Single component photoresponsive fluorescent organic nanoparticles: a smart platform for improved biomedical and agrochemical applications

Article abstract of DOI:10.1039/d0cc07183h

In the last two decades, light responsive nano drug delivery systems (DDSs) have gained considerable importance, particularly in the area of biology and medicine. In general, light responsive nano DDSs are bicomponent and constructed using two ingredients, namely a nanocarrier and a phototrigger. The synthesis of these bicomponent nano DDSs requires multiple steps, which limits their applications. Hence, we have reported single component light responsive nano DDSs using fluorescent organic nanoparticles (FONPs) which acted both as a nanocarrier and a phototrigger. This feature article provides an overview of recently developed light responsive single component FONPs and their applications in the regulated release of anticancer drugs, gasotransmitters, antibacterial agents, and pesticides, and also as efficient PDT agents. We have summarised the synthesis, characterisation, and photophysical, photochemical, andin vitrobehaviours of these light responsive FONPs. In addition, we also discussed the advantages of using FONPs as a nano DDS for cellular studies like: excellent biocompatibility, efficient cellular internalisation, real time monitoring of the drug release ability inside the cells, and enhanced cytotoxicity due to regulated release of bioactive molecules inside the cells.

Full text of DOI:10.1039/d0cc07183h

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FEATURE ARTICLE
Single component photoresponsive fluorescent
organic nanoparticles: a smart platform
for improved biomedical and agrochemical
applications
Cite this: Chem. Commun., 2021,
57, 1715
Amrita Chaudhuri, Amrita Paul, Antara Sikder and N. D. Pradeep Singh
*
In the last two decades, light responsive nano drug delivery systems (DDSs) have gained considerable
importance, particularly in the area of biology and medicine. In general, light responsive nano DDSs are
bicomponent and constructed using two ingredients, namely a nanocarrier and a phototrigger. The
synthesis of these bicomponent nano DDSs requires multiple steps, which limits their applications.
Hence, we have reported single component light responsive nano DDSs using fluorescent organic
nanoparticles (FONPs) which acted both as a nanocarrier and a phototrigger. This feature article
provides an overview of recently developed light responsive single component FONPs and their
applications in the regulated release of anticancer drugs, gasotransmitters, antibacterial agents, and
pesticides, and also as efficient PDT agents. We have summarised the synthesis, characterisation,
and photophysical, photochemical, and in vitro behaviours of these light responsive FONPs. In addition,
Received 29th October 2020,
Accepted 8th January 2021
we also discussed the advantages of using FONPs as
a nano DDS for cellular studies like:
excellent biocompatibility, efficient cellular internalisation, real time monitoring of the drug release
ability inside the cells, and enhanced cytotoxicity due to regulated release of bioactive molecules
inside the cells.
DOI: 10.1039/d0cc07183h
rsc.li/chemcomm
Introduction
Photoresponsive nanocarriers have emerged as a promising
tool for targeted therapy, as they can regulate the release
of caged bioactive molecules in a spatio-temporal fashion using
Department of Chemistry, Indian Institute of Technology Kharagpur, 721302
Kharagpur, West Bengal, India. E-mail: ndpradeep@chem.iitkgp.ac.in
Amrita Chaudhuri received her
bachelor’s degree in Chemistry
Honours (2013) and master’s
degree in Chemistry (specialization
in Organic Chemistry, 2015) from
Amrita Paul completed her
bachelor’s degree in Chemistry in
the year 2011 and then achieved
her master’s degree in Chemistry
with specialization in Organic
Chemistry in 2013 from the
Vidyasagar University, Midnapore,
India. In 2020, August, she
achieved her PhD degree in organic
the
Vidyasagar
University,
Midnapore, India. In July 2015,
she joined as a PhD student in
organic photochemistry under the
supervision of Prof. N. D. Pradeep
Singh in the department of
chemistry, IIT Kharagpur, India.
Her current research interests
include design, synthesis and
photochemistry
under
the
supervision of Prof. N. D. Pradeep
Singh in the Department of
Chemistry, IIT Kharagpur, India.
Her research interests involve
Amrita Chaudhuri
Amrita Paul
photochemical studies of new photoremovable protecting groups, and development of new strategies for the light-triggered release of caged
photoresponsive gasotrasmitter donors and development of light- active molecules and synthesis of new photoremovable protecting groups
responsive therapeutic systems.
in order to produce efficient delivery systems for biological applications.
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non-invasive light as an external stimulus.1 Their distinctive
features, such as smaller size, higher surface area to volume ratio,
and better ability to penetrate deeper into tissue, have helped
to improve the pharmacokinetics and biodistribution of several
chemotherapeutic agents.2–5 The essential characteristics that a
photoresponsive nanocarrier requires for its application in the area
of drug and biomolecule delivery are (i) biocompatibility and easy
functionalizability, (ii) high caging efficiency and no premature
degradation of delivery agents, (iii) being soluble or colloidal in
aqueous medium in order to increase the effectiveness of the
delivery agents in the biological medium, (iv) extended circulatory
half-life and long shelf life, (v) high biodegradability to reduce
undesirable side effects, and (vi) the ability to regulate the release
of caged bioactive molecules at the target site.6,7
Fig. 1 Single component photoresponsive FONPs for the regulated
release of anticancer drugs, antibacterial agents, gasotransmitters, and
agrochemicals and as effective PDT agents.
In general, photoresponsive nanocarriers can be classified into
two main categories: organic nanocarriers (polymers, dendrimers,
liposomes and DNA scaffolds) and inorganic nanocarriers (metal
oxides, quantum dots, nanotubes, and semiconducting nano-
crystals).8–10 In both types of photoresponsive nanocarriers the active synthesis, which are (i) synthesis of a biocompatible nanocarrier,
biomolecules are loaded by two strategies: (i) physical entrapment by (ii) surface functionalization of the nanocarrier, and (iii) attach-
the nanocarrier or (ii) attachment to the nanocarrier through ment of the phototrigger to the nanocarrier.20–22 To overcome
covalent bonding. In the case of physical entrapment, the active the aforementioned synthetic steps for the construction
molecules are trapped in the porous channel or container of the of photoresponsive nanocarriers, which limited their scope of
nanoparticles, and the pores of these nanoparticles are closed or applications, we introduced for the first time single-component
sealed with a phototrigger (phototriggers are small organic photoresponsive organic nanocarriers in which the nanoparticle-
chromophores that initiate the release of active molecules on sized fluorescent phototrigger itself acts as a nanocarrier for
irradiation with light).11–15 With covalent anchoring, the active the delivery of bioactive molecules. For the fabrication of
molecules are covalently attached directly to the phototrigger, these phototriggers as light responsive fluorescent organic
which is then connected to the nanocarrier.16–19 In both cases, nanoparticles (FONPs), we adapted the effective ‘‘reprecipitation
the activity of the caged active molecule is temporarily switched technique’’.23,24
off. During photolysis, the active molecule gets released from
the phototrigger or removed from the porous system by opening opment of single component photoresponsive fluorescent organic
the light-activated gate.
nanoparticles (FONPs) as drug delivery systems (DDSs) for the
This feature article is mainly focused on the design and devel-
The photoresponsive nanocarriers synthesized by the above regulated release of anticancer drugs, antibacterial agents, gaso-
discussed strategies are generally bicomponent systems; they transmitters, and agrochemicals. In addition, we also developed
require two ingredients for their construction, namely a nano- single component FONPs as photosensitizers for efficient PDT in
particle and a phototrigger. They need three crucial steps for their cancer treatment (Fig. 1).
Antara Sikder obtained bachelor’s
degree in Chemistry in the year
2014 from Calcutta University,
Kolkata, India, and master’s degree
in chemistry from IIEST Shibpur,
Howrah, India, in 2016. She is
currently a PhD student in organic
chemistry under the supervision of
Prof. N. D. Pradeep Singh in the
department of chemistry, IIT
Kharagpur, India. Her current
research interests involve the
N. D. Pradeep Singh obtained his
BSc and MSc Degrees in Chemistry
from University of Madras, Chennai,
and conducted research with Prof. A.
Anandhan. He completed his PhD
study in 2001 at the same institute
with Prof. Geetha Gopalakrishnan
in
Organic
Photochemistry.
Currently, he is a Professor of
Chemistry at the Indian Institute of
Technology, Kharagpur, India, and
received numerous awards. The
Pradeep lab is engaged in the
synthesis of new fluorescent
design,
synthesis
and
Antara Sikder
N. D. Pradeep Singh
photochemical studies of new
photolabile protecting groups and synthesis of light-responsive probes photoremovable protecting groups for biological applications and
for applications in therapy and diagnostics.
studying visible-light-induced photoredox catalysis.
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Feature Article
1 Single component photoresponsive
fluorescent organic nanoparticles as a
drug delivery system for cancer
treatment
1.1 Single component photoresponsive fluorescent organic
nanoparticles for regulated release of anticancer drugs
The phototrigger is an important ingredient used in the construc-
tion of light responsive drug delivery systems (DDSs). The photo-
trigger is an organic chromophore which cages the drug molecule
in such a way that the activity of the drug molecule is temporarily
masked and on exposure to light releases the drug molecule in a
controlled manner.25 In accordance, we also developed several
visible light and two-photon activated fluorescent phototriggers
based on perylen-3-yl methyl,26 acetyl pyrene,27 acridine-9-
ylmethyl,28 carbazole,29,30 bimane,31 stilbene,32 rhodamine,33
naphthalene,34 and carboline35 derivatives for the release of
important bioactive molecules. Though phototriggers provide
immense scope to control drug release at a specific time and
space, thereby providing localised treatment, they also face certain
limitations like low solubility in the physiological medium, a lack
of targeting ability, and low drug imposing capacity. In order to
overcome the limitations of phototriggers and use them efficiently
as a DDS, we introduced the strategy of converting these photo-
triggers to single-component photoresponsive FONPs. Herein, the
phototriggers were directly converted into organic nanoparticles
by a simple reprecipitation technique, which made them capable
of serving both as a nanocarrier and a delivery agent. Photo-
responsive organic nanoparticle based DDSs were designed
utilising fluorescent phototriggers, and therefore they provided
the added advantage of image-guided delivery of drug molecules
and the opportunity to monitor the drug release in real-time. In
this section, we will illustrate our efforts in designing light-
responsive single-component FONPs as anticancer DDSs
which were synthesised using phototriggers developed by
our group viz., perylene-3-yl methyl,36 acridin-9-methyl,37 and
p-hydroxyphenacyl38–40 (Fig. 2).
Fig. 2 Single-component photoresponsive fluorescent organic nano-
particles based on perylene-3-yl methyl, acridin-9-methyl, and p-hydro-
xyphenacyl as drug delivery systems for cancer treatment.
to be quite different (Fig. 3Ci and ii), which was attributed to
the difference in their particle size i.e., 30 nm and 20 nm,
respectively. The Pe–Cbl NPs were well dispersed in aqueous
medium due to their nano size and hence showed efficient
photorelease ability in aqueous physiological medium
(Fig. 3D), with about 95% drug release after 30 min of irradia-
tion. On the other hand, the Pe–Cbl conjugate was not able to
efficiently photorelease the anticancer drug in aqueous medium
due to its insolubility in water. Another fascinating property of
Pe–Cbl NPs was that they showed a distinct fluorescence colour
change from red (625 nm) to blue (445 nm) upon photolysis.
This noticeable blue shift in the emission spectra after the
photolysis was due to the difference in the sizes of Pe–Cbl NPs
and the photoproduct, perylene-3-ylmethanol NPs. The fluo-
rescence colour change during the photorelease was exploited
to monitor and quantify the drug release in real-time. However,
the photocaged Pe–Cbl conjugates failed to exhibit the ability of
real-time monitoring of drug release.
In vitro studies revealed that our DDS, Pe–Cbl NPs, showed
excellent cellular uptake and good biocompatibility. We also
explored the unique real-time monitoring ability of Pe–Cbl NPs
for the quantification of the drug release inside HeLa cells
1.1.1 Perylene-3-ylmethanol: single-component photore- using the fluorescence imaging technique (Fig. 3E). The MTT
sponsive fluorescent organic nanoparticles with real-time mon- [MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
=
itoring of anticancer drug release. In the year 2012, our group bromide, a yellow tetrazole] assay revealed that Pe–Cbl NPs
for the first time showed the application of single-component showed enhanced cytotoxicity compared to free chlorambucil
FONPs based on perylene-3-ylmethyl for the in vitro release of due to precise drug release upon irradiation causing the
the anticancer drug chlorambucil (Cbl) (Fig. 3A).36 Photo- killing of the cancer cells (Fig. 3F). Hence, we have clearly
responsive organic NPs fabricated using the phototrigger demonstrated that the nanoparticles of photocaged perylene–
(perylene-3-ylmethanol) exhibited three important features: (i) chlorambucil seem to be an effective anticancer DDS in com-
efficient photorelease in aqueous physiological medium, (ii) a parison to Pe–Cbl conjugates.
highly fluorescent nature, which helped in image-guided
1.1.2 Acridin-9-methanol: single component photorespon-
release of drug molecules, and (iii) a distinct fluorescence sive fluorescent organic nanoparticles for nuclear-targeted drug
colour change (red to blue) upon photorelease, which aided delivery. Though the Pe–Cbl NPs showed excellent properties as
in monitoring and quantifying the drug release in real-time.
a DDS, still they face certain limitations which need to be
Nanoparticles of perylene–chlorambucil, Pe–Cbl (DDS), were addressed. One of the main limitations is that the Pe–Cbl NPs
prepared by the reprecipitation technique, which were globular were found to be localised mainly in the cellular membrane or
in shape with an average particle size of B30 nm (Fig. 3B). in the cytoplasm. It has been well studied that anticancer drugs
Interestingly, the absorption and emission spectra of Pe–Cbl which work based on a DNA alkylating mechanism need to be
NPs and perylene-3-ylmethanol NPs (phototrigger) were found delivered into the cell nucleus, in order to efficiently damage
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Fig. 3 (A) Schematic representation of photoinduced in vitro anticancer drug release from Pe–Cbl NPs with real time monitoring. (B) TEM images of
(a) Pe–Cbl NPs before photolysis, (b) Pe–Cbl NPs after photolysis, and (c) perylene-3-ylmethanol NPs. (C) Normalized absorption and emission spectra of
(i) Pe–Cbl NPs and (ii) perylene-3-ylmethanol nanoparticles. (D) HPLC profile of Pe–Cbl NPs at regular time intervals of photolysis under visible-light
irradiation (Z410 nm). The vertical axes were offset by 15 mAU units and the horizontal axes by 15 s to facilitate better visualization. (E) Real-time drug
release studies using confocal microscopy. Shown are overlays of the bright-field image and the fluorescence images in the 445 and 625 nm emission
channels (i) before photolysis, (ii) after 10 min of photolysis, and (iii) after 20 min of photolysis. (F) Cell viability tests of Pe–Cbl NPs, perylene-3-ylmethanol
nanoparticles, and chlorambucil on the HeLa cell line: (i) before irradiation, (ii) after irradiation in the presence of different concentrations of Pe–Cbl NPs,
and (iii) after regular time intervals of irradiation in the presence of 2 ꢁ 10ꢀ5 M Pe–Cbl NPs. Values are presented as means ꢂ standard deviations. (B–F)
are reprinted with permission from ref. 36. Copyright 2012: American Chemical Society.
the DNA and cause cell death. Based on this idea, in the year of the Acr–Cbl NPs was studied using ethidium bromide (EB)
2013, we fabricated new single-component fluorescent organic displacement assay and docking analysis. The docking analysis
nanoparticles which have high affinity to DNA based on an revealed that the acridine moiety acted as a minor groove
acridine phototrigger for nuclear-targeted and regulated deliv- binder (Fig. 4Ci and ii), whereas the ester carbonyl group
ery of anticancer drug chlorambucil (Cbl).37 The acridin-9- formed hydrogen bonds with two guanine N7–H atoms. Ethi-
methyl based FONPs performed three important roles: as a (i) dium bromide displacement assay (Fig. 4Di) revealed that the
‘‘nuclear-targeted nanocarrier’’ for drug delivery, (ii) ‘‘photo- Acr–Cbl NPs preferred to bind DNA in an intercalative manner.
trigger’’ for regulated drug release, and (iii) fluorescent chro- Interestingly, the affinity constant of the Acr–Cbl NPs was
mophore for cell imaging (Fig. 4A).
found to be 1.59 ꢁ 106 Mꢀ1, which was similar to that of other
Acridine–chlorambucil (Acr–Cbl) NPs (DDS) and acridin-9- strong acridine DNA binders (Fig. 4Dii). Real-time cellular
methanol NPs (phototrigger) were found to be spherical in uptake and imaging studies of Acr–Cbl NPs were carried out
shape with an average particle size of 60 nm (Fig. 4Ba–d). using the cancerous HeLa cell line. The nuclear colocalization
The Acr–Cbl NPs and acridin-9-methanol NPs exhibited quite of the Acr–Cbl NPs was compared with the Acr–Cbl conjugate
different absorption spectra; however, their emission spectra using propidium iodide; the results revealed that the Acr–Cbl
were found to be similar, showing a strong blue emission band NPs showed profound accumulation in the cell nucleus com-
at 480 nm. Similar to Pe–Cbl nanoparticles, Acr–Cbl NPs were pared to the Acr–Cbl conjugate (Fig. 4E). MTT assay results
also well dispersed in aqueous medium and showed efficient strongly supported the argument that nuclear targeted delivery
photorelease ability in aqueous physiological medium under of chlorambucil (an alkylating anticancer agent) using Acr–Cbl
soft visible light irradiation (Z410 nm), with about 90% drug NPs in regulated release resulted in enhanced anticancer
release after 35 min of irradiation. The nuclear targeting ability activity.
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Fig. 4 (A) Schematic presentation of photocontrolled nuclear-targeted delivery of chlorambucil by photoresponsive organic nanoparticles of Acr–Cbl.
(B) TEM images of (a and b) Acr–Cbl nanoparticles before photolysis, and (c) Acr–Cbl nanoparticles after photolysis, and (d) TEM image of acridin-9-
methanol nanoparticles. (C) Docked conformations of the DNA–Acr–Cbl caged conjugate (i) surface representation showing the binding pockets of the
conjugate, and (ii) stereoview of the docked conformations of the Acr–Cbl conjugate showing that the ester carbonyl is forming H-bonds with guanine
N7 H-atoms, thereby facilitating DNA-alkylation. (D) (i) Fluorescence quenching spectra of EB-ct-DNA with Acr–Cbl nanoparticles. The red line indicates
the fluorescence emission spectrum of EB in the absence of ct-DNA and the top blue lines indicate the fluorescence emission spectra of EB bound to
ct-DNA; [EB] = 2 mM, [ct-DNA] = 20 mM, [Acr–Cbl nanoparticles] = 0–38 mM. (ii) The quenching of EB bound to ct-DNA by Acr–Cbl nanoparticles is in
good agreement with the linear Stern–Volmer equation. (E) (ai–biii) Confocal fluorescence and brightfield images of HeLa cells: (ai–aiii) cells +
nonaggregated Acr–Cbl (4 ꢁ 10ꢀ5 M) after 4 h of incubation: (ai) brightfield image, (aii) fluorescence image and (aiii) overlay image of (ai) and (aii). Cells
were incubated with 20 mM Acr–Cbl nanoparticles. (B–E) are reprinted with permission from ref. 37 American Chemical Society.
1.1.3 p-Hydroxyphenacyl conjugated benzothiazole: single using H2O2-mediated oxidation of the boronate fluorophore. In
component photoresponsive fluorescent organic nanoparticles the next step, precise spatio-temporal irradiation with light only
as an environment activatable nanoprodrug with two-step of identified tumor sites results in the release of anticancer drug
surveillance in anticancer drug release. Recent studies have chlorambucil. Real time information on drug release was
shown that DDSs which can be localized to cancer and deliver achieved by a second fluorescence colour change (signal 2, green
the chemotherapeutic agent on demand resulted in enhanced to blue fluorescence). The detection of H2O2 and specific activa-
therapeutic activity. A higher level of intrinsic hydrogen peroxide tion of ANPD-X (mediated by the boronate trigger) were mon-
(5 mM to 1.0 mM) has been observed in cancer cells due to the itored by the spectral change under physiological conditions by
overproduction of reactive oxygen species (ROS) in comparison UV-vis and fluorescence spectroscopy (Fig. 5Ba). Interestingly, in
to normal cells.41–46 This exclusive feature of the cancer cell the presence of H2O2, a dramatic increase of the emission band
microenvironment provided an advantage toward the develop- at 518 nm corresponding to the keto emission (Fig. 5C and Bb)
ment of cancer cell selective chemotherapeutic nano DDSs. was observed, along with a concomitant decrease in the fluores-
Keeping this in mind, our group has developed a microenviron- cence intensity at 448 nm (enol emission) (Fig. 5C). This large
ment sensitive nanoprodrug, ANPD-X (Activatable Nano Pro- bathochromic shift of 70 nm caused a distinct fluorescence
Drug-X), using a phototrigger p-hydroxyphenacyl (pHP) group colour change from blue to green. The TEM and DLS data
tagged with a benzothiazole moiety for targeted cancer therapy showed that ANPD-X nanoparticles were globular in shape with
(Fig. 5A).38 The unique ability of nanoprodrug ANPD-X is its dual a size of B78 nm, but after H2O2 treatment the particle size was
surveillance in two successive steps in anticancer drug delivery. reduced by B20 nm. On the other hand, with an increase in the
In the first step, the nanoprodrug identifies the tumor sites by a irradiation time (0–15 min) a gradual decrease of the intense
fluorescence colour change (signal 1, blue to green fluorescence) green emission band was observed along with the appearance of
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Fig. 5 (A) Schematic representation of the H2O2 activable nanoprodrug exhibiting two-step surveillance in drug release. (B) (a) Fluorescence response of
ANPD-X against time after addition of H2O2 (50 equiv.), (b) change in the fluorescence spectral profile with increasing irradiation time, and (c) comparison of
the fluorescence spectral profile in the absence of H2O2, in the presence of H2O2 and after photoirradiation. (C) Emission spectra of benzothiazole
conjugated p-hydroxyphenacyl–chlorambucil in different solvents, showing ESIPT. (D) Cellular fluorescence images of HeLa cells after cellular uptake study
with ANPD-X: (ai) bright field image, (aii) fluorescence image, and (aiii) merge of the bright field and fluorescence images, and (bi) cells treated with ANPD-X,
(bii) ANPD-X pretreated cells after treatment with H2O2, and incubating for 12 h, and (biii) after visible light (Z410 nm) irradiation. (B and D) are reprinted with
permission from ref. 40. Copyright 2017: American Chemical Society. (C) is reprinted with permission from ref. 40. Copyright 2016: Willey-VCH.
a new blue emission band at 450 nm corresponding to the high signal to noise ratio, excellent photostability in the
photoproduct pHP-Benz-COOH (Fig. 5Bb and c). The hypso- solution phase and a chance to look into the interesting
chromic shift in the emission spectra was due to the disruption emission properties of a molecule in the aggregated state.47,48
of conjugation (phenolic hydroxyl group to the carbonyl group) AIE fluorogens are highly emissive in their aggregated state but
occurring during photolysis. Fascinatingly, ANPD-X showed exhibit weak fluorescence in solution. The restricted intra-
almost 90% drug release after 15 minutes of visible light molecular motion in the aggregated state of AIE fluorogens
irradiation.
plays a significant role in their photophysical behaviour.49 To
The H2O2-mediated activation and light triggered release of the best of our knowledge, photoresponsive organic nano-
chlorambucil of nano DDS ANPD-X (Fig. 5D) was performed particles as a DDS based on the AIE process have not been
in vitro using HeLa cell lines. MTT assay revealed that enhanced developed. Hence, our group designed a photoresponsive nano
cytotoxicity was noted upon photoirradiation of ANPD-X DDS, which exhibits both AIE and ESIPT phenomena, by
incubated cells in comparison with pHP-Benz–Cbl and chlorambucil simple incorporation of a salicylaldazine moiety into a pHP
because of the efficient bio-distribution of ANPD-X nanoparticles (p-hydroxyphenacyl) phototrigger (Fig. 6A).39 Our newly
and photorelease of the anticancer drug inside the cancer cells.
designed nano DDS (nanoparticulate drug delivery system-5,
Therefore, our single-component organic nanoparticles NDD5) showed excellent properties like (i) the excitation wave-
ANPD-X showed very fascinating properties like good tumor length is extended greater than 410 nm, (ii) it releases drugs
microenvironment sensitivity, high cellular uptake capability only in the aggregated state, (iii) it produces a distinct change
and spatiotemporal control over the drug delivery, which made in the fluorescence colour on drug release, thus providing
them a highly efficient photoresponsive nano DDS.
immense scope to monitor the drug release profile in real-
1.1.4 p-Hydroxyphenacyl conjugated salicylaldazine: single time (Fig. 6B) and (iv) it has the ability to release two different
component photoresponsive fluorescent organic nanoparticles drugs sequentially so that it can be used effectively in the area
showing ‘AIE + ESIPT’ assisted photorelease for dual anticancer of combination chemotherapy (Fig. 6C). Transmission electron
drug delivery with real-time monitoring ability. In recent years, microscopy (TEM) revealed that our nano DDS (NDD5) was
the AIE process has captured great attention since it provides a globular in shape with a size of B68 nm (Fig. 6D). It was also
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Fig. 6 (A) AIE + ESIPT0 assisted photorelease from NDDS-5. (B) Comparison between the fluorescence spectra of NDDS-5 before and after photolysis
(inset: fluorescence colour change during photolysis). (C) HPLC overlay chromatogram of NDDS-5 during photolysis using visible light (Z410 nm) in an
ACN–water mixture (fw = 99 vol%). a = 5, b = photoproduct 6, c = released chlorambucil, d = released ferulic acid, and e and f are singly protected
species. (D) TEM images of NDDS-5 before photolysis. (E) Exploration of the real-time (fluorescence confocal images captured at different intervals of
time during irradiation) monitoring capability of NDDS-5 during photorelease of drugs upon visible light (Z410 nm) irradiation. Irradiation time: (a) 0 min
and (b) 5 min. (A–E) are reprinted from ref. 39: The Royal Society of Chemistry.
noted that our nano DDS NDDS-5 was quite stable in the dark cytotoxicity was observed upon irradiation of NDDS-5 with
in culture medium but B90% of one of the caged drugs respect to free chlorambucil, which was owing to efficient
(chlorambucil and ferulic acid) was released within 5 min of bio-distribution and the synergistic effect of ferulic acid and
visible light irradiation.
chlorambucil inside the cancer cells. In short, our nano NDD5
We carried out in vitro studies of NDDS-5 using HeLa exhibited unique properties like excellent cellular uptake
cells, which showed a uniform distribution of NDDS-5 inside capability, appreciable biocompatibility, and great spatiotem-
the cells, indicating good cellular internalization. The co- poral precision of the drug release, which makes it an excellent
localization experiment revealed that NDDS-5 went selectively photoresponsive nano DDS for dual release.
into the lysosomes of the HeLa cells. Real time monitoring of
1.2 Single component photoresponsive fluorescent organic
nanoparticles for microenvironment targeted PDT and
combination therapy
drug release by our NDDS-5 was followed through confocal
microscopy. We noted that initially the cells exhibited yellow
fluorescence due to the internalization of NDDS-5 having an
AIE effect (Fig. 6Eai). After 3 min, both yellow and blue Photodynamic therapy (PDT) has emerged as a promising
fluorescence were observed on exposure to light, intimating treatment modality for cancer. PDT is a well-established ther-
partial release of the drugs. The cells showed a complete apeutic causing cellular and tissue damage with spatio-
fluorescence colour change from yellow to blue after 5 min of temporal selectivity and specificity.50,51 In PDT, singlet oxygen
irradiation with visible light (410 nm), suggesting a greater acts as a potential killing agent and is generated from the
quantity of photoreleased drugs (chlorambucil and ferulic acid) ground state triplet molecular oxygen in the presence of a
(Fig. 6Ebii). MTT assay clearly revealed that amplified suitable photosensitizer upon irradiation. Therefore, for
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Nowadays, researchers have utilized the difference in the
microenvironment between healthy and cancer cells to target
and selectively kill cancer cells.55,56 Considering this fact, we
reported a few single component FONPs (Fluo-Mor NPs and
ANT-pen NPs, Fig. 7A) to target the microenvironment of cancer
cells [e.g., low pH and high Cu(II) concentration] and result in
enhanced PDT activity. In this case, the structural modifica-
tions of the photosensitizers enable the development of
targeted specific PDT efficacy by suitably organizing the excited
energy states. We also introduced another strategy of light
induced drug release along with PDT by single component
fluorescent organic NPs for combination therapy (Fig. 7B). In
this section, we will illustrate our efforts to discuss the develop-
ment of such FONPs and their effectiveness in cancer therapy.
1.2.1 Fluorescent organic nanoparticles for microenviron-
ment targeted PDT
Fig. 7 Single component photoresponsive fluorescent organic NPs for
(a) microenvironment targeted PDT and (b) combination therapy (PDT and
chemotherapy).
1.2.1.1 Fluorene–morpholine: single-component photorespon-
sive fluorescent organic nanoparticles for lysosome targeted
effective singlet oxygen production, apart from light and oxygen, pH-triggered photodynamic therapy with fluorescence switch on–
suitable photosensitizers are required. To serve the purpose, off. The significantly increased acidity in the lysosome of cancer
inorganic nanoparticles such as Au nanoparticles, graphene cells (pH 4.5–5.0) provides a promising pH-sensitive site for pH
oxide, and transition metal oxides have been extensively activatable therapy.57,58 Keeping this in mind, in 2016 our
explored as nanocarriers for PDT agents.52–54 However, these group designed fluorene-morpholine NPs (Flu-Mor-NPs)
inorganic nanoparticles are often associated with cytotoxicity, (Fig. 8A), for targeted and real-time monitored cancer
which limits their use in biomedical applications. Hence, our treatment.59 In this system, a functionalized fluorene moiety
group designed single component fluorescent organic nano- was chosen to construct the nanosystem for the following
particle (FONP) based photosensitizers, which can efficiently reasons: (i) strong 2PE absorption to induce NIR-activated
penetrate and accumulate in cancer cells via the EPR effect. PDT, (ii) extended p-conjugation with a push–pull effect, and
With precise modifications in the photosensitizer systems, we (iii) moderate hydrophobicity. Functionalization of fluorene
achieved effective anticancer activity employing target specific with the morpholine unit made it specific to the acidic
PDT activity and combination therapy (PDT + chemotherapy).
organelles, e.g., lysosomes (pH 4.0–6.0), of the cells with
Fig. 8 (A) Fluo-Mor NPs for lysosome targeted PDT. (B) pH-dependent Zeta Potential change in Fluo-Mor organic NPs. (C) Photodegradation study of
DPBF (418 nm) in presence of Fluo-Mor NPs at different pH. (D) Live/dead assay for the HT-29 colon cancer cell line in the presence of Fluo-Mor NPs:
(a) before irradiation with light, and (b) after 5 min of light irradiation: (1) bright-field images, (2) fluorescence image of calcein AM at 520 nm, (3)
fluorescence image of ethidium homodimer 1 at 617 nm, and (4) overlays of the bright-field images and the fluorescence images. Scale bar =100 mm.
(B–D) are reprinted from ref. 59: The Royal Society of Chemistry.
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pH-regulated fluorescence emission for real-time monitoring of acidic environment turned on the fluorescence response and
the PDT action. also enhanced the cancer cell killing efficiency with minimum
These NPs were synthesized by a simple reprecipitation side-effects.
technique with a uniform spherical shape of B43 nm in size.
The zeta potential values of the Fluo-Mor NPs were largely
positive upon lowering the pH, which in turn helped these NPs
to act as an endosomolytic and lysosomolytic pH-responsive
PDT agent (Fig. 8B). Interestingly, at neutral pH, morpholine
was observed to behave like a donor unit indulging in
effective photoinduced electron transfer (PET) to the acceptor
fluorene unit, but, in acidic pH, the PET process was quenched,
resulting in the turn-on green fluorescence of the fluorene
moiety. Accordingly, effective PDT was observed in acidic pH
compared to physiological pH.
1.2.1.2 Anthracene-N-phenylethylenediamine: single compo-
nent photoresponsive fluorescent organic nanoparticles for a
chemosensor of Cu(II) with selective switching on and off photo-
dynamic therapy. Targeting the enhanced copper concentration
in cancer cells has emerged as an imperative technique for
tumour microenvironment targeted treatment of multiple
melanomas.60 It was known from the literature that the average
copper content is 2–3 times higher in cancer-affected patients
compared to their healthy counterparts.61 Hence, a photosen-
sitizer which is otherwise inactive for PDT but could show
efficient singlet oxygen generation upon specific coordination
with Cu(II) will be an excellent choice for intracellular hetero-
geneity responsive targeted PDT.
Keeping this in mind, in 2016, we designed fluorescent
organic NPs based on anthracene-N-phenylethylenediamine
(ANT-pen), which selectively detected Cu(II) ions and concur-
rently exhibited efficient PDT activity only from their Cu(II)-
chelation state (Fig. 9A).62 ANT-pen NPs were also synthesized
by a simple reprecipitation technique, being globular in shape
and B37 nm in size. It is noteworthy that the large red shift
The 1O2 quantum yields of the Fluo-Mor NPs decreased with
increasing pH of the solution. The maximum quantum yield
was found in the region of pH 3.0–4.0 (FD = 0.65 and 0.58,
respectively), whereas the value decreased up to 0.09 at pH 7.4
(Fig. 8C). Thus, the generation of 1O2 by the Fluo-Mor NPs was
almost negligible in non-cancerous cells. The live/dead assay
with calcein AM and ethidium homodimer-1 showed the loss of
cancer cell membrane integrity or membrane rupture, confirm-
ing the light triggered cancer cell death (Fig. 8D). Hence,
selectively switching the activity of Fluo-Mor NPs under an
Fig. 9 (A) ANT-pen organic nanosystem for Cu(II) targeted PDT. (B) Fluorescence spectra of ANT-pen NPs (10 mM) with different amounts of Cu(II)
(0–2.0 equiv. i.e. 0–3.7 mM) in an EtOH-HEPES buffer (60 : 40) solution upon excitation at (a) 450 nm, and (b) 710 nm. (C) Cellular internalization study of
ANT-pen NPs (9 mM) in HeLa (a1–c2) and NIH 3T3 cell lines (a3–c4) using confocal microscopy; fluorescence images of ANT-pen NPs in the (a) control,
(b) green channel (460 nm emission channel) and (c) red channel (620 nm emission channel) after 2 h (lanes 1 and 3) and 4 h (lanes 2 and 4) incubation.
(D) Comparative cell viability study of ANT-pen NPs on HeLa (red bar) and NIH 3T3 cell lines (green bar) (a) before irradiation, and (b) after irradiation for
10 min with visible light (4410 nm) at different concentrations. Values are presented as means ꢂ standard deviations of three different observations.
(B–D) are reprinted with permission from ref. 62. Copyright 2016: Wiley-VCH.
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(green to red) in fluorescence for the ANT-pen NPs upon and chemotherapy using a single nanoplatform has been effec-
complexation with Cu(II) made them useful for real-time monitoring tively applied to achieve enhanced therapeutic efficacy with
of singlet oxygen generation (Fig. 9B). The comparative studies by minimal side effects in cancer treatment.63,64 In particular,
confocal laser scanning microscopy (CLSM) analysis between a combining PDT with chemotherapy is a self-regulating approach
cancerous HeLa cell line having high Cu(II) concentration and a since noninvasive PDT and chemotherapy follow two different
non-cancerous NIH3T3 cell line having low Cu(II) concentration mechanistic pathways in the destruction of cancer cells.
confirmed the selective sensing property of ANT-pen NPs towards
Cu(II) (Fig. 9C).
1.2.2.1 Squaric acid–coumarin–chlorambucil: single component
photoresponsive fluorescent organic nanoparticles for self-monitored
combination therapy. In 2018, we introduced squaric acid–
coumarin–chlorambucil (Sq–cou–cbl) conjugates as single com-
ponent photoresponsive NPs (Fig. 10A) for combination therapy.65
Here, a coumarin based phototrigger was chosen for caging the
anticancer drug chlorambucil (Cbl), whereas the squarin unit
was chosen as a sensitizer for PDT activity. The Sq–Cou–Cbl NPs
(photosensitizer–phototrigger nanoconjugate) and Sq–Cou–OH
NPs (photoproduct after drug release) were found to be globular
in shape with B60 nm and B140 nm sizes, respectively.
It is noteworthy that the Sq–Cou–Cbl NPs released chlorambucil
in a spatio-temporal manner and generated singlet oxygen
upon visible light irradiation. HPLC study showed that nearly
85% anticancer drug was released upon 60 min of irradiation
(l Z 410 nm) (Fig. 10B). During photolysis, the blue fluorescence
intensity of the Sq–Cou–Cbl NP solution increased by 5 fold owing
to the conversion to Sq–Cou–OH NPs. The quantum yield for
singlet oxygen generation by Sq–Cou–Cbl NPs and Sq–Cou–OH
NPs was monitored by DPBF degradation study and found to be
Also, the comparative in vitro biological studies in cancerous
HeLa cells and non-cancerous NIH3T3 cells manifested selec-
tive cytotoxicity towards HeLa cells (Fig. 9D), owing to
the formation of ANT-pen-Cu(II) complexes. Our designed
ANT-pen NPs were enabled to visualize the presence of excess
Cu(II) in cancer cells and exhibited profound singlet oxygen
generation with a quantum yield of 0.70 only upon chelation
with Cu2+. Therefore, the ANT-pen NPs showed tumor selectivity
by a distinct fluorescence colour change (green to red) method
and acted as a potential PDT agent in the presence of Cu(II) in
cancer cells. In the above mentioned two studies, we discussed
our developed single component FONPs for targeted PDT activity
based on the tumour microenvironment i.e. lower pH concen-
tration and excess Cu(II) availability in cancer cells. In the next
section, we will discuss single component FONPs for combi-
nation therapy (PDT and chemotherapy).
1.2.2 Single component photoresponsive fluorescent
organic nanoparticles for combination therapy (PDT and
chemotherapy). In the last few years, the combination of PDT
Fig. 10 (A) Combination therapy (anticancer drug + PDT) of Sq–Cou–Cbl upon irradiation. (B) HPLC study for light triggered drug release by Sq–Cou–
Cbl nanoparticles. (C) Singlet oxygen generation of Sq–Cou–Cbl and Sq–Cou–OH nanoparticles by DPBF degradation study. (D) Confocal images of
internalization of Sq–Cou–Cbl nanoparticles in HeLa cells: (a) DIC (differential interference contrast), (b) before photolysis (0 min), and (c) after photolysis
(60 min). Scale bar = 20 mm. (E) Cell viability assay of Sq–Cou–Cbl and Sq–Cou–OH nanoparticles and chlorambucil in the HeLa cell line: (a) before
photolysis, and (b) after photolysis. Values are presented as mean ꢂ SD. (B–E) are reprinted with permission from ref. 65. Copyright 2018: American
Chemical Society.
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0.51 and 0.46, respectively (Fig. 10C). The above study indicates that of the AIE phenomenon (Fig. 11Ba). Most importantly, the
the photoproduct after the drug release, i.e. Sq–Cou–OH NPs, was TPE–pHP–Cbl NPs showed a fluorescence colour change (yellow
also able to generate singlet oxygen. Further, abilities like the to green) on photolysis, thus helping in real-time visualization
in vitro cellular uptake efficiency (through a lysosomal pathway), of drug release by fluorescence imaging. The blue shift of the
singlet oxygen generation, and self-reporting of drug delivery of the emission spectra was due to the disruption of the conjugation
Sq–Cou–Cbl NPs were demonstrated by confocal cellular imaging from the –OH moiety to the keto group of TPE–pHP–Cbl NPs.
studies (Fig. 10D). The cytotoxicity test revealed that the increased
The HPLC study confirmed that B90% drug was released by
anticancer activity of Sq–Cou–Cbl NPs compared to Sq–Cou–OH TPE–pHP–Cbl NPs within 5 min of irradiation under visible
NPs was due to the synergistic effect of the released anticancer drug light (l Z 410 nm) (Fig. 11C). The singlet oxygen generation
chlorambucil and PDT (Fig. 10E). Interestingly, PDT from the quantum yield was found to be 0.28 and 0.24 for TPE–pHP–Cbl
photoproduct Sq–Cou–OH NPs was an additional benefit for NPs and TPE–pHP–OH NPs (photoproduct), respectively
improved therapeutic efficacy. Overall, the single-component (Fig. 11D). The in vitro cell imaging study confirmed the real-
visible light responsive Sq–Cou–Cbl NPs were found to be highly time monitoring drug release ability of TPE–pHP–Cbl NPs. As
effective for reducing the cancer cell viability due to the synergistic expected, before irradiation, the confocal image exhibited a
chemo-photodynamic therapeutic effect.
yellow fluorescence colour of TPE–pHP–Cbl NPs due to the AIE
effect, while after irradiation a green fluorescence colour was
observed due to the disruption in conjugation (Fig. 11E). The
significant features of the TPE–pHP–Cbl NPs are the real-time
monitoring ability during drug release, and enhanced PDT
activity due to singlet oxygen generation by the photoproduct.
1.2.2.2 Tetraphenylethylene-p-hydroxyphenacyl–chlorambucil:
single component fluorescent organic nanoparticles for chemo-
photodynamic therapy with real time cellular imaging. We have
discussed the advantages of incorporating AIE
+ ESIPT
phenomena in the construction of drug delivery systems. In
continuation, in the year 2018, we developed an AIE based
1.2.2.3 Four armed tetraphenylethylene: single component
nanosystem (TPE–pHP–Cbl NPs) by combining tetraphenylethy- fluorescent organic nanoparticles for chemo-photodynamic therapy
lene and a p-hydroxyphenacyl phototrigger for the combination and cellular imaging. In the same way, in 2019, we developed
of PDT and chemotherapy (Fig. 11A).66
visible light-responsive FONPs, tetraphenylethelene–chlorambucil
The TPE–pHP–Cbl NPs were synthesized by the reprecipita- [TPE(Cbl)4], using the TPE chromophore for an improved
tion technique from the TPE–pHP–Cbl conjugate. HR-TEM combination of chemo-photodynamic therapy (Fig. 12A).67
revealed that the nanoparticles were spherical and B38 nm The most interesting feature of this work was the release of
in size. The yellow fluorescence of TPE–pHP–Cbl NPs was found 4 equivalents of active drugs from a single nanosystem along
to be increased with an increase in the water fraction as a result with PDT activity, which potentially increased the therapeutic
Fig. 11 (A) AIE–ESIPT based fluorescent TPE–pHP–Cbl NPs for combination therapy. (B) (a) The yellow fluorescence intensity of TPE–pHP–Cbl NPs
increases with an increase in the water fraction and (b) the fluorescence color change (yellow to green) of TPE–pHP–Cbl NPs upon photoirradiation. (C)
Photorelease of the drug (chlorambucil) by TPE–pHP–Cbl NPs upon irradiation. (D) Singlet oxygen generation of TPE–pHP–Cbl NPs and TPE–pHP–OH
NPs by DPBF degradation study. (E) Confocal microscopy images captured at different intervals of time during irradiation of TPE–pHP–Cbl NPs in HeLa
cells: (a) bright-field image, (b) 0 min, (c) 3 min and (d) 5 min. Scale bar = 20 mm. (A–E) are reprinted with permission from ref. 66. Copyright 2018:
American Chemical Society.
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Fig. 12 (A) ‘‘AIE’’-based TPE(Cbl)4 nanoparticles for combination (chemo-photodynamic) therapy. (B) Increase in the green fluorescence intensity of
TPE(Cbl)4 with an increase in the water fraction. (C) Singlet oxygen generation of TPE(Cbl)4 NPs and TPE(OH)4 NPs by DPBF degradation study. (D) Cell
viability assay of (Cbl)4, TPE(Cbl)4 NPs, and TPE(OH)4 NPs in the cancer cell line after photoirradiation. (E) Cell viability assay study of TPE(Cbl)4 NPs at
different time intervals of photoirradiation. (A–E) are reprinted with permission from ref. 67. Copyright 2019: American Chemical Society.
efficacy by lowering the drug dose. The TPE(Cbl)4 NPs were and hydrogen sulfide (H2S) are a family of molecules known as
spherical in shape with a B42 nm size, synthesized by the same gasotransmitters.69 Cell signalling by gasotransmitters is
reprecipitation technique from the TPE(Cbl)4 conjugate. The mostly complex and is dependent on their local concentration.
TPE(Cbl)4 NPs displayed strong green fluorescence with an This indicates the need for controlled generation of gasotrans-
increase in the water fraction because of the AIE effect mitters under physiological conditions. However, site specific
(Fig. 12B). Upon exposure to visible light (l Z 410 nm), our controlled administration of gasotransmitters to their target
TPE(Cbl)4 NPs simultaneously produced singlet oxygen and sites in the physiological environment is challenging because of
uncaged 4 equivalents of the anticancer drug sequentially in their high diffusion rate and low solubility in blood. This
their aggregated state (Fig. 12C). On the other hand, the formed problem was eliminated using light-responsive gasotransmitter
photoproduct also exhibited PDT activity upon light irradiation, donors.70,71
which was an added advantage for effective anticancer activity.
Hence, our group developed single component fluorescent
In vitro studies exhibited properties like self-tracking, cellular organic nanoparticles for regulated release of gasotransmitters
imaging, photoregulated synergistic chemo-photodynamic ther- (H2S and NO) with enhanced transportation and ‘‘on-demand’’
apy, and biocompatibility of TPE(Cbl)4 NPs (Fig. 12D and E).
Overall, the above discussed fluorescent organic TPE(Cbl)4
NPs exhibited unique properties like appreciable biocom-
patibility and simultaneous singlet oxygen generation with four
equivalents of anticancer drug release, making them a potential
combination therapeutic agent for cancer treatment.
‘‘tunable’’ release (Fig. 13).
2.1 Tetraphenylethylene conjugated p-hydroxyphenacyl:
single component fluorescent organic nanoparticles for the
release of hydrogen sulfide with real-time cellular imaging
H2S has a wide range of physiological functions such as anti
inflammatory, antioxidative, vasodialation and cytoprotective
effects.72,73 H2S has tunable activity depending on the concen-
tration: at low concentrations it has cytoprotective ability
against cellular damage, while at higher concentrations it
2 Single component photoresponsive
fluorescent organic nanoparticles as a
gasotransmitter donor
Of late, biological functions of gasotransmitters have captured
great attention in the scientific community. Although they are
not recognized as classical signaling molecules, still they
regulate a number of important cellular functions through
complex chemical interactions with each other (including
metabolites) and targeting proteins. Gasotransmitters control
various physiological functions, including regulation of the
cardiovascular, nervous, gastrointestinal, excretory, and
immune systems, and many other cellular functions, including
apoptosis, proliferation, inflammation, cell metabolism, gene
transcription, etc.68 Nitric oxide (NO), carbon monoxide (CO),
Fig. 13 Single component light responsive fluorescent organic nano-
particles for regulated release of (a) H2S and (b) NO.
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exhibits cytotoxicity. Hence, the monitoring of H2S release is demonstrated the controlled and real time release of H2S under
necessary for treatment.
visible light without the help of any external agent.
In 2018, we developed visible light activatable single com-
ponent FONPs by integrating a tetraphenylethylene (TPE)
moiety and p-hydroxyphenacyl (pHP) phototrigger for the
release of hydrogen sulfide (H2S) with real time monitoring
2.2 Perylenetetracarboxylate ester: single component NIR
fluorescent organic nanoparticles for photoinduced nitric oxide
delivery with self monitoring and real time reporting abilities
ability (Fig. 14A).74 Our TPE–pHP–H2S NPs provided advantages The therapeutic efficacy of nitric oxide (NO) is found to be a
like (i) aggregation-induced emission enhancement, (ii) a large dual-edged sword like H2S: in high concentrations, NO is
Stokes shift, (iii) an excitation wavelength extended to cytotoxic and is used to treat cancer, while in low concentra-
4410 nm, (iv) unlocking of photorelease of H2S in the aggre- tions it promotes tumor growth. Therefore, on-demand spatio-
gated state, (v) real-time monitoring of H2S release by a simple temporal NO release has captured great attention.75 In this
fluorescence colour change, and (vi) no requirement of an regard, several photoresponsive NO-donating nanoparticulate
additional reagent for the release of H2S.
systems based on metallic, up-conversion NPs etc. have been
The TPE–pHP–H2S NPs were synthesized by the reprecipita- developed. Since the activity of NO is concentration-dependent,
tion technique from the TPE–pHP–H2S conjugate. HR-TEM it is absolutely necessary to estimate the amount of NO release.
revealed that the nanoparticles were spherical in shape with a To solve this problem, photoresponsive NO donor NPs with the
B35 nm size. Irradiation of TPE–pHP–H2S NPs under visible ability to release along with fluorescence reporting were devel-
light (l Z 410 nm) released H2S only in the aggregated state via oped. In this context, our group developed nitric oxide photo-
ESIPT assisted photo-Favorskii rearrangement. Methylene donor (NOD) conjugated perylenetetracarboxylate ester (TPT)
blue assay showed that 100 mM of TPE–pHP–H2S NPs released based fluorescent organic nanoparticles, TPT(NOD)4, with
B40 mM of H2S after 20 min of irradiation. Our TPE–pHP–H2S aggregation induced NIR emission for photoinduced nitric
NPs aided in real-time monitoring of photorelease of H2S by a oxide delivery along with a red to green emission transition
fluorescence colour change (yellow to blue) (Fig. 14B).
(Fig. 15A).76 Fluorescent TPT-(NOD)4 NPs were prepared via a
The cellular uptake and real-time monitoring of H2S release reprecipitation technique, and their sizes were determined
from the TPE–pHP–H2S NPs was established in vitro using from TEM and DLS studies. From TEM images it was observed
cervical cancer cells (Fig. 14C). MTT assay clearly suggested that the TPT-(NOD)4 NPs were globular in shape, with an
that there was no inhibition of proliferation of HeLa cells by the average diameter of B55 nm (Fig. 15B), whereas from a DLS
TPE–pHP–H2S NPs before and after photolysis, which clearly study the average size of the TPT-(NOD)4 NPs was found to be
shows that the H2S donor TPE–pHP–H2S NPs are not cytotoxic B60 nm. These NPs have a zeta potential of +58 mV, representing
at the studied concentration. Therefore, the TPE–pHP–H2S NPs the surface charge of the NPs. Further, the polydispersity of the
Fig. 14 (A) AIE and ESIPT assisted H2S release from TPE–pHP–H2S NPs. (B) Fluorescent spectral change of the TPE–pHP–H2S NP donor during
photolysis at regular intervals of time (0–20 min). (C) Confocal microscopy images of internalization of the TPE–pHP–H2S NP donor in HeLa cells:
(i) bright-field image, (ii) 0 min, (iii) 10 min and (iv) 20 min. Scale bar = 50 mm. (B and C) are reprinted from ref. 74: The Royal Society of Chemistry.
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Fig. 15 (A) The working principle of the NO photodonor [TPT (NOD)4 NPs] with self monitoring internalization and real time monitoring of NO release.
(B) TEM images of TPT-(NOD)4 NPs, (i) before photolysis, and (ii) after photolysis. (C) Confocal images of U87MG cells, incubated with DAPI for nuclear
staining and 6 mM TPT-(NOD)4 NPs, obtained during exposure to visible light irradiation: (i) DAPI nucleus staining; (ii) 0 min – excimer emission (red
fluorescence); (iii) 30 min – monomer emission; and (iv) a merged image. (D) Dose-dependent photoinduced anticancer activity of TPT-(NOD)4 NPs,
obtained (i) before irradiation and (ii) after irradiation. The values are presented as means ꢂ standard deviations from three different observations. (B–D)
are reprinted from ref. 76: The Royal Society of Chemistry.
TPT-(NOD)4 NPs was measured to be 0.144, indicating the uni- contamination of medical devices, food spoilage, equipment
formity and monomodal dispersion of the NPs in aqueous solution. contamination, multi-drug resistance etc.77 The use of excessive
Importantly, the emission spectrum of TPT-(NOD)4 showed mono- antibiotic agents causes the problem of drug resistance and
mer emission, whereas the TPT-(NOD)4 nanoparticles exhibited leads to several side effects due to overdoses.78 In this context,
red-shifted 627 nm emission, which was attributed to aggregation stimuli-responsive antibiotic-loaded nanoparticles with
induced excimer emission. Interestingly, our spherical TPT-(NOD)4 improved therapeutic efficacy were developed.79 Within the
NPs exhibited red emission, and upon photolysis using visible light reported system, the light-responsive bicomponent nano DDS
they released NO with a concomitant shift in fluorescence from red seems to be much more effective as it provides a higher degree
to green. The excimer (NIR) emission of TPT-(NOD)4 NPs was of spatio-temporal control during the drug release. However,
utilised in monitoring the cellular internalization of these NPs in the main concerns about these systems are the (i) biocompat-
U87MG cells. Further, the NO photorelease associated dual colour ibility, (ii) real-time tracking ability and (iii) drug loading
fluorescence transition was exploited for the real time reporting of efficiency, which depends on the nanoparticle surface. All these
NO release inside the cells (Fig. 15C). MTT assay showed high shortcomings can be addressed if we use single component
cytotoxicity in HeLa cells for our TPT-(NOD)4 NPs, which is due to fluorescent organic nanoparticle systems, because of their
the controlled release of 4 equivalents of NO (Fig. 15D). Thus, our inherent advantageous properties as discussed previously.
TPT-(NOD)4 NPs were the first reported organic nanoparticulate
3.1 1-Acetylpyrene–salicylic acid: single component
photoresponsive fluorescent organic nanoparticles for the
regulated release of a natural antimicrobial compound,
system that can self-monitor in vitro cellular internalization and
report photoinduced nitric oxide delivery in real time.
salicylic acid
3 Single component photoresponsive
In 2014, we developed 1-acetylpyrene (AcPy) as single com-
organic nanoparticles as a drug
ponent photoresponsive FONPs for the regulated in vitro release
delivery system for antibacterial
treatment
of a natural antimicrobial agent salicylic acid (SA) under visible
light irradiation (l Z 410 nm) (Fig. 16A).80 AcPy–SA NPs were
prepared from the AcPy–SA conjugate by a simple reprecipita-
Bacterial infection has become a serious threat to public health tion technique. TEM analysis revealed that the photoresponsive
globally owing to the numerous problems created by it, such as AcPy–SA NPs were B68 nm in size. Photophysical studies
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showed that the AcPy–SA NPs exhibited good fluorescence and NPs caused a progressive increase in surface roughness (from
absorbance above 400 nm. The zeta potential of the AcPy–SA 66.86 nm to 228.97 nm). AFM analysis also showed that AcPy–
NPs was about ꢀ48.8 mV (Fig. 16B). The high negative zeta SA NPs which were bound to a bacterial cell membrane
potential value indicated that the AcPy–SA NPs were sufficiently appeared with a different surface topography (Fig. 16F) after
stable in aqueous dispersions and can be used for in vitro irradiation with visible light. Hence, the development of such
antimicrobial drug delivery. AcPy–SA NPs dispersed in aqueous photoresponsive fluorescent organic NPs will be highly bene-
solution released 90% of salicylic acid in 1 h on irradiation with ficial for targeted and regulated antimicrobial drug release
visible light (Z410 nm) (Fig. 16C).
because of their biocompatible nature, efficient cellular uptake,
Both the broth dilution method and live/dead Baclight and light-induced drug release ability.
viability stain studies showed that the AcPy–SA NPs kill bacter-
ial cells upon exposure to visible-light irradiation (Z410 nm)
4 Single component photoresponsive
(Fig. 16D). Finally, a change in the topography of the bacterial
cell membrane by AcPy–SA NPs upon visible-light irradiation
was studied using FESEM and AFM. From the FESEM images
(Fig. 16E) it was found that nanoparticles adhere to the surface
of the bacterial cells (Fig. 16Ef) and lead to cell-lysis of the
fluorescent organic nanoparticles as a
drug delivery system for agrochemical
release
bacterial cells when exposed to visible light (Fig. 16Ed and e). Using excess pesticides on plants leads to many undesirable
The high resolution AFM revealed that the binding of AcPy–SA side effects, including phototoxicity and environmental
Fig. 16 (A) Working principle of AcPy–SA NPs inside bacterial cells. (B) TEM and DLS studies of AcPy–SA NPs. (C) HPLC overlay chromatogram of AcPy–
SA nanoparticles at different intervals of light irradiation (Z410 nm). (D) Live/dead Baclight bacterial viability assay on Pseudomonas aeruginosa-ATCC
27853 ((a): control; and (b and c): treated with AcPy–SA nanoparticles (0.5 ꢁ 10ꢀ6 M) after 10 min and 20 min visible light irradiation, respectively).
Imaging was done using a fluorescence microscope (IX 51, Olympus) with a high-performance CCD camera and Image-Pro discovery 5.1 software. Scale
bar represent 10 mm. (E) FE-SEM micrograph of Pseudomonas aeruginosa-ATCC 27853 ((a–c): control; and (d–f): treated with visible light in the presence
of AcPy–SA nanoparticles). (F) Atomic force microscopy view of Pseudomonas aeruginosa-ATCC 27853. Images were captured using a scanning probe
microscope, Multiview-1000TM, at a scan rate of 0.8–1.0 Hz, and were first-order flattened. The bacteria appear to be hydrated and the outer surface
appears (a) smooth for bacteria grown without NPs and (b) a rough surface treated with visible light in the presence of AcPy–SA NPs. (B–F) are reprinted
with permission from ref. 80. Copyright 2014: American Chemical Society.
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pollution.81 Here, the concept of controlled release formula- precise control over the release of pesticides, including location,
tions (CRFs) of pesticides has gained a lot of interest as they timing, and doses.
allow the use of minimum amounts of pesticides for the same
4.1 Perylene-3-yl methanol: single component
photoresponsive fluorescent organic photoresponsive
nanoparticles for controlled release of 2,4-D and real time
monitoring of morphological changes induced by 2,4-D in
period of time, thereby reducing the risk of environmental
pollution. Within the various strategies of CRFs, nano-
pesticide formulations have recently gained considerable
importance in agriculture as they offer unique advantages such
plant systems
as (i) increasing the solubility of poorly soluble pesticides, (ii)
slow/targeted release of pesticides and (iii) protecting pesticides
against premature release and degradation.82 The main goal in
developing nano-pesticide formulations is to develop intelligent
nanocarriers that contain properties such as fluorescence, tar-
geting ability, and precise control of pesticide release. To date,
several nanocarriers have been developed for the delivery of
pesticides based on polymeric nanoparticles, solid lipid nano-
particles, mesoporous silica nanoparticles, metals, and metal
oxide nanoparticles. Although the reported nanopesticide for-
mulations exhibited low toxicity and good biocompatibility, their
use is mainly limited as they are incapable of monitoring the
amount of pesticides released and tracking the morphological
changes occurring in the plants after pesticide release.83 In this
context, we intend to develop light responsive nanopesticide
formulations based on fluorescent organic nanoparticles that
can provide the following advantages: the ability to (i) visualize
and track the transportation and deposition of nanopesticides
within the plant system, (ii) help to monitor the morphological
changes induced by nanopesticides for the plant, and (iii) have
For the first time, our group developed a nano-pesticide for-
mulation of 2,4-D using perylene-3-yl methanol as a nanocarrier
for performing dual abilities: (i) tracking of morphological
changes induced by the pesticide 2,4-D and (ii) controlled
release of 2,4-D (Fig. 17A).84 Pe-2,4-D nano-pesticides and
perylen-3-ylmethanol nanoparticles were prepared using a
similar reprecipitation technique. TEM images showed that the
Pe-2,4-D nano-pesticides and perylen-3-ylmethanol nano-
particles were globular in shape with an average particle size
of B25 nm and B10 nm, respectively (Fig. 17B). The broad
absorption of the Pe-2,4-D nano-pesticides from 350 nm to
550 nm and strong emission at 625 nm indicated that our
nano-pesticides can be used for both cell imaging (Fig. 17C)
and the regulated release of 2,4-D under visible light irradiation.
The distinct fluorescence colour change on photolysis of the
Pe-2,4-D nano-pesticides was exploited for real time monitoring
of 2,4-D release inside the plant (Fig. 17D). A ten day growth
experiment using different concentrations (10ꢀ4 to 10ꢀ6 M) of
Pe-2,4-D nano-pesticides manifested the growth inhibition effect
Fig. 17 (A) Working principle of Pe-2,4-D nano-pesticides in plant cells. (B) TEM analysis of Pe-2,4-D nano-pesticides (a) before and (b) after photolysis.
(C) Confocal fluorescence and brightfield images of the plant root cross section under (i) control conditions and (ii) treatment with 2 ꢁ 10ꢀ6 M Pe-2,4-D
nano-pesticide in the dark for 6 h [(a) brightfield, (b) fluorescence and (c) overlay]. (D) Confocal microscope images showing real time nano-pesticide
release in plants under (i) 15 min irradiation (Z410 nm) after treatment with 2 ꢁ 10ꢀ6 M Pe-2,4-D nano-pesticide in the dark for 6 h and (ii) control
conditions [(a) brightfield, (b) fluorescence (625 nm emission channels), (c) fluorescence (445 nm emission channels) and (d) overlay]. (E) Effect of 2,4-D,
Pe-2,4-D nano-pesticides and perylene-3-ylmethanol nano-particles (10ꢀ4 to 10ꢀ6 M) on the root and shoot length of C. Arietinum at regular time
intervals. (F) Effect of 6 ꢁ 10ꢀ6 M Pe-2,4-D nano-pesticides after regular time intervals of irradiation under visible light (in each set the left side represents
the control and the right side the treatment). (G) Anatomical changes induced in the root tissues of C. Arietinum by photoreleased 2,4-D (10ꢀ6 to 10ꢀ4 M).
Here Pc, Ph and Xy represent the pericycle, phloem and xylem, respectively. (B–G) are reprinted from ref. 84: The Royal Society of Chemistry.
1730 | Chem. Commun., 2021, 57, 1715ꢀ1733
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Table 1 Photophysical and photochemical properties of phototriggers used as FONPs
Quantum yield
Real-time
Phototrigger
Irradiation wavelength (125 W) fF
FP
fD
monitoringd
Ref.
a
b
c
Perylene-3-ylmethyl
Acridin-9-methyl
Benzothiazole conjugated p-hydroxyphenacyl
Salicylaldazine conjugated p-hydroxyphenacyl
Fluorene–morpholine
Anthracene-N-phenylethylenediamine
Squaric acid–coumarinyl
Tetraphenylethylene-p-hydroxyphenacyl
Four armed tetraphenylethylene p-
hydroxyphenacyl
Z410 nm
Z365 nm
Z410 nm
Z410 nm
Z365 and 740 nm
Z410 nm
Z410 nm
Z410 nm
Z410 nm
0.9 0.09
0.03 0.02
—
—
—
—
O
NO
O
36 and 84
37
38
39
59
62
65
66
67
—
—
—
—
0.46
0.48
—
O
0.65 (Z365 nm) O
0.70
—
O
NO
O
0.039 0.083 0.51
—
—
0.51 0.28
0.52 0.31
NO
1-Acetylpyrene
Z410 nm
0.04 0.39
—
NO
80
a
b
Fluorescence quantum yield using quinine sulphate/9,10 diphenyl anthracene as a standard. Photochemical quantum yield for the release of
c
bioactive molecules using potassium ferrioxalate as an actinometer. Singlet oxygen generation quantum yield using rose bengal as a standard
(error limit within ꢂ5). Fluorescence color change during photorelease; O = observed, NO = not observed.
d
of released 2,4-D on the root and shoot systems of brown pea inside cells. In vitro studies clearly suggest that using prodrugs in
(C. arietinum) plants (Fig. 17E). Further, our Pe-2,4-D nano- the form of light responsive FONPs provided important advan-
pesticides helped us to track the anatomical changes in root tages like (i) excellent biocompatibility, (ii) efficient cellular
tissues induced by released 2,4-D. The development of the roots internalization, (iii) real time monitoring of drug release inside
was almost completely inhibited after 6 days of treatment with the cells and (iv) enhanced cytotoxicity due to regulated release
the nano-pesticides although the roots of the control plants of bioactive molecule inside the cells.
appeared to be healthy and well grown (Fig. 17F). Additionally,
we also observed a change in the phloem structure and a cell
death like phenomenon in the root tip regions of brown pea
Future work
seeds (Fig. 17G). Thus, our newly developed nano-pesticide
formulation provided the following advantages compared to the
simple 2,4-D, like (i) it was easily absorbed by plant cells because
of its nano size, (ii) it was formulated in water so that it can
overcome the limitation of low water solubility of 2,4-D and (iii) it
exhibited a distinct fluorescence colour change of Pe-2,4-D nano-
pesticides upon irradiation that was utilized for real time mon-
itoring of 2,4-D release.
Finally, we have summarized in a table the different proper-
ties of phototriggers used as FONPs including the wavelength
of light used for excitation, fluorescence quantum yield, photo-
chemical quantum yield for deprotection, singlet oxygen genera-
tion, and real time monitoring ability (Table 1).
Despite the emerging progress on light responsive FONPs,
there are still many challenges in order to fulfil their potential
applications. For instance, these nanoparticles should meet the
requirement of the safety, effectiveness, and quality control
standards for clinical application before they can be translated
as DDSs. In future, we are interested to design light responsive
FONPs for regulated release of neurotransmitters, as
FONPs can be easily prepared in the nanorange so that they
can effectively release the neurotransmitters in brain cells by
removing the blood–brain-barrier. Secondly, we are also interested
in developing light responsive FONPs which can release two
different neurotransmitters in a sequential or stepwise manner so
that we can understand the cross-talk between these neurotrans-
mitters. Third, we also want to construct NIR responsive FONPs for
drug delivery applications. Finally, we want to develop agrochemical
formulations based on FONPs for the controlled release of sex
pheromones and plant growth regulators. Hence, we believe that
the above said interesting features of light responsive FONPs will
attract several research groups to explore their applications in the
fields of biology, medicine, materials chemistry and agriculture in
the coming years.
5. Conclusions
In summary, we have shown that light responsive single com-
ponent fluorescent organic nanoparticles can act as efficient
drug delivery systems (DDSs). Light responsive FONPs can be
easily prepared from their corresponding conjugates using a
simple reprecipitation technique. The light responsive FONPs
prepared using the reprecipitation technique were mostly in the
range of 20–60 nm. The absorption and emission behaviour of
these FONPs was strikingly different from their corresponding
conjugates. Light responsive FONPs provide the advantage of
releasing the biological molecules in physiological medium,
curtailing the limitation of solubility of photocages in aqueous
medium. The most remarkable property of these light responsive
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
FONPs is their change in fluorescence colour during photolysis, We thank the DST SERB (Grant No. EMR/2016/005885 & DIA/
which was utilised for the real time monitoring of drug release 2018/000019), and CSIR (Sanction Letter No. 02(367)/19/EMR-II)
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for funding. AC is thankful to UGC, New Delhi, and AP and AS
are thankful to IIT Kharagpur for financial support.
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