Synthesis, characterization and in vitro photodynamic antimicrobial activity of basic amino acid-porphyrin conjugates

Article abstract of DOI:10.1088/0957-4484/21/6/065102

Photodynamic antimicrobial chemotherapy (PACT), as a novel and effective modality for the treatment of infection with the advantage of circumventing multidrug resistance, receives great attention in recent years. The photosensitizer is the crucial element in PACT, and cationic porphyrins have been demonstrated to usually be more efficient than neutral and negatively charged analogues towards bacteria in PACT. In this work, three native basic amino acids, l-lysine, l-histidine and l-arginine, were conjugated with amino porphyrins as cationic auxiliary groups, and 13 target compounds were synthesized. This paper reports their syntheses, structural characterizations, oil-water partition coefficients, singlet oxygen generation yields, photo-stability, as well as their photo inactivation efficacies against methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli and Pseudomonas aeruginosa in vitro. The preliminary structure-activity relationship was discussed. Compound 4i, with porphyrin bearing four lysine moieties, displays the highest photo inactivation efficacy against the tested bacterial strains at 3.91 1/4M with a low light dose (6 J/cm²), and it is stable in serum and lower cytotoxicity to A929 cells. These basic amino acid-porphyrin conjugates are potential photosensitizers for PACT.

Full text of DOI:10.1088/0957-4484/21/6/065102

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Rose Bengal-decorated silica nanoparticles as photosensitizers for inactivation of gram-
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IOP PUBLISHING
NANOTECHNOLOGY
Nanotechnology 21 (2010) 065102 (7pp)
doi:10.1088/0957-4484/21/6/065102
Rose Bengal-decorated silica nanoparticles
as photosensitizers for inactivation of
gram-positive bacteria
Yanyan Guo1, Snezna Rogelj2 and Peng Zhang1,3
1
Department of Chemistry, New Mexico Tech, Socorro, NM 87801, USA
Department of Biology, New Mexico Tech, Socorro, NM 87801, USA
2
E-mail: pzhang@nmt.edu
Received 11 August 2009, in final form 21 December 2009
Published 11 January 2010
Online at stacks.iop.org/Nano/21/065102
Abstract
A new type of photosensitizer, made from Rose Bengal (RB)-decorated silica (SiO2–NH2–RB)
nanoparticles, was developed to inactivate gram-positive bacteria, including
Methicillin-resistant Staphylococcus aureus (MRSA), with high efficiency through
photodynamic action. The nanoparticles were characterized microscopically and
spectroscopically to confirm their structures. The characterization of singlet oxygen generated
by RB, both free and immobilized on a nanoparticle surface, was performed in the presence of
anthracene-9,10-dipropionic acid. The capability of SiO2–NH2–RB nanoparticles to inactivate
bacteria was tested in vitro on both gram-positive and gram-negative bacteria. The results
showed that RB-decorated silica nanoparticles can inactivate MRSA and Staphylococcus
epidermidis (both gram-positive) very effectively (up to eight-orders-of-magnitude reduction).
Photosensitizers of such design should have good potential as antibacterial agents through a
photodynamic mechanism.
(Some figures in this article are in colour only in the electronic version)
towards the development of alternative approaches for the
treatment and prevention of MRSA infections.
1. Introduction
Staphylococci are spherically shaped gram-positive bacteria
that are usually arranged in grape-like microscopic clusters.
Although more than 20 species of Staphylococcus exist,
Staphylococcus aureus and Staphylococcus epidermidis are
two of the most significant species as they are involved
in a large number of human-related diseases. Although S.
epidermidis is usually non-pathogenic, it can cause infections
in patients with a compromised immune system or a long-term
indwelling catheter. S. aureus is pathogenic, causing a wide
variety of infections especially in burn wound patients. In
particular, Methicillin-resistant strains of S. aureus (MRSA),
which are resistant to all but one or two antibiotics, are one
of the major causes of hospital-acquired infections causing
significant infections and morbidity world-wide. An increasing
concern about the growing resistance of MRSA to conventional
antimicrobial agents is leading to tremendous efforts aimed
Photodynamic therapy (PDT) has been identified as one
of the viable approaches for bacterial photoinactivation [1–3].
First introduced in the 1990s to treat cancers [4–6], PDT
involves the delivery of lethal drugs to the targets (tumors or
microbes) by the combination of a light-activatable chemical
(photosensitizer), light, and oxygen [7–9]. Upon exposure to
illumination of appropriate wavelengths, the photosensitizer
is excited from a lower-energy ‘ground state’ to a higher-
energy ‘triplet state’, which can then react with molecular
oxygen in the surroundings, generating reactive oxygen
species (ROS) [10]. Such ROS, and singlet oxygen in
particular, can cause damage to the plasma membranes and
DNA, eventually leading to cell death [11, 12]. Examples of
photodynamic inactivation of various gram-positive and gram-
negative bacteria, such as S. aureus [13–16], Streptococcal
species [17, 18], Escherichia coli [17], Porphyromonas
gingivalis [19], and Pseudomonas aeruginosa [20], have been
documented in the literature. The advantage of using PDT for
3
Author to whom any correspondence should be addressed.
0957-4484/10/065102+07$30.00
1
© 2010 IOP Publishing Ltd Printed in the UK

Nanotechnology 21 (2010) 065102
Y Guo et al
treating and controlling MRSA infections over conventional by centrifuging at 14 000 rpm for 10 min, and washed with
antimicrobials lies in the fact that MRSA is unlikely to develop acetone three more times and twice with dionized (DI) water.
resistance to photochemically induced killing, which, among The nanoparticles were finally dispersed in DI water.
other ROS, is mediated predominantly by singlet oxygen [21].
The synthesis and development of photosensitizer, the key
The silica surface was functionalized with amine groups
by the following procedures. 10 mg of silica nanoparticles
element in effective PDT, has drawn tremendous academic were dispersed into 20 ml of isopropyl alcohol, and the mixture
and industrial interest in recent years. For antimicrobial
applications, a good photosensitizer should ideally possess
such features as: (1) high quantum yield of generating singlet
oxygen or other ROS; (2) minimal or no dark toxicity, and
(3) good specificity or selectivity towards the target(s).
The object of the present study was to develop
nanoparticle-based photosensitizers that would display high
efficacy in inactivating a group of bacteria under in vitro
was sonicated for 30 min. Next, 1 ml of NH4OH (29.6 wt%)
was added into the mixture under stirring for 20 min. 5 μl
of APTS was subsequently added under stirring. Amine-
functionalized silica (SiO2–NH2) nanoparticles were collected
via centrifugation after 3 h. Alternatively, the silica surface
could be functionalized with amine groups directly in one-
pot during the synthesis of the nanoparticles. In that case,
before breaking the microemulsion during the synthesis with
ethanol, 5 μl of APTS was added to the microemulsion while
stirring and was further incubated overnight. The SiO2–NH2
nanoparticles were then recovered by adding ethanol to break
the microemulsion and centrifuging, followed by rigorous
washing with acetone and DI water.
The conjugation of RB to the SiO2–NH2 nanoparticles
was carried out as follows. 10 μl of RB solution (1.6 mM)
was added to 3 ml of MES buffer (0.1 M, pH 6.0), followed
by adding 5–8 mg of EDC into the mixture. The RB–
EDC conjugation reaction was allowed to proceed at room
temperature for 20 min. Separately, 1 ml (∼12 mg ml−1) of
SiO2–NH2 nanoparticles were washed twice with 1 ml of the
above MES buffer. After the second wash, the pellet was re-
dispersed in 2 ml of MES buffer. Subsequently, the SiO2–NH2
nanoparticle dispersion and RB solution were combined under
stirring for 3 h at room temperature. The mixture was then
centrifuged and pellet washed with DI water. After the
third wash, the pellet of SiO2–NH2–RB nanoparticles was re-
dispersed in 1 ml DI water and was ready for use.
conditions.
We found silica nanoparticles decorated
with Rose Bengal (RB), a well-known photosensitizing
molecule [22, 23], to be highly efficient in inactivating
gram-positive bacteria, MRSA. and S. epidermidis, through
photodynamic action. The results show promise for these
nanoparticles to be tested under in vivo conditions.
2. Experimental details
2.1. Chemicals and materials
Rose Bengal (4,5,6,7-tetrachloro-2ꢀ,4ꢀ,5ꢀ,7ꢀ-tetraiodofluoresce
in disodium salt) (RB), tetraethyl orthosilicate (TEOS), and
3-aminopropyltriethoxysilane (APTS) were purchased from
Sigma-Aldrich. 2-(4-morpholino)-ethane sulfonic acid (MES),
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (EDC),
Triton X-100, ammonium hydroxide (29.6 wt%), cyclohexane,
n-hexanol, isopropyl alcohol, LB Broth, and LB Agar
were purchased from Fisher Scientific.
of 9,10-anthracenedipropionic acid (ADPA) was purchased
from Invitrogen. Phosphate buffered saline (PBS) was
purchased from GIBCO. All chemicals were used as-received
without further purification. The bacteria used in this
Disodium salt
2.3. TEM characterization of SiO2–NH2–RB nanoparticles
A drop of nanoparticle suspension was deposited on a Formvar-
covered carbon-coated copper grid, and allowed to dry at room
temperature. TEM images were taken on a JEOL 2010 high
resolution transmission electron microscope.
study were MRSA (gram-positive, ATCC No. BAA-44),
and S. epidermidis. (gram-positive, ATCC No. 35984).
Transmission electron microscopy (TEM) grids were from
Electron Microscopy Sciences, PA.
2.4. IR characterization of SiO2–NH2–RB nanoparticles
2.2. Synthesis and characterization of Rose Bengal-decorated
silica nanoparticles
A drop of nanoparticle suspension in ethanol was deposited
on a plate of NaCl, and allowed to dry at room temperature.
Infrared absorption spectra were taken on a Nicolet Nexus
8700 Fourier transform infrared (FTIR) spectrometer.
The Rose Bengal-decorated silica nanoparticles (denoted as
SiO2–NH2–RB hereafter) were prepared in three steps. First,
pure SiO2 nanoparticles were synthesized by hydrolysis of
TEOS in reverse microemulsion. Second, the silica surface was
functionalized with amine groups. Lastly, RB dye molecules
were covalently conjugated to the silica surface. To begin with,
1.77 g of Triton (X-100) was mixed with 1.6 ml of n-hexanol,
7.5 ml of cyclohexane, and 480 μl of deionized water under
vigorous stirring. After the solution became transparent, 60 μl
2.5. Measurement of singlet oxygen (1O2)
The detection of singlet oxygen, generated by free RB dye in
solution, was similar to had been described previously [24]. In
brief, 10 μl of 1.6 mM RB solution and 3 ml of 5 μM ADPA
solution were mixed in a cuvette under stirring, and placed onto
of ammonium hydroxide (29.6 wt%) was added to the solution. a Photon Technology International (PTI) spectrofluorometer.
The solution was subsequently sealed and stirred for 20 min, The fluorescence intensity of ADPA at 400 nm, when excited
followed by adding 100 μl of TEOS and stirring for 24 h. A at 374 nm, was recorded. The solution was then irradiated at
large amount of ethanol (∼20 ml) was then added to break 525 nm for 2 min, and another reading at 400 nm (excited at
the microemulsion. Silica nanoparticles were then recovered 374 nm) was taken. The irradiation/measurement cycle was
2

Nanotechnology 21 (2010) 065102
Y Guo et al
repeated for 20–30 min. The intensity of the 525 nm light
from the xenon lamp associated with the spectrofluorometer
and used to irradiate the solution was 60 μW, as measured
by a power meter (SPER Scientific Laser Power Meter
840011). The measurement of singlet oxygen generated by
the SiO2–NH2–RB nanoparticles was carried out in a similar
manner, with the concentration of RB on the nanoparticles kept
the same as that in the case of free RB solution.
2.6. Bacterial culture
Bacteria were grown in sterile LB broth in an orbital shaker at
37 ◦C. Following a 20 h incubation period, the bacteria were
grown to ∼108 CFU ml−1 and confirmed by a colony count.
Bacteria were washed twice with PBS solution before they
were used in photosensitization tests.
Figure 1. Schematic of the design of SiO2–NH2–RB nanoparticles.
2.7. In vitro photosensitization tests
10 μl of bacterial suspension (∼108 CFU ml−1) and 20 μl
of SiO2–NH2–RB (∼1013 NPs ml−1, 6 mg ml−1) were added
to 70 μl of LB broth, and then the suspension incubated at
37 ◦C for 30 min with shaking. The following control groups
were treated in a similar manner: bacteria only, bacteria treated
with pure SiO2 nanoparticles, bacteria treated with NH2-
functionalized SiO2 nanoparticles (SiO2–NH2) and bacteria
treated with free RB solution. All tests were performed in
duplicate.
photosensitizers in solution. Therefore, in this study, we chose
silica nanoparticles as the carriers for the photosensitizers.
Silica nanoparticles are generally synthesized by Sto¨ber’s
sol–gel method [25, 26], where the alkoxysilane compounds,
such as TEOS and tetramethyl orthosilicate (TMOS), hy-
drolyze under basic or acidic conditions. The subsequent con-
densation reaction forms a stable alcosol in an ammonia/HCl–
ethanol–water mixture.
However, in this study, silica
nanoparticles were synthesized by the controlled hydrolysis of
TEOS in reverse microemulsion systems, which allows for the
subsequent NP surface functionalization with amine groups to
be carried out in a one-pot reaction.
A light source (Lumacare, LC122A, MBG Technologies
Inc., Newport Beach, CA) with a 525 nm bandpass filter,
which has a measured ∼14 mW cm−2 output (equivalent to 1×
10−4 einstein m−2 s−1), was used for illumination. During the
lamp illumination period, all samples were placed on ice so as
to slow down the growth of bacteria and avoid any overheating.
The surviving fraction of the bacteria was characterized by
a colony count. The colony count was performed by serial
dilution and a spread plate. Each sample was done in duplicate
and each experiment was repeated three times. The average
CFU value was calculated.
Microemulsions are transparent solutions formed by spon-
taneously mixing oil and water with appropriate surfactants,
sometimes with the assistance of a cosurfactant [27, 28].
These systems comprise a large number of oil-in-water (o/w)
or water-in-oil (w/o) droplets of uniform nanometer sizes.
By controlling the reactions that take place within such
droplets, one can synthesize monodisperse nanoparticles using
microemulsions. Since the hydrolysis of TEOS takes place in
the aqueous phase, reverse (w/o) microemulsions were adopted
in this study.
3. Results and discussion
The photosensitizing molecule used in this study is
3.1. Design of nanoparticles
4,5,6,7-tetrachloro-2ꢀ,4ꢀ,5ꢀ,7ꢀ-tetraiodofluorescein (Rose Ben-
Silica-based nanoparticles function as good carriers for the gal, RB), a widely used anionic photosensitizing molecule with
delivery of photosensitizers due to their following attractive a good quantum yield of singlet oxygen generation4. The
features. First, silica nanoparticles are water dispersible. covalent attachment of RB to the silica nanoparticle surface
Second, they are chemically and photodynamically stable. was realized through the conjugation between the carboxylic
groups (COO−) of the RB dye molecules and the amine groups
Third, the silica surface can be easily modified with
(–NH2) pre-functionalized on the nanoparticle surface. The
schematic diagram of the resulting SiO2–NH2–RB nanoparti-
cle is shown in figure 1.
different functional groups.
Various types of target-
recognition molecules can then be effectively decorated onto
the silica nanoparticle surface following well-known and facile
conjugation chemistry. Moreover, silica nanoparticles are
transparent and usually do not alter the spectral characteristics
of the photosensitizers. Finally, and equally importantly,
silica nanoparticles help decrease the self-quenching of the
photosensitizers, possibly by immobilizing and appropriately
spacing the dye molecules. This results in higher photostability
of the immobilized photosensitizers as compared to free
3.2. Characterization
A TEM image of the SiO2–NH2–RB nanoparticles is shown
in figure 2(a). The results showed that the diameters of most
4
Fluorescein, 3,4,5,6-tetrachloro-2ꢀ,4ꢀ,5ꢀ,7ꢀ-tetraiodo-, dianion (Rose Bengal
dianion, RB). Available from [29].
3

Nanotechnology 21 (2010) 065102
Y Guo et al
(a)
(b)
Figure 2. (a) TEM image of SiO2–NH2–RB nanoparticles. (b) IR spectra of SiO2–NH2–RB nanoparticles (dot line) and free RB solution
(solid line).
Figure 3. Excitation and emission spectra of free RB solution and
SiO2–NH2–RB nanoparticles.
Figure 4. Change of ADPA fluorescence due to singlet oxygen
generated by free RB dye and SiO2–NH2–RB nanoparticles under
illumination.
of the particles were in the range of 50–80 nm. The covalent
binding between RB and the SiO2–NH2 nanoparticle surface
1
detect the generation of O2 from the photosensitizers. Note
was confirmed by IR measurement as shown in figure 2(b). that there was no interference between the wavelength used
The sharp band around 1190 cm−1 in the SiO2–NH2–RB to excite ADPA (374 nm) and that used to illuminate the
nanoparticles could be assigned to the C–N bond between RB photosensitizer (525 nm).
and the surface amine group. The excitation and emission
It has been shown in previous reports that the intensity
spectra of RB are shown in figure 3. There were two decrease of ADPA emission follows an exponential decay over
excitation peaks at wavelengths around 508 and 546 nm. Their time, as ADPA is being quenched by the generated singlet
emission was observed at around 562 nm. SiO2–NH2–RB oxygen. The data points in figure 4 could be fitted into
displayed similar excitation and emission bands as free RB exponential decay functions, suggesting that the kinetics of the
dye in solution. Through back titration using ultraviolet– singlet oxygen generation of both free RB and RB-decorated
visible (UV–vis) absorption, we estimated that ∼50% of RB nanoparticles is very similar to that of other silica-based
added to the conjugation mix had actually attached to the silica nanoparticles described in the literature [24, 30, 31]. It can
nanoparticles.
be seen from figure 4 that the fluorescent intensity of ADPA
The characterization of RB, free or decorated on SiO2 initially decreased faster for free RB than for SiO2–NH2–RB.
nanoparticles, as a source of singlet oxygen (1O2) under This indicates that free RB has a higher quantum yield of
illumination, was performed through the photobleaching of generating singlet oxygen than SiO2–NH2–RB in the first
ADPA in aqueous solution. When the ∼375 nm-absorbing few minutes of irradiation. The quantum yield of generating
1
ADPA molecule reacts with O2 to form endoperoxide, its singlet oxygen of free RB was reported to be approximately
fluorescence at 400 and 420 nm decreases. By monitoring the 0.75 in H2O and air (see footnote 4).
The quantum
disappearance of the ADPA fluorescence, one could indirectly yield of generating singlet oxygen of SiO2–NH2–RB was
4

Nanotechnology 21 (2010) 065102
Y Guo et al
thus determined to be approximately 0.6, using ꢀꢁ(U) =
ꢀꢁ(St) × S(U)/S(St) [32], where St represents the standard,
U the unknown, ꢀ the quantum yield, and S the slope
of the linear fit for the initial data points. This value for
SiO2–NH2–RB NPs is higher than 0.43 that was reported
earlier for RB bound to micron-size polymer beads [33].
The higher quantum yield of generating singlet oxygen using
SiO2–NH2–RB nanoparticles, as compared to the polymer-
based RB microparticles, is probably due to the smaller
SiO2–NH2–RB particle size, leading to more surface area and
easier access of RB to the molecular oxygen present in the
solution.
3.3. Bactericidal action of SiO2–NH2–RB
Figure 5. Viability count of gram-positive bacteria treated with
SiO2–NH2–RB nanoparticles and controls. (a) S. epidermidis;
(b) S. epidermidis + SiO2–NH2; (c) S. epidermidis + RB;
(d) S. epidermidis + SiO2–NH2–RB; (e) S. aureus; (f) S. aureus +
SiO2–NH2; (g) S. aureus + RB; (h) S. aureus + SiO2–NH2–RB.
The use of free RB solution in photodynamic inactivation
of a number of bacterial species had long been reported
in the literature [23, 34]. It was found that some gram-
positive bacteria (Bacillus subtilis, S. aureus, Streptococcus
faecalis and Streptococcus salivarius) were inactivated by free
RB dye ∼200 times more quickly than the gram-negative
Salmonella typhimurium. RB immobilized on polystyrene
beads also displayed photodynamic inactivation of E. coli [35]
with a typical ∼99.99% killing efficiency after 1–2 h of
illumination depending on the conditions. This suggested that
the penetration of the photosensitizer molecules into the cell’s
interior may be not necessary for triggering of the bacteria
inactivating mechanism.
In our study, the in vitro photodynamic inactivation
of SiO2–NH2–RB nanoparticles on gram-positive bacteria
has been investigated, using MRSA and S. epidermidis
as the models for gram-positive bacteria. Gram-positive
bacteria were treated with SiO2–NH2–RB nanoparticles and
various controls, followed by 40 min of light illumination.
The controls included bacteria only, bacteria treated with
SiO2–NH2, and bacteria treated with free RB dye. As
shown in figure 5, it appeared that both S. epidermidis and
S. aureus could survive in the presence of amine-functionalized
silica nanoparticles (SiO2–NH2), when the same dosage
of SiO2–NH2 nanoparticles was used as compared with
SiO2–NH2–RB nanoparticles (∼105 NPs/bacterium). Free
RB showed good killing efficiency (approximately six-orders-
of-magnitude reduction in the viable count) on gram-positive
bacteria.
More significantly, SiO2–NH2–RB were shown to be more
potent than free RB in inactivating the gram-positive bacteria,
with an additional improvement in killing efficiency: a two-
orders-of-magnitude reduction in the viability count. This
is an intriguing result, considering that the quantum yield
of generating 1O2 of SiO2–NH2–RB nanoparticles is lower
than that of the free RB and that the amount of RB in
SiO2–NH2–RB nanoparticles used in this set of experiments
is only approximately half of that in the free RB solution. We
suggest that this could be due to the higher localization of RB
to the cell surface when attached to the SiO2 nanoparticles, as
compared to free RB in solution. The locally concentrated
1O2 generated by the SiO2–NH2–RB nanoparticles would
likely be more efficient in causing damage to the bacteria
even though free RB in solution may generate overall more
1O2. This is consistent with the previous observation that
the protoporphyrin IX-loaded silica particles showed a higher
efficiency of singlet oxygen generation than the corresponding
free porphyrins [38]. The result demonstrates the advantages
of nanoparticle-based photosensitizers over the corresponding
free photosensitizing molecules in solution.
3.4. Effects of SiO2–NH2–RB dosage and illumination time
We noticed that the free RB solution in our study ([RB]
∼3 μM) displayed significantly higher efficiency in killing
gram-positive bacteria than reported in the literature [34]
([RB] ∼5 μM; less than two-orders-of-magnitude reduction
in the viable count; white light illumination of 1.1 ×
The effects of the SiO2–NH2–RB nanoparticle dosage and
the illumination time were investigated. The same number
of gram-positive bacteria were either treated with different
amounts of SiO2–NH2–RB under a constant illumination time
or treated with the same amounts of SiO2–NH2–RB while
varying the illumination time. Control bacterial groups were
treated in a similar manner. Ambient light was completely
shielded throughout the treatment until the lamp illumination
step. The results, shown in figures 6 and 7, indicated that
for 10 μl of bacterial suspensions (∼108 CFU ml−1) under
10−3 einstein m−2 s−1). Such improved killing efficiency of
RB observed in our experiments was probably due to the
higher illumination intensity of our light source (∼33 J cm−2
in 40 min of 525 nm illumination, equivalent to 1 ×
10−4 einstein m−2 s−1). We also note that, compared to
other reports using SnCe6 as the photosensitizer and a similar a 525 nm light source of ∼14 mW cm−2, approximately
illumination intensity (21 J cm−2 in 5 min exposure, four– 6 mg ml−1 of SiO2–NH2–RB nanoparticles and 40 min
five-orders-of-magnitude reduction in the viability count of exposure would be required to achieve an eight-orders-of-
MRSA) [36, 37], SiO2–NH2–RB nanoparticles again show magnitude reduction in the viability count. This corresponds
superior killing efficacy.
to a ratio of approximately 105 nanoparticles per bacterium.
5

Nanotechnology 21 (2010) 065102
Y Guo et al
some molecular dyes that are otherwise insoluble in water,
can still be used as photosensitizers in aqueous media.
Furthermore, if the nanoparticle surfaces can be modified
to become positively charged, or be decorated with target-
recognition elements [39], they can become more effective in
photodynamically inactivating gram-negative bacteria or more
specific toward certain targets. Efforts in this direction are
currently underway.
Acknowledgment
This work was partially supported by grant number RR-016480
from the National Center for Research Resources (NCRR) of
the National Institutes of Health (NIH).
Figure 6. Viability count of gram-positive bacteria treated with
different dosages of SiO2–NH2–RB nanoparticles, under a 525 nm
light source of 14 mW cm−2 for 40 min illumination.
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In summary, we report the development of a new type
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dermidis through photodynamic action. The advantages of
these nanoparticle-based photosensitizers over the free pho-
tosensitizing molecules include the following: (1) association
of the dyes with NPs makes the dyes more resistant toward
photobleaching; (2) by concentrating the photosensitizing
molecules onto the nanoparticle surface, the locally generated
singlet oxygen may reach a higher concentration than when
free molecules act individually or in solution; this causes
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