggregated state, (iii) large stokes shift due to the
ESIPT process, and (iv) produces a distinct fluorescent colour
change on drug release, which enables real-time monitoring
ability.
a. Department of Chemistry, Indian Institute of Technology Kharagpur, 721302
Kharagpur, West Bengal, India. E-mail: ndpradeep@chem.iitkgp.ac.in
b. School of Medical Science and Technology, Indian Institute of Technology
Kharagpur, Kharagpur-721302, India
c. Department of Physical Sciences, Indian Institute of Science Education and
Research, Kolkata, Mohanpur, Nadia 741246, West Bengal, India
† Footnotes relating to the title and/or authors should appear here.
Electronic
Supplementary
Information
(ESI)
available:
See
DOI: 10.1039/x0xx00000x
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Furthermore, we also investigated the AIE properties of our
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DOI: 10.1039/D0CC01903H
photoresponsive DDS, pHP-Naph-Cbl. The emission spectra
of our DDS (1 X 10-4 M) were recorded in an ACN–Water,
binary mixtures with different water fractions (fw). In pure ACN
solution, the DDS exhibited very weak fluorescence. But with a
slight increment in the water fraction, the fluorescence
intensity of the DDS increased slowly. The emission intensity of
Fig.1 (a) Christopher G. Elles and co-workers reported two-photon activated pHP
phototriggers for uncaging of the bioactive molecule, and (b) Working protocol
of DDS pHP-Naph-Cbl in phototherapeutic window.
Fig.2 (a) Existence of keto and enol forms of pHP-Naph-Cbl, (b) Normalized UV-
Vis, and (c) Emission spectra of pHP-Naph-Cbl (1
x
10−4 M) in different
solvents).Excitation wavelength 350 nm.
Our DDS, pHP-Naph-Cbl, was synthesized according to the
following protocol (Scheme S1). The synthesis of
compound 2 was carried out as reported in our earlier work.10
The condensation reaction of 2 with 1-Naphthylamine on
heating at 130 C for 2 h in benzene solvent furnished the
desired compound 3. The desired DDS 3 was characterized by
NMR (1H and 13C) (Fig. S1-S2), and mass spectrometry (Fig. S3).
the DDS abruptly increased from fw = 85 to 95 vol % (Fig. 3a),
which is associated with the formation of aggregates of
DDS due to its poor solubility in the aqueous medium, and
fluorescence quantum yield of DDS (1 x 10-4 M) in the above
ACN-Water binary (1:9 v/v) mixture was found to be 0.22
(Table S1). We also estimated the stability of DDS in the
biological medium and at different pH in dark conditions
(Table S2).
o
Scheme 1. Synthesis of photoresponsive DDS, pHP-Naph-Cbl.
The photophysical properties of our DDS, pHP-Naph-Cbl,
was noted to be sensitive with respect to different solvents.
The UV spectrum of our DDS (1 x 10-4 M) showed an
absorption maximum at around 350 nm (Fig. 2b) in studied
solvents. The influence of the ESIPT process was demonstrated
by emission spectroscopy (Fig. 2c). In the case of non-
hydrogen bonding solvents(benzene, chloroform), our DDS
showed emission maximum at around 550 nm, due to the keto
form (5) which can be attributed to the ultrafast proton
transfer from the hydroxyl group of pHP to imine nitrogen
atom of Napthyl moiety (ESIPT). Whereas, in the polar aprotic
solvents (DMSO, EtOAc), our DDS showed blue-shifted
emission maximum at 425 nm, due to the enol form (4) of
pHP-Naph-Cbl. Interestingly, in polar protic solvents (MeOH,
ACN) our DDS provided two emission maxima, one at 550 nm
and another at around 425 nm because of the keto-enol
tautomerization of pHP-Naph-Cbl. However, in MeOH and
DCM our DDS exhibited two humps around 425 nm which can
be due to the presence of both cis- and trans-enolic forms
(Fig. S4).
Fig.3 (a) Fluorescence spectrum of DDS, pHP-Naph-Cbl (1 × 10−4 M) in ACN–
water binary mixture with different water fractions (fw), and (b) Fluorescence
spectrum of DDS, pHP-Naph-Cbl (1 × 10−4 M) in ACN–PBS buffer of pH 7.4 at
different intervals of photoirradiation (0−15 min).
To demonstrate the photorelease capability of our
photoresponsive DDS (1 × 10−4 M), in ACN–PBS buffer of pH
7.4 (1:ꢀ9 v/v) was exposed to a medium pressure mercury lamp
(125 W) as the source of light (λ ≥ 365 nm) using a 1 M
aqueous solution of CuSO4 as the UV cut-off filter with
constant stirring for 15 min. The photorelease was monitored
using reverse-phase HPLC (Fig. 4). The gradual depletion of the
peak at retention time (tR) 4.7 min with increasing time of
irradiation indicates the photodecomposition of 3, whereas,
the appearance of two new peaks at 3.9 min and 3.2 min
denotes the formation of the photoproduct (pHP-Naph-COOH)
and chlorambucil (assigned by injecting the authentic sample),
respectively. Formation of photoproduct Cbl and pHP-Naph-
COOH from pHP-Naph-Cbl, after 15 min of photoirradiation
was characterized by HRMS (Fig. S5-S6).
2 | J. Name., 2012, 00, 1-3
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Interestingly, we noted a distinctive fluorescence colour
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To support our argument that the drug release occurs from
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change from greenish-yellow to blue during the photolysis of T1 state, we carried out the photolysisDiOnI: t1h0.e10p39r/eDs0eCnCc0e19o0f3Ha
our DDS, pHP-Naph-Cbl (Fig.ꢀ3b). Initially, the emission triplet quencher, potassium sorbate (PoS). We noted the
spectrum showed the band at around 550 nm (due to AIE) complete arrest of drug release at 200 µM concentration of
corresponding to pHP-Naph-Cbl (at 0ꢁmin). When the PoS (Fig. S8).
irradiation time was gradually increased (0–15ꢁmin), the
Further, we were also interested to investigate whether the
intensity of the yellow-emission band was gradually decreased drug release happens only in the aggregate state. Hence, we
and a new blue-emission band at 430ꢁnm (Stokes Shift 120 nm) performed irradiation of our DDS in different water fractions
corresponding to the photoproduct, pHP-Naph-COOH gradual (fw). We observed that the anti-cancer drug release reached a
increased in intensity. The blue shift in the fluorescence maximum of 95 % (photochemical quantum yield 0.49) with a
spectrum of the photoproduct, pHP-Naph-COOH, is due to the gradual increase in fw, in compound 3 (1 × 10−4 M) in ACN–PBS
loss of internal charge transfer (ICT) from Napthyl to phenacyl buffer, at pH 7.4. We noted no substantial anti-cancer drug
group (as the photoproduct has no phenacyl functionality). release was observed below fw 80 % and in pure ACN (Fig. S9).
Thus our DDS is capable of monitoring the drug release in real- This indicated that the aggregation phenomenon initiates the
time.
anti-cancer drug release (Table S3). We also evaluated the
photolysis of pHP-Naph-Cbl at different pH medium (Table S4),
the results support that ESIPT process assist the photorelease
(Fig. S10).
After a single-photon uncaging of pHP-Naph-Cbl, we were
interested to study the two-photon uncaging cross-section
(δu)25 of our DDS, pHP-Naph-Cbl (1 × 10−4 M) in DMSO/Water
(1:9 V/V). First δa of our DDS (1 x 10-4 M) in DMSO/Water, (1:9
V/V) was determined using open aperture Z-scan technique
with pulsed laser (pulse width 100 fs, repetition rate 80 MHz at
700 nm with 1.0-Watt power) (Fig. S11), and the result was
found to be δa ≥ 20 GM (Fig. 5). Then δu of pHP-Naph-Cbl was
calculated using the formula δu = ϕuδa, thus δu of pHP-Naph-
Cbl was found to be 10 GM (1 GM = 10-50 cm4 s/photon). The
two-photon release of our DDS was performed with a laser of
100 fs pulse at the 80 MHz repetition rate at the wavelength of
700 nm. The result indicated that 25 % of drug release was
noted after 3 h of irradiation (Fig. S12). Furthermore, we also
evaluated the δu of our DDS at different wavelengths and we
noted that δu of DDS was maximum at 700 nm (Fig. S13).
Fig.4 Overlay HPLC chromatogram of pHP-Naph-Cbl in ACN/PBS buffer of pH 7.4
(1:9 v/v) at different time intervals (0−15 min) of photoirradiation.
We also noted that the drug release solely depends upon the
incident light, thus we carried out a ‘light on-off’ experiment,
in which we exposed our DDS, pHP-Naph–Cbl solution to light
and dark conditions for different time intervals.23 We observed
that our DDS released anti-cancer drug chlorambucil only on
exposure to light. In addition, we also found that our DDS on
exposure to light for 15 min released 95% of anti-cancer drugs
(Fig. S7 a and b).
The mechanism of photorelease by pHP-Naph-Cbl is shown
in scheme 2. Upon exposure to light (λ ≥365 nm), our DDS,
pHP-Naph-Cbl (1 × 10−4 M) gets excited from the ground state
(S0) to its singlet excited state (S1). In the S1 state, DDS
undergoes strong intramolecular hydrogen bonding between
the imine nitrogen and -OH group of pHP (CH=N----HO), which
facilitates ESIPT behaviour in the excited state, thus resulting
in deprotonation of the pHP group to give intermediate 6. The
intermediate 6 then, undergoes efficient ISC to its triplet
excited state 7 (T1). From the T1 state, DDS undergoes photo-
Favorskii rearrangement24 to give spirodiketone intermediate
8 leading to the concomitant release of anti-cancer drugs. The
intermediate spirodiketone is then subjected to hydrolysis in
the aqueous medium to give the rearranged photoproduct 9
(pHP-Naph-COOH).
Fig.5 Open aperture (OA) Z-scan measurement (at 700 nm) of pHP-Naph-Cbl of
normalized transmittance. The discrete points represent experimental data and
the solid line represents theoretical fitting.
Based on the promising photorelease ability of our DDS, we
were keen to carry out the in vitro studies of the DDS using
breast cancer cell line MCF-7. Cellular uptake studies showed
that DDS, pHP-Naph-Cbl got internalized in the cell after 12 h
of incubation. Fig. 6 (ia, ib, and ic), shows that pHP-Naph-Cbl is
uniformly distributed inside the cell. Real-time monitoring of
Scheme 2 Proposed photorelease mechanism of the pHP-Naph–Cbl.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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drug release by our DDS (pHP-Naph-Cbl) inside breast cancer fellowship. We are grateful to Swathi S and VTiehwaArrtuicnleikOanlinSe
cell line MCF-7 was analyzed by fluorescence confocal images (Biotechnology Department, St. JoDsOeI:p1h0’.s1039C/Do0llCeCg0e1903oHf
(Fig. 6). Initially, the cells showed yellow fluorescence due to Engineering) for their help to perform biological experiment.
cellular uptake of pHP-Naph-Cbl. After exposure to light of
wavelength ≥365 nm for 15 min, we observed a complete
fluorescent colour change from yellow to blue, suggesting a
References
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Further, we evaluated the cytotoxicity of pHP-Naph-Cbl
using the MTT assay [MTT=3-(4, 5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, a yellow tetrazole] in the cell
line MCF-7. Cell viability remained above 95 % at different
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line MCF-7 (IC50 31 µM) as validated by the MTT toxicity data
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showed enhanced cytotoxicity in the presence of light
compared to the free Cbl at given concentration.
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Fig.6 Fluorescence confocal images of cellular internalization of pHP-Naph-Cbl in
breast cancer cell line MCF-7 (i)a bright-field image, at 0 min., (i)b fluorescence
image of bright field at 475-550 nm, (i)c fluorescence image of bright field in blue
channel, (emission wavelength range 420-450 nm), (i)d merge image of bright-
field, yellow-green and blue channel, (ii)a bright-field image at 0 min. (ii)b
fluorescence image of bright field at 475-550 nm after 15 min. of
photoirradiation, (ii)c fluorescence image of bright field in blue channel after 15
min. of photoirradiation (emission wavelength range 420-450 nm), and (ii)d
merge images of bright-field, yellow-green and blue channel of pHP-Naph-Cbl.
Excitation wavelength for yellow-green and blue channels are 460 nm and 350
nm, respectively. Scale bar = 200 µM.
In conclusion, two-photon responsive p-hydroxyphenacyl
based DDS with uncaging ability in the phototherapeutic
window was developed by simple tagging with Napthyl moiety.
The designed DDS showed the TPA cross-section to be greater
than 20 GM and TP uncaging of 10 GM at 700 nm region. The
DDS released the anti-cancer drug only in the aggregated state
and exhibited real time monitoring ability of drug release. In
future, we want to explore our pHP-Naph-Cbl as a two-photon
responsive nano DDS for the efficent release of anticancer
drug for in vivo applications.
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Conflicts of interest
There are no conflicts to declare.
Notes and references
We thank DST SERB (Grant No. DIA/2018/000019) for financial
support and DST (SR/FST/CSII-026/2013) for the 400 MHz NMR
spectrometer. A. K. Singh is thankful to UGC-New Delhi for the
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Two-photon responsive drug delivery system having two-photon absorption (TPA) in the phototherapeutic window with two-
photon uncaging cross-section ≥ 10 GM and exhibiting real-time monitoring of anti-cancer drug release.
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