pubs.acs.

org/bc Article

“On-Demand” Antimicrobial Photodynamic Activity through

Supramolecular Photosensitizers Built with Rose Bengal and (p-
Vinylbenzyl)triethylammomium Polycation Derivatives
Cecilia Vera,* Mauro N. Gallucci, Juliana Marioni, Marcelo C. Sosa Morales, Debora M. Martino,
Susana Nuñez Montoya, and Claudio D. Borsarelli*
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ABSTRACT: The antimicrobial photodynamic activity (aPDA) in fungal and

bacterial strains of supramolecular adducts formed between the anionic
photosensitizer (PS) Rose Bengal (RB2−) and aromatic polycations derived
from (p-vinylbenzyl)triethylammonium chloride was evaluated. Stable supra-
molecular adducts with dissociation constants Kd ≈ 5 μM showed photosensitizing
properties suitable for generating singlet oxygen (ΦΔ = 0.5 ± 0.1) with the added
advantage of improving the photostability of the xanthenic dye. However, the
aPDA of both free and supramolecular RB2− was highly dependent on the type of
microorganism treated, indicating the importance of specific interactions between
the different cell wall structures of the microbe and the PSs. Indeed, in the case of
Gram-positive Staphylococcus aureus, the aPDA of molecular and supramolecular PSs was highly effective. Instead, in the case of
Gram-negative Escherichia coli, only the RB2−:polycation adducts showed aPDA, while RB2− alone was inefficient, but in the case of
Candida tropicalis, the opposite behavior was observed. Therefore, the present results indicate the potential of supramolecular
chemistry to obtain aPDA à la carte depending on the target microbe and the PS properties.

■ INTRODUCTION
Microbial infections represent a serious global health threat
Scheme 1. Chemical Structure of the Anionic Dye Rose
Bengal (RB2−) and the Polycation Poly-
that has worsened in recent decades due to the development of Vinylbenzyltriethylammonium Chloride [VBA4Cl4]n≈40
microbial resistance mechanisms to pharmacological treat- (pVBA) and the Copolymer Combining VBT and VBA
ments.1 Infectious diseases cause a large number of morbidity Monomers in a 1:4 M Ratio [(VBT-VBA4Cl4)]n≈40 (VBT-
and mortality and significant health care costs.2 Therefore, the VBA)
current postantibiotic era requires the exploration of new
microbial control strategies without generating microbial
resistance mechanisms. Among the new antimicrobial
approaches,3 antimicrobial photodynamic therapy (aPDT)
has been highlighted as an effective antimicrobial treatment,
involving the elimination of microbes through oxidative
damage of biomolecules due to reactive oxygen species
(ROS), mainly singlet oxygen 1O2, which are generated after
light absorption by a molecule called photosensitizers
(PSs).4−6
Many organic and inorganic molecules are recognized as reflectance, internal filtering, and lower penetration capacity
efficient PSs, with a high quantum yield of 1O2 generation in microbes, among others.11 In the particular case of aPDT,
(ΦΔ), adequate solubility, chemical stability, low dark toxicity, the structural and compositional characteristics of the
and good light absorption properties, among others.7,8 Rose
Bengal (RB2−, Scheme 1) is a classical anionic xanthenic dye
largely used as a PS for in vitro anticancer and antimicrobial Received: December 17, 2021
photodynamic studies.9,10 However, due to the intrinsic Revised: January 25, 2022
complex nature of the biological media, the use of PSs in Published: February 9, 2022
photodynamic bioapplications is often hampered as a result of
several factors, such as photochemical stability, self-aggregation
due to compartmentalization effects, light scattering or

© 2022 American Chemical Society https://doi.org/10.1021/acs.bioconjchem.1c00596

463 Bioconjugate Chem. 2022, 33, 463−472
Bioconjugate Chemistry pubs.acs.org/bc Article

microbial wall surface are other factors that also significantly ultrapure water and phosphate-buffered saline (PBS) solutions.
influence treatment efficacy.11 This is the case for Gram- As an example of the observed spectroscopic behavior, Figure
negative bacteria, which are particularly resistant to photo- 1A shows the absorbance changes produced in a 10 μM RB2−
dynamic inactivation using neutral or anionic PS molecules
due to the structural complexity of their membrane barrier.
Gram-negative bacteria, in addition to peptidoglycan, have an
additional layer in their wall, which is composed of negatively
charged lipopolysaccharides that prevent effective PS pene-
tration.12
To overcome these problems, conjugate and supramolecular
chemistries in combination with different macromolecules have
been successfully applied to design PS systems with improved
properties for photodynamic applications.13−16 Covalent
binding of polar and nonpolar PSs to biomolecules with
bacterial surface binding affinities, such as small peptides,
exopolysaccharides, chitosan, and functionalized cholesterol,
showed good efficacy for inactivation of both planktonic strains
and biofilms.13−15,17 Despite the higher functionality for aPDT
of covalently linked PS, synthetic routes for their preparation
could be costly and time-consuming. Thus, the concept of
noncovalent coassembly of a supramolecular structure that
combines a PS molecule with other entities through
intermolecular interactions ranging from the weakest, such as
van der Waals, to the strongest, such as electrostatic attractive
forces, emerges as a suitable tool for aPDT applications.18,19 In
general, these interactions act cooperatively by reducing the
free energy to obtain the most thermodynamically stable
supramolecular aggregate through reversible equilibria that can
be modulated by different external stimuli, for example, pH,
ionic strength, temperature, light, and so on.20 Therefore, the
functionality of supramolecular PSs in aPDT can also be
modulated by the microbial medium itself.18,19 Figure 1. Changes caused in the UV−vis absorbance (A) and
Taking into account the latter concepts, in this work, we fluorescence emission (B) spectra of a 10 μM RB2− aqueous solution
explored the interaction of RB2− with polycationic (p- by increasing the total concentration of the binding sites [Θ]T of the
vinylbenzyl)triethylammonium derivatives (Scheme 1) to polycation VBT-VBA. The blue and red sets of spectra represent
form stable supramolecular adducts with significant ΦΔ values concentration conditions meeting [Θ]T/[RB2−] < 0.5 and [Θ]T/
and evaluated their antimicrobial photodynamic activity [RB2−] > 0.5, respectively. Insets show the semilog plots for the
against typical strains of Gram-positive and Gram-negative variation of the maximum absorbance wavelength (λmax, ●), FWHM
(■) of the visible absorbance band of the dye, and the fluorescence
bacteria and a unicellular fungus. The selection of these
quantum yield (ΦF, ■) of RB2− as a function of the molar
aromatic polycations is based on their ability to interact with concentration ratio [Θ]T/[RB2−].
dye molecules through various types of intermolecular
interactions, for example, from π-stacking to electrostatic aqueous solution by adding small aliquots of VBT-VBA, which
attractive forces.21 Furthermore, in the case of the copolymer only absorbs light below 300 nm. A similar result was observed
obtained by combining vinylbenzylthymine (VBT) and in PBS and also for the interaction of RB2−with the polycation
vinyltriethylammonium chloride (VBA) monomers, the VBT
pVBA under both conditions (Figure S1 of the Supporting
moiety can also produce multiple hydrogen-bonding inter-
Information). As the concentration of polycations increases,
actions and photo-cross-linking through the formation of
thymine dimers to obtain polymer films,22,23 core-cross-linked there is a bathochromic shift of the visible RB2− band along
micelles,24 and shell-cross-linked hollow microcapsules,25 with with changes in the absorbance value and bandwidth.
different capabilities such as bacteriostatic, drug delivery, and Assuming that the dye molecule interacts with a binding site
selective molecular permeation. Θ of the polycations, whose operating total concentration is
The present results confirmed the formation of stable calculated taking into account the molecular weight of the
RB2−:polycation adducts with good photosensitizing properties minimum polymer block unit containing four positive charges
and higher photostability of the dye compared to the free RB2− including the chloride counteranion, for example, [VBA+Cl−]4
molecule. However, the antimicrobial photodynamic activity and [VBT(VBA+Cl−)4] for pVBA and VBT-VBA, respectively,
(aPDA) of RB2− as a free molecule and as supramolecular the inset of Figure 1A shows the red shift of the absorbance
adducts was highly dependent on the type of treated maximum (λmax) and the bell-shaped downward variation of
microorganism, suggesting that aPDA can be tuned à la carte the full width at half maximum (FWHM) of the absorbance
using supramolecular chemistry. band produced as a function of the molar ratio [Θ]T/[RB2−].

■ RESULTS AND DISCUSSION

Supramolecular Formation of Dye−Polycation Ad-
ducts. The binding of RB2− to both polycations was studied in
464
The sigmoidal fit of λmax showed an inflection point at [Θ]T/
[RB2−] ≈ 0.5, coinciding with the highest value of FWHM.
Furthermore, the absorbance spectral changes indicate the
presence of a single isosbestic point at 558 nm in the range of
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Bioconjugate Chemistry
[Θ]T/[RB2−] < 0.5, while two new isosbestic points were
formed at 548 and 574 nm at [Θ]T/[RB2−] > 0.5. This spectral
behavior suggests that at least two binding equilibria with
different dye stoichiometries occur with increasing polycation
concentration.26 A value of [Θ]T/[RB2−] = 0.5 matches with
the stoichiometric ratio in which two RB2− molecules
neutralize the four positive charges of the binding site Θ of
each polycation. Consequently, it can be assumed that when 0
< [Θ]T/[RB2−] ≤ 0.5, up to two dye molecules bind
electrostatically to each binding site, resulting in an intense
spectral hypochromic and band broadening due to the
formation of dye−dye aggregates on the polycation chain.27
In such a case, intense fluorescent self-quenching of RB2− is
expected, as confirmed by the decreases in the fluorescence
quantum yield (ΦF) of the dye, as shown in Figure 1B.
To confirm the stoichiometry in the dye-binding site under
this condition, the continuous variation (or Job’s) method was
used by fixing the total molar concentration ([Θ]T + [RB2−] =
30 μM) in the mixtures with different mole fraction values.28
UV−vis absorption spectra were recorded before and after
mixing the dye and polycation solutions using a tandem
cuvette with a 5 mm optical path for each reservoir, as shown
in Figure 2A for the aqueous solution with a binding site mole
fraction of xΘ = 0.3. The dashed gray line represents the
difference spectrum obtained by subtracting the spectrum of
Figure 2. (A) Absorption spectra registered in a tandem absorption
cuvette before (blue line) and after (red line) mixing equal aliquots of
RB2− and VBT-VBA aqueous solutions to yield a molar fraction of
binding sites of the polycation of xΘ = 0.3. The gray dashed line
represents the differential spectrum (ΔAbs). Inset: Job’s plot for
ΔAbs values at 548 and 573 nm, respectively. (B) Binding isotherms
obtained using the absorbance changes of RB2− at 563 nm produced
by the addition of aliquots of pVBA and VBT-VBA, respectively. Solid
black lines represent the data fitting with eq 2 (see text).
465
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the mixed solution (red line) from that of the individual
unmixed solutions in the tandem cell (blue line), showing
intense negative and positive bands at 548 and 573 nm,
respectively. The largest absorbance change observed at both
wavelengths analyzed is compatible with an average value of xΘ
= 0.35 ± 0.05 (inset of Figure 2A), which matches the
theoretical value of 0.33 for a 2:1 complex.28
In contrast, under polycation concentration conditions
where [Θ]T/[RB2−] ≥ 0.5, both the absorbance and
fluorescence emission bands of the dye are red-shifted by
about 14 nm, with a hyperchromic and narrowing effect of the
absorbance band and a sharp increase in the ΦF value of RB2−.
This behavior has been extensively reported for dye binding to
proteins29 and polyelectrolytes,26,30 due to the association of a
single dye molecule per binding site, inducing the loss of the
dye aggregation on the polycation by the presence of more
available binding sites as the polycation concentration
increases.26 Under this condition, the ground- and excited-
state spectroscopic properties of the dye resemble those found
for monomeric RB2− in organic solvents sensing a less-polar
local environment,31 supporting the formation of a 1:1 adduct
between a free dye molecule (RB2−free) and a free binding site
(Θ) in the polycation as described by the following
pseudophase equilibrium with a dissociation constant Kd
given by eq 1.
[RB2 −]free [Θ]
RB bound 2 − V RBfree 2 − + Θ kd =
[RB2 −]bound (1)
In this equation, [RB2−]bound = f b[RB2−]0, where f b is the
fraction of dye bound to the polycation and was estimated
using the maximum absorbance value of the red-shifted band at
563 nm as f b = (A − Af)/(Ab − Af), where Af and Ab are the
absorbance values of the fully free and bound dye, respectively.
Ab can be estimated by extrapolation of the absorbance to the
“infinite” concentration of the free polycation binding sites.
The binding isotherm was obtained by plotting the average
number of dye molecules bound to each polycation molecule
(n = [RB2−]bound/[Θ]) as a function of free dye concentration
[RB2−]free = (1 − f b)[RB2−]0, and a typical sigmoidal shape was
observed for adducts prepared with both polycations, Figure
2B. The binding isotherm was analyzed with a modified Hill
equation, eq 2, where nmax, νH, and Kd represent the maximum
average number of bound dye per binding site Θ, the
cooperative Hill factor, and the dissociation constant of the
adduct, respectively.32
ij [RB2 −]free yz
n = n0 + nmax jjj zz
j K + [RB2 −] zz
νH
k d free { (2)
Solid lines in Figure 2B represent the best fit of the binding
isotherm of eq 2 for the formation of both adducts in ultrapure
water, resulting in Kd (in μM) values of 5.9(±0.1) and
5.2(±0.1), with nmax values of 0.93 (±0.13) and 0.95 (±0.06)
for the adducts (RB 2− :pVBA) and (RB 2−:VBT-VBA),
respectively. In PBS, Kd values of 4.0 (±0.1) and 4.2 (±0.1),
with an nmax of 0.96 (±0.03) and 1.07 (±0.04) were obtained,
respectively (Figures S2 and S3 of the Supporting
Information). In all cases, nmax ≈ 1 in agreement with the
binding of a single RB2− molecule per Θ of each polycation to
form highly stable supramolecular adducts.
Supramolecular Adduct Photostability and Photo-
sensitizing Capability. Following the binding behavior
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Bioconjugate Chemistry
described above, the concentration condition where [Θ]T/
[RB2−] = 5 was selected to ensure the formation of 1:1
supramolecular adducts using both polycations, and their
photostability and singlet oxygen (1O2) photogeneration ability
were evaluated in comparison with those of free RB2− in PBS
solutions. Figure 3A compares the relative absorbance changes
Figure 3. (A) Photobleaching kinetics observed upon green LED
excitation of air-saturated PBS solutions of free RB2− (green solid
line) and the adducts with the polycations pVBA (orange dot line)
and VBT-VBA (blue dash line) prepared in a fivefold molar
concentration excess of polycations relative to RB2−. (B) Kinetic
traces for the consumption of dissolved oxygen in PBS occurred by
the singlet oxygen-mediated oxidation of 9 mM furfuryl alcohol (FA)
in the presence of RB2− (green solid line) and supramolecular adducts
RB2−:pVBA (orange dot line) and RB2−:VBT-VBA (blue dash line).
The black solid lines represent the linear fitting of the kinetic traces to
calculate the initial consumption rate of molecular oxygen.
(A/A0)max monitored at the maximum dye band (λmax = 548
and 560 nm for free RB2− and both adducts, respectively)
Figure 4. Variation of the colony-forming unit (CFU·mL−1) of planktonic strains in the dark as a function of the molar concentration of the PS
Rose Bengal (RB2−, green ●) or both polycations pVBA (orange ■) and VBT-VBA (blue ▲). The arrows indicate the concentration conditions
selected for the preparation of the supramolecular adducts used in the aPDA experiment for each strain to avoid toxicity effects in the dark.
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during illumination with a green LED (523 ± 20 nm) under
aerobic conditions. After irradiation, only the free dye solution
showed intense photobleaching compared to the
(RB2−:pVBA) and (RB2−:VBT-VBA) adducts, in which almost
no dye degradation was observed.
Photobleaching of RB2− in aqueous solutions has been
recently revisited, eqs 3−6.33 According to the reaction
scheme, dye degradation can occur by the reaction of a
molecule of RB2− with singlet oxygen 1O2 (eq 5), which is
generated by the energy-transfer reaction from the dye triplet
excited state 3RB2− to the molecular oxygen 3O2 (eq 4), and
also by electron-transfer reaction between 3RB2− and RB2− to
generate semioxidized RB3•− and semireduced RB•− radicals,
which finally recombine to produce the bleaching products (eq
6).
hν 1 2 − k isc 3 2 −
RB2 − ⎯→
⎯ RB ⎯⎯⎯→ RB (3)
kq
3
RB2 − + 3O2 → RB2 − + 1O2 kq = 2 × 109M−1s−1
(4)
kox
1
O2 + RB2 − ⎯⎯⎯→ oxidized dye kox = 1.5 × 107M−1s−1
(5)
ket
3
RB2 − + RB2 − ⎯→
⎯ RB3 •− + RB•− → degraded dye
ket = 8 × 108M−1s−1 (6)
The intense photobleaching reduction of the dye associated
with the polycations can be associated with the electrostatic
repulsion effect caused by the polycation backbone excess
positive charge, preventing the diffusion-controlled electron-
transfer reaction to produce the semioxidized and semireduced
radical ions, eq 6.
RB2− is a recognized type II PS with a quantum yield of 1O2
photogeneration ΦΔ = 0.75 in aqueous media.7 Figure 3B
compares the consumption kinetics of dissolved oxygen
concentration in the presence of 9 mM FA during the green-
LED irradiation of RB2− or adduct solutions in a sealed
cuvette. FA is a water-soluble molecule used for 1O2 quantum
yield determination (ΦΔ), as it reacts with a singlet oxygen
quenching rate constant of ≈1 × 108 M−1 s−1 to produce 6-
hydroxy-(2H)-pyran-3-one, an oxidation product incorporating
an O2 molecule,34 as shown in the inset of Figure 3B.
Calculating the initial O2 consumption rate (v0 = −d[O2]0/dt)
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Figure 5. Variation of the colony-forming unit (CFU.mL−1) of planktonic S. aureus (A), E. coli (B), and C. tropicalis (C) at different photolysis
times with white LED light coupled to a 495 nm cutoff filter in the presence of Rose Bengal as a free molecule (RB2−, green●) or forming
supramolecular adducts with the polycations pVBA (orange■) and VBT-VBA (blue▲). The insets show the controls for the microbial suspensions
irradiated without the additive (◯) or only with the presence of pVBA (orange□) and VBT-VBA (blue△). (D) Variation of S. aureus survival
after 15 min of photodynamic treatment with PS solutions previously photolyzed up to 30 min in the absence of bacteria to achieve a ratio [Θ]T/
[RB2−] = 5 with polycation concentrations that do not produce strain toxicity in the dark.

for the adducts and free RB2− (the latter used as a reference
with ΦΔ = 0.75), eq 7 gives ΦΔ values of (0.40 ± 0.05) and
(0.60 ± 0.05) for (RB2−:pVBA) and (RB2−:VBT-VBA)
adducts, respectively, with ARB2−/Aadduct being the correction
factor for the absorbance changes between the sample and
reference solutions in the spectral region illuminated with the
green LED.
v0,adduct ARB2−
ΦΔ,adduct = − × × ΦΔ,RB2−
v0,RB2− A adduct (7)
The results indicate that both adducts are efficient
supramolecular PSs. Thus, the minimal photobleaching
observed in the adducts is driven by 1O2-mediated oxidation
of the dye in the adduct, eq 5.
Therefore, the high photostability and good efficiency in
singlet oxygen photogeneration for both supramolecular
adducts turn out to be interesting advantages for their use in
photodynamic applications.18
aPDA of RB2− and Adducts. Before performing aPDT
assays with Staphylococcus aureus, Escherichia coli, and Candida
tropicalis strains, the dark toxicity of the dye and polycations on
microbial strains was evaluated. Under dark conditions, the
free RB2− was not active at all concentrations tested on all
strains studied. On the other hand, both polycation
concentrations were matched to maintain the molar ratio
[Θ]T/[RB2−] = 5 in the adducts and showed some effect on
the bacterial strains but were not active on C. tropicalis. S.
aureus (Gram-positive) was more susceptible than E. coli
467
(Gram-negative) to both polycations (Figure 4). These results
indicate that the antimicrobial activity in the dark is produced
on bacteria solely by the polycations, as expected for
quaternary ammonium derivatives.35
Based on these results, to avoid the intrinsic antimicrobial
effect of polycations observed in the dark over each strain, the
aPDA experiments were conducted with supramolecular
adducts formed at concentrations of polycations below each
active threshold in the dark and keeping the ratio [Θ]T/[RB2−]
= 5, that is, corresponding to RB2− concentrations of 0.5, 1,
and 4 μM for S. aureus, E. coli, and C. tropicalis, respectively.
Figure 5A−C shows the variation of CFU·mL−1 in dilute
microbial suspensions containing free RB2− and the adducts
(RB2−:pVBA) and (RB2−:VBT-VBA), respectively, caused by
irradiation with a white LED coupled to a 490 nm cutoff filter
to selectively excite the dye. The aPDA strongly depends on
the combination of the type of PS and microbe. In the case of
S. aureus (Gram-positive), despite using the lowest dye
concentration (0.5 μM), both RB2− and adducts achieved
about 6-log inactivation after 2 min of irradiation, indicating
that this strain is highly sensitive to all PS preparations (Figure
5A). An interesting result was observed for E. coli (Gram-
negative), Figure 5B. While in the presence of 1 μM RB2−, no
photodynamic effect was observed even after 15 min of
irradiation, both (RB2−:VBT-VBA) and (RB2−:pVBA) pre-
pared with the same dye concentration were highly active in
reducing also 6-log E. coli viability after 2 and 5 min of
irradiation, respectively. In contrast, for the fungal strain, only
RB2− (4 μM) achieved more than efficient inactivation after 5
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min of irradiation, whereas both adducts produced a 1-log bound

Fem
reduction of CFU·mL−1 after 20 min of irradiation (Figure f bPS = bound free
5C). Fem + Fem (8)
Control experiments (graphs inserted in Figure 5A−C) in
the absence of RB2−confirmed that the viability of all
microorganisms was not affected by irradiation, even in the
presence of polycations, confirming that the photodynamic
effect in all cases was exerted by photoexcitation of the dye to
produce ROS. Thus, the order of susceptibility of aPDA using
RB2− was S. aureus > C. tropicalis > E. coli, while for adducts, it
was S. aureus > E. coli > C. tropicalis.
The effect of increasing the photostability of the dye in the
adducts on the photoinactivation of S. aureus was also
evaluated since all the PS preparations showed the same
aPDA profile on this strain (Figure 5A). For this purpose,
sterile 0.5 μM solutions of RB2− as a free molecule or forming
adducts at [Θ]T/[RB2−] = 5 were irradiated at different times
without the presence of bacteria. Subsequently, each dye
solution was incubated for 1 h in the dark with a suspension of
S. aureus (≈3 × 108 CFU·mL−1), and finally, the photo-
dynamic treatment was applied for 15 min. Figure 5D shows
the survival of S. aureus as a function of the photobleaching
time exposed to the dye.
For free RB2−, a recovery of the original viability of the
microorganism of 7 and 28% was observed after 15 and 30 min
of photobleaching treatment, respectively, corresponding to a
reduction of about ≤1 log CFU·mL−1. This result implies that
photobleaching of free RB2− prevents efficient aPDA after
prolonged exposure to light. In contrast, both adducts showed
a viability reduction of S. aureus of approximately 3- and 4-log Figure 6. (A) Percentage of binding of molecular and supramolecular
even after 30 min of preirradiation, in agreement with the PSs to microbial cells. (B) Rose Bengal fluorescence profiles of linear
higher photostability of the dye in the adducts (Figure 2A). ROI traced over the equatorial axis of C. tropicalis cells on
PS Binding to Microorganisms. Taken together, the epifluorescence images stained with the different PSs. Insets: (left)
abovementioned results indicate that the modulation of aPDA Representative epifluorescence and phase-contrast images of C.
is governed by the degree of interaction of the PS with the tropicalis after 1 h of dark incubation with the free RB2− solutions
microbe cells, which is highly dependent on the composition of (scale bar = 2.5 μm). (Right) Relative integrated fluorescence Frelative
calculated from the linear ROI traces.
the PS and the structure of the outer wall of the microbe.36
Given that microbial surfaces are negatively charged, positively
charged PSs have a wide activity range, being able to inactivate S. aureus showed a similar degree of binding (≈60%) to all
both Gram-positive and Gram-negative bacteria and patho- PSs, which explains the same aPDA profile obtained for this
genic yeasts.37 In addition, the porosity of the peptidoglycan bacterium in all cases. On the other hand, in the case of Gram-
wall located outside the cytoplasmic membrane of Gram- negativeE. coli, both supramolecular adducts bind almost twice
positive bacteria allows the transfer of complex nutrients with as much compared to free RB2−, which explains the increased
molecular weight between 30 and 60 kDa, making them aPDA with both adducts. In this case, the lower binding of free
equally permeable to supramolecular PSs of similar sizes. RB2− may be associated with a highly negatively charged outer
Gram-negative bacteria such as E. coli are characterized by the wall ofE. coli, which may prevent the penetration of small
presence of an additional outer membrane, composed of negative molecules.
lipopolysaccharides and negatively charged phospholipids. This In contrast, lower adduct binding was observed forC.
highly organized extra barrier is only permeable to small tropicalis compared to free RB2−, which was similar to that
hydrophilic molecules (up to ∼700 Da).38 The presence of observed forE. colibut half that ofS. aureus. The yeast cell wall
divalent cations such as Mg2+ and Ca2+ attached to adjacent has a layer of β-glucan and chitin, which confers a lower
polyanionic lipopolysaccharide molecules on the surface of the permeability than that of the Gram-positive strain.41 Therefore,
outer membrane derived the thermodynamic integrity and the highest relative binding of free RB2− to yeast cells explains
stability of the membrane.39 However, it has been reported the larger aPDA obtained as compared to both supramolecular
that polycations bind to the negatively charged surface of the adducts.
lipopolysaccharide layer, displacing the divalent cations and Furthermore, equatorial axial epifluorescence quantification
modifying the organization and permeability of the outer ofC. tropicalisimages confirmed a higher accumulation of free
membrane.40 RB2− within cells compared to both adducts, Figure 6B.
In an attempt to rationalize the aPDA behavior observed in Indeed, the (RB2−:pVBA) adduct was more accumulated at the
Figure 5A−C, the fraction of PSs bound to the microbial cells yeast cell edge than the (RB2−:VBT-VBA) adduct, suggesting
(fbPS) was calculated with eq 8 by measuring the area under the that the presence of the VBT copolymer slightly enhances the
fluorescence emission spectrum (Fem), and the percentage of penetration of the supramolecular PS, but this did not produce
binding of each PS is shown in Figure 6A. any differential effect on aPDA efficiency (Figure 5C).
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Bioconjugate Chemistry
Morphological Alterations after aPDT. The morpho-
logical alterations on the surface of microbial cells caused after
photodynamic treatments of the three strains with free RB2− or
with the adduct (RB2−:VBT-VBA) were analyzed by scanning
electron microscopy (SEM), Figure 7. In the case of untreated
Figure 7. Morphological cell modifications observed by SEM of S.
aureus, E. coli, and C. tropicalis produced after irradiation with free
RB2− and the adduct (RB2−:VBT-VBA) under the same conditions
used for the aPDA experiments. Irradiation times were 10 and 20 min
for the bacterial and yeast strains, respectively.
S. aureus (control), isolated cells were observed, in pairs or
clusters, with round shapes and well-defined contours with less
than 1 μm diameter.48 After photodynamic treatment, the
bacterial cells became highly aggregated structures. Although S.
aureus retained its round shape after photoinactivation with
free RB2−, individual contours were less evident among cells
irradiated in the presence of (RB2−:VBT-VBA). Also, the SEM
micrograph of S. aureus exposed to hypocrelin A combined
with a halogen−tungsten lamp showed gaps in the cell
membrane, and the bacteria were disorganized in several parts,
indicating that the microbial cells were damaged.42 In the case
of E. coli, both control and irradiated cells in the presence of
free RB2− showed a similar rod-like shape with uniform surface
roughness, consistent with the inefficient photodynamic action
observed using the free dye as the PS.
On the other hand, the photodynamic action produced by
the adduct caused clear damage to the surface of the Gram-
negative bacteria, which showed an irregular structure with
cleavage and vesicular alterations of the membrane. The
production of vesicles at the membrane level is associated with
a stress response.43 Similar alterations on the E. coli cellular
surface were observed after aPDT with a meso-tetra(N-methyl-
4-pyridyl)porphine tetratosylate derivate.44 In contrast, the
typically smooth, rounded surface of C. tropicalis was heavily
cracked and rougher after irradiation in the presence of free
RB2−. Similar morphological defects, such as a heterogeneous
and warty cell surface, have been described in Candida albicans
after aPDT with dimethylaminopropoxyphenyl porphyrin
derivates.45 In addition, after photoinactivation with aloe
emodin, twisted and broken surfaces were found on C. albicans
cells, suggesting damage to the cell envelope.46
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pubs.acs.org/bc Article
After phototreatment in the presence of adducts, the yeast
surface was mostly covered by vesicles of various sizes. Similar
morphological changes were also observed after exposure of C.
albicans to fungicidal concentrations of miconazole, where the
cells had smooth surfaces but were covered by vesicular
structures that would represent cytoplasmic debris derived
from ruptured cells.47
■ CONCLUSIONS
The present results on aPDA in fungal and bacterial strains
confirmed the direct relationship between microbial photo-
inactivation efficiency and specific interactions between PSs
and microbial cells, which can modify PS binding and/or
incorporation. In this case, the anionic dye RB2− was able to
form stable supramolecular adducts (Kd ≈ 5 μM) with [(p-
vinylbenzyl)triethylammonium-derived polycations] such as
pVBA and VBT-VBA, with good photosensitizing properties to
generate singlet oxygen (ΦΔ = 0.4−0.6), with the additional
advantage of enhanced dye photostability.
Depending on the microbe, differential binding and
distribution of RB2− as a free molecule or adducts were
achieved, modulating the impact of the photodynamic activity.
Gram-positiveS. aureus was the most susceptible micro-
organism to aPDT using RB2− or adducts (RB2−:pVBA and
RB 2− :VBT-VBA), probably as a result of the higher
permeability of the outer membrane of Gram-positive
bacteria.41 In contrast, free RB2− was not successful in
photoinactivation of Gram-negative E. coli due to its poor
binding to microbial cells, basically attributable to electrostatic
repulsions with the highly negatively charged and structured
cell walls. However, significant aPDA of E. coli was achieved
with both cationic adducts, due to modifications of membrane
permeability by binding of the polycations to the anionic
lipopolysaccharide layer, which allowed better fixation and
distribution of the PS in the cytoplasm or membrane of
bacterial cells, photosensitizing singlet oxygen at both sites,
which can diffuse into the cell, oxidizing the target
biomolecules and inducing cell death.48
The supramolecular strategy for photoinactivation of C.
tropicalis was less effective than treatment with free RB2− due
to the substantially smaller accumulation of adducts in fungal
cells compared to the smaller dye molecule. In addition, the
larger size ofCandidacells compared to that of bacterial cells
implies that the amount of singlet oxygen required to inactivate
the yeasts must be greater than that of bacteria, so higher doses
of light must be used.49 In such a case, the high photostability
of the present adducts may be an advantage for treatments
with prolonged irradiation times.
■
EXPERIMENTAL SECTION
Materials. All reagents were purchased from Sigma-Aldrich
in the purest form available and used as received. Two types of
polycations derived from vinylbenzyltriethylammonium chlor-
ide (VBA) were synthesized as previously described.50 The
homopolymer of VBA, with approximately 40 monomeric
units [VBACl]n≈40 (named as pVBA), and copolymer
combining VBT and VBA monomers in a 1:4 M ratio [VBT-
(VBACl)4]n≈40 (named as VBT-VBA) were used. All solutions
were prepared in 10 mM PBS solutions at pH = 7.4 using
ultrapure water.
Microorganisms and Culture Conditions. E. coli ATCC
25922 (Gram-negative), S. aureus subsp. aureus Rosenbach
https://doi.org/10.1021/acs.bioconjchem.1c00596
Bioconjugate Chem. 2022, 33, 463−472
Bioconjugate Chemistry
ATCC 29213 (Gram-positive), and C. tropicalis NCPF 3111
were used. Microbe stock cultures were suspended in glycerol
20% and trypticase soy broth (TSB) for bacteria or Sabouraud
dextrose broth (SDB) for the fungus and kept at −20 °C.
Microbial cultures were incubated aerobically at 37 °C
overnight with orbital agitation, and cells were harvested by
centrifugation for 10 min at 4500 rpm, washed twice, and
resuspended in PBS.
■ METHODS
RB2−:Polycation Adduct Characterization. The binding
isotherms of RB2− to each polycation were calculated from the
changes of the absorbance spectrum of RB2− (8−10 μM)
recorded with an Agilent 8453 spectrophotometer upon
addition of increasing concentrations of the polycation either
in ultrapure water or PBS.29 The operating molar concen-
trations of each polycation were calculated taking into account
the molecular weight of the minimum polymer block
containing four positive charges including the chloride
counteranion, for example, [VBA+Cl−]4 and [VBT(VBA+Cl−)-
4] for pVBA and VBT-VBA, respectively. The same titration
was also monitored by fluorescence changes of RB2− using an
Agilent Cary Eclipse fluorescence spectrophotometer. Binding
stoichiometry was determined also by Job’s continuous
variation method,28 by measuring the absorption spectra of
solutions with different molar ratios of [polycation]/[dye] with
the same final molarity (30 μM).
Photostability studies of RB2− as a free molecule and
supramolecular adducts at the molar ratio of polycation/dye of
1:5 were performed in air-saturated PBS by irradiation with a
green LED (Luxeon, 1 W, emission at 523 ± 20 nm) in a 1 cm
quartz cuvette under magnetic stirring.
The quantum yield of singlet oxygen formation (ΦΔ) was
determined by measuring the depletion of dissolved molecular
oxygen in PBS by reaction with FA (9 mM) with a Clark-type
microelectrode.34 The value = 0.75 for RB2− in air-saturated
water was used as a reference.51 Corrections were made due to
differences in the absorption of green LED light between RB2−
and the dye−polymer adduct solution.
aPDT Assays. The highest nonactive concentration of
compounds that can be used under dark conditions without
generating antimicrobial activity was determined in microbial
cell suspensions with an optical density of 620 nm, OD620 =
0.1, in 96-well plates (100 μL/well) and mixed with 100 μL of
treatment solutions. Microbial suspensions without added
compounds served as controls. Microplates were incubated at
37 °C in the dark for 1 h with orbital agitation. Then, 10 μL of
10−1−10−6 dilutions of treated and control microbial
suspensions were plated on trypticase soy agar (TSA) for
bacterial strains and Sabouraud dextrose agar (SDA) for C.
tropicalis. Finally, plates were incubated at 37 °C for 24 h for
assessing microbial growth.
For the aPDA tests, microbial suspensions (100 μL of 106
CFU.mL−1) in TSB for bacteria or SDB for fungus were
incubated in a microplate under dark conditions for 1 h at 37
°C with 100 μL of the corresponding PS solution. The amount
of polycation and dye utilized were below the concentration
threshold to induce dark toxicity. Microbial suspensions
treated with the same concentration of each polycation
without RB2− were included as controls. In all cases, the
microplate was irradiated using a homemade illuminator
constructed with a white LED (100 W) as the light source
and coupled to a 490 nm cutoff filter to produce an incident
470
pubs.acs.org/bc Article
irradiance of 13 mW/cm2. Aliquots (20 μL) with and without
PS were taken at different irradiation times (up to 20 min), and
serial 1:10 dilutions in PBS (10 μL) were placed in the
corresponding agar culture (TSA or SDA) and incubated
overnight at 37 °C, and finally, the number of colony-forming
units (CFUs) per mL was counted.52 Cell-free solutions of
PBS, polycations, and PS-polycation adducts were also plated
as a sterility control.
The effect of dye photobleaching on aPDA assays was
analyzed by prephotolysis with a homemade illuminator during
5, 15, and 30 min with 0.5 μM RB2− in the absence and
presence of polycations. Subsequently, photolyzed and non-
photolyzed solutions were incubated with a suspension ofS.
aureus (≈2 × 108 CFU.mL−1) for 1 h in the dark and irradiated
during 15 min. The efficiency of the aPDT treatment was
evaluated by CFU·mL−1 counting as described above.53
PS Binding to Microorganisms. A 250 μL aliquot of
microbial suspension (≈106−107 CFU.mL−1) was mixed with
250 μL of PS solutions at the same concentration used for each
strain as in the aPDT assays. After 1 h of incubation in the dark
at 37 °C with orbital shaking, the cell suspensions were
harvested by 10 min of centrifugation at 10,000 rpm. Microbial
pellets were washed twice with PBS, and quantification of
unbound and bound RB2− was performed by measuring the
fluorescence of the dye in the supernatant and the resuspended
pellet in 500 μL of PBS, with excitation and emission slits at 10
nm (λexc = 545 nm; λem = 560−660 nm) using the Cary Eclipse
apparatus.
The binding of RB2− to C. tropicalis cells, either as a free
molecule or adducts, was additionally studied by epifluor-
escence microscopy. An overnight culture of the fungus was
harvested at room temperature for 20 min at 10,000 rpm and
washed with PBS up to OD620 ≈ 0.3. Cell suspensions were
incubated for 1 h at 37 °C with the PS solution protected from
light. Then, 10 μL of the suspensions was placed onto a glass
slide with a coverslip and analyzed with a Carl Zeiss Axio-
Observer Z1 inverted microscope. Images were acquired using
a filter Set 43 HE (excitation 550/25; emission 605/70) and
phase contrast. Different fields were analyzed for each sample
condition, comprising a total number of 61 cells incubated
only with RB2− and about 109 and 120 cells with the adducts
RB2−:pVBA and RB2−:VBT-VBA. Fluorescence images were
analyzed with free ImageJ software.54 The regions of interest
(ROI) of 8 μm length were plotted along the equatorial axis of
the cells, and fluorescence intensity plots were obtained, and
the mean values of the area under the curve (AUC) were
calculated and normalized to the value obtained for the cell
stained only with RB2−.
Scanning Electron Microscopy (SEM). SEM images of
microbial suspensions before and after aPDT experiments
treated with RB2− and the adduct RB2−:VBT-VBA were
obtained. Cells were harvested by centrifugation for 8 min at
10,000 rpm, washed with PBS, and fixed in Karnovsky solution
(4% glutaraldehyde + 2.5% paraformaldehyde in 10 mM
sodium cacodylate buffer, pH 7.4) for 24 h.55 Dehydration was
carried out in consecutive stages starting with 30% ethanol
solution for 10 min, until reaching 100% ethanol. After that,
acetone was added for 10 min, and the drying critical point was
processed with CO2 (Denton Vacuum equipment model DCP-
1). Finally, the samples were mounted on an aluminum stub
and coated with gold in a JEOL Ion Sputter, model JFC-1100.
Samples were observed with a scanning electron microscope
(SEM Phenom ProX).
https://doi.org/10.1021/acs.bioconjchem.1c00596
Bioconjugate Chem. 2022, 33, 463−472
Bioconjugate Chemistry
pubs.acs.org/bc
All statistical analysis of biological assays was performed by
two-way ANOVA using GraphPad Prism software, over data
obtained from at least two independent experiments performed
in duplicate.
■
ASSOCIATED CONTENT
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.bioconjchem.1c00596.
UV−vis absorption and fluorescence emission spectra
changes of Rose Bengal by adding vinylbenzyltriethy-
lammonium chloride (pVBA) in ultrapure water
solutions, UV−vis absorption and fluorescence emission
spectra changes of Rose Bengal by adding the copolymer
formed with vynylbenzylthymime and vinylbenzyltrie-
thylammonium chloride (VBT-VBA) in PBS solution,
and binding isotherm plots obtained of RB2− to pVBA or
VBT-VBA in PBS (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
Cecilia Vera − Instituto de Bionanotecnolgía del NOA
(INBIONATEC), CONICET, Universidad Nacional de
Santiago del Estero (UNSE), Santiago del Estero
G4206XCP, Argentina; Email: cevera90@gmail.com
Claudio D. Borsarelli − Instituto de Bionanotecnolgía del
NOA (INBIONATEC), CONICET, Universidad Nacional
de Santiago del Estero (UNSE), Santiago del Estero
G4206XCP, Argentina; Facultad de Agronomía y
Agroindustrias. UNSE, Santiago del Estero G4200,
Argentina; orcid.org/0000-0003-0120-645X;
Phone: +54-385-155-951531; Email: cdborsarelli@
gmail.com
Authors
Mauro N. Gallucci − Instituto de Bionanotecnolgía del NOA
(INBIONATEC), CONICET, Universidad Nacional de
Santiago del Estero (UNSE), Santiago del Estero
G4206XCP, Argentina
Juliana Marioni − CONICET, Instituto Multidisciplinario de
Biología Vegetal (IMBIV), Cordoba X5000HUA, Argentina
Marcelo C. Sosa Morales − Instituto de Bionanotecnolgía del
NOA (INBIONATEC), CONICET, Universidad Nacional
de Santiago del Estero (UNSE), Santiago del Estero
G4206XCP, Argentina
Debora M. Martino − Instituto de Física del Litoral (IFIS
Litoral), CONICET, Universidad Nacional del Litoral
(UNL), Santa Fe S3000GLN, Argentina
Susana Nuñez Montoya − CONICET, Instituto
Multidisciplinario de Biología Vegetal (IMBIV), Cordoba
X5000HUA, Argentina; Universidad Nacional de Córdoba,
Facultad de Ciencias Químicas, Dpto. Cs. Farmacéuticas,
Córdoba X5000HUA, Argentina; orcid.org/0000-0002-
2030-0890
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.bioconjchem.1c00596
Notes
The authors declare no competing financial interest.
471
■
Article
ACKNOWLEDGMENTS
We thank the following Argentinean Institutions for financial
support: Universidad Nacional de Santiago del Estero (UNSE,
grant 23A/254), Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET, grants PUE-2018-035
and PIP-2020-101043CO), and Fondo para la Investigación
Científica y Tecnológica (FONCyT grants PICT-2019-02052,
PICT 2016-1697 and PICT 2018-0476) and SeCyT-UNC,
Proyecto Consolidar 2018-2021. We also thank Dr. Jorge
Gómez Rojas (INBIONATEC) for technical assistance in the
microscopy experiments.
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https://doi.org/10.1021/acs.bioconjchem.1c00596
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