Photosensitizing properties of hollow microcapsules built by multilayer self-assembly of poly(allylamine hydrochloride) modified with rose Bengal

Article abstract of DOI:10.1039/c9ra03153g

A polymeric photosensitizer based on poly(allylamine hydrochloride) (PAH) and rose Bengal (RB) was synthesized. The modified polycation PAH-RB was demonstrated to be suitable for construction of microcapsules via a layer-by-layer (LbL) assembly technique, using sodium poly(styrene sulfonate) (PSS) as counter-polyelectrolyte and CaCO₃ microcrystals as templates. After CaCO₃ core removal, a stable suspension of hollow microcapsules with shells incorporating RB (HM-RB) was obtained. The spectroscopic and photophysical behavior of both PAH-RB and HM-RB in aqueous environments were studied and described in terms of dye-dye interactions and dye hydrophobicity. Only HM-RB was able to generate singlet molecular oxygen with similar efficiency to free RB in air-saturated solutions upon green light irradiation. In order to explore possible practical applications as a supramolecular photosensitizer, experiments of HM-RB irradiation in the presence of chemically and biologically relevant target molecules were carried out. It was observed that is possible to use visible light to initiate the photooxidation of biological compounds in water, with many interesting advantages compared to low-molecular-weight photosensitizers such as an enhancement of the photosensitizing effect, due to a significant reduction of dye-dye interaction, or improved reuse given the straightforward size-based separation from the reaction mixture without loss of efficiency.

Full text of DOI:10.1039/c9ra03153g

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Photosensitizing properties of hollow
microcapsules built by multilayer self-assembly of
poly(allylamine hydrochloride) modified with rose
Bengal†
Cite this: RSC Adv., 2019, 9, 19226
a
a
a
*
Mariana P. Serrano,
Matıas Rafti,
Andres H. Thomas
´
´
and Claudio D. Borsarellib
A polymeric photosensitizer based on poly(allylamine hydrochloride) (PAH) and rose Bengal (RB) was
synthesized. The modified polycation PAH-RB was demonstrated to be suitable for construction of
microcapsules via a layer-by-layer (LbL) assembly technique, using sodium poly(styrene sulfonate) (PSS)
as counter-polyelectrolyte and CaCO3 microcrystals as templates. After CaCO3 core removal, a stable
suspension of hollow microcapsules with shells incorporating RB (HM-RB) was obtained. The
spectroscopic and photophysical behavior of both PAH-RB and HM-RB in aqueous environments were
studied and described in terms of dye–dye interactions and dye hydrophobicity. Only HM-RB was able
to generate singlet molecular oxygen with similar efficiency to free RB in air-saturated solutions upon
green light irradiation. In order to explore possible practical applications as
a supramolecular
photosensitizer, experiments of HM-RB irradiation in the presence of chemically and biologically relevant
target molecules were carried out. It was observed that is possible to use visible light to initiate the
photooxidation of biological compounds in water, with many interesting advantages compared to low-
molecular-weight photosensitizers such as an enhancement of the photosensitizing effect, due to
a significant reduction of dye–dye interaction, or improved reuse given the straightforward size-based
separation from the reaction mixture without loss of efficiency.
Received 27th April 2019
Accepted 11th June 2019
DOI: 10.1039/c9ra03153g
rsc.li/rsc-advances
the production of singlet oxygen (1O2) through an energy
transfer process (type II mechanism).5–7
Introduction
Among the main drawbacks of using homogeneous photo-
sensitized reactions is the non-straightforward removal and
reutilization procedures needed for continuous operation. As
a consequence, an increasing interest in the development of
strategies directed towards immobilization of photosensitizers
on different platforms has been recently observed;8,9 e.g.,
modication of synthetic and natural polymers with organic
dyes were used in the development of new photoactive materials
for water treatment,8,10 PDI11 and PDT.12
Rose Bengal (RB) (Fig. 1) is a xanthene dye intensively used
as photosensitizer for the 1O2 production,13 due to its high
solubility in water, high radiation absorption in the visible
region, and high quantum yield of triplet-state production.14
Additionally, RB can be easily anchored to different polymers
using; e.g., the method introduced by Merrield,15 which has
been already employed in the development of polymer-based
photosensitizers for photooxidation.16–18 Photocatalytic mate-
rials based on immobilized dyes on different platforms were
already reported for environmental applications.19,20 However,
the main drawback in the development of such photosensitizer
materials is the tendency to form molecular aggregates driven
Photosensitized reactions constitute one of the possible prac-
tical uses of solar radiation. In these processes, a chemical
species called a photosensitizer, absorbs solar radiation in
a certain spectral range and induces chemical changes in
a target molecule, which does not absorb radiation. Photo-
sensitized processes have a wide range of applications such as
synthesis with controlled regio- and stereo-selectivity,1 photo-
dynamic therapy (PDT),2 photoinactivation of microorganisms
(PDI),3 and wastewater treatment for pollutant removal.4 In
general, the photosensitization process may involve two types of
mechanism: (i) the generation of radicals (type I mechanism,
e.g., via electron transfer or hydrogen abstraction); and/or (ii)
aInstituto de Investigaciones Fisicoquımicas Teoricas
y Aplicadas (INIFTA),
´
´
´
Departamento de Quımica, Facultad de Ciencias Exactas, Universidad Nacional de
La Plata, CCT La Plata-CONICET, La Plata, Argentina. E-mail: mariserr@inia.
unlp.edu.ar
b
´
Instituto de Bionanotecnologıa del NOA (INBIONATEC), CONICET. Universidad
Nacional de Santiago del Estero (UNSE), RN9, Km 1125, G4206XCP Santiago del
Estero, Argentina
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c9ra03153g
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Fig. 1 Molecular structure of (a) rose Bengal (RB) and the polymers: (b) poly(allylamine hydrochloride) (PAH), (c) sodium poly(styrene sulfonate)
(PSS), and (d) poly(ethyleneimine) (PEI).
by dye-to-dye interactions caused by high local concentrations, (EDC), anthracene-9,10-dipropionic acid disodium salt (ADPA)
thus provoking an enhancement of non-radiative deactivation and 20-deoxyguanosine 50-monophosphate (dGMP) were
pathways and the consequent decrease of photosensitizing purchased from Sigma-Aldrich (Sigma-Aldrich, Argentina).
efficiency.21,22
Poly(ethyleneimine) solution 50 wt% in H2O (PEI, average Mw ꢀ
It is therefore highly desirable, to attain control over the 750 000) and N-hydroxysulfo succinimide sodium salt (sulfo-
spatial distribution of dye molecules in order to improve the NHS) were purchased from Fluka (Argentina). Pro-analysis
design of photo-active polymeric materials. Such spatial control anhydrous sodium carbonate, calcium chloride dihydrate,
can be achieved for lms using layer-by-layer (LbL) assembly sodium chloride, hydrochloric acid, sodium hydroxide, and
technique, which offers a simple and versatile construction ethanol were obtained from Cicarelli (Argentina). Ultrapure
method based on the sequential adsorption of interacting deionized water was used for all experiments.
species; e.g., oppositely charged polyelectrolytes.23 Sodium
poly(styrene sulfonate) (PSS), poly(ethyleneimine) (PEI), and
Synthesis of PAH-RB and microcapsule preparation
poly(allylamine hydrochloride) (PAH) are typical synthetic
polyelectrolytes used for multilayer formation (Fig. 1).24,25 Such
lms can be conveniently functionalized via post-synthetic
modication with selected molecules26 or nanoparticles,27,28 or
by using already functionalized polyelectrolytes.29–31 LbL
assembly can be carried out to create lms, but also grown over
different templates, such as carbonates microparticles, which
aer removal with suitable chelating agents, give raise to hollow
microcapsules (HM).32
Although the use of RB in the assembly of HM was already
reported,30,33 the inuence of different anchoring strategies and
its assembly into LbL architectures on their photophysical and
photosensitization properties was not studied in detail. The
goals of the present work can be summarized as follows: (i) to
synthesize a RB modied PAH polycation (PAH-RB); (ii) to use
such PAH-RB polycation for microcapsule formation via LbL
with CaCO3 microspheres as sacricial templates and PSS as
counter polyelectrolyte; (iii) to evaluate the effect of polymer
conformation and spatial distribution in the photophysical
properties of RB; and nally (iv) to assess the photosensitizing
properties of such supramolecular assembly using chemical
and biological oxidizable targets.
The EDC/NHS mediated synthesis of PAH-RB was inspired in
the bioconjugation and labelling of proteins.34 Briey, EDC and
sulfo-NHS solution was added to an RB buffered solution and
stirred evenly. Then, 20 mL of PAH solution was added and
stirred for 15 h. The nal product was puried by dialysis. The
whole reaction and dialysis process were protected from light.
Polymeric microcapsules were built using CaCO3 templates
sequentially coated with oppositely charged polyelectrolytes
layers (LbL assembly),23,24 see Scheme 1 and ESI.† Removal of
CaCO3 template was carried by re-suspension of the coated
microparticles with 0.1 M EDTA solution at pH 7 during 30 min
Experimental methods
Materials
All reagents were used as received without further purication.
Rose Bengal (RB), sodium poly(styrene sulfonate) (PSS, average
Mw ꢀ 70 000), poly(allylamine hydrochloride) (PAH, average Mw
Scheme 1 Preparation of hollow microcapsules (HM) using layer by
layer self-assembly technique onto CaCO3 template. Inset: proposed
58 000), 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide molecular structure of the PAH-RB polycation.
ꢀ
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at 25 ꢁC while shaking at 1100 rpm. A nal wash was performed NaCl 500 mM buffer solutions at pH 7 and 25 ꢁC, using a Zeta-
using 50 mM NaCl solution to remove the remaining EDTA sizer Nano ZS apparatus from Malvern.
(Scheme 1). Finally, the obtained HM were stored at 8 ꢁC before
use.
Transient absorption experiments. Laser-ash photolysis
(LFP) experiments were performed using a homemade LFP
system with laser pulse excitation at 532 nm (7 ns full width at
half maximum, 7 mJ per pulse) as previously described.37 The
transient absorption decays of RB and PAH-RB solutions or HM
suspensions were acquired with a Tektronix TDS3032B 300 MHz
instrument, and up to 10 single-shot signals averaged. Prior
each measurement, the solutions were degassed by Ar bubbling
for 20 min. The signal analysis of the transient decays was done
with the OriginPro 8.5 soware from OriginLab Corporation
using a mono and/or bi-exponential tting function.
Fluorescence microscopy. Optical microscopy images were
acquired on a Carl Zeiss Axio-Observer Z1 inverted microscope.
Samples were illuminated using bright eld or an HBO Xe lamp
complete with excitation and emission bandpass lters of 550 ꢄ
50 nm and 605 ꢄ 70 nm, respectively, acquired on an AxioCam
camera. Zen imaging soware was used for image acquisition
and analysis. All uorescence microscopy images were digitally
colored post acquisition.
Polymer and microcapsule characterization
UV-vis spectroscopy. UV-vis absorption spectra were regis-
tered with a Shimadzu UV-1800 spectrophotometer, using
quartz cells of 1 cm optical path length. All the absorption
spectra registered for HM were corrected using cubic poly-
nomial extrapolations of the spectral baseline in the visible
region, between 400–800 nm, as a simple approximation of
scattering contribution of the sample.35
Fluorescence emission. Steady-state and time-resolved uo-
rescence measurements were performed using a single-photon-
counting equipment FL3TCSPC-SP (Horiba Jobin Yvon),
described elsewhere.36 All the experiments were carried using
1 cm optical path quartz cells. The uorescence quantum yields
(FF) were determined from the corrected uorescence spectra
using eqn (1).
AR Icorr
FF ¼ FRF
(1)
A
IR
Steady-state photolysis experiments
where Icorr is the corrected maximum intensity, A is the absor-
bance at the excitation wavelength (lexc) and the superscript R
refers to the reference uorophore. To avoid inner lter effects,
an RB aqueous solution of absorbance below 0.1 at the excita-
Aqueous suspensions containing the target molecule and HM
were irradiated in 1 cm optical path quartz cells at room
temperature in continuous stirring. Two different irradiation
systems were employed: (I) a Xenon arc lamp (300 W, Newport)
coupled to a motorized monochromator UV-VIS (Oriel Corner-
stone 130 1/8 m) by a Mounting Kit (Model 74017) and (II)
a Xenon arc lamp (150 W, Luzchem) with a 475 nm lter
(GG475). UV-vis absorption spectral changes of anthracene-
9,10-dipropionic acid disodium salt (ADPA) were registered
with a CCD-USB2000 UV-vis spectrometer (OceanOptics), with
the analyzing beam placed at right angle of the photolysis beam
from the irradiation setup (II).
A high-performance liquid chromatograph Prominence from
Shimadzu (solvent delivery module LC-20AT, on-line degasser
DGU-20A5, communications bus module CBM-20, auto sampler
SIL-20A HT, column oven CTO-10AS VP and photodiode array
detector SPD-M20A) was employed for monitoring the photo-
chemical process of 20-deoxyguanosine 50-monophosphate. A
Synergi Polar-RP column (ether-linked phenyl phase with polar
end-capping, 150 ꢂ 4.6 mm, 4 mm, Phenomenex) was used for
products separation. Solutions containing 3% of methanol and
97% of 25 mM formic acid (pH ¼ 3.2) were used as mobile
phase.
tion wavelength was used as uorescence reference (FF
¼
0.018). The Icorr values were calculated at the maximum of the
uorescence band registered between 530 to 700 nm. The
uorescence intensity from RB in the HM was corrected by
scattering using eqn (2).
exc+Aemi)/2
Icorr ¼ Iobs ꢂ 10(A
(2)
where Icorr and Iobs were the corrected and observed uores-
cence maximum intensities, Aexc and Aemi were the absorbance
of the system at excitation and emission wavelengths,
respectively.
Fluorescence decays were registered with a time-correlated
single photon counting (TSCPC) apparatus from Horiba. In
time-resolved experiments a NanoLED source (maximum at 575
nm) was used for excitation and the emitted photons were
registered by a TBX-04 detector and counted by a FluoroHub-B
module. The uorescence intensity decays were tted with the
Fluorescence Decay Analysis Soware DAS6® from Horiba by
deconvolution of the NanoLED pulse using multi-exponential
model function.
Infrared spectroscopy. Fourier-transform infrared spectra
were obtained with a Jasco 4600 spectrometer with an attenu-
ated total reectance (ATR-FTIR) accessory containing a single
Results and discussion
Characterization of PAH-RB polycation
reection monolithic diamond crystal. Spectral resolution was Aer covalent functionalization of PAH with RB, the resulting
4 cmꢃ1 and background corrections were performed by PAH-RB polycation was characterized thoroughly, Fig. 2 shows
assuming an average refractive index of 1.45 for all the samples. a comparison of ATR-FTIR absorbance spectra of RB, PAH, and
Dynamic light scattering and zeta-potential. All dynamic PAH-RB. The carbonyl stretching mode corresponding to RB
light scattering (DLS) and zeta-potential measurements were appears at 1608 cmꢃ1, whereas absorption peaks at 1543, 1442,
performed with a cell drive voltage of 3.3 V in 10 mM HEPES and 1334 cmꢃ1 correspond to aromatic C]C stretching
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appeared with the concomitant disappearance of carboxylic
acid peak at 1608 cmꢃ1, indicating the amide bond formation
with carboxylate group of RB. Additionally, the sharp peak
corresponding to C–halogen stretching at 950 cmꢃ1 is preserved
but broadened aer functionalization.
The UV-vis absorption spectrum of PAH-RB in aqueous
solutions was registered and compared with free RB (PAH is
completely transparent in the visible region). PAH-RB spectrum
displays one band in the green spectral region with well-dened
maximum (l1 ¼ 558 nm) and a shoulder (l2 ¼ 523 nm), as ex-
pected for xanthene dyes (Fig. 3a).40 However, these bands
appear broader and red-shied in comparison to those of free
RB. Such behavior can be associated with the self-aggregation of
xanthene dyes resulting in a red shi of the absorption maxima
and a decrease of the maximum-to-shoulder absorbance ratio
(A1/A2).40 Hence, a comparison of normalized absorbance
spectra of RB and PAH-RB in Fig. 3a suggests that aqueous
solution of PAH-RB features the expected effect corresponding
to dye–dye interactions in the polymeric structure.
Fig. 2 ATR-FTIR spectra for RB, PAH and PAH-RB polycation.
The excitation/emission spectra of PAH-RB appear red-
shied compared to RB (Fig. 3b and c). Such spectral shis
are expected for RB in a less protic and polar environment.41 In
addition, the uorescence quantum yield (FF) value of PAH-RB
is more than one-order of magnitude lower than that corre-
sponding to free RB (Table 1), which can be explained assuming
a strong static quenching of RB uorescence as a consequence
of the intense dye–dye interactions in the functionalized
modes.38 For PAH, a signal at 1595 cmꢃ1 corresponding to N–H
in-plane bending mode appears. The spectrum obtained for
PAH-RB shows bands that can be assigned to amide bond
between ꢃP1AH and RB; e.g., C–O stretching mode (amide I) at
1620 cm , and N–H in-plane bending mode (amide II)39 at
1535 cmꢃ1. Amide I and amide II bands present in PAH-RB
Fig. 3 (a) UV-vis spectra (b) excitation fluorescence spectra (c) emission fluorescence spectra for RB in aqueous solution, PAH-RB and HM-RB in
NaCl (50 mM) aqueous solution. (d) Emission fluorescence decays registered at 580 nm for HM-RB and free RB in air-equilibrated aqueous
solutions.
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Table 1 Summary of the results obtained for RB in aqueous medium, bound to the polymer (PAH-RB) and in hollow microcapsules (HM-RB)
sF
sT
Sample
l
(nm) ꢄ 2
A1/A2 ꢄ 0.2
l
(nm) ꢄ 2
l
max
(nm) ꢄ 2
FF
(ps) ꢄ 10
(ms) ꢄ 0.2
kTOq2 (Mꢃ1 sꢃ1) ꢂ 109 ꢄ 0.02
abs
max
a
emi
max
exc
RB
PAH-RB
HM-RB
549
558
568
3.1
1.5
3.7
546
556
564
564
570
577
0.02b
#10ꢃ3
$0.4
98
ND
27
60.0
14.0
0.9
1.50
0.40
0.97
0.04
786
63.2
a
Ratio of the absorbance at the maximum of the RB peak to the absorbance at the absorption shoulder. b Quantum yield corresponds to RB in the
monomeric form in solution.41
polymer. In time-resolved experiments, uorescence lifetime percentage of amine groups that reacts with RB. That is, the
(sF) of RB in aqueous environments showed a mono-exponential positive charge of PAH is due to the protonated amino groups
decay with a sF 98 ꢄ 10 ps, as expected for monomeric RB in and, according to the ratio of reactants used in the synthesis of
water (see Fig. 3d).42 Although there is some minor proportion PAH-RB (see Experimental section), the resulting fraction of
of non-aggregated PAH-RB responsible for the observed steady- amino groups linked to RB molecules is very low, which means
state emission, such contribution is not enough to be deter- that the decrease of the total charge of the polymer is negligible.
mined in the time-resolved experiments.
The above discussed results conrm that synthesized PAH-
DLS analysis showed an increased hydrodynamic diameter RB features covalently attached RB, but also suggest the asso-
from 122 ꢄ 19 nm to 250 ꢄ 50 nm, when comparing PAH and ciation between dye molecules on neighboring polymeric
PAH-RB respectively. Considering that covalent binding of RB to chains, which are likely to be favored by hydrophobic interac-
PAH involve a negligible increase in average molecular weight, tions; and cause a strong effect on the spectroscopic properties
the observed size increment can be understood in terms of of RB.
association of neighboring polymer chains caused by dye
functionalization.
Hollow PAH-RB microcapsules
Zeta potential was determined for PAH and PAH-RB in
aqueous solution (pH 7.0, [NaCl] ¼ 500 mM) and the values
obtained were +14.0 mV and +15.0 mV, respectively. The posi-
tive value of PAH-RB is crucial for the construction of LbL
architectures. The fact that the same zeta potential value was
determined for both systems is consistent with the low
To evaluate the integrity of microcapsules constructed via LbL
with PAH-RB and PSS, optical and uorescence microscopy
images were recorded before and aer removal of CaCO3
template with EDTA. This procedure leads to the formation of
hollow microcapsules, hereaer referred as HM-RB. As it can be
observed in Fig. 4a and c, template removal did not signicantly
change the size of microcapsules, but affected the shape regis-
tered, which was due to the loss of the rigid ller material and
was expected considering previous reports. In addition, micro-
capsules with and without CaCO3 core exhibit the characteristic
uorescence of RB dye upon excitation at 545 nm (Fig. 4b and d,
respectively), thus conrming the presence of dye in the
multilayered coating.
Fig. 3a also shows the UV-vis spectrum of HM-RB aer
baseline correction by scattering. RB spectrum when supported
on HM features a red-shied narrower band with A1/A2 ratio
higher than the corresponding to PAH-RB (see Table 1). These
spectral features strongly suggest that supramolecular organi-
zation aer LbL formation hinders dye–dye interactions.
Accordingly, both excitation and emission spectra of HM-RB
suspensions showed red-shied bands when compared to
those corresponding to PAH-RB aqueous solutions (Fig. 3a and
b). More importantly, FF of HM-RB was determined to be twenty
times greater than the corresponding to dye aqueous solution
(Table 1). In time-resolved experiments, the uorescence decay
can be tted assuming a bi-exponential decay with an increased
sF value of 786 ꢄ 10 ps (compared to 98 ps, Table 1), together
with a 27% decrease on the additional decay process, as can be
observed in Fig. 3d. This complex decay for HM-RB indicates
that the behavior of dye singlet excited states is strongly
Fig. 4 Optical (a and c) and fluorescence (b and d) microscopy images
of microcapsules with CaCO3 (a and b) and after the dissolution of the
mold with EDTA (c and d). Excitation was at 545 nm and emission was
collected at 605 nm. The image color does not correspond to the light
emitted by the sample. It was digitally colored post-acquisitions.
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Fig. 5 (a) Time-evolution of UV-vis spectra of ADPA in the presence of HM-RB under irradiation. Inset: normalized absorbance changes of ADPA
at 378 nm vs. irradiation time for an air-equilibrated suspension of HM-RB (red), in comparison to the same sample in the absence of oxygen
(black). (b) Time evolution of absorbance at 378 nm over different periods on anthracene endoperoxide thermolysis. Inset: UV-vis spectra of
ADPA after different heating time at 80 ꢁC. Irradiation was carried out with the irradiation setup I.
inuenced by the heterogeneous environment of LbL assembly, corresponding values are reported in Table 1 (Fig. S2†), where sT0
which is in line with the observed red shi in steady state and sT are the triplet lifetime of RB in the absence and in the
uorescence experiments (see Fig. 3c).
presence of O2, respectively.
Spectral analysis of HM-RB suggests that LbL assembly on
the CaCO3 template surface favors two effects. On the one hand
the disaggregation of the dye, that is, the separation of the RB
molecules, avoiding the interactions observed in PAH-RB. On
the other hand the immobilization of the dye, similar to what
already reported for proteins43 and other thin lms.44,45 It is
expected that both effects hinder the deactivation pathways of
the excited states of the dye, which would enhance its photo-
sensitizing properties. This point will be analyzed in detail in
the next paragraphs.
It is known that triplet excited states play a dominant role in
the initiation of photosensitized processes, therefore the
formation of triplet RB (3RB*) species was investigated in both
PAH-RB and HM-RB Ar-saturated aqueous suspensions by laser-
ash photolysis experiments. In PAH-RB suspensions a mono-
exponential decay of 14 ms was observed, almost 23% shorter
than values corresponding to free RB. Differently, for HM-RB,
sT0
¼ 1 þ kTOq2 s0T½O2ꢅ
(3)
sT
In this case, an increase of complexity in the assembly (from
PAH-RB to HM-RB) leads to a decrease in kTOq2 value; which can
be rationalized considering that O2 free diffusion is partially
hindered in the heterogeneous assembled multilayer
environment.
The results shown in this section suggest that RB molecules
are accommodated into the microcapsule shell in a spatial
arrangement that hinders dye–dye interaction. This supramo-
lecular array is compatible with the observation that, upon
irradiation, singlet and triplet excited states of RB (1RB* and
3RB*) are efficiently generated. The larger the local molecular
rigidity around RB in the multilayer, the more favored an effi-
cient radiative deactivation of 1RB*; thus, conferring an intense
uorescence in HM-RB suspensions. Furthermore, the forma-
tion of 3RB* suggests that HM-RB can feature photosensitizing
properties, as it is analyzed in the next section.
the measured transient absorption changed into
a bi-
exponential decay featuring longer lifetime compared to PAH-
RB and similar to free RB (within experimental error, see
Table 1). The above described transients can be assigned to the
triplet excited state of RB in different environments; based on
the observed decay rates increase in presence of O2. The cor-
responding total bimolecular quenching rate constant ðkTOq2 Þ was
calculated according to the Stern–Volmer eqn (3), and the
Photosensitizing properties of HM-RB
Although RB molecules remain immobilized into the multilayer
of the HM-RB and generates 3RB* upon irradiation, which is
quenched by molecular oxygen, it is not straightforward to
assume that this supramolecular system can act as an efficient
1
photosensitizer. Generation of O2 upon irradiation of HM-RB
was detected using anthracene-9,10-dipropionic acid diso-
dium salt (ADPA) acting as chemical-trap reaction of 1O2, since
it becomes photo-bleached to give an endoperoxide.46 Fig. 5a
shows the UV-absorbance decreases of ADPA dissolved in the
supernatant of HM-RB suspension as a function of the irradi-
ation time using the irradiation setup I (see Experimental
section). The rst-order kinetic decay of ADPA relative
Scheme
sensitized by HM-RB.
2 Degradation mechanism of ADPA or dGMP photo-
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Fig. 6 (a) Chromatograms of supernatant of air-equilibrated aqueous suspensions containing dGMP and HM-RB, recorded using the UV/vis
detector, before and after 60 min of irradiation. Inset: normalized absorption spectra of oxidation product of retention time 5.5 min. [dGMP]0
¼
80 mM, pH ¼ 6.5, lirr ¼ 567 nm, lan ¼ 260 nm. (b) Concentration profiles of dGMP in aqueous suspension of HM-RB with different number of
bilayers at different irradiation time. Inset: dGMP concentration after 60 min of consecutive irradiation cycles. pH ¼ 7, lirr ¼ 567 nm, irradiation set
up II.
absorbance at 378 nm is shown in Fig. 5a inset, conrming 1O2, that ultimately will produce the observed substrate
1
a constant steady-state concentration of O2. A similar signi- oxidation.
cant decrease of ADPA absorbance was observed in HM-RB
Taking into account previous results, 20-deoxyspir-
suspensions, as well as in aqueous RB solutions aer 180 s oimidantoin 50-monophosphate (dSpMP) was identied as the
1
irradiation (no ADPA consumption was observed in control primary product of O2 mediated reaction.49 Analyzing the UV
experiments without irradiation).
spectra and tr of the reaction products using irradiated HM-RB
In contrast, Ar-bubbling in HM-RB suspensions strongly and dGMP suspension, the principal product identity can be
1
reduces the consumption of ADPA upon illumination, sug- assigned to dSpMP, which suggests the participation of O2 in
1
gesting that ADPA oxidation occurs by O2-mediation to yield the reaction mechanism. Additionally, an increase of dGMP
the corresponding endoperoxide. To conrm such assumption, consumption was observed as pH was increased from 7 to 9
oxidized ADPA solutions were heated while the absorbance was (Fig. S3†). It is noteworthy that dGMP chemical reactivity
monitored in order to provoke thermolysis of endoperoxides, towards 1O2 is higher as pH becomes alkaline, which conrms
1
thus regenerating the parent aromatic anthracene ring.47 Fig. 5b that photosensitization mechanism of dGMP by HM-RB is O2
shows the recovery of typical anthracene ring absorption bands mediated.
upon heating at 80 ꢁC. These results conrm the effective
The dGMP consumption aer 60 min of irradiation in
generation of 1O2 by irradiation with visible light of HM-RB successive irradiation-centrifugation cycles for HM-RB and
suspensions.
dGMP suspensions was evaluated. For each subsequent irradi-
ation experiment, pellets were re-suspended in dGMP fresh
solutions. It is possible to reuse the same HM-RB batch since
dGMP consumption was proportional to the irradiation time,
and additionally, similar consumption was obtained for at least
three irradiation cycles of 60 minutes each (Fig. 6b inset). This
behavior showed two interesting advantages of HM-RB over RB
in solution. First, the supramolecular organization prevents the
dye photobleaching reactions, and second HM-RB can be easily
separated by centrifugation from the reaction mixture to be
reused. On the contrary, RB solution requires more sophisti-
cated and expensive separation techniques such as
chromatography.
HM-RB as photosensitizer for the oxidation of dGMP
The possible application of polymeric photosensitizers bearing
RB chromophores in microcapsules was tested with the model
reaction of 20-deoxyguanosine 50-monophosphate (dGMP)
degradation as a biological target. We chose this nucleotide
because in DNA guanine is the only nucleobase that reacts
signicantly with 1O2.48 Therefore, aqueous suspensions of HM-
RB and dGMP were exposed to UV-vis radiation using the irra-
diation setup II (see Experimental section). Supernatants ob-
tained from the irradiated samples were analyzed by HPLC
using the UV/vis detector. The decrease observed for peak area
corresponding to dGMP was used as an indicator of nucleotide
consumption in the process. In addition, several peaks with
Conclusions
shorter retention times (tr) were detected (Fig. 6a). It was found In the present work we have presented a strategy for the
that HM-RB featuring 3 or 6 bilayers of polyelectrolytes can assembly of hollow microcapsules (HM) using sacricial CaCO3
effectively photosensitize dGMP degradation (Fig. 6b). This templates, via LbL approach constituted by PSS (sodium poly(-
result suggests that O2 can diffuse across the HM-shells with styrene sulfonate)) polyanion and a modied polycation, PAH-
different number of bilayers and interact with 3RB* to generate RB, (poly(allylamine hydrochloride)) covalently modied with
19232 | RSC Adv., 2019, 9, 19226–19235
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rose Bengal dye. The presence of attached bulky aromatic RB
molecule on modied PAH induces dye–dye interactions,
presumably driven by hydrophobicity. Such aggregation
modies supported RB both, spectroscopic and photochemical
properties, reducing its uorescence quantum yield (FF). The
spectroscopic analysis of HM-RB revealed that the observed
behavior corresponds to what can be expected for free RB in less
protic and polar environments, without signicant dye–dye
interactions.
Irradiation with visible light of HM-RB aqueous suspensions
induces the efficient production of singlet oxygen (1O2)
comparable to what produced by RB in aqueous solutions. Such
reactive oxygen species was demonstrated to be responsible for
degradation of a biological target molecule used (20-deoxy-
guanosine 50-monophosphate, dGMP). A proposed degradation
mechanism is described in Scheme 2 (reactions (1)–(4)). Aer
subsequent irradiation cycles and oxidation of dGMP, the
reutilization of HM-RB as photosensitizer was also proved
indicating that the supramolecular organization of the RB
molecules into the HM shell prevents the photobleaching
reactions induced by dye self-interactions.
3 M. C. DeRosa and R. J. Crutchley, Photosensitized singlet
oxygen and its applications, Coord. Chem. Rev., 2002, 233–
234, 351–371.
4 O. Legrini, E. Oliveros and A. M. Braun, Photochemical
processes for water treatment, Chem. Rev., 1993, 93, 671–698.
5 C. S. Foote, Denition of type I and type II photosensitized
oxidation, Photochem. Photobiol., 1991, 54, 659.
6 P. Di Mascio, M. R. Hamblin, A. H. Thomas, M. Vignoni,
C. Lorente, A. A. Ghogare, S. C. Nunez, J. Cadet,
M. S. Baptista, M. S. Ribeiro, A. Greer and
T. M. Yoshimura, Type I and Type II Photosensitized
Oxidation Reactions: Guidelines and Mechanistic
Pathways, Photochem. Photobiol., 2017, 93, 912–919.
7 I. E. Kochevar and R. W. Redmond, Photosensitized
production of single oxygen, Methods Enzymol., 2000, 319,
20–28.
8 A. Valkov, K. Raik, Y. Mualem-Sinai, F. Nakonechny and
M. Nisnevitch, Water Disinfection by Immobilized
Photosensitizers, Water, 2018, 11, 26.
9 C. Spagnul, L. C. Turner and R. W. Boyle, Immobilized
photosensitizers for antimicrobial applications, J.
Photochem. Photobiol., B, 2015, 150, 11–30.
Summarizing, we have demonstrated that by controlling the
supramolecular assembly and spatial arrangement of RB 10 M. Nowakowska and K. Szczubiałka, Photoactive polymeric
attached to a polymeric structure; the photophysical and
photochemical characteristics of the dye can be modulated,
and hybrid systems for photocatalytic degradation of water
pollutants, Polym. Degrad. Stab., 2017, 145, 120–141.
remarkably recovering photosensitization properties of RB in 11 A. Parthasarathy, S. Goswami, T. S. Corbitt, E. Ji, D. Dascier,
homogeneous solution with the advantage of high photo-
stability and an easier separation for its subsequent use.
D. G. Whitten and K. S. Schanze, Photophysics and Light-
Activated Biocidal Activity of Visible-Light-Absorbing
Conjugated Oligomers, ACS Appl. Mater. Interfaces, 2013, 5,
4516–4520.
Conflicts of interest
12 M. Kepczynski, M. Dzieciuch and M. Nowakowska,
Nanostructural Hybrid Sensitizers for Photodynamic
Therapy, Curr. Pharm. Des., 2012, 18, 2607–2621.
13 R. W. Redmond and J. N. Gamlin, A Compilation of Singlet
Oxygen Yields from Biologically Relevant Molecules,
Photochem. Photobiol., 1999, 70, 391–475.
There are no conicts to declare.
Acknowledgements
14 P. Murasecco-Suardi, E. Gassmann, A. M. Braun and
E. Oliveros, Determination of the Quantum Yield of
Intersystem Crossing of Rose Bengal, Helv. Chim. Acta,
1987, 70, 1760–1773.
15 R. B. Merrield, Solid Phase Peptide Synthesis. I. The
Synthesis of a Tetrapeptide, J. Am. Chem. Soc., 1963, 85,
2149–2154.
16 A. P. Schaap, A. L. Thayer, E. C. Blossey and D. C. Neckerszd,
Polymer-Based Sensitizers, J. Am. Chem. Soc., 1975, 97, 3741–
3745.
17 E. C. Blossey, D. C. Neckers, A. L. Thayer and A. P. Schaap,
Polymer-Based Sensitizers for Photooxidations, J. Am.
Chem. Soc., 1973, 95, 5820–5822.
The present work was partially supported by the Consejo
´
´
Nacional de Investigaciones Cientıcas y Tecnicas (CONICET-
Grant PIP 112-200901-0304, PIP 145-201401-00012CO), Agen-
´
´
´
cia de Promocion Cientıca y Tecnologica (ANPCyT-Grants PICT
2015-2173, PICT-2012-0508, PICT-2015-1988, PICT-2015-0828),
Universidad Nacional de La Plata (UNLP-Grant X712), Uni-
versidad Nacional de Santiago del Estero (UNSE-Grant 23A215).
C. D. B. also thanks the Alexander von Humboldt Foundation
for equipment grant (ATR-FTIR Jasco 4600). All authors are
research members of CONICET.
References
˜
18 G. V. Ferrari, M. E. Andrada, J. Natera, V. A. Munoz,
˜
M. Paulina Montana, C. Gambetta, M. L. Boiero,
1 E. L. Clennan, New Mechanistic and Synthetic Aspects of
Singlet Oxygen Chemistry, Tetrahedron, 2000, 56, 9151–9179.
2 P. Agostinis, K. Berg, K. A. Cengel, T. H. Foster, A. W. Girotti,
S. O. Gollnick, S. M. Hahn, M. R. Hamblin, A. Juzeniene,
D. Kessel, M. Korbelik, J. Moan, P. Mroz, D. Nowis,
´
M. A. Montenegro, W. A. Massad and N. A. Garcıa, The
employment of a removable chitosan-derivatized polymeric
sensitizer in the photooxidation of polyhydroxylated water-
pollutants, Photochem. Photobiol., 2014, 90, 1251–1256.
¨
J. Piette, B. C. Wilson and J. Golab, Photodynamic therapy 19 B. Braun, J. Ortner, K. H. Funken, M. Schafer, C. Schmitz,
of cancer: an update, Ca-Cancer J. Clin., 2011, 61, 250–281.
G. Horneck and M. Fasdni, in Proceedings of the 8th
This journal is © The Royal Society of Chemistry 2019
RSC Adv., 2019, 9, 19226–19235 | 19233

View Article Online
RSC Advances
Paper
International Symposium on Solar Thermal Concentrating
Technologies, 1997, pp. 1391–1401.
20 M. Nowakowska, M. Kepczynski and K. Szczubialka, New
B. L. Pentelute, Systematic Investigation of EDC/sNHS-
Mediated Bioconjugation Reactions for Carboxylated
Peptide Substrates, Bioconjugate Chem., 2016, 27, 994–1004.
polymeric photosensitizers, Pure Appl. Chem., 2001, 73, 35 A. Salinaro, A. V Emeline, J. Zhao, H. Hidaka, V. K. Ryabchuk
491–495.
and N. Serpone, Terminology, relative photonic efficiencies
and quantum yields in heterogeneous photocatalysis. Part
II: experimental determination of quantum yields, Pure
Appl. Chem., 2007, 71, 321–335.
21 D. Xu and D. C. Neckers, Aggregation of rose Bengal
molecules in solution, J. Photochem. Photobiol., A, 1987, 40,
361–370.
´
´
´
22 S. D. Ezquerra Riega, H. B. Rodrıguez and E. San Roman, 36 M. P. Serrano, M. Vignoni, M. L. Dantola, E. Oliveros,
Rose bengal in poly(2-hydroxyethyl methacrylate) thin
lms: self-quenching by photoactive energy traps, Methods
Appl. Fluoresc., 2017, 5, 014010.
C. Lorente and A. H. Thomas, Emission properties of
dihydropterins in aqueous solutions, Phys. Chem. Chem.
Phys., 2011, 13, 7419–7425.
23 J. Borges and J. F. Mano, Molecular Interactions Driving the 37 M. A. Montenegro, A. de O. Rios, A. Z. Mercadante,
Layer-by-Layer Assembly of Multilayers, Chem. Rev., 2014,
114, 8883–8942.
24 E. Donath, G. B. Sukhorukov, F. Caruso, S. A. Davis and
M. A. Nazareno and C. D. Borsarelli, Model Studies on the
Photosensitized Isomerization of Bixin, J. Agric. Food
Chem., 2004, 52, 367–373.
¨
H. Mohwald, Novel Hollow Polymer Shells by Colloid- 38 J. Coates, Interpretation of Infrared Spectra, A Practical
Templated Assembly of Polyelectrolytes, Angew. Chem., Int.
Ed., 1998, 37, 2201–2205.
Approach, Encycl. Anal. Chem., 2006, pp. 1–23.
39 J. H. Kim, P. K. Park, C. H. Lee and H. H. Kwon, Surface
modication of nanoltration membranes to improve the
removal of organic micro-pollutants (EDCs and PhACs) in
drinking water treatment: gra polymerization and cross-
linking followed by functional group substitution, J.
Membr. Sci., 2008, 321, 190–198.
´
25 C. Dejugnat and G. B. Sukhorukov, pH-responsive properties
of hollow polyelectrolyte microcapsules templated on
various cores, Langmuir, 2004, 20, 7265–7269.
26 Y. Xu, J. Fei, G. Li, T. Yuan and J. Li, Compartmentalized
Assembly of Motor Protein Reconstituted on Protocell
Membrane toward Highly Efficient Photophosphorylation, 40 O. Valdes-Aguilera and D. C. Neckers, Aggregation
ACS Nano, 2017, 11, 10175–10183.
Phenomena in Xanthene Dyes, Acc. Chem. Res., 1989, 22,
27 A. S. Angelatos, B. Radt and F. Caruso, Light-Responsive
171–177.
ˇ
´
´
ˇ
´
Polyelectrolyte/Gold Nanoparticle Microcapsules, J. Phys. 41 L. Ludvıkova, P. Fris, D. Heger, P. Sebej, J. Wirz and P. Klan,
Chem. B, 2005, 109, 3071–3076.
28 W. Cui, A. Wang, J. Zhao, X. Yang, P. Cai and J. Li, Layer by
layer assembly of albumin nanoparticles with selective
Photochemistry of rose Bengal in water and acetonitrile:
a comprehensive kinetic analysis, Phys. Chem. Chem. Phys.,
2016, 18, 16266–16273.
recognition of tumor necrosis factor-related apoptosis- 42 M. A. J. Rodgers, Picosecond uorescence studies of rose
inducing ligand (TRAIL), J. Colloid Interface Sci., 2016, 465,
11–17.
Bengal in aqueous micellar dispersions, Chem. Phys. Lett.,
1981, 78, 509–514.
¨
¨
˜
´
29 Z. Dai, L. Dahne, E. Donath and H. Mohwald, Downhill 43 M. B. E. Turbay, V. Rey, N. M. Arganaraz, F. E. Moran Vieyra,
´
Energy Transfer via Ordered Multichromophores in Light-
Harvesting Capsules, J. Phys. Chem. B, 2002, 106, 11501–
11508.
A. Aspee, E. a Lissi and C. D. Borsarelli, Effect of dye
localization and self-interactions on the photosensitized
generation of singlet oxygen by rose Bengal bound to
bovine serum albumin, J. Photochem. Photobiol., B, 2014,
141C, 275–282.
30 Y. Yang, H. Liu, M. Han, B. Sun and J. Li, Multilayer
Microcapsules for FRET Analysis and Two-Photon-
´
Activated Photodynamic Therapy, Angew. Chem., Int. Ed., 44 M. Mirenda, L. E. Dicelio and E. San Roman, Effect of
2016, 55, 13538–13543.
31 Q. Bricaud, R. M. Fabre, R. N. Brookins, K. S. Schanze and
J. R. Reynolds, Energy transfer between conjugated
Molecular Interactions on the Photophysics of Rose Bengal
in Polyelectrolyte Solutions and Self-Assembled Thin
Films, J. Phys. Chem. B, 2008, 112, 12201–12207.
´
polyelectrolytes in layer-by-layer assembled lms, 45 M. Mirenda, C. A. Strassert, L. E. Dicelio and E. S. Roman,
Langmuir, 2011, 27, 5021–5028.
Dye-Polyelectrolyte
Layer-by-Layer
Self-Assembled
32 A. a. Antipov, D. Shchukin, Y. Fedutik, A. I. Petrov,
Materials: Molecular Aggregation, Structural Stability, and
Singlet Oxygen Photogeneration, ACS Appl. Mater.
Interfaces, 2010, 2, 1556–1560.
¨
G. B. Sukhorukov and H. Mohwald, Carbonate
microparticles for hollow polyelectrolyte capsules
fabrication, Colloids Surf., A, 2003, 224, 175–183.
46 B. A. Lindig, M. A. J. Rodgers and A. P. Schaaplc,
Determination of the Lifetime of Singlet Oxygen in D20
Using 9,10-Anthracenedipropionic Acid, a Water-Soluble
Probe, J. Am. Chem. Soc., 1980, 102, 5590–5593.
33 X. L. Wang, Y. Zeng, Y. Z. Zheng, J. F. Chen, X. Tao,
L. X. Wang and Y. Teng, Rose Bengal-graed
biodegradable microcapsules: singlet-oxygen generation
and cancer-cell incapacitation, Chem.–Eur. J., 2011, 17, 47 N. J. Turro, M. F. Chow and J. Rigaudy, Mechanism of
11223–11229.
Thermolysis of Endoperoxides of Aromatic Compounds.
Activation Parameters, Magnetic Field, and Magnetic
Isotope Effects, J. Am. Chem. Soc., 1981, 103, 7218–7224.
34 K. A. Totaro, X. Liao, K. Bhattacharya, J. I. Finneman,
J. B. Sperry, M. A. Massa, J. Thorn, S. V. Ho and
19234 | RSC Adv., 2019, 9, 19226–19235
This journal is © The Royal Society of Chemistry 2019

View Article Online
Paper
RSC Advances
´
48 J. Cadet, J.-L. Ravanat, G. R. Martinez, M. H. G. Medeiros and 49 M. P. Serrano, S. Estebanez, M. Vignoni, C. Lorente,
P. Di Mascio, Singlet oxygen oxidation of isolated and
cellular DNA: product formation and mechanistic insights,
Photochem. Photobiol., 2006, 82, 1219–1225.
P. Vicendo, E. Oliveros and A. H. Thomas, Photosensitized
oxidation of
20-deoxyguanosine 50-monophosphate:
mechanism of the competitive reactions and product
characterization, New J. Chem., 2017, 41, 7273–7282.
This journal is © The Royal Society of Chemistry 2019
RSC Adv., 2019, 9, 19226–19235 | 19235