molecules

Review
Revisiting Current Photoactive Materials for
Antimicrobial Photodynamic Therapy
Mariana Q. Mesquita 1,2,† , Cristina J. Dias 1,† , Maria G. P. M. S. Neves 1, * ,
Adelaide Almeida 3, * and M. Amparo F. Faustino 1, *
1 Department of Chemistry and QOPNA, University of Aveiro, 3810-193 Aveiro, Portugal;
marianamesquita@ua.pt (M.Q.M.); cristina.jesus.dias@ua.pt (C.J.D.)
2 Department of Biomedical Sciences and iBiMED, University of Aveiro, 3810-193 Aveiro, Portugal
3 Department of Biology CESAM, University of Aveiro, 3810-193 Aveiro, Portugal
* Correspondence: gneves@ua.pt (M.G.P.M.S.N.); aalmeida@ua.pt (A.A.); faustino@ua.pt (M.A.F.F.);
Tel.: +351-234-401-406 (M.A.F.F.)
† These authors contributed equally to this work.

Academic Editors: Scott Reed and Jung-Jae Lee




Received: 2 August 2018; Accepted: 18 September 2018; Published: 21 September 2018

Abstract: Microbial infection is a severe concern, requiring the use of significant amounts
of antimicrobials/biocides, not only in the hospital setting, but also in other environments.
The increasing use of antimicrobial drugs and the rapid adaptability of microorganisms to these
agents, have contributed to a sharp increase of antimicrobial resistance. It is obvious that the
development of new strategies to combat planktonic and biofilm-embedded microorganisms is
required. Photodynamic inactivation (PDI) is being recognized as an effective method to inactivate a
broad spectrum of microorganisms, including those resistant to conventional antimicrobials. In the
last few years, the development and biological assessment of new photosensitizers for PDI were
accompanied by their immobilization in different supports having in mind the extension of the
photodynamic principle to new applications, such as the disinfection of blood, water, and surfaces.
In this review, we intended to cover a significant amount of recent work considering a diversity of
photosensitizers and supports to achieve an effective photoinactivation. Special attention is devoted
to the chemistry behind the preparation of the photomaterials by recurring to extensive examples,
illustrating the design strategies. Additionally, we highlighted the biological challenges of each
formulation expecting that the compiled information could motivate the development of other
effective photoactive materials.

Keywords: antimicrobial photodynamic therapy; photosensitizers; drug delivery; nanoparticles;

silica; chitosan; cellulose; liposomes; hydrogels; nanotubes

1. Introduction
Nowadays bacterial infections are still considered a severe burden worldwide, an issue that
unfortunately tends to increase, not only in the clinic area, but also in other areas. This type of
infection is one of the most serious causes of mortality of patients with traumatic lesions or after being
subjected to surgeries, and also of animals, namely farmed ones [1,2]. Microorganisms replicate very
fast and a mutation that benefits a microbe survival in the presence of an antimicrobial drug will
rapidly become prevalent throughout the population. The inappropriate prescription of antimicrobials
or their overuse exacerbates the problem [3]. The formation of biofilms is also responsible for an
increase in the level of microbial resistance [4]. In fact, several microorganisms, such as bacteria,
fungi, green algae, cyanobacteria, and lichen, possess the ability to grow in biofilm form and they are
usually incorporated in a matrix of extracellular polymeric substances (EPS) auto-produced by the

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Molecules 2018, 23, x FOR PEER REVIEW 2 of 47
Molecules 2018, 23, 2424 2 of 47
produced by the microorganisms, which makes the penetration of antimicrobial drugs difficult [5,6].
The organization in biofilms makes an organism more resilient to various environmental conditions
microorganisms, which makes the penetration of antimicrobial drugs difficult [5,6]. The organization
and allows for its growth, even in hostile places [6–8]. The growth of biofilms in clinical settings, on
in biofilms makes an organism more resilient to various environmental conditions and allows for
buildings, monuments, and in technical installations, causes important health concerns as well as
its growth, even in hostile places [6–8]. The growth of biofilms in clinical settings, on buildings,
aesthetic, ecological, and economical damage. Additionally, spore-forming microorganisms, like
monuments, and in technical installations, causes important health concerns as well as aesthetic,
bacteria and filamentous fungi, can cause severe problems to human health and their eradication is
ecological, and economical damage. Additionally, spore-forming microorganisms, like bacteria and
harder to achieve than in planktonic form [9,10]. Thus, significant amounts of antimicrobials/biocides
filamentous fungi, can cause severe problems to human health and their eradication is harder to
are being used to combat planktonic and biofilm-embedded microorganisms’ growth in
achieve than in planktonic form [9,10]. Thus, significant amounts of antimicrobials/biocides are being
physiological fluids, waters, and on solid surfaces. However, the release of antimicrobials/biocides
used to combat planktonic and biofilm-embedded microorganisms’ growth in physiological fluids,
into the environment may induce new hazards to human health and environmental problems [11].
waters, and on solid surfaces. However, the release of antimicrobials/biocides into the environment
The adaptability of microorganisms caused by their fast reproduction rate is responsible for an
may induce new hazards to human health and environmental problems [11]. The adaptability of
increase in their resistance to the antimicrobials/biocides [6]. Therefore, it is of general consensus that
microorganisms caused by their fast reproduction rate is responsible for an increase in their resistance
the development of new strategies to combat planktonic, as well as biofilm-embedded
to the antimicrobials/biocides [6]. Therefore, it is of general consensus that the development of new
microorganisms, is required.
strategies to combat planktonic, as well as biofilm-embedded microorganisms, is required.
Photodynamic inactivation (PDI) is an antimicrobial strategy that has been reported as an
Photodynamic inactivation (PDI) is an antimicrobial strategy that has been reported as an effective
effective method to inactivate a broad spectrum of pathogens [12–14], including microorganisms that
method to inactivate a broad spectrum of pathogens [12–14], including microorganisms that are highly
are highly resistant to conventional antimicrobials [15] and those that form biofilms [4,16–18].
resistant to conventional antimicrobials [15] and those that form biofilms [4,16–18].
This methodology requires the activation of a dye called a photosensitizer (PS) by an adequate
This methodology requires the activation of a dye called a photosensitizer (PS) by an adequate
light wavelength in the presence of molecular oxygen (Figure 1). The activated dye (PS*) then
light wavelength in the presence of molecular oxygen (Figure 1). The activated dye (PS*) then
transfers its excess of energy or electrons to molecular oxygen (O2) or substrate molecules, generating
transfers its excess of energy or electrons to molecular oxygen (O2 ) 1 or substrate molecules,
highly cytotoxic reactive oxygen species (ROS), especially singlet oxygen ( O2) that 1oxidize different
generating highly cytotoxic reactive oxygen species (ROS), especially singlet oxygen ( O2 ) that oxidize
biomolecules (multi-target action) of planktonic or biofilm-associated microorganisms [19,20]. The
different biomolecules (multi-target action) of planktonic or biofilm-associated microorganisms [19,20].
lipids and proteins of the external structures of the microorganisms, cytoplasmic membrane, cell
The lipids and proteins of the external structures of the microorganisms, cytoplasmic membrane,
walls, capsids, and lipidic envelopes, are considered to be the main targets in PDI [21,22]. This
cell walls, capsids, and lipidic envelopes, are considered to be the main targets in PDI [21,22].
oxidative stress induced by PDI leads to irreparable damage of these vital cellular components,
This oxidative stress induced by PDI leads to irreparable damage of these vital cellular components,
causing microbial inactivation, avoiding the development of photoresistant strains [21–24].
causing microbial inactivation, avoiding the development of photoresistant strains [21–24].

Figure 1. Illustration of the photochemical mechanisms of different reactive oxygen species (ROS)
Figure 1. Illustration of the photochemical mechanisms of different reactive oxygen species (ROS)
produced during photodynamic action.
produced during photodynamic action.
In the last few years, the development and biological assessment of new molecules for PDI
In the last few years, the development and biological assessment of new molecules for PDI has
has grown considerably. Some classes of PSs, such as phenothiazinium dyes (e.g., methylene
grown considerably. Some classes of PSs, such as phenothiazinium dyes (e.g., methylene blue and
blue and toluidine blue O), psoralens, perylenequinonoid pigments (e.g., hypocrellin), natural and
toluidine blue O), psoralens, perylenequinonoid pigments (e.g., hypocrellin), natural and synthetic
synthetic based tetrapyrrolic macrocycles (e.g., chlorophylls, bacteriochlorophylls, porphyrins, corroles,
based tetrapyrrolic macrocycles (e.g., chlorophylls, bacteriochlorophylls, porphyrins, corroles, and
and phthalocyanines), and fullerenes have been effectively tested, demonstrating themselves to be good
phthalocyanines), and fullerenes have been effectively tested, demonstrating themselves to be good
antimicrobial agents (Figure 2) [25–31]. For instance, the studies showed that the photoinactivation of
antimicrobial agents (Figure 2) [25–31]. For instance, the studies showed that the photoinactivation
Gram-(+) and Gram-(−) bacterial strains is highly dependent on the PS structure. In general, neutral,
of Gram-(+) and Gram-(−) bacterial strains is highly dependent on the PS structure. In general,
cationic, or anionic PS can easily inactivate Gram-(+) bacteria, but an effective photoinactivation of
neutral, cationic, or anionic PS can easily inactivate Gram-(+) bacteria, but an effective
Gram-(−) bacteria, without the presence of membrane disrupting agents, requires the presence of a
photoinactivation of Gram-(−) bacteria, without the presence of membrane disrupting agents,
cationic PS. This inactivation difference is associated with the cell wall constitution. Most Gram-(+)
requires the presence of a cationic PS. This inactivation difference is associated with the cell wall
bacteria have a single cell wall constituted by peptidoglycan, with lipoteichoic and teichuronic
constitution. Most Gram-(+) bacteria have a single cell wall constituted by peptidoglycan, with
acids, which display a relatively high degree of porosity, while the Gram-(−) bacteria, other than
lipoteichoic and teichuronic acids, which display a relatively high degree of porosity, while the Gram-
the peptidoglycan layer, possess an additional highly organized outer membrane composed of
(−) bacteria, other than the peptidoglycan layer, possess an additional highly organized outer
Molecules
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2424PEER REVIEW 3 of 47 47
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membrane composed of lipopolysaccharides that are strongly negatively charged, phospholipids,
lipopolysaccharides
membrane and
lipoproteins, composed thatofare
proteins strongly
(Figurenegatively
lipopolysaccharides
[31] 3). charged,
that phospholipids,
are strongly lipoproteins,
negatively charged,and proteins [31]
phospholipids,
(Figure 3).
lipoproteins, and proteins [31] (Figure 3).

Figure 2. Structures of cores and some natural and synthetic PSs commonly used as antimicrobial
Figure 2.2. Structures
agents. of cores
Structures and some
of cores and natural
some and synthetic
natural and PSs commonly
synthetic PSs used as antimicrobial
commonly used as
agents.
antimicrobial agents.

Figure
Figure3. 3.
Schematic representation
Schematic ofof
representation thethecellular
cellularenvelope
envelopeofofGram-(+)
Gram-(+)(left)
(left)and
andGram-(−)
Gram-(−bacteria
) bacteria
Figure
(right).
(right). 3. Schematic
Reproduced
Reproduced representation
from Reference
from Reference of
[21]
[21]the
with cellular
with envelope
permission ofof
permission of Gram-(+) (left) and Gram-(−) bacteria
Elsevier.
Elsevier.
(right). Reproduced from Reference [21] with permission of Elsevier.
TheThe possibility
possibility ofof using
using thethe photodynamic
photodynamic approach
approach toto improve
improve the
the microbiological
microbiological quality
quality ofof
water
water and
The
and food,
food, toto
possibilitycontrol
control insect
of using
insect thepests, andto to
photodynamic
pests, and disinfect and
approach
disinfect and sterilize
to improve
sterilize materials
the and
materials and surfaces
microbiological
surfaces in in different
quality
different of
contexts
water and
contexts (industrial,
(industrial, household,
food, to household,
control insect and hospital)
pests,
and has
and tohas
hospital) been
disinfectthe research
andresearch
been the subject
sterilizesubject of
materials several groups
and surfaces
of several groups [23,32–34].
in[23,32–
different
Additionally,
contexts
34]. the the
use household,
(industrial,
Additionally, of
usethis
of approach
this and to inactivate
hospital)
approach hasmicroorganisms
been the
to inactivate researchhassubject
microorganisms become
hasofincreasingly
several groups
become achievable
[23,32–
increasingly
achievable in practice if the PS is immobilized on inert solid supports [18]. Consequently, this willfor
in practice
34]. if the
Additionally, PS is
the immobilized
use of this on inert
approach solid
to supports
inactivate [18]. Consequently,
microorganisms has this
become will allow
increasingly
achievable
allow for the in practice
reuse and ifthe
thematerials
PS is immobilized
recycling, on inert this
making solidmethodology
supports [18].economic,
Consequently, this will
sustainable,
allow for the reuse and the materials recycling, making this methodology economic, sustainable,
Molecules 2018, 23, 2424 4 of 47

the reuse and the materials recycling, making this methodology economic, sustainable, durable,
and environmentally friendly [23,35,36]. Thus, the selection of the solid supports must be based
on a few items: (i) compatibility with the PS, (ii) stability and mechanical strength towards light,
(iii) easy and reproducible immobilization procedures; (iv) good oxygen permeability for efficient
1 O production with minimum quenching, (v) commercial availability and low-cost, and (vi) high
2
biocompatibility to maximize the interaction between the support and the microorganisms [37].
To this extent, some different supports have been created to immobilize PSs. The importance of
this subject is demonstrated by the number of works and reviews that are appearing in the last
decade [38–40]. Herein, we decided to revisit several recent innovative formulations, molecular
designs, and modifications that demonstrated effective PDI of microorganisms, giving a special
attention to the design of the synthetic strategies behind the preparation of the photoactive materials.
In order to facilitate the discussion, the review is organized considering the type of support, namely
metal nanoparticles (NPs), silica, biopolymers (chitosan and cellulose), liposomes, nanogels, and
carbon materials. In terms of PS, special attention is given to some of the most studied options such
as tetrapyrrolic macrocycles derivatives, phenothiazinium dyes, and fullerenes. However, when
pertinent, some older works and research with other PSs will also be mentioned.

2. Metal Nanoparticles
The versatility of metal nanoparticles (MNPs) is well recognized in different areas such as
engineering, chemistry, physics, biology, and medicine [41–43]. Some of their biological applications are
related with their potential in the PDT of cancer, as drug delivery systems [44] and also as antibacterial
and antifungal agents [45]. In fact, due to their small size, MNPs can attach to the cell wall of the
bacteria or fungi through self-assembly causing cell death [46]. Additionally, some metals, like silver
or zinc, are known for their natural antibacterial activity [47–49]. However, the MNPs must have
the adequate dimension in order to avoid agglomeration/aggregation phenomena, which is related
with a significant reduction of the antimicrobial effect. The mechanism of MNP action has not been
fully understood. However, numerous theories were proposed based on the damage of the microbial
enzymes by the release of metal ions, modifications in the membrane integrity with penetration into
the bacterial cytoplasm, and accumulation in the periplasmic space, or due to the direct action of ROS
generated by the effect of MNPs [50]. In this section, we will give an update on the antimicrobial
properties of different types of MNPs associated with PS dyes on different microbial strains.

2.1. Gold Nanoparticles

Among all the MNPs, gold NPs (AuNPs) have received particular attention due to their
distinctive properties that can make them suitable for several applications such as labeling, delivery,
heating, and sensing [51–53]. Recently, AuNPs also earned significant attention in the photodynamic
approach due to their properties, such as, biocompatibility, size, unique surface, and also optical
properties [54–56]. Furthermore, the conjugation of PSs on the surface of AuNPs may increase the PDI
efficacy as a result of the localized surface plasmon resonance (SPR) of AuNPs upon light exposure.
This situation will then lead to a PS-efficient activation (an increase in the PS excitation rate) and an
improvement in the ROS production (Figure 4) [56,57].
In light of this, and considering that the colonization of catheters by microorganisms can be related
with the development of some resistant biofilm-related infections, in 2009, Wilson and co-workers [58]
prepared antimicrobial materials based on polysiloxane (a polymer used in catheters), methylene
blue (MB; Figure 2), and 2 nm AuNPs and compared their photoefficacy with the polymeric matrix
alone [58]. Small squares of the polymer were left for 24 h in the dark in solutions containing just
MB (acetone–water solution) or MB and/or AuNPs. Then, the swelled polymers were removed from
the solution and the solvents were allowed to evaporate in the dark for 24 h. The antimicrobial
activity of prepared polymers was tested against the Gram-(−) Escherichia coli and the Gram-(+)
methicillin-resistant Staphylococcus aureus (MRSA) and the results showed that the presence of AuNP
Molecules 2018, 23, 2424 5 of 47

potentiated approximately 2-fold the efficacy of MB. The polymer containing MB and AuNP showed
Molecules 2018, 23, x FOR PEER REVIEW 5 of 47
1.0 log reduction of E. coli after 5 min of irradiation with a red light (660 nm) provided from a handheld
light system light
a handheld operating
system at 250 mW, versus
operating at 2500.5mW,log with the 0.5
versus polymer
log withjust the
containing
polymer MB.justSimilarly,
containingthe MB.
best
bacterial reductions were observed for MRSA in the presence of
Similarly, the best bacterial reductions were observed for MRSA in the presence of the polymer the polymer containing MB and
AuNP (3.5 log reduction), whereas with the MB polymer, a reduction
containing MB and AuNP (3.5 log reduction), whereas with the MB polymer, a reduction of just 1.0 of just 1.0 log was attained.
No
log phototoxicity
was attained.was No detected eitherwas
phototoxicity withdetected
the polymereitheralone
withor thewith just AuNP
polymer aloneembedded.
or with just The best
AuNP
performance of the polymer containing both additives (MB and
embedded. The best performance of the polymer containing both additives (MB and AuNP) was AuNP) was explained by recognizing
the possibleby formation of other 1 O , potentiated by the presence of AuNP [58]. The good
explained recognizing the ROS besides
possible formation
2 of other ROS besides 1O2, potentiated by the
results impelled the authors to consider
presence of AuNP [58]. The good results impelled the the use of MB and AuNPtoembedded
authors consider the in a use
polysiloxane
of MB and polymer
AuNP
to reduce the formation of Staphylococcus epidermidis biofilms [59]. The
embedded in a polysiloxane polymer to reduce the formation of Staphylococcus epidermidis biofilms study was performed under
the
[59].same laser was
The study irradiation
performed (660under
nm), thebut same
different
laserexposure
irradiation times
(660 and
nm),light regimes exposure
but different were assessed.
times
The results showed that the best PDI combination (a reduction in
and light regimes were assessed. The results showed that the best PDI combination (a reductionbiofilm coverage greater than 50% in
when
biofilmcompared
coverage to untreated
greater polysiloxane
than 50% when compared polymer) was attained
to untreated when thepolymer)
polysiloxane light waswas applied for
attained
10 minthe every −2 ). Other combinations,
when light60wasminapplied
for a total period
for 10 of 6 h 60
min every (total
minenergy doseperiod
for a total of 117.0of 6J cm
h (total energy dose of 117.0
like
J cmshort irradiation
−2). Other periods (5like
combinations, min) but frequent
short irradiation (every 30 min),
periods or longer
(5 min) irradiation
but frequent periods
(every (20 min)
30 min), or
but infrequent
longer irradiation(every 120 min),
periods were but
(20 min) much less efficient,
infrequent (everyending up in were
120 min), 75% andmuch 60%, respectively,
less of the
efficient, ending
surface
up in 75% covered
and 60%,by the biofilm compared
respectively, to 100%covered
of the surface coverage bywhen no light
the biofilm was used.toThe
compared 100% mechanical
coverage
properties of the photoactive material (e.g., elasticity) were not affected,
when no light was used. The mechanical properties of the photoactive material (e.g., elasticity) confirming the possibility
were
to
notbeaffected,
incorporated
confirmingin catheters in orderto
the possibility tobereduce catheter in
incorporated acquired
catheters infections
in order[58,59].
to reduce Since then,
catheter
and following the same approach, this group prepared other photoactive
acquired infections [58,59]. Since then, and following the same approach, this group prepared other materials based on polymers
(polyurethane,
photoactive materials silicone)based
and other materials used
on polymers in catheterssilicone)
(polyurethane, and in hospital
and other touch surfacesused
materials (screenin
protectors for telephones and tablets, covers, keyboards, and hand
catheters and in hospital touch surfaces (screen protectors for telephones and tablets, covers,dryers) embedded with PSs [MB,
toluidine
keyboards,blue andOhand (TBO), crystal
dryers) violet] and
embedded withAuNPPSs [MB,against methicillin-resistant
toluidine blue O (TBO), crystalS. aureus (MRSA),
violet] and
S. epidermidis,
AuNP againstSaccharomyces cerevisiae,
methicillin-resistant E. coli, Bacteriophage
S. aureus MS2, theSaccharomyces
(MRSA), S. epidermidis, fungus-like organismcerevisiae,Pythium
E. coli,
ultimum, and the
Bacteriophage filamentous
MS2, the fungus-like Botrytis cinereal,
fungusorganism Pythium and the results
ultimum, andshowed an effective
the filamentous inactivation
fungus Botrytis
of all microorganisms [60–68].
cinereal, and the results showed an effective inactivation of all microorganisms [60–68].

Figure 4.
Figure 4. Plasmonic
Plasmonic AuNP. The local
AuNP. The local electric
electric field
field caused
caused by
by conductance
conductance electrons
electrons potentiates
potentiates the
the
optical field
optical fieldnear
nearthethe surface
surface andand enhances
enhances the fluorescence
the fluorescence or photoactivity
or photoactivity of the attached
of the attached PS
PS (adapted
(adapted
from from Reference
Reference [69]). [69]).

In 2015, Morsy and co-workers [70] also reported that the presence of 13 nm AuNP could
improve the phototoxicity of MB towards an S. aureusaureus strain
strain isolated
isolated from
from childrens’
childrens’ impetigo
impetigo lesions;
lesions;
this strain is the most common organism found in impetigo infections and resistance to conventional
antibiotics starts
starts to
tobe
beproblematic.
problematic.TheTheauthors prepared
authors preparedthethe
PS-AuNP
PS-AuNP nanoconjugates
nanoconjugates by using the
by using
swell–encapsulation–shrink
the swell–encapsulation–shrink method. Succinctly,
method. anan
Succinctly, aqueous
aqueoussolution
solutioncontaining
containing colloidal
colloidal AuNPs
(obtained from a hot hot solution
solution of
of HAuCl
HAuCl44 using trisodium citrate as as aa capping
capping and reduction agent)
and MB was maintained under stirring and ultra-sonication in order to improve the electrostatic
interaction between both components. The MB-AuNP nanoconjugate prepared was assessed against
and compared
S. aureus and compared with
with the
the photodynamic
photodynamic effect of AuNP
AuNP and and MBMB alone.
alone. As in the previous
studies by Perni [58], the highest inhibitory effect on S. aureus was obtained with the conjugate MB-
AuNP when irradiated for 2 min at 660 nm. Djavid and colleagues [71] extended the biological
assessment of MB-AuNP nanoconjugate towards mature biofilms of MRSA. They prepared 5 nm MB-
AuNP nanoconjugates and the successful conjugation between the positively charged MB dye and
Molecules 2018, 23, 2424 6 of 47

studies by Perni [58], the highest inhibitory effect on S. aureus was obtained with the conjugate
MB-AuNP when irradiated for 2 min at 660 nm. Djavid and colleagues [71] extended the biological
assessment
Molecules 2018, of
23, MB-AuNP nanoconjugate towards mature biofilms of MRSA. They prepared 56 ofnm
x FOR PEER REVIEW 47
MB-AuNP nanoconjugates and the successful conjugation between the positively charged MB dye and
the citrate-stabilized AuNPs (Figure 5) at aa molar
molar ratio
ratio of
of 20/1 was confirmed
20/1 was confirmed by the presence of the
characteristic AuNP absorbance peak at 520 nm in the the UV-Vis
UV-Visspectrum.
spectrum.

5. Schematic
Figure 5. Schematic representation
representation of
of the
the preparation
preparation of
of MB-AuNP
MB-AuNP conjugates (adapted from
Reference [71]).

The results showed that the MB-AuNP nanoconjugates were able to efficiently photoinactivate
1- and 4-day-old biofilms of MRSA after being exposed to light (650 nm) for 10 min (light irradiance
of 22.93 J cm −2 In this case, a reduction higher than 5
cm−2).).In colony-forming unit
this case, a reduction higher than 5 log colony-forming unit (CFU)
(CFU) was
was attained,
attained,
while
while with
withthe non-conjugate
the non-conjugate MB,MB,
the reduction was smaller
the reduction than 1 log
was smaller CFU.1 This
than log greater performance
CFU. This greater
of the MB-AuNP nanoconjugate was attributed to two important features of AuNP:
performance of the MB-AuNP nanoconjugate was attributed to two important features of AuNP: a a high production
of ROS
high due its localized
production of ROSsurface
due itsplasmon
localizedresonance
surface and to its resonance
plasmon role as a carrier
and to allowed forasdelivering
its role a carrier
monomeric MB into the deeper part of the mature biofilm. The potential of the prepared
allowed for delivering monomeric MB into the deeper part of the mature biofilm. The potential of the conjugates to
deal with chronic wound healing was also highlighted considering the minimal
prepared conjugates to deal with chronic wound healing was also highlighted considering the damage observed in
the fibroblast
minimal cellsobserved
damage [71]. in the fibroblast cells [71].

2.2. Silver Nanoparticles

2.2. Silver Nanoparticles
Silver NPs (AgNPs)
Silver NPs (AgNPs) areare also
also being
being considered
considered asas an
an interesting
interesting alternative
alternative to to antibiotic
antibiotic resistance
resistance
due to powerful antimicrobial activity against both Gram-(+) and
due to powerful antimicrobial activity against both Gram-(+) and Gram-(−) bacteria of Gram-( − ) bacteria of silver,
silver,
accompanied by their low toxicity to mammalian cells at low concentrations [72–75].
accompanied by their low toxicity to mammalian cells at low concentrations [72–75]. Besides, AgNPs Besides, AgNPs
have
have also
also been
been proved
proved to to disrupt
disrupt the
the biofilm
biofilm formation
formation [76].
[76]. In
In fact,
fact, the
the possibility
possibility ofof developing
developing aa
new generation of antibiotics based on AgNPs merits special attention from the scientific
new generation of antibiotics based on AgNPs merits special attention from the scientific community. community.
Although, the mechanisms underlying the action of AgNPs towards
Although, the mechanisms underlying the action of AgNPs towards microorganisms are not microorganisms are not fully
fully
clarified,
clarified, it is accepted
it is accepted that
that the
the possible
possible development
development of of resistance
resistance byby microorganisms
microorganisms is is unlikely
unlikely duedue
to the multitarget action of silver
to the multitarget action of silver [41].[41].
In
In 2012,
2012, Nyokong
Nyokong and and co-workers reported that
co-workers reported that the
the conjugation
conjugation ofof spherical
spherical 77 nm
nm AgNP
AgNP to to the
the
zinc(II) complex of phthalocyanines bearing poly-lysine chains (ZnPcPyPL and
zinc(II) complex of phthalocyanines bearing poly-lysine chains (ZnPcPyPL and ZnPc-SO2PL) (Figure ZnPc-SO 2 PL) (Figure 6)
significantly improved the S. aureus growth inhibition when compared to the results
6) significantly improved the S. aureus growth inhibition when compared to the results achieved with achieved with
the
the corresponding
corresponding phthalocyanines alone. The
phthalocyanines alone. The conjugation
conjugation ontoonto the
the AgNP
AgNP surface
surface was
was potentiated
potentiated
by
by the presence of amino groups in the ZnPc poly-lysine chains, and it was achieved by
the presence of amino groups in the ZnPc poly-lysine chains, and it was achieved stirring both
by stirring both
components at room temperature for 15 min (Figure
components at room temperature for 15 min (Figure 6) [77]. 6) [77].
As an extension of the previous studies, the same group reported the synthetic access to a series
of conjugates involving the non-symmetric metallophthalocyanines MPc bearing a cysteinyl unit
(Figure 6) with different shapes of AgNPs (spherical, cubes and triangles) [78]. The presence of the –
NH2 and –COOH groups on the phthalocyanine was important in the stabilization of the MPc-AgNP
nanoconjugates. The antimicrobial activity of the prepared nanoconjugates was evaluated against S.
aureus in the dark and after irradiation with visible light for 90 min. The highest antimicrobial activity
found for the spherical AgNPs alone was explained by considering their smaller size (15 nm) and
consequently more surface area when compared to triangles (size 54 nm) and cubes (60 nm) shaped
AgNPs. The results also showed that the toxicity of the MPc derivatives was improved by irradiation
and the photodynamic inactivation efficiency of all MPc-AgNPs nanoconjugates was slightly higher
Molecules 2018, 23, x FOR PEER REVIEW 7 of 47

MPc-AgNPs nanoconjugates also presented the best antimicrobial performance, and the high efficacy
of the germanium
Molecules 2018, 23, 2424 phthalocyanine
complex was associated to its high efficacy to generate 1O2, either
7 of 47
alone or after AgNP conjugation [78].

6. Structures
Figure 6. Structures of metal phthalocyanines used in the preparation
preparation of
of phthalocyanine-AgNPs
phthalocyanine-AgNPs
conjugates (adapted from References
References [66,67]).
[66,67]).

As an extension
Promising of the
results previous studies,
concerning the same group
the combination reported
of AgNPs the synthetic
with a PS towards access to a series
Gram-(−)
of conjugatesaeruginosa
Pseudomonas involvingwere the also
non-symmetric
reported by metallophthalocyanines
Lyutakov et al. [79]. The study MPc involved
bearing athe cysteinyl unit
preparation
(Figure
of films6)based
with ondifferent shapes of AgNPs (spherical,
poly(methylmethacrylate) (PMMA)cubes doped and triangles)
with [78]. The presence of the
5,10,15,20-tetraphenylporphyrin
–NH and –COOH
(TPP;2 Figure 2) andgroups
AgNPson theThese
[79]. phthalocyanine was important in were
films (TPP-AgNPs/PMMA) the stabilization
prepared byofdissolving
the MPc-AgNPsilver
nanoconjugates. The antimicrobial activity
nitrate, TPP, N-methylpyrrolidone, and PMMA of theinprepared nanoconjugates
dichloroethane. Then, after was evaluated against
spin-coating at 1000
S.
rpmaureus
for in
15the darksolvent
s, the and after wasirradiation
evaporated withinvisible
a hot light
platefor 90 min.
(200 °C). A The highest
similar antimicrobial
approach activity
was used to
found
preparefor the spherical
PMMA films with AgNPs alone
just TPP or was
AgNPs explained by considering
for comparison. their smaller
The antimicrobial size (15
results nm) and
showed that
consequently
the presence ofmore bothsurface
componentsarea when comparedfor
was important to an
triangles
effective (size 54 nm) of
reduction and cubes (60
Gram-(−) nm) shaped
bacteria under
AgNPs.
blue (405Thenm)results also irradiation.
LED light showed thatThe the authors
toxicity considered
of the MPc derivatives was antimicrobial
that the specific improved by irradiation
properties
and the photodynamic
of AgNPs/PMMA filmsinactivation
towards theefficiency
Gram-(−) of all MPc-AgNPs
bacteria nanoconjugates
(through silver release) and was theslightly higher
photoactivity
than that of MPcs
of TPP/PMMA alone,S.although
against following
aureus (through theformation)
ROS same ordercould (Sn < justify
OTi < Zn the<better
(OH)performance
2 Ge). The spherical
of the
MPc-AgNPs nanoconjugates also presented
antimicrobial material comprising both components [79]. the best antimicrobial performance, and the high efficacy of
the germanium phthalocyanine complex was 1
A similar approach was developed by associated
Moor et al.to[80]its high efficacy
to create to generate O2antimicrobial
multifunctional , either alone
or after AgNP
materials, whereconjugation [78].
two independent components (fullerenes and AgNP) were combined for a
Promising
synergistic results concerning
enhancement the combination
of antimicrobial characterof AgNPs
of thinwithfilmsa PS towards
against and−bacteriophage
Gram-(
E. coli ) Pseudomonas
aeruginosa
PR772. The were also reported
authors by Lyutakov et al. [79]. The study involved
used polystyrene-block-poly-4-vinylpyridine the preparation
(PS-P4VP) copolymers of films
withbased
two
on poly(methylmethacrylate) (PMMA) doped with 5,10,15,20-tetraphenylporphyrin
domains to integrate fullerene molecules (C60 and C70) into the polystyrene domain and the in situ (TPP; Figure 2)
and
formedAgNPs
AgNP [79].
intoThese
P4VPfilms (TPP-AgNPs/PMMA)
domains (Figure 7). The results were prepared
revealed thatbyCdissolving silver nitrate,
70 loaded PS-P4VP TPP,
films were
N-methylpyrrolidone,
able to produce an upper andamount
PMMAof in1Odichloroethane.
2 and displayed Then, after spin-coating
a significantly at 1000 rpm for
higher antimicrobial 15 s,
activity
the solvent was evaporated in a hot plate (200 ◦ C). A similar approach was used to prepare PMMA films
with just TPP or AgNPs for comparison. The antimicrobial results showed that the presence of both
Molecules 2018, 23, 2424 8 of 47

components was important for an effective reduction of Gram-(−) bacteria under blue (405 nm) LED
light irradiation. The authors considered that the specific antimicrobial properties of AgNPs/PMMA
films towards the Gram-(−) bacteria (through silver release) and the photoactivity of TPP/PMMA
against S. aureus (through ROS formation) could justify the better performance of the antimicrobial
material comprising both components [79].
A similar approach was developed by Moor et al. [80] to create multifunctional antimicrobial
materials, where two independent components (fullerenes and AgNP) were combined for a synergistic
enhancement of antimicrobial character of thin films against E. coli and bacteriophage PR772.
The authors used polystyrene-block-poly-4-vinylpyridine (PS-P4VP) copolymers with two domains
to integrate fullerene molecules (C60 and C70 ) into the polystyrene domain and the in situ formed
AgNP
Moleculesinto
2018, P4VP domains
23, x FOR (Figure 7). The results revealed that C70 loaded PS-P4VP films 8were
PEER REVIEW of 47
able to produce an upper amount of 1 O2 and displayed a significantly higher antimicrobial activity
(against bacteriophage PR772PR772 and
and E.
E. coli)
coli) after
after light
light activation
activation than
than the
the corresponding
corresponding analogous
analogousCC70 70

loaded homopolymer film (without AgNP incorporated), which confirmed the dual functionality of
the produced film [80].

Figure 7. Schematic representation of the procedure to prepare PS-P4VP multifunctional antimicrobial

Figurecontaining
films 7. Schematic
C70 andrepresentation
in situ prepared ofAgNPs.
the procedure to from
Reproduced prepare PS-P4VP
Reference multifunctional
[80] with permission
antimicrobial films containing C
from the American Chemical Society.
70 and in situ prepared AgNPs. Reproduced from Reference [80] with
permission from the American Chemical Society.
2.3. Platinum Nanoparticles
2.3. Platinum
ConsideringNanoparticles
the antibacterial features of platinum, the development of materials based on
platinum NPs (PtNPs) associated to features
Considering the antibacterial PS molecules also merited
of platinum, some attentionoffrom
the development severalbased
materials research on
groups.
platinum Platinum
NPs (PtNPs)is known to inactivate
associated to PS microbes
moleculesby interacting
also merited withsometheir enzymes,
attention fromproteins, or DNA,
several research
and to restrain
groups. Platinum cell is
proliferation or cell division
known to inactivate [81]. Thus,
microbes the combined
by interacting with action of PSs with
their enzymes, PtNPs for
proteins, or
photodynamic inactivation of microorganisms may cause a synergistic effect,
DNA, and to restrain cell proliferation or cell division [81]. Thus, the combined action of PSs with resulting in improved
antibacterial activity.
PtNPs for photodynamic inactivation of microorganisms may cause a synergistic effect, resulting in
The studies of Nyokong
improved antibacterial activity. and co-workers are in accordance with the beneficial effect of combining
PtNPs with PSs [46,82].
The studies of Nyokong and The authors reported
co-workers arethe conjugation
in accordance of different
with shapes
the beneficial of of
effect PtNP (cubic,
combining
hexagonal,
PtNPs withand PSsunshaped)
[46,82]. The with 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrinatogalium(III)
authors reported the conjugation of different shapes of PtNPchloride (cubic,
(1, ClGaTCPP) and evaluated their antimicrobial activity towards a drug-resistant
hexagonal, and unshaped) with 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrinatogalium(III) Gram-(+) S. aureus
strain
chloridein (1,
solution and embedded
ClGaTCPP) in polystyrene
and evaluated nanofibers
their antimicrobial (Figure
activity 8). TheaPS
towards units were covalently
drug-resistant Gram-(+)
conjugated to theinPtNP
S. aureus strain through
solution and an amide bond
embedded involving the
in polystyrene oleylamine
nanofibers amine 8).
(Figure groups
The PSpresent
unitson the
were
PtNP surface and the carboxylic groups of the porphyrin (Figure 8). The
covalently conjugated to the PtNP through an amide bond involving the oleylamine amine groups different shaped conjugates
1-PtNPs
present on werethethen
PtNP suspended
surface andin athe
dimethylformamide/tetrahydrofuran
carboxylic groups of the porphyrin(DMF/THF) (1:1) different
(Figure 8). The solution
containing polystyrene polymer (1:10) and were electrospun into
shaped conjugates 1-PtNPs were then suspended in a dimethylformamide/tetrahydrofuran nanofibers with diameters ranging
6–14 µm. The (1:1)
(DMF/THF) studies showedcontaining
solution a better antimicrobial
polystyrene activity of the (1:10)
polymer conjugatesand than
werewhen the porphyrin
electrospun into
ClGaTCPP was used
nanofibers with alone ranging
diameters and the higher
6–14 µm.reduction was attained
The studies showed whena bettertheantimicrobial
porphyrin was conjugated
activity of the
to cubic PtNPs
conjugates than(4.64
when log the
indicating
porphyrinthat 99.99%
ClGaTCPP of the bacteria
was used havealonebeen
andkilled); ClGaTCPP
the higher (1) alone
reduction was
caused
attaineda when
reduction of 3 log. was conjugated to cubic PtNPs (4.64 log indicating that 99.99% of the
the porphyrin
bacteria have been killed); ClGaTCPP (1) alone caused a reduction of 3 log.
The same authors extended the strategy to another tetracarboxylic porphyrin derivative 2
(Figure 8), which was also covalently immobilized into PtNPs through an amide bond, and assessed
its photoefficacy against S. aureus, E. coli, and Candida albicans in solution and after being embedded
in polystyrene nanofibers [83]. Once again, an increased antimicrobial photodynamic effect was
Molecules 2018, 23, 2424 9 of 47
Molecules 2018, 23, x FOR PEER REVIEW 9 of 47

Figure 8.8.Synthetic access

Synthetic to conjugates
access involving
to conjugates acid porphyrins
involving and PtNP
acid porphyrins (adapted
and PtNP from Reference
(adapted from
[82]).
Reference [82]).

TheSupports
3. Silica same authors extended the strategy to another tetracarboxylic porphyrin derivative 2
(Figure 8), which was also covalently immobilized into PtNPs through an amide bond, and assessed
Among the nanocarriers used as drug delivery systems, silica has been extensively studied due
its photoefficacy against S. aureus, E. coli, and Candida albicans in solution and after being embedded in
to its biocompatibility, versatility, and easy preparation under mild conditions from readily available
polystyrene nanofibers [83]. Once again, an increased antimicrobial photodynamic effect was observed
precursors [84,85]. The approaches usually used to immobilize the PS in this type of support can
in the presence of the porphyrin conjugated 2-PtNP when compared with the porphyrin 2 and PtNP
involve adsorption, entrapment during inorganic matrix preparation, or covalent bonding to the
tested alone for all microorganisms [83].
matrix via surface OH or other present groups.
Some
3. Silica of the prepared materials appeared as an opportunity for water treatment purposes [86–
Supports
88]. In fact, materials based on non-magnetic and magnetic silica NPs with different PSs covalently
Among the
immobilized havenanocarriers
been recently used as drug delivery
developed, in order systems, silica
to facilitate theirhas been extensively
removal from the water studied due
matrix,
to its
for biocompatibility,
subsequent versatility,
reuse [23]. and easy
Additionally, preparation
silica NPs loaded under
with mild conditions
antimicrobial fromhave
agents readily
beenavailable
shown
precursors [84,85]. The approaches usually used to immobilize the PS in this type
capable to revert antibiotic resistance [84,89–91]. In particular, the immobilization of a PS on a silicate of support can
involve adsorption, entrapment during inorganic matrix preparation, or covalent
matrix possesses some benefits when compared with organic matrices since it is insoluble in water, bonding to the matrix
via surface
resistant OH ormicroorganisms,
towards other present groups. easy to fabricate, and can be successfully developed for the photo-
Some of the prepared materials appeared
disinfection of water or physiological as an opportunity for water treatment purposes [86–88].
fluids [35,92].
In fact,
Under the context of water disinfection, magnetic
materials based on non-magnetic and Orellana silica
and NPs with different
Fresnadillo and their PSsco-workers
covalently
immobilized
developed have been
materials recently
based developed,
on Ru(II) in order
complexes to facilitateintheir
immobilized removal
silicone from the
[37,93–97]. Inwater
one of matrix,
their
studies [96] the authors reported the synthetic access to photosensitizing materials based onbeen
for subsequent reuse [23]. Additionally, silica NPs loaded with antimicrobial agents have the
ruthenium complexes tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride, (RDP ) anda
shown capable to revert antibiotic resistance [84,89–91]. In particular, the immobilization of a PS
2+ on
silicate matrixtris(1,10-phenanthrolinyl-4,7-bis(benzenesulfonate)ruthenate(II)
tetrasodium possesses some benefits when compared with organic matrices since (RSDit4−is
) insoluble
(Figure 9) in
water, resistant towards microorganisms, easy to fabricate, and can be successfully
electrostatically immobilized, respectively, on commercially available anionic and cationic porous developed for the
photo-disinfection
silicone of water
(pSil− and pSil or physiological
+). Then, fluids [35,92].
the authors evaluated the efficiency of prepared materials against a
Under the context of water disinfection, Orellana
Gram-(+) bacterium Enterococcus faecalis. Several photosensitizing and Fresnadillo materials
and their co-workers
based on the developed
RDP2+
materials based on Ru(II) complexes immobilized in silicone [37,93–97].
complex and pSil− were prepared in the load range of 0.75–4.40 g m−2 without achieving surface In one of their studies [96]
the authorsbut
saturation, reported
with thethe synthetic
anionic RSD4− access to photosensitizing
sensitizer, materials
due to its solubility in polarbased on theand
solvents, ruthenium
the load
complexes tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride, (RDP 2+ ) and tetrasodium
on pSil+ was limited to 0.03 g m−2. The photodisinfection assays performed under solar-simulated
tris(1,10-phenanthrolinyl-4,7-bis(benzenesulfonate)ruthenate(II) (RSD4with− ) (Figure 9) electrostatically
radiation (20 W m−2) showed that the best performance was obtained the RDP2+/pSil− material
immobilized, respectively, on commercially available anionic and cationic
with the highest sensitizer load (4.40 g m ), reaching a bacterial reduction of 2 orders
−2 porous silicone (pSil− and
of magnitude;
+
pSil ). sunlight
Then, the(ca. authors
under 400 Wevaluated the efficiency
m−2) the same material of prepared
was able to materials
provide aagainst a Gram-(+)
full disinfection bacterium
after 45 min
Enterococcus faecalis. Several photosensitizing materials based on the RDP 2+ complex and pSil − were
of irradiation while a decrease of 50% was observed with the support pSil alone. The antibacterial −
prepared indifferences
the load range of 0.75–4.40 gm −2
efficiency observed for the two without
prepared achieving
materialssurface
(RDPsaturation,
2+/pSil− andbut RSD with the+)anionic
−/pSil under
RSD 4− sensitizer, due to its solubility in polar solvents, and the load on pSil+ was limited to 0.03 g m−2 .
solar-simulated radiation was explained by the 1O2 production differences, which is probably related
The
to thephotodisinfection
limited load of the assays
RSD4−performed
PS. Thus, itunder solar-simulated
was concluded that theradiation (20 W and
1O2 generation m−2the ) showed
stabilitythat
of
the material, and consequently the bacteria photoinactivation efficiency, were related with the
sensitizer charge, its load, and the ionic character of the porous silicone.
Molecules 2018, 23, 2424 10 of 47

the best performance was obtained with the RDP2+ /pSil− material with the highest sensitizer load
(4.40 g m−2 ), reaching a bacterial reduction of 2 orders of magnitude; under sunlight (ca. 400 W m−2 )
the same material was able to provide a full disinfection after 45 min of irradiation while a decrease
of 50% was observed with the support pSil− alone. The antibacterial efficiency differences observed
for the two prepared materials (RDP2+ /pSil− and RSD− /pSil+ ) under solar-simulated radiation was
explained by the 1 O2 production differences, which is probably related to the limited load of the RSD4−
PS. Thus, it was concluded that the 1 O2 generation and the stability of the material, and consequently
the bacteria photoinactivation efficiency, were related with the sensitizer charge, its load, and the ionic
character of the
Molecules 2018, 23, xporous silicone.
FOR PEER REVIEW 10 of 47

2+2+ and RSD4−

Figure 9.9. Structure of
Figure of the
the ruthenium(II)
ruthenium(II) complexes
complexesRDP
RDP and RSD4− with
with polyazaheterocyclic
polyazaheterocyclic
(adapted
(adapted from
from Reference
Reference[96]).
[96]).

Under
Under the the same
same context
context of of water
water disinfection,
disinfection, the the use
use of of silica
silica magnetic
magnetic nanoparticles
nanoparticles as as aa
template to disperse the PS also deserved some attention from the scientific
template to disperse the PS also deserved some attention from the scientific community considering community considering
the
the photobactericidal
photobactericidal material
material is is easily
easily recovered
recoveredjust justbybyapplying
applyingaamagnetic
magneticfield field[35,98,99].
[35,98,99].
In
In 2010, considering
2010, considering the remarkable photodynamic
the remarkable efficiency of 5,10,15-tris(1-methylpyridinium-
photodynamic efficiency of 5,10,15-tris(1-
4-yl)-20-(pentafluorophenyl)porphyrin + -Me-PF) towards a wide range of
tri-iodide (Tri-Pytri-iodide
methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin (Tri-Py+-Me-PF) towards a wide
microorganisms under mild conditions [100], our
range of microorganisms under mild conditions [100], our group reported group reported its immobilization in cationized
its immobilization in
silica-coated magnetic nanoparticles of Fe O (Figure 10) [35]. The
cationized silica-coated magnetic nanoparticles of Fe3O4 (Figure 10) [35]. The multi-charged
3 4 multi-charged nanomagnet Hybrid
1, based on Tri-Py + -Me-PF, was effective+in the photoinactivation of both Gram-(+) and Gram-(−) fecal
nanomagnet Hybrid 1, based on Tri-Py -Me-PF, was effective in the photoinactivation of both Gram-
bacteria, causing afecal5 logbacteria, for E. faecalis
decreasecausing and E. coli at + -Me-PF when using white
(+) and Gram-(−) a 5 log decrease for20E.µM of Tri-Py
faecalis and E. coli at 20 µM of Tri-Py+-
light irradiation (64.8 J cm − 2 ). The full inactivation of the T4-like bacteriophage (≈7 log of
Me-PF when using white light irradiation (64.8 J cm ). The full inactivation −2 of inactivation)
the T4-like
in the presence of the same hybrid also confirmed its efficacy as
bacteriophage (≈7 log of inactivation) in the presence of the same hybrid also confirmed an antiviral agent [35]. Theitscapability
efficacy
of
as this hybrid material
an antiviral agent [35].to beTherecycled and reused,
capability of this and alsomaterial
hybrid the potentialto beofrecycled
the analogue with a CoFe
and reused, 2 O4
and also
core (Hybrid 2), were posteriorly evaluated using water contaminated
the potential of the analogue with a CoFe2O4 core (Hybrid 2), were posteriorly evaluated using waterwith the Gram-( − ) bacterium
Allivibrio
contaminatedfischeri (Figure
with 10) [99]. The
the Gram-(−) results obtained
bacterium Allivibrio demonstrate
fischeri (Figure that10)both
[99].hybrids were obtained
The results effective
in the bacterial inactivation withwereaccumulative values after a six-cycle of ≈accumulative
reusewith 42 log CFU mL −1 in
demonstrate that both hybrids effective in the bacterial inactivation values
21.5 ≈38 log − 1
afterhaand
six-cycle CFU
reuse of mL≈42 log inCFU
27 h,mLrespectively,
−1 in 21.5 hforandHybrids
≈38 log1CFU and mL2. It−1was
in 27 commented that the
h, respectively, for
efficacy
Hybridsof1the andrecycling
2. It wasprocess
commented could be thatimproved if bigger
the efficacy of the active particles
recycling were could
process used tobebeimproved
completely if
retained by a magnetic field [99].
bigger active particles were used to be completely retained by a magnetic field [99].
Durantini and co-workers [98] also selected magnetic SiNP of Fe3O4 functionalized with
aminopropyl groups to covalently link the 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin (TCPP)
(Figure 11) in order to evaluate their potential as an antimicrobial material. In the material referred
to as MN-SiNP-NH-TCPP, the magnetic core was previously coated with silica before
functionalization with (3-aminopropyl)trimethoxysilane (APTS), while in the other material,
designed as MN-NP-NH-TCPP, the grafting of the amino functionalities was performed directly on
the magnetic core. In both cases, the conjugation of porphyrin derivative in the magnetic NP was
successful attained via carbodiimide activation of the carboxylic groups (Figure 11). The biological
studies revealed that both materials were effective against S. aureus, E. coli, and C. albicans in the first
cycle of inactivation but the efficiency of MN-NP-NH-TCPP rapidly decreased in further cycles. A
different situation was observed with the magnetic coated material MN-SiNP-NH-TCPP; the efficient
Molecules 2018, 23, 2424 11 of 47
Molecules 2018, 23, x FOR PEER REVIEW 11 of 47

Figure
Figure Preparation
10.10. of silica
Preparation nanomagnet-porphyrin
of silica hybrids
nanomagnet-porphyrin 1 and
hybrids 2 (adapted
1 and from
2 (adapted References
from [35,99]).
References
[35,99]).
Durantini and co-workers [98] also selected magnetic SiNP of Fe3 O4 functionalized with
aminopropyl
Considering groupsthe to covalently
suitability of link the 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin
the pyrrolidine-fused chlorin derivative (Chl-TPFPP) obtained (TCPP)
(Figure 11) in order to evaluate their potential as an antimicrobial
from 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin) (TPFPP) material.
to act In asthe material referred
nucleophile, to as
our group
MN-SiNP-NH-TCPP, the magnetic core was previously coated with silica before
envisaged its immobilization on the commercial available 3-bromopropyl-functionalized silica and functionalization with
(3-aminopropyl)trimethoxysilane
also to Merrifield resin-based materials, (APTS), while
as an in economically
the other material,
viable designed as MN-NP-NH-TCPP,
and environmental friendly
the grafting
approach toof the amino
allow functionalities
successive recovery and wasremoval
performedof thedirectly on theafter
PS material magnetic core. In both
photodynamic cases,
treatment
the conjugation of porphyrin derivative in the magnetic NP was successful attained
(Figure 12A,B) [92,101]. The study indicated that the materials resulting from the immobilization of via carbodiimide
activation
the chlorinofderivative
the carboxylic
on both groups (Figurematerials
commercial 11). Thefollowed
biologicalbystudies
furtherrevealed
treatmentthat both
with materials
pyridine had
were
high effective
potentialagainst S. aureus,
as PSs for E. coli, andofC.E.albicans
the inactivation coli (ca in
3.0the
logfirst cycle of inactivation
reductions) but theof
with an irradiance efficiency
4.0 mW
of
cmMN-NP-NH-TCPP rapidly decreased
−2 after 180 min. Additionally, in further(silica
both materials cycles.and
A different
Merrifield situation was observed
resin based) showed goodwith
the magnetic coated
photostability undermaterial
PAR whiteMN-SiNP-NH-TCPP;
light (380–700 nm) theand
efficient
after decrease for S. of
of 2.5 logcycles
three successive aureus and
bacterial
C. albicans and 3.0 log for E. coli after 30 min of irradiation (90 mW cm −2 ) at 3.0 µM of TCPP observed
photoinactivation, the bacterial reduction was kept constant. So, the prepared photoactive materials
in the
can befirst inactivation
considered as ancycle was maintained
inexpensive in theoption
and friendly following two cycles. in
for application In clinic
this study, it was proved
and environmental
that 1
areasO[85].
2 was the main cytotoxic species involved in the photodynamic process and the presence of the
silica coating layer in the MN-SiNP-NH-TCPP was important to avoid the magnetic core oxidation
and decomposition, and to prevent aggregation [98].
Molecules 2018, 23, 2424 12 of 47
Molecules 2018, 23, x FOR PEER REVIEW 12 of 47

11. Representation of the

Figure 11. the synthetic
synthetic access
access to
to nanomagnet
nanomagnet materials
materials MN-SiNP-NH-TCPP
MN-SiNP-NH-TCPP and
MN-NP-NH-TCPP (adapted from from Reference
Reference [98]).
[98]).

Considering
Kuznetsova etthe al. suitability
[102] selectedof the
the pyrrolidine-fused
commercially availablechlorin derivative silica
aminopropyl (Chl-TPFPP) obtained
to immobilize the
from 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin)
zinc and aluminum complexes of phthalocyanines tetrasubstituted (TPFPP) toatact as nucleophile,
non-peripheral our group
positions with
envisaged its immobilization
chloromethylated thiophenylon the commercial
groups available
as potential 3-bromopropyl-functionalized
photobactericidal materials (Figure silica 13). and
The
also to Merrifield
grafting resin-based materials,
of the phthalocyanines in silica was as performed
an economically
in DMFviable
at ≈85 and
°C and environmental friendly
further reaction with
approach to allow successivegave
N,N-dimethylaminoethanol recovery
rise toand removalcharged
positively of the PS material after
D/MPc(SPh) 4Chol photodynamic treatment
7. Sodium taurinate was
(Figure 12A,B) [92,101]. The study indicated that the materials resulting from
selected to obtain negatively charged phthalocyanines. The photodynamic efficiency of the different the immobilization
of the chlorin
materials wasderivative
evaluatedon bothacommercial
using bioluminescentmaterials
E. colifollowed
and theby further treatment
biological assays were with pyridine
performed
had
underhigh potential
white asmW
light (75 PSs cm
for−3the
) at ainactivation of E. coli
PS concentration (caµM.
of 20 3.0 The
log results
reductions)
showed with anthe
that irradiance
aluminum of
4.0 mW cm −2 after 180 min. Additionally, both materials (silica and Merrifield resin based) showed
positively charged phthalocyanine D/AlPc(SPh)4Chol7 had the highest bacterial activity
good photostability
(bioluminescence under PAR
reduction whitefollowed
> 80%), light (380–700 nm) and
by neutral after three
D/AlPc(SPh) successive
4Clm 7 (80%), cycles of bacterial
and finally by the
photoinactivation,
negatively chargedthetaurinate
bacterial reduction was kept
phthalocyanines constant. So, the reduction
(bioluminescence prepared photoactive
≈60%). Thematerials
authors
can be considered
commented that aasmore
an inexpensive and friendly
effective interaction option for
between theapplication in clinic and
Gram-(−) bacteria and environmental
the positively
areas
charged[85].PS was probably responsible for this fact. The dependence observed on the cationic
D/ZnPc(SPh)4Chol7 to generate 1O2 with the load in the support (less PS load means better efficiency)
was not reflected in its photodynamic action, since a similar bioluminescence reduction of ≈70% was
found for materials with different zinc(II) phthalocyanine loads [102].
Molecules 2018, 23, 2424 13 of 47
Molecules 2018, 23, x FOR PEER REVIEW 13 of 47

Figure 12.
12.(A)
(A) Schematic
Schematic representation
representation of theofimmobilization
the immobilization of a pyrrolidine-fused
of a pyrrolidine-fused chlorin
chlorin derivative
derivative
on on asilica
a modified modified silica gelwith
gel support support withcationization
further further cationization with pyridine
with pyridine units.
units. (B) (B) Diagram
Diagram of the
of the pyrrolidine-fused
pyrrolidine-fused chlorinchlorin derivative
derivative immobilized
immobilized on a cationized
on a cationized Merrifield
Merrifield resin (adapted
resin (adapted from
from Reference
Reference [101]).[101]).

Kuznetsova
Light-activated et al.silica
[102]NPs
selected the commercially
adequately functionalizedavailable aminopropyl
with acids silica used
groups were to immobilize
as templatesthe
zinc and aluminum
to immobilize complexes
Toluidine Blue Oof phthalocyanines
(Figure tetrasubstituted
14) giving rise to materials with at non-peripheral positions with
promising photobactericidal
chloromethylated
activity against S.thiophenyl
epidermidis,groups
E. coli,asandpotential
MRSAphotobactericidal materials
[103]. After irradiating the(Figure
samples 13). Theagrafting
with light of
of the phthalocyanines in silica was performed in DMF at ≈ 85 ◦ C and further reaction with
630 nm, delivered by a 500 mW laser, a bacterial reduction of 2 log was observed for S. epidermidis
N,N-dimethylaminoethanol
and E. coli after 2 and 3 min gave rise to positively
of treatment, respectively.charged
Also,D/MPc(SPh)
the number 4ofChol 7 . Sodium
viable MRSA cellstaurinate
after
was
laserselected
irradiationto obtain negatively
in the presence charged
of the phthalocyanines.
SiNP-TBO nanoconjugates Thedecreased
photodynamicwith the efficiency
increase of of the
the
different
treatmentmaterials
time. Thewas higherevaluated
difficulty using a bioluminescent
to photoinactivate E. when
E. coli coli and the biological
compared assays were
to S. epidermidis and
performed −3 ) at a PS concentration of 20 µM. The results showed
MRSA was under explainedwhiteby light (75 mW
considering thecmmore complex cell wall structure of Gram-(−) bacteria when
that the aluminum
compared positively
with Gram-(+). charged no
Moreover, phthalocyanine
antimicrobialD/AlPc(SPh)
activity was4 Chol 7 hadby
observed thethehighest bacterial
unconjugated
activity
silica or(bioluminescence
by the laser lightreduction
alone. With > 80%),
thesefollowed by neutral
results, the authorsD/AlPc(SPh)
concluded that 4 Clmthe7 (80%), and finally
conjugates could
by
be the
used negatively
to prepare charged taurinate
materials, suchphthalocyanines
as hydrogels and (bioluminescence
topical creams,reduction
that exhibit≈60%). The authors
light-dependent
commented
antimicrobialthat a more
activity [103].effective interaction between the Gram-(−) bacteria and the positively
charged PS was
Lacombe andprobably responsible
their co-workers [104]foralsothis fact. The
reported dependence
the synthesis and observed on theefficiency
the disinfection cationic
D/ZnPc(SPh) Chol to generate 1 O with the load in the support (less PS load means better efficiency)
towards E. coli 4 of 7silica-based materials
2 covalently linked to other non-porphyrinic PSs as 9,10-
was not reflected in its photodynamic
anthraquinone-4-carboxylic acid (ANT) action,
and since a similar bioluminescence reduction of ≈70% acid
9,14-dicyanobenzo[b]triphenylene-3-carboxylic was
found for materials
(DBTP-COOH) with15).
(Figure different zinc(II) phthalocyanine
The immobilization of ANT loads [102].a preliminary functionalization
involved
withLight-activated silica NPs adequately
(3-aminopropyl)triethoxysilane followedfunctionalized withinacids
by the grafting groups were
commercial silica used
beadsasbytemplates
reflux in
to immobilize
toluene Toluidine
affording SiO2-ANT.Blue OThe (Figure
DBTP 14)was
giving rise to
grafted materials
directly in with promising photobactericidal
a commercially available amino-
activity
functionalized S. epidermidis,
againstsilica powder giving E. coli,rise
andtoMRSA [103]. After
the material irradiating
designed as SiNH the samples
2-DBTP. with a light
Although of
a slow
630 nm, delivered by a 500 mW laser, a bacterial reduction of 2 log was observed
but effective inactivation of E. coli (7 log after 360 min of irradiation) was observed with pure silica for S. epidermidis
and E. coliany
(without after
PS)2 under
and 3 UV-A
min of(3.85
treatment,
mW cmrespectively.
−2, 365 nm), aAlso, the number
noticeably of viable
improvement wasMRSA cellswhen
detected after
laser irradiationtook
the irradiation in the
placepresence
in the of the SiNP-TBO
presence of the samenanoconjugates
amount (2.5decreased
g L−1) of SiOwith2-ANTthe increase
and to a of the
lesser
extent with SiNH2-DBTP at the same concentration. After exposure to UV-A for 110 min and 270 min,
respectively, SiO2-ANT and SiNH2-DBTP were able to decrease the bacterial concentration to
undetectable levels (decreases of > 6.0 log). It was also demonstrated that the mode of PS introduction
Molecules 2018, 23, 2424 14 of 47

treatment time. The higher difficulty to photoinactivate E. coli when compared to S. epidermidis and
MRSA was explained by considering the more complex cell wall structure of Gram-(−) bacteria when
compared with Gram-(+). Moreover, no antimicrobial activity was observed by the unconjugated silica
Molecules 2018, 23, x FOR PEER REVIEW 14 of 47
or by the laser light alone. With these results, the authors concluded that the conjugates could be used
to
in prepare
silica and materials, such of
the amount as silica
hydrogels and
in the topical creams,
suspension that exhibit light-dependent
were fundamental parameters forantimicrobial
the material
activity
efficiency[103].
[104].

13. Synthetic
Figure 13. Synthetic route for the preparation
preparation of the
the metallophthalocyanines
metallophthalocyanines supported on silica
silica
materials (adapted
materials (adapted from
from Reference
Reference [102]).
[102]).

Lacombe and theirofco-workers

The development surfaces based[104] onalsobridged
reported the synthesis andfilms
polysilsesquioxane the disinfection
doped withefficiency
the non-
towards E. coli
symmetric porphyrin 5-(4-carboxyphenyl)-10,15,20-tris(4-methylphenyl)porphyrin (P-acid, PSs
of silica-based materials covalently linked to other non-porphyrinic as
Figure
9,10-anthraquinone-4-carboxylic
16) is another approach, givingacid rise(ANT) and 9,14-dicyanobenzo[b]triphenylene-3-carboxylic
to materials with promising antifungal activity [105]. acid The
(DBTP-COOH) (Figure 15). The immobilization of ANT involved a preliminary
precursor of silsesquioxane was prepared from a (glycidoxypropyl)trimethoxysilane (GPTMS) and functionalization with
(3-aminopropyl)triethoxysilane
dodecylamine (DA) reaction, and followed by the grafting
the incorporation ofin commercial
the porphyrin,silica beads by reflux
predominantly in toluene
as monomer,
affording SiO2 -ANT.
occurred during The hydrolysis
the acid DBTP was grafted directly in a commercially
of the trimethoxysilane available
groups [Si(OCH amino-functionalized
3)] and polycondensation
silica
reaction powder
to formgiving rise topolysilsesquioxane
bridged the material designed as films.
plastic SiNH2The-DBTP. Although
absence and athe slow but effective
presence of the
inactivation
porphyrin in E. coli
of the (7 log after
flexible films360 min
was of irradiation)
confirmed by thewastonality
observed with pure
acquired. silica
The (without activity
antifungal any PS)
under UV-A (3.85
photoinduced by the cm−2 , 365
mWprepared nm),was
films a noticeably
evaluatedimprovement was detected
against C. albicans in aqueouswhen the irradiation
suspensions and
took place in the presence of the same amount (2.5 g L −1 ) of SiO -ANT and to a lesser extent with
on its surface. It was observed a 99.7% of fungal inactivation after 260 min of irradiation (90 mW cm−2)
SiNH -DBTP light.
with 2visible at the Also,
same concentration.
they were effective After exposure to UV-A for 110
in photoinactivating min and dropped
C. albicans 270 min, respectively,
on the film
SiO -ANT
surface,
2 and SiNH
with a 95% decrease
2 -DBTP were able to decrease the bacterial concentration to undetectable
in cells survival after 30 min of irradiation. The results showed that levels
the
(decreases
inactivationofof> C.6.0albicans
log). Itwas
wasmostly
also demonstrated
mediated by that 1 the mode of PS introduction in silica and the
O2 [105].
amount of silica in the suspension were fundamental parameters for the material efficiency [104].
Molecules 2018, 23, 2424 15 of 47
Molecules
Molecules 2018,
2018, 23,
23, xx FOR
FOR PEER
PEER REVIEW
REVIEW 15
15 of
of 47
47

Figure
Figure 14.
14. Structure
Structure of
Structure of Toluidine
Toluidine Blue
Blue O silica nanoparticle
O silica nanoparticle (adapted
(adapted from
from Reference
from Reference [103]).
Reference [103]).
[103]).

Figure
Figure 15. Structures of
15. Structures
Structures of the
the two different PSs
two different PSs covalently
covalently linked
linked to
to silica
silica [104].
[104].

The development of surfaces based on bridged polysilsesquioxane films doped with

the non-symmetric porphyrin 5-(4-carboxyphenyl)-10,15,20-tris(4-methylphenyl)porphyrin (P-acid,
Figure 16) is another approach, giving rise to materials with promising antifungal activity [105].
The precursor of silsesquioxane was prepared from a (glycidoxypropyl)trimethoxysilane (GPTMS)
and dodecylamine (DA) reaction, and the incorporation of the porphyrin, predominantly as monomer,
occurred during the acid hydrolysis of the trimethoxysilane groups [Si(OCH3 )] and polycondensation
reaction to form bridged polysilsesquioxane plastic films. The absence and the presence of the
porphyrin in the flexible films was confirmed by the tonality acquired. The antifungal activity
photoinduced by the prepared films was evaluated against C. albicans in aqueous suspensions and on
its surface. It was observed a 99.7% of fungal inactivation after 60 min of irradiation (90 mW cm−2 )
Molecules 2018, 23, 2424 16 of 47

with visible light. Also, they were effective in photoinactivating C. albicans dropped on the film
surface, with a 95% decrease in cells survival after 30 min of irradiation. The results showed that the
inactivation of C. albicans was mostly mediated by 1 O2 [105].
Molecules 2018, 23, x FOR PEER REVIEW 16 of 47

Figure 16.
16. Preparation
Preparation of
of the
the polysilsesquioxane
polysilsesquioxane flexible
flexible films
films doped
doped with 5-(4-carboxyphenyl)-
5-(4-carboxyphenyl)-
10,15,20-tris(4-methylphenyl)porphyrin (adapted
10,15,20-tris(4-methylphenyl)porphyrin (adapted from
from Reference
Reference [105]).
[105]).

4. Biopolymers
4. Biopolymers
The
The design of new
design of new photodynamic
photodynamic antimicrobial
antimicrobial materials
materials based
based on non-toxic and
on non-toxic and biodegradable
biodegradable
supports, suchasaschitosan,
supports, such chitosan, cellulose,
cellulose, andand
theirtheir derivatives,
derivatives, is alsoishaving
also having some on
some impact impact on the
the research
research concerning the recovery and reuse of PSs in PDI, as it is evident in the works reported
concerning the recovery and reuse of PSs in PDI, as it is evident in the works reported in the following
in the following sections.
sections.

4.1. Chitosan
4.1. Chitosan
Chitosan
Chitosan is
is aa poly(
poly(D -glucosamine) that is easily isolated from the chitin exoskeleton of crustacea
D-glucosamine) that is easily isolated from the chitin exoskeleton of crustacea
using acid hydrolysis [106]. Chitosan displays
using acid hydrolysis [106]. Chitosan displays attractive
attractive properties
properties to
to be
be used
used as
as a
a PS
PS support
support such
such
as
as its
its film-forming
film-forming ability,
ability, biodegradability,
biodegradability, and
and an
an inherent
inherent antimicrobial
antimicrobial activity
activity related
related with
with the
the
protonated amino groups. The antimicrobial activity of this polymer can be improved through the
covalent or non-covalent incorporation of bioactive materials/compounds like PS, phenolic
compounds, antibiotics, and tetrahydrocurcominoid derivatives [30,106–111].
One of the first references where chitosan was recognized to have the potential to immobilize
PSs for the photodynamic inactivation approach was reported by Artarsky and co-workers in 2006
Molecules 2018, 23, 2424 17 of 47

protonated amino groups. The antimicrobial activity of this polymer can be improved through
the covalent or non-covalent incorporation of bioactive materials/compounds like PS, phenolic
compounds, antibiotics, and tetrahydrocurcominoid derivatives [30,106–111].
One of the first references where chitosan was recognized to have the potential to immobilize
PSs for the photodynamic inactivation approach was reported by Artarsky and co-workers in
Molecules 2018, 23, x FOR PEER REVIEW 17 of 47
2006 [106]. The authors selected chitosan membranes (simple or reinforced in nylon) to immobilize
two porphyrins
porphyrins and aand a phthalocyanine
phthalocyanine (Figure (Figure
17) as17) as a proof
a proof of concept
of concept that thethat the photodynamic
photodynamic effect
effect could
could
be used betoused
lower to microbial
lower microbial
levels inlevels
water in under
water flow
under flow conditions.
conditions. The authors The authors
considered considered
that the
that the hydrophilicity (wettable) of the polymer would facilitate
hydrophilicity (wettable) of the polymer would facilitate the contact between the PS and the contact between the the
PS
and the microorganisms and the incorporation of those PSs involved
microorganisms and the incorporation of those PSs involved three different approaches. The three different approaches.
The 5,10,15,20-tetrakis(4-hydroxyphenyl)porphyrin
5,10,15,20-tetrakis(4-hydroxyphenyl)porphyrin (TPP-OH),
(TPP-OH), waswas incorporated
incorporated via via adsorption
adsorption fromfrom
an
an
alkaline solution; the 5,10,15,20-tetrakis(4-aminophenyl)porphyrin (TPP-TetraNH2),2 due to its
alkaline solution; the 5,10,15,20-tetrakis(4-aminophenyl)porphyrin (TPP-TetraNH ), due to its
insolubility
insolubility in in aqueous
aqueousalkali,
alkali,was
wasincorporated
incorporatedinto intochitosan
chitosanviavia dissolution
dissolution andand homogenization
homogenization in
in mixed solvents (aqueous acetic acid, chloroform) followed by casting.
mixed solvents (aqueous acetic acid, chloroform) followed by casting. Meanwhile, the zinc(II) Meanwhile, the zinc(II)
phthalocyanine
phthalocyanine tetrasulfonic
tetrasulfonic acid
acid (ZnPcS)
(ZnPcS) was was covalently
covalently attached
attached to to the
the amino functions after
amino functions after being
being
converted
converted intointo the
the corresponding
corresponding sulphonyl
sulphonyl chloride.
chloride. The The absorption
absorption spectrum
spectrum of of the
the different
different
materials
materials was an important tool to confirm and to estimate the amount of the different dyes
was an important tool to confirm and to estimate the amount of the different dyes
incorporated
incorporated into into the
the polymeric
polymeric membrane.
membrane. A A preliminary
preliminary evaluation
evaluation of of their
their photomicrobicidal
photomicrobicidal
activity
activity inin static
static systems against E.
systems against E. coli
coli allowed
allowed for for the
the selection
selection of ofthe
thephthalocyanine/chitosan
phthalocyanine/chitosan
membrane as the most effective one, showing no detectable
membrane as the most effective one, showing no detectable microorganisms after microorganisms after 30
30 min
min under
under
irradiation with aa halogen
irradiation with halogen lamp
lamp (500
(500 W, W, 230
230 V,
V, 5050 Hz).
Hz). In
In the
the photomicrobicidal
photomicrobicidal studies studies using
using the
the
circulating water photoreactor system designed by the authors, the nylon
circulating water photoreactor system designed by the authors, the nylon reinforced ZnPcS/chitosanreinforced ZnPcS/chitosan
5 −1
membrane
membrane showedshowed aa significant
significantphotoinactivation
photoinactivation(>2 (>2log) E. E.
log)ofof colicoli present
present at 510
at 10 cellscells
mL−1mL. The.
The authors
authors highlighted
highlighted thatphotodynamic
that the the photodynamic effecteffect was maintained
was maintained (although
(although at a reduced
at a reduced level)level)
after
after the membrane had been kept in the
the membrane had been kept in the dark for 9 months. dark for 9 months.

17. Structures
Figure 17. Structuresofof
thethe porphyrins
porphyrins andand phthalocyanine
phthalocyanine derivatives
derivatives used
used by by Bonnett
Bonnett and co-
and co-workers
workers (adapted
(adapted from Reference
from Reference [106]). [106]).

Based onon the

theeasy
easyreplacement
replacement of of
thethe fluorine
fluorine atom
atom the p-position
in p-position
in the of pentafluorophenyl
of pentafluorophenyl units
units by nucleophiles
by nucleophiles [112,113],
[112,113], Barata
Barata et al.
et al. [30][30] reported
reported thethesynthetic
syntheticaccess
accessto
to transparent
transparent and
fluorescent chitosan films bearing corrole units obtained from 5,10,15-tris(pentafluorophenyl)corrole
with bacteriostatic
bacteriostatic action. The graft
action. The graft of
of 5,10,15-tris(pentafluorophenyl)corrole
5,10,15-tris(pentafluorophenyl)corrole to to chitosan was
performed in dimethyl sulfoxide at 80 ◦ C (Figure 18A,B) and it was verified that the amount of corrole
performed in dimethyl sulfoxide at 80 °C
linked to chitosan was dependent on the reaction time. Additionally,
Additionally, it was proved that the presence
of the corrole units did not affect the film-forming ability and all films with good thermomechanical
properties and thermal stability showed a bacteriostatic effect even in the dark against S. S. aureus
aureus
(reduction of 2 log).
Molecules 2018, 23, 2424 18 of 47
Molecules 2018, 23, x FOR PEER REVIEW 18 of 47

(A)

(B)

Figure 18. (A) Conditions used to graft 5,10,15-tris(pentafluorophenyl)corrole in chitosan. (B) Visual
appearance of chitosan (a) and of corrole grafted-chitosan derivatives obtained after different reaction
times [(b) 6 h, (c) 24 h, and (d) 48 h], and of the corresponding films. Reproduced from Reference [30]
from the
with permission from the American
American Chemical
Chemical Society.
Society.

Considering
Considering thethe suitable
suitable features
features of of chitosan
chitosan to
to act
act as a support for PS as well as its antimicrobial
antimicrobial
activity and film forming ability,
ability, Neves
Neves and colleagues reported
reported the synthetic access to new materials
obtained
obtained through
throughthe thenon-covalent incorporationofofmeso-tetraarylporphyrins
non-covalentincorporation meso-tetraarylporphyrins P1–P4
P1-P4 bearing
bearing phenyl
phenyl or
pentafluorophenyl
or pentafluorophenyl groups at theatmeso
groups the positions with or
meso positions without
with acid groups
or without acid in chitosan
groups (Figure 19)
in chitosan [109].
(Figure
The effectiveness
19) [109]. of the non-immobilized
The effectiveness porphyrins
of the non-immobilized and of the
porphyrins porphyrinic-chitosan
and films (PS-CF)
of the porphyrinic-chitosan films
to photoinactivate
(PS-CF) Listeria innocua
to photoinactivate Listeriaandinnocua and L.toinnocua
to prevent preventbiofilm development
L. innocua biofilm was evaluated was
development and
it was verified
evaluated and itthat
was the photodynamic
verified inactivation was
that the photodynamic dependentwas
inactivation on dependent
the porphyrin structure
on the and
porphyrin
also on their 1 O . The biofilm
structure andability
also onto produce
their ability 2 to produce 1O development
2. The biofilm was almost absolutely
development inhibited
was almost in the
absolutely
presence
inhibited ofin PS-CFs containing
the presence of PS-CFsporphyrins
containingP1 porphyrins
and P2 afterP1 being irradiated
and P2 with
after being white light
irradiated forwhite
with 24 h
(irradiance
light for 24of h 10 mW cm−of
(irradiance 2 ) followed by−2incubation in the dark for 48 h. The authors highlighted that
10 mW cm ) followed by incubation in the dark for 48 h. The authors
the good results
highlighted thatassociated
the goodwith the easy
results recovery
associated withof the
thefilms
easymerit further
recovery of investigation
the films merit using other
further
investigation using other PSs and new biofilm-forming microorganisms with potential application as
an anti-fouling coating materials for the food industry [109].
Molecules 2018, 23, 2424 19 of 47

PSs and new biofilm-forming microorganisms with potential application as an anti-fouling coating
materials for23,
Molecules 2018, the foodPEER
x FOR industry [109].
REVIEW 19 of 47

Figure 19. Structure of meso-tetraarylporphyrin P1–P4, bearing phenyl or pentafluorophenyl groups

at the meso positions with or without acid groups used to prepare chitosan films (adapted from
Reference [109]).

Considering
Considering the thepotential
potentialofofthethe PDI PDI approach
approach to treat
to treat infected
infected dentaldental hard tissues
hard tissues in a
in a clinical
clinical scenario [114–116], but also under other contexts, several
scenario [114–116], but also under other contexts, several groups evaluated the antibacterial groups evaluated the antibacterial
performance
performance of ofconjugates
conjugatesinvolving
involvingdyes dyes like
likeRoseRoseBengal
Bengalandand methylene
methylene blueblue
andandchitosan [117–121].
chitosan [117–
For instance, Kishen and co-workers [120] reported the photodynamic
121]. For instance, Kishen and co-workers [120] reported the photodynamic efficacy against dental efficacy against dental biofilms
of the conjugate
biofilms Rose Bengal-chitosan
of the conjugate (CS-RB) obtained
Rose Bengal-chitosan from the from
(CS-RB) obtained reaction
the between RB and chitosan
reaction between RB and
in the presence of the coupling agent EDC (N-ethyl-N 0 -(3-dimethylaminopropyl)carbodiimide).
chitosan in the presence of the coupling agent EDC (N-ethyl-N′-(3-
The photodynamic efficacy of CS-RB, with a high efficiency to generate 1 O , was evaluated towards
dimethylaminopropyl)carbodiimide). The photodynamic efficacy of CS-RB, 2 with a high efficiency to
planktonic
generate 1Oand in vitro
2, was biofilms
evaluated of E. faecalis
towards after activation
planktonic and in vitro withbiofilms
green light of (540 nm) using
E. faecalis after light doses
activation
between 5 to 60 J cm −2 . The results indicated that the in vitro CS-RB against E. faecalis in planktonic
with green light (540 nm) using light doses between 5 to 60 J cm . The results indicated that the in
−2

cells
vitro gave
CS-RB a much
against higher inactivation,
E. faecalis eithercells
in planktonic in the gavedark or in the
a much presence
higher of light,either
inactivation, whenin compared
the dark
with RB presence
or in the alone (>7oflog of reduction)
light, when compared in both withlightRBregimes.
alone (>7However, in the presence
log of reduction) of E.regimes.
in both light faecalis
biofilms, the bacterial reduction was not so drastic, achieving in both
However, in the presence of E. faecalis biofilms, the bacterial reduction was not so drastic, achievingcases (CS-RB and RB alone) a
reduction of at least 3 log after photodynamic treatment. Nevertheless,
in both cases (CS-RB and RB alone) a reduction of at least 3 log after photodynamic treatment. when CS-RB was employed,
the biofilm photoinactivation
Nevertheless, when CS-RB was was light-dose
employed, thedependent and was also significantly
biofilm photoinactivation higherdependent
was light-dose than that
by RB at 40 and 60 J cm −2 . Moreover, in the assays concerning cytotoxicity and photocytotoxicity in
and was also significantly higher than that by RB at 40 and 60 J cm . Moreover, in the assays −2

fibroblasts
concerningcells, it was found
cytotoxicity that the presence of
and photocytotoxicity in CS-RB gavecells,
fibroblasts higher fibroblast
it was foundcell survival
that than did
the presence of
RB alone. Additionally, the dentin collagen tests used to evaluate the
CS-RB gave higher fibroblast cell survival than did RB alone. Additionally, the dentin collagen tests chemical changes, resistance to
enzymatic degradation,
used to evaluate and mechanical
the chemical changes, properties
resistance demonstrated
to enzymatic that degradation,
the CS-RB-cross-linked
and mechanicaldentin
collagen
properties showed higher resistance
demonstrated to collagenase degradation
that the CS-RB-cross-linked and superior
dentin collagen showed mechanical properties.
higher resistance to
Thus, it was envisaged that the photoactivated CS-RB conjugate
collagenase degradation and superior mechanical properties. Thus, it was envisaged that the could be a targeted treatment strategy
to deal with infected
photoactivated CS-RBdental hard could
conjugate tissuesbe inaa targeted
clinical scenario,
treatment where
strategybothtodisinfection and structural
deal with infected dental
integrity
hard tissuesneedin to abeclinical
concomitantly
scenario, addressed.
where both disinfection and structural integrity need to be
As an extension
concomitantly addressed.of the previous work, the same group [121] developed NPs based on polymeric
chitosan
As an covalently
extensionlinked to Rose Bengal
of the previous work, the (CSNP-RB)
same group (Figure
[121] 20) and evaluated
developed NPs based their
on efficacy
polymericin
reducing the viability of E. faecalis biofilms to disrupt biofilm structure
chitosan covalently linked to Rose Bengal (CSNP-RB) (Figure 20) and evaluated their efficacy in and their ability to stabilize
the dentin-collagen
reducing the viability structural integrity.
of E. faecalis The chitosan
biofilms to disrupt nanoparticles
biofilm structure (CSNP) and were prepared
their bystabilize
ability to treating
chitosan, previouslystructural
the dentin-collagen dissolvedintegrity.
in acetic acid, with a solution
The chitosan of sodium
nanoparticles (CSNP)hydroxide in order by
were prepared to increase
treating
the pH to ≈ 5 and then with sodium tripolyphosphate under stirring.
chitosan, previously dissolved in acetic acid, with a solution of sodium hydroxide in order to increase The NPs were obtained by
centrifugation at 20,000 rpm for 30 min followed by the conventional
the pH to ≈5 and then with sodium tripolyphosphate under stirring. The NPs were obtained by work-up. The conjugation of
CSNP to RB acid
centrifugation group rpm
at 20,000 was performed
for 30 min in the presence
followed by theof EDC and N-hydroxysuccinimide
conventional work-up. The conjugation (NHS),
of
CSNP to RB acid group was performed in the presence of EDC and N-hydroxysuccinimide (NHS),
as previously described. The study showed that the CSNP-RB was also less toxic to fibroblasts and
had significant antibacterial activity (reduction > 8 log) even in the presence of bovine serum albumin.
The authors explained that CSNP-RB exerted an antibacterial mechanism by adhering to a bacterial
cell surface, permeabilizing the membrane and lysing the cells subsequent to photodynamic
Molecules 2018, 23, 2424 20 of 47

as previously described. The study showed that the CSNP-RB was also less toxic to fibroblasts and
had significant antibacterial activity (reduction > 8 log) even in the presence of bovine serum albumin.
The authors explained that CSNP-RB exerted an antibacterial mechanism by adhering to a bacterial
Molecules 2018, 23, x FOR PEER REVIEW 20 of 47
cell surface, permeabilizing the membrane and lysing the cells subsequent to photodynamic treatment.
Incorporation
treatment. of CSNP-RB
Incorporation ofand photocrosslinking
CSNP-RB significantlysignificantly
and photocrosslinking improved resistance to resistance
improved degradation to
and the mechanical strength of dentin-collagen.
degradation and the mechanical strength of dentin-collagen.

Conditions used to graft Rose Bengal in CSNP (adapted from Reference [121]).
Figure 20. Conditions

Interestingly, in
Interestingly, in 2016,
2016, Mashayekhan
Mashayekhan and and co-workers
co-workers reported
reported that that the the presence
presence of of chitosan
chitosan
nanoparticles (CSNPs)
nanoparticles (CSNPs) couldcould improve
improve the the photodynamic
photodynamic action action ofof methylene
methylene blue blue towards
towards S. S. aureus
aureus
and P. aeruginosa biofilms, and human fibroblasts [122]. The CSNPs were
and P. aeruginosa biofilms, and human fibroblasts [122]. The CSNPs were prepared using the previous prepared using the previous
ionic gelation
ionic method and
gelation method and the
the dynamic
dynamic light light scattering
scattering (DLS),
(DLS), andand field-emission
field-emission scanningscanning electron
electron
microscope (FESEM)
microscope (FESEM) results
resultsconfirmed
confirmedtheir theirnanometric
nanometricsize. size.The
Thephotodynamic
photodynamic assays
assays performed
performed in
the presence of MB + CSNPs at a final concentration of 50 µM MB and an irradiance of 22.93 J cm −−22
in the presence of MB + CSNPs at a final concentration of 50 µM MB and an irradiance of 22.93 J cm
showed aa significant
showed significant anti-biofilm
anti-biofilmphotoinactivation
photoinactivation(>3 (>3andand>2>2 loglog CFU
CFU reduction
reduction S. S.
in in aureus
aureus andandP.
P. aeruginosa
aeruginosa biofilms,
biofilms, respectively)
respectively) whilewhile MB alone
MB alone led toled to a reduction
a reduction of approximately
of approximately < 1 log
< 1 log CFU. At
CFU.
the At the
same same experimental
experimental conditions, conditions,
only 25.1% onlyof25.1% of the fibroblasts
the fibroblasts were photoinactivated
were photoinactivated by MBby +
MB + CSNPs
CSNPs and the and the efficacy
efficacy of MB-PDIof MB-PDI was explained
was explained by considering
by considering that the that the polycationic
polycationic CSNPs CSNPs
were
were
able toable to disrupt
disrupt biofilm
biofilm structure
structure allowing
allowing a deeper
a deeper andand higher
higher penetration
penetration of MB
of MB intointo
thethe biofilms.
biofilms.
In a previous study, Durantini and co-workers [123] also reported the effect of chitosan (CS) (CS)
In a previous study, Durantini and co-workers [123] also reported the effect of chitosan and
and other
other additives
additives (divalent
(divalent cations cations and EDTA)
and EDTA) on theon the uptake
uptake and onand theon the photoinactivation
photoinactivation E.
of in
of E. coli
coli in the presence 5,10,15,20-tetrakis(4-N,N,N-trimethylammoniumphenyl)porphyrin (TAAP 4+ )
the presence 5,10,15,20-tetrakis(4-N,N,N-trimethylammoniumphenyl)porphyrin (TAAP ) (Figure 2), 4+

(Figure 2), 5,10-di(4-methylphenyl)-15,20-di(4-N,N,N-tri-methylammoniumphenyl)porphyrin

5,10-di(4-methylphenyl)-15,20-di(4-N,N,N-tri-methylammoniumphenyl)porphyrin (MPAP2+), and
(MPAP 2+ ), and 5,10,15,20-tetrakis(4-sulphonatophenyl)porphyrin 4 −
5,10,15,20-tetrakis(4-sulphonatophenyl)porphyrin (TPPS4−) (Figure(TPPS 21). The) authors
(Figure 21). The that
verified authors
the
verified that the addition of Ca 2+ or Mg2+ to the cultures enhanced the cell2+uptake of MPAP 2+ and
addition of Ca or Mg to the cultures enhanced the cell uptake of MPAP and TPPS , but not of
2+ 2+ 4−

TPPS 4− 4+ . Concerning the other two additives, EDTA produced an increase in the
TMAP4+,. but not of TMAP
Concerning the other two additives, EDTA produced an increase in the uptake of all
uptake of all porphyrins, while CS mainly enhanced 4− to E. coli.of
porphyrins, while CS mainly enhanced the amount of the
TPPS amount
4− bound of to
TPPSE. coli.bound
In the presence InCa
the2+

presence of Ca 2+ and Mg2+ , the phototoxic activity mediated by MPAP 2+ and TPPS4− towards E. coli
and Mg , the phototoxic activity mediated by MPAP and TPPS towards E. coli was increased but
2+ 2+ 4−

was 4+
was increased
reduced in butthewaspresence
reduced of in the
TMAPpresence
4+. EDTA of TMAP
did not. EDTA
affect didthe not affect the photoinactivation
photoinactivation induced by
induced by cationic porphyrins, while a small enhancement was found for TPPS 4− . In the presence of
cationic porphyrins, while a small enhancement was found for TPPS . In the presence of CS, using a 4−

CS, using a slightly toxic CS concentration, an photoinactivation

enhanced photoinactivation E. coli4−by
of TPPS TPPS 4− was
slightly toxic CS concentration, an enhanced of E. coli by was observed,
observed,tocontrary to observed
what waswith observed with 4+
TMAP . Therefore, the cytotoxic
combinedactivities
cytotoxicofactivities
contrary what was TMAP 4+. Therefore, the combined CS and
of CS and PDI mediated by TPPS 4− could be beneficial for the inactivation of E. coli.
PDI mediated by TPPS could be beneficial for the inactivation of E. coli.
4−
Molecules 2018, 23, 2424 21 of 47
Molecules 2018, 23, x FOR PEER REVIEW 21 of 47

Figure 21. Structures

Figure 21. of 5,10-di(4-methylphenyl)-15,20-di(4-N,N,N-tri-methylammoniumphenyl)porphyrin
Structures of 5,10-di(4-methylphenyl)-15,20-di(4-N,N,N-tri-
(MPAP 2+ ) and 5,10,15,20-tetrakis(4-sulphonatophenyl)porphyrin (TPPS4− ) (adapted from Reference [123]).
methylammoniumphenyl)porphyrin (MPAP )
2+ and 5,10,15,20-tetrakis(4-
sulphonatophenyl)porphyrin (TPPS )(adapted from Reference [123]).
4−
4.2. Cellulose
4.2. Cellulose
Cellulose is the most abundant natural biopolymer, consisting of high molecular weight
Celluloseanhydro-
β-1,4-linked is the most D -glucose polymer chains, and therefore, is also considered a great
abundant natural biopolymer, consisting of high molecular weight β-1,4-
linked anhydro-D-glucose polymernew
starting material for developing chains,andand more sustainable
therefore, is alsomaterials
considered from renewable
a great startingresources.
material
As cellulose owns a carbohydrate nature, it has inherent compatibility
for developing new and more sustainable materials from renewable resources. As cellulose owns with biological tissues and,a
consequently, possesses unique utility with respect to their bioavailability,
carbohydrate nature, it has inherent compatibility with biological tissues and, consequently, biocompatibility, and
biodegradability
possesses unique considerations.
utility with respect to their bioavailability, biocompatibility, and biodegradability
Considering that cellulose could be an excellent material to develop germicidal surfaces
considerations.
at low cost, in that
Considering 1994, Bonnett
cellulose andbeGalia
could [124] reported
an excellent material tothe photobactericidal
develop properties
germicidal surfaces at low of
“cellophane” films (regenerated
cost, in 1994, Bonnett and Galia cellulose)
[124] reportedimpregnated with the cationic
the photobactericidal porphyrins
properties 5,10,15,20-tetrakis
of “cellophane” films
(1-methylpyridinium-4-yl)porphyrin tetra-tosylate (TMPyP)
(regenerated cellulose) impregnated with the cationic porphyrins 5,10,15,20-tetrakis(1- and with 5,10,15,20-tetrakis(N,N,N-
trimethylanilinium-4-yl)porphyrin tetra-tosylate (TAAP 4+ ) (Figure 2). The impregnation time required
methylpyridinium-4-yl)porphyrin tetra-tosylate (TMPyP) and with 5,10,15,20-tetrakis(N,N,N-
trimethylanilinium-4-yl)porphyrin tetra-tosylate (TAAP ) (Figure sheets
was performed by maintaining commercially available cellophane
4+ 2). The of impregnation
50 µm thickness in
time
contact with the porphyrins dissolved in aqueous solution or aqueous
required was performed by maintaining commercially available cellophane sheets of 50 µm thickness methanol solution at about
◦ C. The amount of absorbed porphyrin was estimated by UV-Vis spectroscopy after washing
50contact
in with the porphyrins dissolved in aqueous solution or aqueous methanol solution at about
thoroughly.
50 °C. The amountThe authors verified porphyrin
of absorbed that the films was containing
estimatedTMPyP, by UV-Visafter spectroscopy
exposure to a after1500 W xenon
washing
lamp for 50 h, retained their flexibility and strength and were not
thoroughly. The authors verified that the films containing TMPyP, after exposure to a 1500 W xenon photobleached (although some
photochemical
lamp changes occurred
for 50 h, retained such asand
their flexibility a slightly
strength opacity).
and were No not
bacterial growth was
photobleached verified when
(although some
the films impregnated with TMPyP (18 µg cm −2 ) were maintained in contact with bacterial inoculum
photochemical changes occurred such as a slightly opacity). No bacterial growth was verified when
of S.
the aureus,
films Proteus vulgaris
impregnated and E.(18
with TMPyP coliµg for
cm24 −2)hwere
under visible light
maintained (fluorescent
in contact lamp) irradiation.
with bacterial inoculum
Similar photobactericidal results were reported with the TAAP 4+ only towards S. aureus. The results
of S. aureus, Proteus vulgaris and E. coli for 24 h under visible light (fluorescent lamp) irradiation.
with P. aeruginosa strain were not conclusive. The authors highlighted
Similar photobactericidal results were reported with the TAAP only towards S. aureus. The results
4+ that the attempts to impregnate
the cellophane
with P. aeruginosa films with
strain anionic
were porphyrinsThe
not conclusive. were not successful.
authors highlighted that the attempts to impregnate
In 2016, Rassa and co-workers [125] also
the cellophane films with anionic porphyrins were not successful.selected the tetracationic porphyrin TAPP4+ (Figure 2)
and In the2016,
corresponding Zn(II) complex
Rassa and co-workers [125]toalso prepare
selected cellulosic fabrics for
the tetracationic antimicrobial
porphyrin TAPPapplications.
4+ (Figure 2)
The impregnation was performed by soaking a 100% cellulosic fabric,
and the corresponding Zn(II) complex to prepare cellulosic fabrics for antimicrobial applications. pretreated with an aqueous The
solution of Na CO at 50 ◦ C for 30 min, with solutions of the porphyrins in PBS. After 30 min at 50 ◦ C,
2 3
impregnation was performed by soaking a 100% cellulosic fabric, pretreated with an aqueous
the unbound
solution of Naporphyrin was thoroughly washed, and the molar grafting ratio was calculated using
2CO3 at 50 °C for 30 min, with solutions of the porphyrins in PBS. After 30 min at 50
UV-Vis.
°C, The basicporphyrin
the unbound pretreatment waswas important
thoroughly to activate
washed, and thethe cellulose
molar graftinghydroxyl group
ratio was in order to
calculated
promote electrostatic interactions with the cationic PS. The authors noted
using UV-Vis. The basic pretreatment was important to activate the cellulose hydroxyl group in order that the grafting yield was
dependent on the initial concentration of the PS and its presence in the cellulosic
to promote electrostatic interactions with the cationic PS. The authors noted that the grafting yield fabrics was confirmed
by spectroscopic
was dependent on techniques.
the initialThe antimicrobial
concentration of activity
the PS and of theitsnew materials
presence in thewas tested under
cellulosic fabricsvisible
was
light irradiation (100 W tungsten lamp with an average intensity of ≈ 0.36 mW cm −2 ) towards S. aureus,
confirmed by spectroscopic techniques. The antimicrobial activity of the new materials was tested
P. aeruginosa,
under lightE.irradiation
visibleand coli. The results
(100 W showed
tungsten that lamp
the irradiation period, the
with an average PS concentration,
intensity of ≈0.36 mW andcmthe
−2)

towards S. aureus, P. aeruginosa, and E. coli. The results showed that the irradiation period, the PS
concentration, and the bacterial strain were important factors to be taken in account. S. aureus was
Molecules 2018, 23, 2424 22 of 47

Molecules 2018, 23, x FOR PEER REVIEW 22 of 47

bacterial strain were important factors to be taken in account. S. aureus was fully inactivated with
both
fully prepared
inactivated cellulose
with both fabrics at a PS
prepared concentration
cellulose fabrics of at 100
a PSµM after 30 minofof100
concentration irradiation,
µM after 30 while
minforof
P. aeruginosa,while
irradiation, at theforsame PS concentration,
P. aeruginosa, at the samethe full PSinactivation
concentration, wasthe only attained
full in the was
inactivation presence
only
of the ZnTAPP 4+ after 90 min of irradiation. The E. coli
attained in the presence of the ZnTAPP4+ after 90worse
min ofefficiency
irradiation. of the
Thematerials towards of
worse efficiency the
(58.5% for TAPP 4+ and 30% for ZnTAPP4+ )4+at the same concentration and after 90 min of irradiation
materials towards E. coli (58.5% for TAPP and 30% for ZnTAPP ) at the same concentration and 4+

was
afterexplained by considering
90 min of irradiation was the more complex
explained composition
by considering of this
the more Gram-(-)
complex cell wall, of
composition that seems
this Gram- to
protect this strain more effectively from extracellular 1O .
(-) cell wall, that seems to protect this strain more effectively 2 from extracellular O2. 1

The cationic phthalocyanines 4+ and 4+ with four pyridyl substituents linked

phthalocyanines PcPy PcPy4+ and ZnPcPy4+ with four pyridyl substituents linked via a
direct C-C
C-Cbond
bond to the α-phthalo-positions
to the α-phthalo-positions (Figure 22) were
(Figure 22)also
wereconsidered in the design
also considered of photoactive
in the design of
antimicrobial surface based on cellulose [126]. The synthetic access to those
photoactive antimicrobial surface based on cellulose [126]. The synthetic access to those derivatives derivatives required the
previous
required thepreparation
previous of the pyridine
preparation phthalonitrile
of the from the pyridine
pyridine phthalonitrile from the boronate
pyridine ester and the
boronate triflate
ester and
phthalonitrile (Figure 22).(Figure
the triflate phthalonitrile Then, tetramerization under conventional
22). Then, tetramerization conditions to
under conventional afford thetoneutral
conditions afford
precursors
the neutral PcPy 4 and ZnPcPy
precursors PcPy4 and4 followed
ZnPcPyby methylation
4 followed with iodomethane
by methylation in DMF gave
with iodomethane rise togave
in DMF the
desired cationic salts 4+
(PcPysalts and ZnPcPy 4+ ). The antimicrobial surfaces surfaces
were prepared by soaking
rise to the desired cationic (PcPy 4+ and ZnPcPy 4+). The antimicrobial were prepared by
3.5 × 3.5 cm filter papers (Whatman 1) in aqueous solutions containing
soaking 3.5 × 3.5 cm filter papers (Whatman 1) in aqueous solutions containing the neutral and the the neutral and the cationic
dyes at different
cationic dyes at concentrations (range from(range
different concentrations 0.008 to 0.080.008
from mg cmto−20.08
). Themgphotodynamic antimicrobial
cm−2). The photodynamic
efficacy of theefficacy
antimicrobial dyed filter
of thepaper
dyed samples usingsamples
filter paper bioluminescent E coli and Acinetobacter
using bioluminescent baylyi ADP1
E coli and Acinetobacter
strains allowed for the selection of the zinc complex ZnPcPy 4+ as the best 4+PS; the PDI experiments
baylyi ADP1 strains allowed for the selection of the zinc complex ZnPcPy as the best PS; the PDI
were performed
experiments werefor 60 min using
performed for 60white light (wavelength
min using 485–750 nm)485–750
white light (wavelength of intensity
nm) of mW cm−18
18intensity 2.

The 4+
mWantimicrobial activity of the
cm−2. The antimicrobial filter of
activity paper dyed paper
the filter with ZnPcPy
dyed with achieved
ZnPcPya4+ reduction
achieved of 2.7 log CFU
a reduction of
against E. coliagainst
2.7 log CFU and ofE. 3.4coli
logand of 3.4A.
against logbaylyi.
against A. baylyi.

Figure 22. Synthetic access to the phthalonitrile used in the preparation of phthalocyanines
phthalocyanines (Zn)PcPy
(Zn)PcPy44
and (Zn)PcPy4+4+(adapted
(adaptedfrom
fromReference
Reference [126]).
[126]).

The preparation of
The preparation photoactive materials
of photoactive materials based on the
based on the non-covalent
non-covalent interaction
interaction of
of phenothiazine
phenothiazine
dyes
dyes like TBO and RB (Figure 2) with cellulose also deserved some attention from the
like TBO and RB (Figure 2) with cellulose also deserved some attention from the scientific
scientific
community.
community. For
For instance,
instance, Michael
Michael Wilson
Wilson [127]
[127] evaluated
evaluated the
the efficacy
efficacy of
of cellulose
cellulose acetate
acetate containing
containing
the TBO towards MRSA and P. aeruginosa under light conditions similar to those present in hospitals
(white light, 60 W domestic lamp bulb, for up to 24 h). The antimicrobial coatings were prepared just
by adding the TBO to a solution of cellulose acetate in acetone and then leaving the solvent to
Molecules 2018, 23, 2424 23 of 47

the TBO towards MRSA and P. aeruginosa under light conditions similar to those present in hospitals
(white light, 60 W domestic lamp bulb, for up to 24 h). The antimicrobial coatings were prepared
Molecules 2018, 23, x FOR PEER REVIEW 23 of 47
just by adding the TBO to a solution of cellulose acetate in acetone and then leaving the solvent
to evaporate.
evaporate. The Theapproach
approachwas wasconsidered
consideredtotohave havethethepotential
potentialtotominimize
minimizethe thetransmission
transmission of of
infectious diseases through surfaces in hospitals since substantial bacterial reduction (10 5 CFU cm−2 )
infectious diseases through surfaces in hospitals since substantial bacterial reduction (105 CFU cm−2)
was wasachieved
achieved following
following irradiation
irradiationofofthe thefilm.
film.
In In
2006,
2006, the same group [128] evaluated the
the same group [128] evaluated the efficacy
efficacy ofof the
the previous
previousmaterial
materialandandalsoalsoof of
anan
analogue
analogue containing
containingRB RBtowards
towardsmicroorganisms
microorganisms such such as S. aureus
as S. aureusand MRSA,E.E.coli,
andMRSA, coli,
C.C. albicans,
albicans,
Clostridium
Clostridium difficile,
difficile,and bacteriophageØX174.
andbacteriophage ØX174. The results
results confirmed
confirmedthe thehigh
highefficacy
efficacy ofof
thethe doped
doped
films
films upon
upon illuminationwith
illumination witha awhite
whitelight
lightemitted
emitted from a 28 28 WWfluorescent
fluorescentlamp lampfor
forperiods
periods between
between
2 to2 16
to 16
h, h,
withwith bacterialreductions
bacterial reductionsranging
ranging fromfrom 6.3
6.3 to 6.7 log.
log. C.C.albicans
albicanswas wasfound
foundtoto bebe the least
the least
sensitive
sensitive microorganism
microorganism to theto material
the material photodynamic
photodynamic actionaction
(0.9 log(0.9 log reduction
reduction after 16 hafter 16 h
irradiation).
irradiation).
Considering that aerosols are important vehicles for the transmission of infectious diseases
in hospitalConsidering
setting, that aerosols
the same are important
group vehicles
still evaluated thefor the transmission
efficacy of TBO andofRB infectious diseases
on cellulose in
acetate
hospital setting, the same group still evaluated the efficacy of TBO
towards S. aureus from aerosols [129]. The assays were performed by spraying the photoactive and RB on cellulose acetate
towards
surfaces S. S.
with aureus
aureusfrom
(105 aerosols
CFU m−[129]. The assays
2 ) suspended were performed bysaline,
in phosphate-buffered spraying theorphotoactive
saliva, horse serum.
surfaces with S. aureus (10 CFU m ) suspended in phosphate-buffered saline, saliva, or horse serum.
5 −2
The inactivation ranged from 0.8 log (in horse serum) to 2.5 log (in PBS) after 6 h of exposure with
The inactivation ranged from 0.8 log (in horse serum) to 2.5 log (in PBS) after 6 h of exposure with
white light emitted from a 28 W domestic fluorescent lamp.
white light emitted from a 28 W domestic fluorescent lamp.
The possibility to prepare photoactive cotton fabrics by covalently linking the PS to cellulose
The possibility to prepare photoactive cotton fabrics by covalently linking the PS to cellulose
was also considered in different studies [130]. For instance, Krausz and co-workers [130] reported the
was also considered in different studies [130]. For instance, Krausz and co-workers [130] reported the
synthetic
synthetic access to ato
access new
a antibacterial
new antibacterial material, TTPropP-Cel,
material, by grafting
TTPropP-Cel, throughthrough
by grafting a “Click-Chemistry”
a “Click-
approach the acetylenic porphyrin derivative TTPropP on cellulose
Chemistry” approach the acetylenic porphyrin derivative TTPropP on cellulose functionalized functionalized with azidewith units
(Figure 23). The authors found that under irradiation with visible light,
azide units (Figure 23). The authors found that under irradiation with visible light, the material the material displayed
antibacterial activity against
displayed antibacterial representative
activity strains of E.strains
against representative coli and
of E.S. coli
aureus.
and S. aureus.

Figure
Figure 23.23. Syntheticmethodology
Synthetic methodologyused
usedtoto covalently
covalently graft
graft the
theacetylenic
acetylenicporphyrin
porphyrinTTPropP
TTPropPin in
azido
azido
cellulose
cellulose fabric
fabric (adaptedfrom
(adapted fromReference
Reference[130]).
[130]).

Soon after, the same group [131] reported the covalent grafting of the neutral, anionic and
cationic amino 1–3 porphyrins on cotton fabrics via a 1,3,5-triazine linker without any preliminary
Molecules 2018, 23, 2424 24 of 47

Soon after, the same group [131] reported the covalent grafting of the neutral, anionic 24
Molecules 2018, 23, x FOR PEER REVIEW
and
of 47
cationic amino 1–3 porphyrins on cotton fabrics via a 1,3,5-triazine linker without any preliminary
chemical
chemical modification
modification ofof
thethe
cellulosic material
cellulosic (Figure
material 24).
(Figure When
24). When subjected
subjectedtoto
light irradiation
light irradiationof of
0.16 mW cm −2−2for 24 h (total light dose 13.8 J cm−2−2), the cationic fabric exhibited the best performance
0.16 mW cm for 24 h (total light dose 13.8 J cm ), the cationic fabric exhibited the best performance
(100%
(100%photoinactivation
photoinactivation against S. S.
against aureus while
aureus nono
while photoactivity
photoactivitywas
wasdetected against
detected againstE. coli).
E. coli).

Figure
Figure 24.24. Synthetic
Synthetic methodology
methodology used
used to to covalently
covalently graft
graft thethe amino
amino porphyrins
porphyrins 2-42-4
onon cotton
cotton fabrics
fabrics
viavia 1,3,5-triazine
1,3,5-triazine linker
linker (adapted
(adapted from
from Reference
Reference [131]).
[131]).

AA better
betterefficiency
efficiency towards
towards E. E.
colicoli
was wasfound
found when
when thetheauthors
authors tested
tested thethe
photobactericidal
photobactericidal
surface,
surface,obtained
obtained bybygrafting
grafting a tricationic
a tricationic porphyrin
porphyrin inin filter paper
filter paper through
through thethe
1,3,5-triazine
1,3,5-triazine linker
linker
(Figure 25). The authors verified that the untreated paper in the dark
(Figure 25). The authors verified that the untreated paper in the dark or under irradiation and or under irradiation and thethe
treated
treated paper
paperininthe dark
the dark allowed
allowed a bacterial
a bacterialgrowth
growthofof4 4and and2 2log unitsforforS.S.aureus
logunits aureusandandE.E.coli,
coli,
respectively, when compared with the initial bacterial concentration. However,
respectively, when compared with the initial bacterial concentration. However, no survival of either no survival of either
type
typeof of
bacteria
bacteriawaswasdetected
detected onon thethe
paper
paper bearing
bearing thethe
porphyrin
porphyrin after light
after lightexposure
exposure forfor
2424h, h,
totaling
totaling
a light dose of of
9.59.5
J cm − 2
a light dose J cm−2[132].
[132].
The
TheCu(I)-catalyzed
Cu(I)-catalyzedHuisgen Huisgen1,3-dipolar
1,3-dipolar cycloaddition
cycloaddition was also also selected
selected by byGhiladi
Ghiladiand andco-
co-workers to append
append the tricationic +
workers to tricationic porphyrin
porphyrin TriPy TriPy Me-PhCCH
+ Me-PhCCH bearing bearingan analkynyl
alkynylfunction
functionto to
cellulose
cellulosenanocrystals
nanocrystalscontaining
containingazide azidemoieties
moieties (Figure
(Figure 26)26)
[133,134].
[133,134].It Itwas wascommented
commentedthat that
cellulose nanocrystals (CNC)
cellulose nanocrystals (CNC) possessed possessed some benefits against amorphous cellulose
benefits against amorphous cellulose ester due to their ester due to
their defined
defined surface
surface structure
structure and and dimension
dimension and and a particular
a particular degree degree of molecular
of molecular controlcontrol for
for precise
precise functionalization because of their rigidity.
functionalization because of their rigidity. The authors prepared The authors prepared the cellulose
cellulose nanocrystalsbyby
nanocrystals
hydrolysis
hydrolysis of of
Whatman
Whatman #1#1 filter paper
filter paper (98%
(98% α-cellulose,
α-cellulose, 80%80% crystallinity),
crystallinity), previously
previously blended
blended using
using
aqueous
aqueous HBr. After
HBr. removing
After removing thetheexcess
excess of of
acid, water-soluble
acid, water-soluble fragments
fragments andandultrafine
ultrafineparticles
particles of of
CNC were obtained with an average length of 100–400 nm. The
CNC were obtained with an average length of 100–400 nm. The nanocrystals were then nanocrystals were then functionalized
with the azide moities for aazide
further reaction +
functionalized with the moities for awith the porphyrin
further reaction with TriPytheMe-PhCCH.
porphyrin TriPy Although, only
+Me-PhCCH.

suspended
Although,inonly an aqueous
suspended system,
in anthe CNC-Por
aqueous showed
system, excellent showed
the CNC-Por efficacy excellent
toward the photodynamic
efficacy toward the
inactivation
photodynamic of Mycobacterium
inactivation of smegmatis and S. aureus,
Mycobacterium smegmatisalbeitandwith only aalbeit
S. aureus, slightwith
activity a slightE.activity
onlyagainst coli.
against E. coli.
Molecules 2018, 23, 2424 25 of 47
Molecules2018,
Molecules 2018,23,
23,xxFOR
FORPEER
PEERREVIEW
REVIEW 25of
25 of47
47

Figure 25.
Figure
Figure 25. Structure
25. Structure of
Structure of the
of the antimicrobial
the antimicrobial filter
antimicrobial filter paper
filter paper bearing
paper bearing aaa tricationic
bearing tricationic porphyrin
tricationic porphyrin (adapted
porphyrin (adapted from
(adapted from
from
Reference
Reference [132]).
Reference [132]).
[132]).

Figure26.
Figure 26.Preparation
Preparationof
ofaacellulose
cellulosenanocrystal
nanocrystalconjugated
conjugatedto
toaacationic
cationicporphyrin TriPy+++Me-PhCCH
porphyrinTriPy
TriPy Me-PhCCH
(adapted from
(adapted
(adapted fromReference
from Reference [133,134]).
Reference [133,134]).
[133,134]).

In the firststudy,
study,the thebacterial
bacterialcultures
cultures 8
(≈8810 CFU −1
In the first
first study, the bacterial cultures (≈10
(≈10 CFU
CFU mL−1mL
mL
−1) were
) were ) were irradiated
irradiated
irradiated with
whitewhite
with white
with light
light (400–
light (400–
(400–700 nm; 60 mW cm − 2 ) in PBS with 20 µM of CNC-Por after a dark incubation period and the
700 nm; 60 mW cm −2 ) in PBS with 20 µM of CNC-Por after
700 nm; 60 mW cm−2) in PBS with 20 µM of CNC-Por after a dark incubation period and the results a dark incubation period and the results
results showed
showed aa 66 log
showed a 6 log
log reduction
reduction inreduction
in viable in viable
viable cells cells
cells against
against S.against S.
S. aureus aureus
aureus (5 (5 min (5 min incubation,
min incubation,
incubation, 30 30 min30 min irradiation),
min irradiation),
irradiation), 3.5 3.5
3.5
log log
for for
M. M. smegmatis
smegmatis (45 (45
min min incubation,
incubation, 30 30minmin irradiation),
irradiation),
log for M. smegmatis (45 min incubation, 30 min irradiation), and 2 log for E. coli (60 min incubation, and
and 2 2 log
log for
for E.
E. coli
coli (60
(60 min
min incubation,
incubation,
30 min
30 minirradiation)
irradiation)[133].
irradiation) [133].In
[133]. InInthe
the the second
second
second study,
study,
study, thethe
the efficacy
efficacy
efficacy ofthe
of of photoactive
the the photoactive
photoactive CNC-Por
CNC-PorCNC-Por conjugate,
conjugate,
conjugate, also
also
also
at 20 at
µM, 20 µM,
was was
tested tested
against against
A. A.
baumannii,baumannii,
multidrug multidrug
resistant
at 20 µM, was tested against A. baumannii, multidrug resistant A. baumannii strain, MRSA, and P. resistant
A. A.
baumanniibaumannii
strain, strain,
MRSA, MRSA,
and P.
and P. aeruginosa ( ≈ 10 8 CFU mL −1 ) with visible light (65−2mW cm−2 ) [134]. A decrease of 6 log units
aeruginosa (≈10 8 CFU mL −1) with visible light (65 mW cm
aeruginosa (≈10 CFU mL ) with visible light (65 mW cm ) [134]. A decrease of 6 log units in viable
8 −1 −2 ) [134]. A decrease of 6 log units in viable
in
cellsviable
cells was cells
was observed
observedwas for observed
MRSAfor
for MRSA (5 MRSA
(5 min
min (5 min incubation),
incubation),
incubation), 5–6 log
5–6 log units
units5–6 log
for
for A.units
A. for A.(30
baumannii
baumannii baumannii
(30 (30 min
min incubation)
min incubation)
incubation)
and multidrug
and multidrug andresistant
multidrug
resistant A.resistant
A. baumannii
baumannii A.(15
baumannii
(15 (15 min incubation),
min incubation),
min incubation), and 2.5
and 2.5 log
logand 2.5 for
units
units log units
for P. for P. aeruginosa
P. aeruginosa
aeruginosa (60 min
(60 min
(60 min incubation) at a total light dose of −2118 J cm −2 (30 min irradiation). Confocal laser scanning
incubation) at a total light dose of 118 J cm (30 min irradiation).
incubation) at a total light dose of 118 J cm (30 min irradiation). Confocal laser scanning microscopy
−2 Confocal laser scanning microscopy
microscopy
analysisof
analysis analysisafter
ofCNC-Por
CNC-Por of CNC-Por
after incubation
incubation afterwith
with incubation
A.baumannii
A. baumannii withor A.S.
or S.baumannii
aureussuggested
aureus or S. aureus
suggested aalack
lacksuggested a lack of
ofinternalization
of internalization
internalization
of the
of the PS. of the PS.
PS. Considering
Considering Considering
both
both studies, the
studies, both
the studies,
authors
authors the authorsthat
commented
commented commented
that CNC-Porthat
CNC-Por wasCNC-Por
was able to
able was ablethe
to mediate
mediate to
the
mediate the photodynamic
photodynamic inactivation
photodynamic inactivation of inactivation
of all
all the of all
the tested the tested
tested bacterial bacterial
bacterial strains strains
strains studied.studied.
studied. ConfocalConfocal microscopy
Confocal microscopy
demonstrated that the mode of action for CNC-Por
demonstrated that the mode of action for CNC-Por did not proceed through a PS-binding did not proceed through a PS-binding or or uptake
uptake
mechanism. Rather, the cytotoxic
cytotoxic species (e.g., 1 O22 and ROS) generated
generated by by the
the photodynamic
photodynamic
mechanism. Rather, the cytotoxic species
species (e.g.,
(e.g., 11O and other
other ROS) photodynamic
process were
process were likely likely
likely to to diffuse
to diffuse
diffuse over over short
over short distances
short distances
distances to to ultimately
to ultimately damage the bacteria,
ultimately damage the bacteria, leading leading to to cell
cell
inactivation as
inactivation as already
already stated
stated in
in previous
previous studies
studies [135].
[135]. The
The authors
authors commented
commented the
the potentiality
potentiality of
of
Molecules 2018, 23, 2424 26 of 47

inactivation
Molecules as xalready
2018, 23, stated
FOR PEER in previous
REVIEW studies [135]. The authors commented the potentiality 26 of of
47
the prepared materials to reduce the rates of transmission of a wide range of bacteria, particularly
antibiotic
the preparedresistant strains,
materials which is
to reduce theimportant for hospitals and
rates of transmission of a healthcare-related industries
wide range of bacteria, [134].
particularly
Carpenter
antibiotic et al.
resistant [136]which
strains, also employed
is important cellulose paperand
for hospitals activated with azide industries
healthcare-related moieties to graft
[134].
differently-charged porphyrins and boron dipyrromethenes (BODIPY) bearing
Carpenter et al. [136] also employed cellulose paper activated with azide moieties to graft an alkenyl unit via
the “Click-Chemistry”
differently-charged approach
porphyrins (Figure
and boron27). The bactericidal
dipyrromethenes papers obtained
(BODIPY) bearing anwith a dyeunit
alkenyl loadvia of
≈ 8 mg g −1 (12.4 nmol mg−1 , MW 1 672) were fully characterized and their antibacterial efficacy was
the “Click-Chemistry” approach (Figure 4 27). The bactericidal papers obtained with a dye load of ≈8
evaluated
mg nmolS.mg
against
g−1 (12.4 aureus,
−1, MW ¼ 672) were fully E.
vancomycin-resistant faecium, A. baumannii,
characterized and their P. aeruginosa, and
antibacterial Klebsiella
efficacy was
pneumoniae.
evaluated TheS.best
against results
aureus, were obtained with
vancomycin-resistant the Por
E. faecium, A.(+) -paper conjugate
baumannii, that and
P. aeruginosa, wasKlebsiella
able to
photoinactivate
pneumoniae. The>4best
log results
(99.99+%) of all
were bacterialwith
obtained strainsthestudied upon conjugate
Por(+)-paper illumination forwas
that 30 minablewithto
white light (65 mW − 2 , 400−700
cm(99.99+%) nm). The same (+)
Porstudied
-paper wasillumination
also able to for
photoinactivate
photoinactivate >4 log of all bacterial strains upon 30 min with
dengue-1
white lightvirus by >4
(65 mW cmlog, influenza
−2, 400−700 nm).A The by ≈Por
virussame 2.5(+)log (~99.5%)
-paper andable
was also human adenovirus-5 by
to photoinactivate ≈2 log
dengue-
(1≈virus
99%).by >4 log, influenza A virus by ≈2.5 log (~99.5%) and human adenovirus-5 by ≈2 log (≈99%).

Figure 27. Structure of

of the
theantimicrobial
antimicrobialfilter
filterpaper
paperbearing
bearing different
different porphyrins
porphyrins andand BODIPY
BODIPY (adapted
from Reference
(adapted [136]). [136]).
from Reference

Considering the
Considering the requirement
requirement of of antimicrobial
antimicrobial textile
textile products,
products, such
such as
as gowns,
gowns, beddings
beddings andand
wound dressings for the health industry, in order to decrease the occurrence of
wound dressings for the health industry, in order to decrease the occurrence of hospital-acquiredhospital-acquired
infections [137],
infections [137], several
several synthetic
synthetic and
and natural
natural fibers
fibers have
have been
been manufactured
manufactured in in the
the presence
presence ofof
antimicrobial products. For instance, cellulose has been modified into antimicrobial wound
antimicrobial products. For instance, cellulose has been modified into antimicrobial wound dressings dressings
by introducing
by introducing numerous
numerous antimicrobial
antimicrobial or or disinfecting
disinfecting agents
agents such
such asasantibiotics,
antibiotics,peptides,
peptides,polymers,
polymers,
and inorganic
and inorganic nanoparticles
nanoparticles [138].
[138]. Under
Under this
this context,
context, Chen
Chen and
and colleagues
colleagues concentrated
concentrated their
their efforts
efforts
in developing fabrics with antimicrobial features. The group found out that the efficacy
in developing fabrics with antimicrobial features. The group found out that the efficacy of a fabric of a fabric
only covered with the antimicrobial agent ε-polylysine could be improved by adding
only covered with the antimicrobial agent ε-polylysine could be improved by adding a second layer a second layer
of aa PS
of PS agent
agent (the
(the zinc(II)
zinc(II) complex
complex ofof aa mono-substituted
mono-substituted β-carboxyphthalocyanine)
β-carboxyphthalocyanine) onto onto the
the fabric
fabric
(Figure 28). Therefore, the positively charged bottom layer of ε-polylysine was able to disrupt the
bacterial membrane, while the zinc phthalocyanine derivative was able to inactivate microorganisms
by 1O2 and other ROS, thus compensating the ε-polylysine low antimicrobial efficacy. The authors
found that the fabric was stable, durable, and washable if ε-polylysine was first adsorbed before the
Molecules 2018, 23, 2424 27 of 47

(Figure 28). Therefore, the positively charged bottom layer of ε-polylysine was able to disrupt the
bacterial membrane,
Molecules 2018, whileREVIEW
23, x FOR PEER the zinc phthalocyanine derivative was able to inactivate microorganisms 27 of 47
1
by O2 and other ROS, thus compensating the ε-polylysine low antimicrobial efficacy. The authors
found that thebetween
condensation fabric was thestable, durable,
present PS acidand washable
groups and ifthe amino groups
ε-polylysine was first
ontoadsorbed before
ε-polylysine the
under
condensation between the present PS acid groups and the amino groups onto
mild conditions. This special fabric proved to be a potent antimicrobial against both Gram-(−) and
ε-polylysine under mild
conditions. This special
Gram-(+) bacterial fabric
strains, proved to
decreasing besurvival
the a potentofantimicrobial againstby
E. coli or S. aureus both
99%Gram-( −) and
and 98%, Gram-(+)
respectively.
bacterial strains,
Besides, this fabricdecreasing
was alsothe ablesurvival E. coli
to kill aofdrug or S. aureus
resistant by 99%
bacterial andAt
strain. 98%,
therespectively. Besides,
molecular level, the
this fabric
authors was also
found thatable to kill a drug
conjugated resistant
PS exists bacterial
in both strain. At
monomeric andtheaggregate
molecularforms,
level, the
the authors
former
found that conjugated
accounting PS exists in
for its antimicrobial both monomeric
efficacy, and aggregate
while the latter forms, the
was responsible forformer accounting for
the photostability its
of the
antimicrobial efficacy, while
fabric. Consequently, thisthe latterdemonstrated
work was responsiblethe for the photostability
feasibility of the coatings
of double fabric. Consequently,
with dual
this work demonstrated
antimicrobial mechanisms the feasibility of double
on the material coatings
surface withbacteria,
to kill dual antimicrobial
which canmechanisms
be used inon the
other
antimicrobial
material surfacematerials [137]. which can be used in other antimicrobial materials [137].
to kill bacteria,

28. Design strategy of PS-ε-polylysine-fabric

Figure 28. PS-ε-polylysine-fabric material with dual
dual antimicrobial
antimicrobial mechanisms
mechanisms
(adapted
(adapted from
from Reference
Reference [137]).
[137]).

Krausz
Krausz andand co-workers
co-workers [139,140]
[139,140] developed
developed photobactericidal
photobactericidal plastic
plastic films
films based
based on on esterified
esterified
cellulose with fatty acids and porphyrins (Figure 29). In their first report, they considered
cellulose with fatty acids and porphyrins (Figure 29). In their first report, they considered plastic films plastic films
obtained by esterification of cellulose with protoporphyrin IX and lauric acid (PPIX-Cel),
obtained by esterification of cellulose with protoporphyrin IX and lauric acid (PPIX-Cel), while in the while in
the posterior works, they selectedadequately
adequatelysubstituted meso-tetraarylporphyrins (TriTol-Py
substituted meso-tetraarylporphyrins + -Cel,
posterior works, they selected (TriTol-Py+-Cel,
TriTol-Ph-Cel) with acid groups, namely a cationic one (Figure 29). The biological
TriTol-Ph-Cel) with acid groups, namely a cationic one (Figure 29). The biological results towards E. results towards
E.
colicoli
and S. S.
and aureus
aureus strains
strains showedthat
showed thatthe
thephotobactericidal
photobactericidalactivity
activityofofthe
theplastic
plastic films
films prepared
prepared was was
dependent on the porphyrin grafting percentage, and in the absence of porphyrin,
dependent on the porphyrin grafting percentage, and in the absence of porphyrin, the films allowed the films allowed
the
the full
full growth
growth ofof bacteria.
bacteria.
Molecules 2018, 23, 2424 28 of 47
Molecules 2018, 23, x FOR PEER REVIEW 28 of 47

Figure 29.
29. Structures
Structures of
of photobactericidal
photobactericidal plastic films based on esterified
esterified cellulose
cellulose with fatty
fatty acids
acids
porphyrin (adapted
and porphyrin (adapted from
from Reference
Reference [139,140]).
[139,140]).

4.3.
4.3. Superparamagnetic
Superparamagnetic Iron
Iron Oxide
Oxide Nanoparticles
Nanoparticles (SPION)
(SPION) Functionalized
Functionalized with
with Dextran
Dextran
Superparamagnetic
Superparamagnetic iron iron oxide
oxide nanoparticles (SPIONs) are
nanoparticles (SPIONs) are aa class
class of
of NPs
NPs that
that offer
offer aa variety
variety of
of
applications,
applications, comprising magnetic resonance imaging, drug delivery, cell labeling, tissue targeting,
comprising magnetic resonance imaging, drug delivery, cell labeling, tissue targeting,
tissue
tissue repair,
repair,and
andhyperthermia
hyperthermia [141–143]. They
[141–143]. are found
They in different
are found forms in
in different nature
forms insuch as hematite
nature such as
(α-Fe O
hematite ), maghemite (γ-Fe
2 3 (α-Fe2O3), maghemite O ), magnetite (Fe
2 3 (γ-Fe2O3), magnetite O ), among other forms. However,
3 4 (Fe3O4), among other forms. However, the the most studied
most
SPIONs are γ-Fe2are
studied SPIONs O3 γ-Fe
and 2Fe
O33 O 4 [142,144].
and Fe3O4 [142,144].
As
As SPIONs
SPIONs have
have aa certain
certain propensity
propensity toto agglomerate,
agglomerate, their
their surfaces
surfaces have
have to to be
be adjusted
adjusted with
with
biocompatible molecules and/or polymers in order to obtain stable NPs in physiological
biocompatible molecules and/or polymers in order to obtain stable NPs in physiological media media [144].
[144].
They
They can
can bebe functionalized
functionalized with
with hydrophilic
hydrophilic ligands
ligands such
such as
as polyethylene
polyethylene glycolglycol (PEG),
(PEG), dextran,
dextran,
phospholipids,
phospholipids, chitosan, and catechol derivatives [145]. Therefore, these ligands can be used
chitosan, and catechol derivatives [145]. Therefore, these ligands can be used in
in
combination with PSs to produce a water-stable SPION that can be used
combination with PSs to produce a water-stable SPION that can be used in the photodynamic in the photodynamic
approach. In fact, several groups prepared SPIONs that were stable in water containing PSs, such as
chlorin e6 [146], hematoporphyrin monomethyl ether [147], or a dendrimer porphyrin derivative
Molecules 2018, 23, 2424 29 of 47

approach. In23,
Molecules 2018, fact, several
x FOR groups prepared SPIONs that were stable in water containing PSs, such
PEER REVIEW 29 of as
47
chlorin e6 [146], hematoporphyrin monomethyl ether [147], or a dendrimer porphyrin derivative [148].
Additionally, as SPIONs
[148]. Additionally, are easily
as SPIONs manipulated
are easily manipulated by an external
by an magnetic
external magnetic field,
field,they
theycan
canthen
then be
directed toto the
thelocation
locationofof infection
infection andand where
where the the antimicrobial
antimicrobial agent
agent can becan be activated,
activated, leadingleading
to the
to the inactivation of microbial cells, with the tiniest side effects for the host [144].
inactivation of microbial cells, with the tiniest side effects for the host [144]. Besides, due to their Besides, due
to their superparamagnetic
superparamagnetic features, features,
SPIONs can SPIONs can
also be also be employed
employed to isolate contaminants
to isolate contaminants from wastewaterfrom
wastewater
[149]. [149].
Considering the thepromising
promisingresults
resultsobtained
obtained withwith
thethe
useuse of TCPP
of TCPP incorporated
incorporated in SPIONs
in SPIONs as PS
as PS towards
towards murine murine melanoma
melanoma cells [150],
cells [150], Thandu Thandu
et al.etdecided
al. decided to evaluate
to evaluate theirtheir potential
potential in thein
the photoinactivation of bacteria [151]. Therefore, in 2017, Thandu et al. [151] reported
photoinactivation of bacteria [151]. Therefore, in 2017, Thandu et al. [151] reported the use of SPION- the use of
SPION-MCTPP
MCTPP obtained obtained
from from 5-(4-carboxyphenyl)-10,15,20-triphenylporphyrin
5-(4-carboxyphenyl)-10,15,20-triphenylporphyrin (MCTPP)(Figure
(MCTPP) (Figure 30)
against S. aureus, Streptococcus mutans, and E. coli. It was observed that when exposed to blue light
(470 −2 SPION-MCTPP at 0.5 µM caused an inactivation of S. aureus
(470 nm)
nm) at
at an
an irradiance
irradiance ofof 4.8
4.8 mW
mW cmcm−2, ,SPION-MCTPP at 0.5 µM caused an inactivation
of 2 log after 180 min of irradiation. For S. mutans, only a 1 log decrease decrease was observed under the same
experimental conditions.
experimental conditions. This nanoconjugate
nanoconjugate was not effective against
was not effective against the
the Gram-(
Gram-(−) −) bacterium
bacterium E. E. coli.
coli.
The possibility of recovering this nanoconjugate
nanoconjugate with magnets, post treatment, was an important
advantage when it is considered
considered forfor disinfection
disinfection applications
applications [151].
[151].

Figure 30. Structure of the SPION-MCTPP (adapted from References [150,151]).

5. Liposomes
5. Liposomes
The
The hydrophobic
hydrophobicnature natureof most PSs makes
of most them aggregate
PSs makes in aqueous
them aggregate inmedium,
aqueousthus diminishing
medium, thus
the PDI efficacy because it reduces the 1 O formation by a self-quenching effect in the excited state [152].
2
diminishing the PDI efficacy because it reduces the O2 formation by a self-quenching effect in the
1
Thus,
excitedinstate
order[152].
to preserve
Thus, in the monomeric
order to preservestatetheof the PS, to protect
monomeric stateitoffrom
the PS,aqueous environment,
to protect and to
it from aqueous
increase the efficacy
environment, and toofincrease
photodynamic treatment,
the efficacy several transporters
of photodynamic treatment,and delivery systems, suchand
several transporters as
liposomes, have also been developed in the last few years. Liposomes became
delivery systems, such as liposomes, have also been developed in the last few years. Liposomes known due to their high
loading capacity,
became known biodegradability,
due biocompatibility,
to their high loading and size [153]. In
capacity, biodegradability, fact, these liposomal
biocompatibility, and sizevesicles
[153].
with onethese
In fact, or more concentric
liposomal phospholipid
vesicles with one or bilayer(s) have twophospholipid
more concentric main advantages in PDIhave
bilayer(s) (Figure
two31).
main
First, they
advantages in PDIcan(Figure
accommodate
31). both hydrophobic and hydrophilic PSs and thus can be used
to inactivate
First, they microorganism
can accommodate cells both
afterhydrophobic
irradiation with light. Second,
and hydrophilic the synergistic
PSs and thus can beeffect
used ofto
positively charged and highly fluid components of the liposomes increases
inactivate microorganism cells after irradiation with light. Second, the synergistic effect of positively the PS uptake by
microorganisms,
charged and highly as fluid
well components
as the overall of phototoxicity
the liposomes [154–156].
increases the ThePSrelease
uptakeof byliposome content
microorganisms,
can be as
as well accomplished in several ways
the overall phototoxicity involving
[154–156]. photochemical
The release of liposome activation
content (photoisomerization,
can be accomplished
photocleavage,
in several ways involving photochemical activation (photoisomerization, such
and photopolymerization), as well as by photophysical activation, as photothermal
photocleavage, and
conversion [157]. In addition, the release rate of the drug incorporated into
photopolymerization), as well as by photophysical activation, such as photothermal conversion [157].liposomes depends on the
drug itself and
In addition, thethe properties
release rate ofofthe
thedrug
liposome such as composition,
incorporated into liposomes temperature,
depends on sensitivity
the drugtoitself
changes
and
with the pH, and osmotic gradient. Microwave radiation and pulses of laser
the properties of the liposome such as composition, temperature, sensitivity to changes with the light can also lead topH,
the
and osmotic gradient. Microwave radiation and pulses of laser light can also lead to the release of
drugs from liposomes [157,158]. The possibility of the release of other drugs incorporated into
liposomes using a laser light can be exploited in combination with PDI.
Molecules 2018, 23, 2424 30 of 47

release of drugs from liposomes [157,158]. The possibility of the release of other drugs incorporated
into liposomes
Molecules 2018, 23, xusing a laser
FOR PEER light can be exploited in combination with PDI.
REVIEW 30 of 47

Figure 31. Structure of phospholipids L-α-dioleoylphosphatidylserine (OOPS), L-α-

dimiristoylphosphatidylcholine (DMPC), L-α-dipalmitoylphosphatidylcholine (DPPC), and 1,2-dioleoyl-
Figure 31. Structure of phospholipids
sn-glycerophosphatidylcholine -α-dioleoylphosphatidylserine
(DOPC)L(adapted from Reference [69]). (OOPS), L-α-
dimiristoylphosphatidylcholine (DMPC), L-α-dipalmitoylphosphatidylcholine (DPPC), and 1,2-
In 2007, Jori and co-workers [159] compared
dioleoyl-sn-glycerophosphatidylcholine (DOPC) the(adapted
photodynamic action of
from Reference the monocationic porphyrin
[69]).
5-(1-dodecanoylpyridinium-4yl]-10,15,20-triphenylporphyrin (TDPyP) (Figure 32) against a bacterial
MRSA Instrain
2007,inJori and co-workers
a homogeneous [159]
aqueous compared
solution, the photodynamic
and after action
being incorporated intoof the monocationic
neutral phospholipid
porphyrin
vesicles of 5-(1-dodecanoylpyridinium-4yl]-10,15,20-triphenylporphyrin (TDPyP) (Figure 32) against
DL-α-dipalmitoylphosphatidylcholine (DPPC) and L -α-dimiristoylphosphatidylcholine
a bacterial
(DMPC), orMRSA
into thestrain in liposome
cationic a homogeneous aqueous solution, and after being incorporated into
of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium
neutral phospholipid vesicles of DL-α-dipalmitoylphosphatidylcholine (DPPC) and L-α-
chloride (DOTAP).
dimiristoylphosphatidylcholine
The results showed that the(DMPC), or into the
low photosensitizing cationic
activity liposome
(<1 log after 10 of
min N-[1-(2,3-
of white
dioleoyloxy)propyl]-N,N,N-trimethylammonium
light irradiation) of this PS (with high ability tochloride 1
generate(DOTAP).
O2 ) in aqueous solution or in neutral
lipid vesicles was dramatically improved (>6 log) after its incorporation in the polycationic
liposome. This behavior change of photinactivation was attributed to the action of cationic liposome
(DOTAP) disorganizing the bacterial wall, thus enhancing its permeability to the PS molecules.
A similar effect towards MRSA and E. coli was found when the same porphyrin derivative was
incorporated into the cationic amphiphilic heptakis [2-(ω-amino-O-oligoethyleneglycol)-6-deoxy-
6-hexylthio]-β-cyclodextrin (SC6 NH2 ) (Figure 32) [160].
The improvement of the photodynamic inactivation using a PS stabilized in positively charged
liposomal formulations was corroborated by the results obtained by Girigoswami and co-workers [161].
The authors evaluated the effect of two liposomal formulations of xanthene dye Rhodamine 6G
(R6G) (Figure 33) on a multidrug resistant P. aeruginosa present in a sewage water treatment plant.
The liposomes were prepared from phospholipids extracted from hen eggs with the further addition of
cholesterol. In one of the liposomal formulations, the authors used polyvinyl alcohol (PR6G) to stabilize
the liposome thin films (obtained by the hydration method) and to minimize reticuloendothelial system
uptake. The other positively charged formulation (CR6G) was obtained by covering the films with
the cationic quaternary surfactant amine hexadecyltrimethylammonium bromide (CTAB). The results
showed that, in terms of 1 O2 quantum yield, both formulations are more efficient generators than R6G

Figure 32. Structure of 5-(1-dodecanoylpyridinium-4yl]-10,15,20-triphenylporphyrin (TDPyP) and

the cationic deliver systems DOTAP and SC6NH2 (adapted from Reference [160]).
 Figure 31. Structure of phospholipids L-α-dioleoylphosphatidylserine (OOPS), L-α-
dimiristoylphosphatidylcholine (DMPC), L-α-dipalmitoylphosphatidylcholine (DPPC), and 1,2-
dioleoyl-sn-glycerophosphatidylcholine
Molecules 2018, 23, 2424 (DOPC) (adapted from Reference [69]). 31 of 47

In 2007, Jori and co-workers [159] compared the photodynamic action of the monocationic
alone, leading
porphyrin to a more effective photodynamic inactivation of P. aeruginosa.
5-(1-dodecanoylpyridinium-4yl]-10,15,20-triphenylporphyrin (TDPyP) However,
(Figure despite the
32) against
cationic
Molecules formulation
2018, 23, x FOR being
PEER less
REVIEW efficient than PR6G in generating 1 O , it showed a faster decrease 31 of in
47
a bacterial MRSA strain in a homogeneous aqueous solution, and2 after being incorporated into
bacterial survival
neutral phospholipidthan PR6G. The authors
vesicles explained that the electrostatic interactions
of DL-α-dipalmitoylphosphatidylcholine (DPPC) between
and Lthe -α-
The
superficial results
positive showed
charge that
in the
CR6G low
andphotosensitizing
the cell surface activity
of this (<1
Gram-(
dimiristoylphosphatidylcholine (DMPC), or into the cationic liposome of N-[1-(2,3- log
− ) after 10
bacterium min of
were white light
responsible
irradiation)
by this higher of this PS (with
efficiency high ability to generatechloride
[161].
dioleoyloxy)propyl]-N,N,N-trimethylammonium
1O2) in aqueous solution or in neutral lipid vesicles
(DOTAP).
was dramatically improved (>6 log) after its incorporation in the polycationic liposome. This behavior
change of photinactivation was attributed to the action of cationic liposome (DOTAP) disorganizing
the bacterial wall, thus enhancing its permeability to the PS molecules. A similar effect towards
MRSA and E. coli was found when the same porphyrin derivative was incorporated into the cationic
amphiphilic heptakis [2-(ω-amino-O-oligoethyleneglycol)-6-deoxy-6-hexylthio]-β-cyclodextrin
(SC6NH2) (Figure 32) [160].
The improvement of the photodynamic inactivation using a PS stabilized in positively charged
liposomal formulations was corroborated by the results obtained by Girigoswami and co-workers
[161]. The authors evaluated the effect of two liposomal formulations of xanthene dye Rhodamine
6G (R6G) (Figure 33) on a multidrug resistant P. aeruginosa present in a sewage water treatment plant.
The liposomes were prepared from phospholipids extracted from hen eggs with the further addition
of cholesterol. In one of the liposomal formulations, the authors used polyvinyl alcohol (PR6G) to
stabilize the liposome thin films (obtained by the hydration method) and to minimize
reticuloendothelial system uptake. The other positively charged formulation (CR6G) was obtained
by covering the films with the cationic quaternary surfactant amine hexadecyltrimethylammonium
bromide (CTAB). The results showed that, in terms of 1O2 quantum yield, both formulations are more
efficient generators than R6G alone, leading to a more effective photodynamic inactivation of P.
aeruginosa. However, despite the cationic formulation being less efficient than PR6G in generating
1O2, it showed a faster decrease in bacterial survival than PR6G. The authors explained that the

electrostatic
Figure 32.interactions
32. between
Structureofof
Structure the superficial positive charge in CR6G and the(TDPyP)
5-(1-dodecanoylpyridinium-4yl]-10,15,20-triphenylporphyrin
5-(1-dodecanoylpyridinium-4yl]-10,15,20-triphenylporphyrin cell surface
(TDPyP)
andandof this
the
Gram-(−)
the bacterium
cationic deliver
cationic were
systems
deliver responsible
DOTAP
systems DOTAP by
SC this
andand NH higher
SC6NH(adaptedefficiency
from
2 (adapted
[161].
Reference
from [160]).
Reference [160]).
6 2

Figure Structure
33.33.
Figure of of
Structure Rhodamine 6G6G
Rhodamine that waswas
that encapsulated in liposomes
encapsulated (adapted
in liposomes from
(adapted Reference
from [161].
Reference
[161].
Recent studies also show that the incorporation of the PS in liposomes can accelerate the practical
application
Recent of photodynamic
studies also show inactivation to endodontic
that the incorporation of theinfections as a new
PS in liposomes clinical
can protocol
accelerate [162].
the practical
Azevedo and co-workers [163] evaluated the delivery and the photoxicity of
application of photodynamic inactivation to endodontic infections as a new clinical protocol [162]. aluminum chloride
phthalocyanine
Azevedo and (AlPc) (Figure 2)
co-workers entrapped
[163] evaluated in cationic liposomes
the delivery and the against cariogenic
photoxicity bacteria and
of aluminum pulp
chloride
cells in caries lesions. The study showed that the AlPc-cationic liposome was preferentially
phthalocyanine (AlPc) (Figure 2) entrapped in cationic liposomes against cariogenic bacteria and absorbed
by bacterial
pulp cells incells when
caries compared
lesions. to eukaryotic
The study showeddental
that thepulp cells, and theliposome
AlPc-cationic reductionwasof microbial load
preferentially
from bacterial cultures was evident. The clinical study with volunteers showed
absorbed by bacterial cells when compared to eukaryotic dental pulp cells, and the reduction of a mean reduction of
82% of total bacteria in the treated cavities after the PDT application.
microbial load from bacterial cultures was evident. The clinical study with volunteers showed a mean
Underofthe
reduction 82%same context,
of total the photodynamic
bacteria in the treated efficacy
cavitiesof 5,10,15,20-tetrakis(3-hydroxyphenyl)chlorin
after the PDT application.
(mTHPC)
Under the same context, the photodynamic bacteria
(Figure 34) in liposomes was investigated towards efficacyE. faecalis frequently found in
of 5,10,15,20-tetrakis(3-
persistent endodontic infections [164]. The results showed that the
hydroxyphenyl)chlorin (mTHPC) (Figure 34) in liposomes was investigated towards amount of mTHPC incorporated in
bacteria E.
the liposomal
faecalis formulation
frequently formed byendodontic
found in persistent 9.1 mixtureinfections
of DL-α-dipalmitoyl-phosphatidyl
[164]. The results showed that choline (DPPC)
the amount
of mTHPC incorporated in the liposomal formulation formed by 9.1 mixture of DL-α-dipalmitoyl-
phosphatidyl choline (DPPC) and DL-α-dipalmitoylphosphatidylglycerol (DPPG) (formulation
Foslipos, Biolitec AG, Jena, Germany) and the light dose were important protocol features to consider
during the photoinactivation treatment. E. faecalis was completely suppressed (reduction of > 8 log)
Molecules 2018, 23, 2424 32 of 47

and DL-α-dipalmitoylphosphatidylglycerol (DPPG) (formulation Foslipos, Biolitec AG, Jena, Germany)

Molecules 2018, 23, x FOR PEER REVIEW 32 of 47
and the light dose were important protocol features to consider during the photoinactivation treatment.
E. faecalis
after was completely
incubation suppressed
with liposomes (reduction
enriched with of50 µM
> 8 log) after incubation
mTHPC with liposomes
and illumination enriched
with 100 J cm −2.

with 50 µM mTHPC and illumination with 100 J cm −2 . Irradiation of the non-photosensitized solution
Irradiation of the non-photosensitized solution showed no suppressing impact and incubation of the
showed
PS without no additional
suppressing impact and
irradiation incubation
caused of the
a maximal PS without
reduction additional
of 1.5 log. irradiation
The liposomal caused a
formulation
maximal reduction ofmTHPC
Foslipos containing 1.5 log. The
was liposomal formulation
also able to completely Foslipos containing
eradicate mTHPC
Streptococcus was also
sobrinus able to
(reduction
completely eradicate Streptococcus sobrinus (reduction of 100% at 5 µg mL −1 ) and S. mutants (reduction
of 100% at 5 µg mL ) and S. mutants (reduction of 100% at 1.25 µg mL ) after 15 min of dark
−1 −1

of 100% at 1.25 mLof−1 ) after 15 min of dark incubation and 2 min of blue light (400–505 nm) [165].
incubation and µg 2 min blue light (400–505 nm) [165].

Figure 34. Structures of

34. Structures of PS
PS used
used after
after incorporation
incorporation in
in liposomes
liposomes formulations
formulations to efficiently
photoinactivate bacteria and viruses.
viruses.

As
As an
an extension
extension of of the
the previous
previous study,
study, thethe German
German groupgroup evaluated
evaluated the the photodynamic
photodynamic killing killing
of E.
of E. faecalis
faecalis in
in dentinal
dentinal tubules
tubules using
using thethe mTHPC
mTHPC incorporated
incorporated in in liposome
liposome formulation
formulation Foslipos
Foslipos
(DPPC/DPPG)
(DPPC/DPPG) and andin inflexible
flexibleinvasomes
invasomescontaining
containingsoybean
soybeanphosphatidyl,
phosphatidyl,10% 10%ethanol,
ethanol,and and1% 1%ofofa
a terpene mixture ( D -limonene, citral and 1,8-cineole) [166]. The results
terpene mixture (D-limonene, citral and 1,8-cineole) [166]. The results showed that both mTHPC showed that both mTHPC
formulations
formulations had had aa significant
significant antimicrobial
antimicrobial effect effect and
and were
were able
able to suppress E.
to suppress E. faecalis
faecalis inside
inside the
the
dentinal tubules up to 300 µm. A bacterial reduction of up to 3.6 log mL −−11 was ascertained for the
dentinal tubules up to 300 µm. A bacterial reduction of up to 3.6 log mL was ascertained for the
mTHPC
mTHPC invasomes
invasomesformulation
formulation directly at theatroot
directly thecanal
rootwall. It was
canal alsoItobserved
wall. was also that observed
photodynamic that
treatment using an invasomes formulation was even more
photodynamic treatment using an invasomes formulation was even more effective than 1% effective than 1% chlorohexidine, a
conventional disinfectant, which caused a bacterial reduction of only
chlorohexidine, a conventional disinfectant, which caused a bacterial reduction of only 1.2 log CFU. 1.2 log CFU. A very recent
study
A veryconcerning
recent study the limited use
concerning theoflimited
hypericin
use of (HYP, Figure(HYP,
hypericin 34) asFigure
a PS due
34) as toaits
PShighly
due to lipophilic
its highly
nature, poor solubility in aqueous media, and poor bioavailability
lipophilic nature, poor solubility in aqueous media, and poor bioavailability showed that its showed that its incorporation
in liposomes could
incorporation be a promising
in liposomes could be alternative
a promising[167]. The results
alternative [167]. Therevealed
resultsthat the liposomal
revealed that the
formulations of DOPE/CHEMS/DPPC (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine/
liposomal formulations of DOPE/CHEMS/DPPC (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine/ cholesteryl
hemisuccinate/ L -α-dipalmitoylphosphatidylcholine),
cholesteryl hemisuccinate/ L-α-dipalmitoylphosphatidylcholine), DSPC/DPPC/DSPE-PEG
DSPC/DPPC/DSPE-PEG (1,2-distearoyl-sn-
(1,2-
glycero-3-phosphatidylcholine/
distearoyl-sn-glycero-3-phosphatidylcholine/ L -α-dipalmitoylphosphatidylcholine/1,2-distearoyl-sn-glycero-3-
L-α-dipalmitoylphosphatidylcholine/1,2-distearoyl-sn-
phosphoethanolamine-N-[methoxy(polyethylene
glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] and DPPC/DOTAP
glycol)-2000] and DPPC/DOTAP loaded with HYP
loaded
were able to attain a 2.3–2.5 log reduction of Staphylococcus saprophyticus subsp.
with HYP were able to attain a 2.3–2.5 log reduction of Staphylococcus saprophyticus subsp. bovis after bovis after irradiation
with a 589 nm
irradiation withdiode
a 589lasernm at diode J cm−at
22.2 laser 2 and facilitated
22.2 J cm−2 andthe binding the
facilitated andbinding
uptake of and the PS through
uptake of thethe
PS
bacterial cell wall.
through the bacterial cell wall.
Finally,
Finally, considering
considering that that the
the viral
viral safety
safety of of blood-derived
blood-derived products
products relies
relies onon properly
properly chosen
chosen
inactivation procedures, Sagristá et al. [168] reported a protocol based on
inactivation procedures, Sagristá et al. [168] reported a protocol based on an efficient incorporation an efficient incorporation
of
of chlorin
chlorin derivative
derivative (CHL) (CHL) (Figure
(Figure 34)34) into
intoanionic
anionicunilamellar
unilamellarliposomes
liposomes(POPC/OOPS)
(POPC/OOPS) followed followed
by
by its
its immobilization
immobilization in in the
the chromatographic
chromatographic support support Sephacryl
Sephacryl S-1000
S-1000 beads.
beads. The The photodynamic
photodynamic
efficiency
efficiency of of the
the chlorin-containing
chlorin-containingliposomes liposomesformulation,
formulation, free
free in in solution
solution andand immobilized
immobilized on
on the
the chromatographic support, was evaluated towards bovine
chromatographic support, was evaluated towards bovine viral diarrhea virus (BVDV) and viral diarrhea virus (BVDV) and
encephalomyocarditis
encephalomyocarditis virus virus (EMCV).
(EMCV).The Theresults
resultsindicated
indicatedthatthatthethe enveloped
enveloped virus
virus BVDVBVDV could
could be
be successfully inactivated (>4 log) by both systems in culture medium
successfully inactivated (>4 log) by both systems in culture medium but not the non-enveloped virus but not the non-enveloped
virus
EMCV. EMCV. The results
The results suggested
suggested thattargets
that the the targets
of theofphotodynamic
the photodynamic actionaction of chlorin
of chlorin CHLCHL werewere
the
envelope components. The diminished effectiveness of CHL-containing liposomes in solution or
immobilized in the chromatographic support when the culture media was replaced with human
plasma was justified by the authors considering that plasma components hinder the access of the PS
to the light.
Molecules 2018, 23, 2424 33 of 47

the envelope components. The diminished effectiveness of CHL-containing liposomes in solution

or immobilized in the chromatographic support when the culture media was replaced with human
plasma was justified by the authors considering that plasma components hinder the access of the PS to
the light.

6. Hydrogel Materials
The immobilization of PS in organic synthetic polymers can involve different approaches like the
covalent or non-covalent coupling of the macrocycle with polymers with complimentary functionalities
or the co-polymerization of PS with polymerizable moieties with monomers like styrene or methyl
methacrylate [169–173]. Since in this review the preparation of some photoactive materials containing
organic synthetic polymers was already referred, we decided in this section to give a special attention
to photoactive materials based on hydrogels.
Hydrogels are three-dimensional networks composed of hydrophilic polymer chains. Many research
groups are exploring them due to their efficacy in preserving the loaded cargo and due to their good
responsiveness to the environment stimuli [174,175]. The hydrogel’s features make them good candidates
for wound healing, implant/catheter coatings, skin infections treatment, and even orifice-barrier
applications [176]. Therefore, several hydrogel-based systems have been selected in the last few years to
overcome the low solubility of macrocycles, such as some porphyrin derivatives [177–179]. Their ability
to encapsulate porphyrins by cross-linking them in the hydrogel structure avoids the leakage of the
PS and also prevents their aggregation in aqueous media or physiologic conditions. In addition, the
hydrogel carriers can commonly be designed to be biocompatible and/or biodegradable [180].
Thus, PSs immobilized on hydrogels are being considered to fight bacterial attachment
onto intraocular lenses during cataract extraction and intraocular lens implantation, namely
by bacteria such as S. epidermidis, which is the most frequently responsible for cases of acute
endophthalmitis [181,182], and in some cases leading to the production of a microbial biofilm on the
surface of the intraocular lenses [183]. Bearing this in mind, McGlinchey and co-workers characterized
the physicochemical and the photoantimicrobial properties of hydrogel materials (anionic copolymers
of 2-(hydroxyethyl)methacrylate with methacrylic acid), which have been surface-modified by
impregnation with the tetracationic porphyrin TMPyP (Figure 2) [184]. This study enabled the
discovery of candidate anti-infective hydrogel based intraocular lens materials to improve patient
outcomes in cataract surgery. The anionic hydrogel copolymers were shown to permanently bind
the cationic TMPyP by electrostatic interactions as a thin surface layer. The thermal and mechanical
properties of the materials showed that the porphyrin acts as a surface cross-linking agent, yielding
surfaces that were more hydrophilic. Thus, the microbiological studies showed that S. epidermidis
adherence was reduced by up to 99.02 ± 0.42% relative to the control in intense light conditions,
and 91.76 ± 5.99% in the dark. The ability to concentrate the photocytotoxic effect at a surface, together
with a significant dark effect, was envisaged as a platform for a range of light-activated anti-infective
biomaterial technologies. This was considered a great advantage since the treatment area can be
strongly localized at the required point of bacterial contact with the intraocular lens surface. This can
also avoid the side-effects associated with toxicity arising from accumulation of PS in the tissue [184].
Later, Gorman and colleagues also selected hydrogel materials (methyl methacrylate, methacrylic
acid, 2-(hydroxyethyl)methacrylate, benzoyl peroxide, and ethylene glycol dimethacrylate), modified
with the same tetracationic porphyrin (TMPyP), in order to get a hydrogel based intraocular lens
materials. By doing this, the authors expected to obtain a material with antimicrobial properties,
due to the photodynamic generation of ROS, and a material that was able to protect the retina
from damaging of short wavelength visible light. The incorporation of the PS into hydrogels was
accomplished by immersing samples of the materials in PBS solutions of TMPyP (2 min), followed
by washing with deionized water for seven days. Then, the authors evaluated the resulting materials
against both Gram-(+) S. aureus and Gram-(−) P. aeruginosa. At the end of the study, a significant
reduction of bacterial adherence to the TMPyP-impregnated materials was observed when compared
Molecules 2018, 23, 2424 34 of 47

to untreated control materials, confirming the generation of ROS by the incorporated porphyrin upon
photoactivation [185].
This type of hydrogel materials can also be used against bacterial biofilms. For instance, McCarron
and co-workers [186] developed a poly(vinylalcohol)-borate hydrogel as a delivery system to achieve
a photodynamic inactivation of wound infections. Therefore, they chose two cationic PSs (MB and
TMPyP (Figure 2)) to be loaded into the hydrogel materials in order to photoinactivate an MRSA
strain. After the biological assessment, they found that reductions of bacterial abundance in the biofilm
was 88.19% for MB at a concentration of 10 µg mL−1 and a light dose of 100 J cm−2 , while TMPyP
(10 µg mL−1 ) caused a reduction of 99.71%. Despite the total bacterial biofilm eradication did not
occur in both tested hydrogel materials and bacterial abundance was higher than 105 CFU mL−1 in
many cases, photodynamic inactivation of microorganisms was not associated with the development
of resistance, even upon multiple treatments [186].
All these studies demonstrate that some of the hydrogel-based photoinactivation caused
admirable antibacterial performance against both Gram-(−) and Gram-(+) bacteria in planktonic
and biofilm form. Additionally, this type of treatment can be performed several times at one site due
to the low fluidity of the hydrogels [187].

7. Supports Based on Carbon Nanomaterials

The unique properties of graphene oxide, such as a large surface area decorated with different
functional groups, biocompatibility, a lack of obvious toxicity, photothermal activity, intrinsic high
near-infrared absorbance, and water solubility, are making it an ideal material to carry PSs [188].
Under this context, and considering the attention given to PDI as an effective approach to improve
root canal disinfection, Tayebeh Akbaria et al. [189] used functionalized nano-graphene oxide (GO)
with APTS and then with L-carnitine (GO-APTES-L CA) as a platform to load, via π-π stacking
interactions, the amphiphilic dye indocyanine green (ICG), also known as Cardio-Green® (Figure 35).
This dye, with a unique peak absorbance at 780 nm, has limitations such as little photostability and
concentration-related aggregation. The success of the incorporation was confirmed by adequate
techniques (FT-IR, SEM and UV–Vis spectrometry) and the composite GO-APTES-L CA-ICG obtained
contained 200 µg mL−1 of ICG. The antimicrobial and anti-biofilm potential of GO-APTES-L CA-ICG
was assessed against E. faecalis, both in planktonic and biofilm forms. PDI mediated by GO-APTES-L
CA-ICG at an energy density of 31.2 J cm−2 showed a significant reduction (2.81 log) in the count of
E. faecalis and a reduction of up to 99.4% on the biofilm formation ability. The overall antimicrobial
and anti-biofilm potential of GO-APTES-L CA-ICG was higher than PDI based on ICG (1000 µg
mL−1 ) (47% and 21%, respectively). The authors verified that the composite GO-APTES-L CA-ICG
with a higher incorporation of ICG (400 µg mL−1 ) after a total light dose irradiation of 31.2 J·cm−2
showed a significantly greater reduction in the number of E. faecalis than PDI based on ICG alone
(1000 µg mL−1 ) [190].
Considering the attractive properties of carbon nanotubes to develop ultrathin, flexible,
and transparent films for immobilization, accompanied by their ability to photogenerate ROS, Kane and
co-workers [191] reasoned that their efficiency could be significantly enhanced by functionalization of
the nanotubes with porphyrins due to their high efficiency in generating ROS. The authors developed
nanocomposite films based on the conjugation of multiwalled carbon nanotubes (MWNT) and
protoporphyrin-IX (PPIX) (Figure 36) and evaluated their photoinactivation efficiency towards S. aureus.
The coupling involved the previous functionalization of the acid groups of protoporphyrin-IX with
amino groups followed by their condensation with the acid groups of MWNT after their activation
with SOCl2 . The biological results showed that after 1 h of visible light irradiation, the nanocomposite
films reduced 15–20% of S. aureus survival on plate surface. As an extension of the previous studies,
the authors tested the MWNT-PPIX ability to inactivate the Influenza A virus (Figure 36) [192].
The strain used was Influenza A/X-31, which is a high-yield H3N2 strain, commercially available
and developed for designing vaccines. The conjugate was found to be highly active in reducing the
Molecules 2018, 23, 2424 35 of 47

ability of the influenza virus to infect mammalian cells, and the study showed that the conjugate
can be recovered from an aqueous solution and reused at least three times without any significant
loss of its
Molecules activity.
2018, 23, x FORTherefore, it was proposed that in addition to being effective bactericidal agents,
PEER REVIEW 35 of 47
the MWNT-PPIX conjugates may be applied as reusable antivirals in wastewater treatment, as they
can be easily2018,
Molecules recovered without
23, x FOR PEER leaving any toxic by-products.
REVIEW 35 of 47

Figure
Figure 35. FunctionalizationofofGO
35. Functionalization GOwith
with APTS
APTS and
and LL CA
CAand
andincorporation of ICG
incorporation (adapted
of ICG from from
(adapted
Figure 35. Functionalization
Reference [189]).
of GO with APTS and L CA and incorporation of ICG (adapted from
Reference [189]).
Reference [189]).

Figure 36. Functionalization of MWNT with PPIX (adapted from Reference [191]).

Recently, Gopinath and co-workers [193] also developed a nanocomposite made up of single-
36. Functionalization
Figurenanotubes
walled carbon of MWNT linked
(SWCNTs) covalently with PPIX (adapted
to the from Reference porphyrin
amine-functionalized [191]). 5-(4-
aminophenyl)-10,15,20-triphenylporphyrin (TPP-NH2) and evaluated its antimicrobial potential after
Recently, Gopinath
activation with visible and
light co-workers [193]
(Figure 37). The alsoshowed
results developed a nanocomposite
that the madeamount
use of an appropriate up of of
single-
walled carbon nanotubes (SWCNTs) covalently linked to the amine-functionalized porphyrin 5-(4-
aminophenyl)-10,15,20-triphenylporphyrin (TPP-NH2) and evaluated its antimicrobial potential after
activation with visible light (Figure 37). The results showed that the use of an appropriate amount of
Molecules 2018, 23, 2424 36 of 47

Recently, Gopinath and co-workers [193] also developed a nanocomposite made up of

single-walled carbon nanotubes (SWCNTs) covalently linked to the amine-functionalized porphyrin
5-(4-aminophenyl)-10,15,20-triphenylporphyrin (TPP-NH2 ) and evaluated its antimicrobial potential
Molecules 2018, 23, x FOR PEER REVIEW 36 of 47
after activation with visible light (Figure 37). The results showed that the use of an appropriate
amount of single-walled
single-walled carbon nanotubes
carbon nanotubes (SWCNTs) (SWCNTs) led to
led to cell cell membrane
membrane damage.
damage. Consequently,
Consequently, the
the SWCNT-porphyrin conjugates can be used as an antibacterial
SWCNT-porphyrin conjugates can be used as an antibacterial agent. agent.

Preparation of SWNT and functionalization

Figure 37. Preparation functionalization with
withTPP-NH
TPP-NH22 (adapted
(adapted from
from Reference
Reference[193]).
[193]).
8. Final Remarks
8. Final
PDIRemarks
appears as an effective method to inactivate a broad spectrum of planktonic and biofilm
embedded microorganisms.
PDI appears In addition,
as an effective method the development
to inactivate of photoactive
a broad spectrum materials has provided
of planktonic and biofilman
opportunity to improve several aspects of treatment, namely the possibility to increase
embedded microorganisms. In addition, the development of photoactive materials has provided an the efficiency
of the photodynamic
opportunity to improvetreatment without
several aspects inducing the
of treatment, resistance
namely mechanism
the possibility of microorganisms.
to increase the efficiency
Additionally,
of this type treatment
the photodynamic of technology offersinducing
without the possibility to recovermechanism
the resistance and reuse the photosensitizing
of microorganisms.
system in an eco-friendly
Additionally, manner. Although,
this type of technology offers the the application
possibility of photoactive
to recover and reusematerials in the field
the photosensitizing
of PDI in
system solved some difficulties
an eco-friendly manner.ofAlthough,
this therapy, there is stillofroom
the application for new
photoactive improvements.
materials in the fieldIt of
is
expected that the information compiled in this review can motivate the development of
PDI solved some difficulties of this therapy, there is still room for new improvements. It is expectedother practical
and the
that efficient approaches
information for PDI
compiled in in order
this review to respond to its the
can motivate currently underutilization
development in clinic and
of other practical and
environmental
efficient applications.
approaches for PDI in order to respond to its currently underutilization in clinic and
environmental applications.
Funding: This research received no external funding.
Funding: This research
Acknowledgments: received
The noacknowledge
authors external funding.
the University of Aveiro and FCT/MEC (Fundação para a
Ciência e a Tecnologia, Ministério da Educação e da Ciência), through national founds and, where applicable,
Acknowledgments: The authors
co-financed by the FEDER acknowledge
(European the University
Fund for Regional of Aveirowithin
Development) and FCT/MEC (Fundaç
the PT2020 ão para
Partnership a Ciência
Agreement,
eand
a Tecnologia, Ministério da Educaç ão e da Ciê ncia), through national founds and, where
Compete 2020 for the financial support to the QOPNA research project (FCT UID/QUI/00062/2013) applicable, co-
financed by the FEDER (European Fund for Regional Development) within the PT2020 Partnership Agreement,
and Compete 2020 for the financial support to the QOPNA research project (FCT UID/QUI/00062/2013) and
Institute for Biomedicine–iBiMED (UID/BIM/04501/2013). MQM thanks FCT for her doctoral grant (SFRH/
BD/112317/2015).

Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2018, 23, 2424 37 of 47

and Institute for Biomedicine–iBiMED (UID/BIM/04501/2013). MQM thanks FCT for her doctoral grant
(SFRH/BD/112317/2015).
Conflicts of Interest: The authors declare no conflict of interest.

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