Perylene-3-ylmethanol: Fluorescent organic nanoparticles as a single-component photoresponsive nanocarrier with real-time monitoring of anticancer drug release

Article abstract of DOI:10.1021/ja302482k

We report for the first time the use of perylene-3-ylmethanol fluorescent organic nanoparticles as a drug delivery system. In the present system, perylene-3-ylmethanol nanoparticles performed four important roles: (i) "nanocarriers" for drug delivery; (ii) "phototriggers" for the drug release; (iii) fluorescent chromophores for cell imaging; and (iv) detectors for real time-monitoring of drug release. In vitro biological studies revealed that the newly developed perylene-3-ylmethanol nanoparticles exhibit good biocompatibility and cellular uptake as well as efficient photoregulated anticancer drug release ability. Such fluorescent organic nanoparticles may open up new perspectives for designing a new class of promising photoresponsive nanocarriers for drug delivery.

Full text of DOI:10.1021/ja302482k

Communication
pubs.acs.org/JACS
Perylene-3-ylmethanol: Fluorescent Organic Nanoparticles as a
Single-Component Photoresponsive Nanocarrier with Real-Time
Monitoring of Anticancer Drug Release
Avijit Jana,† K. Sanjana P. Devi,‡ Tapas K. Maiti,‡ and N. D. Pradeep Singh*,†
†Department of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur 721302, India
‡Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur 721302, India
S
* Supporting Information
synthesis of organic nanoparticles have sparked interest in them
in fields ranging from optoelectronics14,15 and sensors to
ABSTRACT: We report for the first time the use of
medicine.16−21 In 2006, Latterini et al.22 synthesized different
perylene-3-ylmethanol fluorescent organic nanoparticles as
a drug delivery system. In the present system, perylene-3-
ylmethanol nanoparticles performed four important roles:
(i) “nanocarriers” for drug delivery; (ii) “phototriggers” for
the drug release; (iii) fluorescent chromophores for cell
imaging; and (iv) detectors for real time-monitoring of
drug release. In vitro biological studies revealed that the
newly developed perylene-3-ylmethanol nanoparticles
exhibit good biocompatibility and cellular uptake as well
as efficient photoregulated anticancer drug release ability.
Such fluorescent organic nanoparticles may open up new
perspectives for designing a new class of promising
photoresponsive nanocarriers for drug delivery.
sizes of perylene nanoparticles by the reprecipitation method
and showed that their spectral properties are markedly different
from those of perylene molecules. Latter, Baba et al.23 applied
perylene nanocrystals for in vitro fluorescence confocal imaging
of living cells. Our group recently demonstrated perylene-3-
ylmethyl24 as an efficient fluorescent phototrigger for carboxylic
acids and alcohols in aqueous media under visible-light
irradiation. On the basis of the above facts, we report for the
first time the use of fluorescent organic nanoparticles of
perylene-3-ylmethanol as a single-component photoresponsive
nanocarrier for in vitro release of the anticancer drug
chlorambucil (Scheme 1).
Scheme 1. Schematic Representation of Photoinduced in
Vitro Anticancer Drug Release
Photoresponsive nanoparticles have recently gained consid-
erable attention in drug delivery research because of their ability
to control the release of bioactive molecules spatially and
temporally by external regulated light stimuli.1 Generally,
photoresponsive nanoparticles for use as a drug delivery system
(DDS) are composed of two main ingredients: a biocompatible
nanocarrier and a “phototrigger”. The phototrigger is a small
organic molecule that performs two important functions: (i)
providing precise control over the drug release and (ii) serving
as a linker between the nanocarrier and the drug.
With the use of both of these ingredients, several
photoresponsive nanoparticles have been developed for use as
DDSs.2−8 The crucial steps involved in the preparation of
photoresponsive nanoparticles are (i) the synthesis of
biocompatible nanoparticles, (ii) surface functionalization of
the nanoparticles, and (iii) attachment of the phototrigger to
the nanoparticles. By any means, if we can synthesize
nanoparticle-sized phototriggers, then the phototrigger by itself
can act as a nanocarrier for the drug delivery, thereby
eliminating the above crucial steps. Hence, we decided to
synthesize organic nanoparticles that can act as both the drug
nanocarrier and the phototrigger.
Unlike inorganic nanoparticles, the progress on organic
nanoparticles has been slow, even though they allow wider
variability and flexibility in materials synthesis and nanoparticle
preparation.9,10 This is mainly due to the difficulties associated
with their synthesis and isolation.11,12 However, the introduc-
tion of the effective “reprecipitation technique”13 for the
First, we synthesized a photocaged perylene−chlorambucil
(Pe−Cbl) conjugate using the procedure reported by us.24
Next, photocaged Pe−Cbl nanoconjugates for use as a DDS
were prepared by a reprecipitation technique involving slow
Received: March 14, 2012
Published: April 20, 2012
© 2012 American Chemical Society
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Journal of the American Chemical Society
Communication
addition of 10 μL of a 3 mM dimethyl sulfoxide solution of Pe−
Cbl caged conjugate into water (20 mL) at room temperature
with controlled stirring.23 The shape and size of the resulting
Pe−Cbl nanoconjugates were determined by transmission
electron microscopy (TEM), which showed the particles of
Pe−Cbl nanoconjugate to be globular in shape with an average
particle size of 30 nm (Figure 1a). Interestingly, the TEM
Figure 2. HPLC profile of Pe−Cbl nanoconjugates at regular time
intervals of photolysis under visible-light irradiation (≥410 nm). The
vertical axes were offset by 15 mAU units and the horizontal axes by 15
s to facilitate better visualization.
chlorambucil after periods of exposure to light and dark
conditions, as shown in the Figure 3 inset. The figure clearly
shows that the drug release proceeded only under illumination.
Figure 1. (a−c) TEM images of (a) Pe−Cbl nanoconjugates before
photolysis, (b) Pe−Cbl nanoconjugates after photolysis, and (c)
perylene-3-ylmethanol nanoparticles. (d, e) Normalized absorption
and emission spectra of (d) Pe−Cbl nanoconjugates and (e) perylene-
3-ylmethanol nanoparticles.
images of perylene-3-ylmethanol phototrigger nanoparticles
synthesized using a similar reprecipitation technique showed
the particles to have a reduced size of 20 nm. The absorption
and emission spectra of Pe−Cbl nanoconjugates were found to
be quite different from those of perylene-3-ylmethanol
nanoparticles (Figure 1d,e). A distinct red shift can be seen
in the emission spectra of Pe−Cbl nanoconjugates. The broad
absorbance of the Pe−Cbl nanoconjugates from 350 to 550 nm
and strong emission at 625 nm indicate that our nano-
conjugates can be used for both cell imaging and the release of
the anticancer drug under visible-light irradiation.
To check the stability of the photocaged Pe−Cbl nano-
conjugates in the culture medium, we dispersed the nano-
conjugates with 10% fetal bovine serum and incubated them at
37 °C in the dark for 48 h. We observed insignificant (2−3%)
release of the drug, which proves that the conjugates are quite
stable in the dark.
Figure 3. Progress of the release of chlorambucil from Pe−Cbl
nanoconjugates under visible-light irradiation (≥410 nm). The inset
shows the progress of chlorambucil release under light and dark
conditions (“ON” and “OFF” indicate the beginning and end of light
irradiation, respectively).
To establish that the photocaged Pe−Cbl nanoconjugates
can be used as a versatile DDS, initial cell imaging studies were
carried out using a HeLa cell line obtained from the National
Centre for Cell Sciences (NCCS; Pune, India), which was
maintained in minimum essential medium containing 10% fetal
bovine serum at 37 °C and 5% CO2. To study the cellular
uptake of Pe−Cbl nanoconjugates, briefly HeLa cells (5 × 104
cells/well) were plated on 12-well plates and allowed to adhere
for 4−8 h. The cells were then incubated with 2 × 10−5
M
A suspension of photocaged Pe−Cbl nanoconjugates in
water (1 × 10−4 M) was irradiated under visible light (≥410
nm) with a 125 W medium-pressure Hg lamp using a suitable
filter (1 M NaNO2 solution), and the time course of the
photorelease of chlorambucil was followed by reversed-phase
HPLC using acetonitrile as the mobile phase, keeping the flow
rate at 1 mL/min. From the HPLC profile (Figure 2), depletion
of the peak at a retention time of 6.17 min indicates the
decomposition of the Pe−Cbl nanoconjugate, and at the same
time, the appearance of two new peaks at retention times of
5.16 and 2.69 min indicates the formation of the photoproduct
perylene-3-ylmethanol and the anticancer drug chlorambucil,
respectively.
photocaged Pe−Cbl nanoconjugate in cell culture medium for
4 h at 37 °C and 5% CO2. Thereafter, the cells were fixed in
paraformaldehyde for 15 min and washed two times with
phosphate-buffered saline. Imaging was done using an Olympus
FV1000 confocal microscope with the appropriate filter. A
cellular uptake study after 4 h of incubation revealed that the
Pe−Cbl nanoconjugates were internalized by the cell
membrane, leading to a uniform distribution of the sample
inside the cell (Figure 4).
The cytotoxicity in vitro was measured using the MTT
assay25 [MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide, a yellow tetrazole] on the HeLa cell line. The
cytotoxic effect of each treatment was expressed as percentage
of cell viability relative to the untreated control cells. The
percentage of cell viability was plotted versus the concentration
We also demonstrated precise control of the photolytic
release of the anticancer drug by monitoring the release of
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Communication
indicating that most of the drugs were released from the
nanoconjugates.
Finally, to demonstrate that perylene-3-ylmethanol nano-
particles can serve as photoresponsive nanocarriers with real-
time monitoring of the drug release, we investigated the
luminescence properties of perylene-3-ylmethanol nanopar-
ticles before and after drug loading. As depicted in Figure 6a,
Figure 4. Confocal bright-field and fluorescence images of HeLa cells
incubated with 2 × 10−5 M Pe−Cbl nanoconjugate for 4 h: (a) bright-
field image; (b) fluorescence image (λex = 625 nm); (c) overlay of (a)
and (b).
of photocaged Pe−Cbl nanoconjugate at different time intervals
(Figure 5). For cells incubated with photocaged Pe−Cbl
Figure 6. Real-time monitoring of the drug release. (a, b) Perylene-3-
ylmethyl nanoparticles (a) before and (b) after drug loading. (c)
Change in fluorescence spectral profile with increasing irradiation
time. (d) Percentage of drug released as a function of fluorescence
intensity change.
Figure 5. Cell viability tests of Pe−Cbl nanoconjugates, perylene-3-
ylmethanol nanoparticles, and chlorambucil on the HeLa cell line: (a)
before irradiation; (b) after irradiation in the presence of different
concentrations of Pe−Cbl nanoconjugates; (c) after regular time
intervals of irradiation in the presence of 2 × 10−5 M Pe−Cbl
nanoconjugates. Values are presented as means standard deviations.
the perylene-3-ylmethyl nanoparticles showed blue fluores-
cence. However, after loading of chlorambucil, the nano-
particles exhibited strong red fluorescence (Figure 6b). In vitro
drug release experiments were then carried out by dialysis.
Figure 6c shows the fluorescence changes of Pe−Cbl
nanoconjugates with different irradiation times. We noticed a
gradual decrease in the red emission band at 625 nm for
photocaged Pe−Cbl nanoconjugates and a concomitant
increase in the blue emission band at 445 nm with increasing
irradiation time. We also calculated the percentage of drug
release as a function of fluorescence intensity change (Figure
6d). The above change in fluorescence color was also employed
to monitor the drug release in the cell in real time using the
fluorescence imaging technique (Figure 7a−c).
nanoconjugates, irradiation for 20 min under visible light
(≥410 nm) resulted in the release of the anticancer drug
chlorambucil, thereby causing cytotoxicity in the cancerous
HeLa cell lines, as validated by the MTT toxicity data given in
Figure 5. There was no significant cell death observed when the
cells without Pe−Cbl nanoconjugates were irradiated. The
cytotoxicity was likely caused by the released chlorambucil
upon light irradiation. In comparison with chlorambucil at the
same concentration as the Pe−Cbl nanoconjugates (Figure 5a),
the Pe−Cbl nanoconjugates showed much lower cytotoxicity.
However, upon irradiation, the Pe−Cbl nanoconjugates
showed an increasing cytotoxicity to cancer cells (Figure 5b)
because of photorelease of chlorambucil inside the cells. Thus,
the photocaged Pe−Cbl nanoconjugates can serve as both the
drug carrier and a versatile photocaged compound for the
release of the anticancer drug.
Furthermore, to validate the ability of photocaged Pe−Cbl
nanoconjugates to be used for externally regulated drug release,
the HeLa cells incubated with 2 × 10−5 M Pe−Cbl
nanoconjugate were exposed to visible light (≥410 nm) for
different time intervals. We found that the cytotoxicity toward
HeLa cells at the irradiation time of 20 min showed the highest
level of toxicity (about 40% in comparison to the control),
Figure 7. 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 (a) before
photolysis, (b) after 10 min of photolysis, and (c) after 20 min of
photolysis.
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In summary, we have developed single-component photo-
responsive nanocarrier based on fluorescent organic nano-
particles consisting of perylene-3-ylmethanol. The nano-
particles acted as both the nanocarrier for the drug and the
phototrigger for the drug release. Interestingly, the perylene-3-
ylmethanol nanoparticles showed good biocompatibility and
cellular uptake as well as precise drug release to kill the cancer
cells upon irradiation. Furthermore, a change in the
fluorescence color of the perylene-3-ylmethanol nanoparticles
indicated loading and unloading of the drug. In the future, we
are looking forward to the development of fluorescent organic
nanoparticles as photoresponsive nanocarriers for targeted drug
delivery.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis details, characterization data, and other experimental
details. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
ndpradeep@chem.iitkgp.ernet.in
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank DST (SERC Fast Track Scheme) for financial
support, DST-FIST for the 400 MHz NMR spectrometer, and
Prof. T. Pal for his valuable suggestions. A.J. is thankful to CSIR
for the fellowship.
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