Light Based Anti Infectives Ultraviolet C Irradiation PDT Blue Light and Beyond

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Curr Opin Pharmacol. Author manuscript; available in PMC 2014 October 01.
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Curr Opin Pharmacol. 2013 October ; 13(5): . doi:10.1016/j.coph.2013.08.009.
Light based anti-infectives: ultraviolet C irradiation,

photodynamic therapy, blue light, and beyond
Rui Yin1,2,3, Tianhong Dai1,2, Pinar Avci1,2,4, Ana Elisa Serafim Jorge1,5, Wanessa CMA de
Melo1,6, Daniela Vecchio1,2, Ying-Ying Huang1,2,7, Asheesh Gupta1,2,8, and Michael R
Hamblin1,2,9
1Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA, USA
2Department of Dermatology, Harvard Medical School, Boston, MA, USA

3Department of Dermatology, Southwest Hospital, Third Military Medical University, Chongqing,
China
4Department of Dermatology, Dermato-oncology and Venerology, Semmelweis University School
of Medicine, Budapest, 1085, Hungary
5Programa de Pós-Graduação Interunidades Bioengenharia - EESC/FMRP/IQSC and Centro de

Pesquisa em Óptica e Fotônica/Instituto de Física de São Carlos - University of São Paulo, Sao
Carlos, SP, Brazil
6University of Sao Paulo, Sao Carlos-SP, Brazil
7Department of Pathology, Guangxi Medical University, Nanning, Guangxi, China
8Defence Institute of Physiology & Allied Sciences, Delhi, India
9Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, USA
Abstract
Owing to the worldwide increase in antibiotic resistance, researchers are investigating alternative
anti-infective strategies to which it is supposed microorganisms will be unable to develop
resistance. Prominent among these strategies, is a group of approaches which rely on light to
deliver the killing blow. As is well known, ultraviolet light, particularly UVC (200–280nm), is
germicidal, but it has not been much developed as an anti-infective approach until recently, when
it was realized that the possible adverse effects to host tissue were relatively minor compared to its
high activity in killing pathogens. Photodynamic therapy is the combination of non-toxic
photosensitizing dyes with harmless visible light that together produce abundant destructive
reactive oxygen species (ROS). Certain cationic dyes or photosensitizers have good specificity for
binding to microbial cells while sparing host mammalian cells and can be used for treating many
localized infections, both superficial and even deep-seated by using fiber optic delivered light.
Many microbial cells are highly sensitive to killing by blue light (400–470 nm) due to
accumulation of naturally occurring photosensitizers such as porphyrins and flavins. Near infrared
light has also been shown to have antimicrobial effects against certain species. Clinical
applications of these technologies include skin, dental, wound, stomach, nasal, toenail and other
infections which are amenable to effective light delivery.
© 2013 Elsevier Ltd. All rights reserved.

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Yin et al. Page 2
1. Introduction
The rising problem of antibiotic resistance has led to fears that medicine will return to the
situation of a century ago when extensive wounds and surgery often led to death due to
uncontrollable infection [1]. These fears have in turn spurred a major research effort to find
alternative antimicrobial approaches which, it is hypothesized, will kill resistant micro-
organisms while being unlikely to cause resistance to develop to themselves. At the present
time many international research efforts to discovery new antimicrobials are underway.
Recently, the emphasis is on how to take precautions against creating, and if possible
eliminate, multidrug resistance in concert with exploring new methods to kill pathogenic
microorganisms. Karen et al. recently pointed out that the investigation of novel non-
antibiotic approaches, which can prevent and protect against infectious diseases should be
encouraged, and should be looked upon as a high-priority for research and development
projects [2].
Prominent among novel non-antibiotic approaches is likely to be the group of light-based

technologies, including ultraviolet C (UVC) irradiation therapy, photodynamic therapy
(PDT), blue light therapy and other light-based therapies. The most attractive advantages of
light-based antimicrobial therapies lie in their ability to eradicate microbes regardless of
antibiotic resistance, and the fundamental improbability of the microbes themselves
developing resistance to these light based therapies due to the rather non-specific nature of
the targets.
In this review, we will provide an overview of recent advances in exploration and

understanding of light-based anti-infective therapies, including UVC, PDT, blue light and
other promising light-based therapies. To our knowledge, this is the first time that there has
been a review covering all these light-based therapies for infectious disease
2. Antimicrobial efficacy of UVC irradiation

2.1 Introduction
UV irradiation (wavelength: 100–400 nm) is electromagnetic irradiation, which is divided
into four distinct spectral areas including: UVA (315–400 nm), UVB (280–315 nm), UVC
(200–280 nm) and vacuum UV (100–200 nm) (Fig 1) [3]. Among these wavelength ranges,
UVC has the best potential ability to inactivate microorganisms because the wavelength of
250–270nm, is strongly and mainly absorbed by the nucleic acids of microbial cells and,
therefore is the most lethal range of wavelengths [4]. The bactericidal mechanism of UVC is
to cause damage to their RNA and DNA, which often leads to the formation of dimers
between pyrimidine residues in the nucleic acid strands. The consequence of this
modification is that the production of cyclobutane pyrimidine dimers (CPDs) causes
deformation of the DNA molecule, which might cause defects in cell replication and lead to
cell death afterwards.
Because tissue penetration of light is limited by the extremely short wavelength and
difficulties in the delivery approach, UVC for anti-infective diseases is likely to be applied
exclusively to superficial infections. Studies that have investigated UVC inactivation of
antibiotic-resistant bacteria have found them to be as equally sensitive as their naive
counterparts [5].
2.2 UVC irradiation for bacterial/fungi infections in vivo

Reports have demonstrated that UVC efficiently inactivated methicillin-resistant
Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VRE),
Yin et al. Page 3
antibiotic-susceptible strains of E. faecalis and S. aureus [5], Enterococci species,

Streptococcus pyogenes [6], Escherichia coli and Pseudomonas aeruginosa in vitro [7]. UVC
irradiation was able to disinfect catheters by destroying associated bacterial biofilms,
including coagulase-negative Staphylococcus, Streptococcus, E. coli, E. faecalis, P.

aeruginosa, Coryneforms and so on [8].
UVC also inactivated 3–5 log10 of dermatophyte suspensions in vitro, including

Trichophyton mentagrophytes, Trichophyton rubrum, Microsporum canis and
Epidermophyton floccosum [9]. When the inactivation efficacies of UVC were compared on
pathogenic microorganisms, including bacteria (P. aeruginosa and Mycobacterium
abscessus) and fungi (Candida albicans, Aspergillus fumigatus), in both mixed suspensions
and single suspensions in vitro, it needed much higher fluences to kill fungi than bacteria
[10]. The diverse structural features of the cell walls of bacteria and fungi are the main
reason for the different killing rates. Different devices that deliver UVC with different
power densities also induced different outcomes. More details about UVC germicidal
efficacies are presented in Table 1.
One of the key questions about the use of UVC as an anti-infective is the following [3]: Can
UVC destroy microbial cells in tissue without causing irreversible and unacceptable severe
damage to the host DNA and host cells? In an animal model infected by fungi with third-
degree burn, C. albicans bioluminescence was showed a significant reduction by UV
irradiation. The efficacy of fungicidal by UV was superior to using nystatin cream, one of
topical antifungal drug [11]. Another animal study showed UVC irradiation (2.59 J/cm2 for
burns and 3.24 J/cm2 for abrasions) significantly reduced Acinetobacter baumannii burden
in UVC-treated wounds by approximately 10-fold compared with non-treated groups (p =
0.019 for burns, p = 0.004 for abrasions) [12].
2.3 Effects of UVC irradiation on host cells and tissues

It is well known that prolonged and repeated exposure to UV irradiation can damage host
cells and be particularly hazardous to human skin. As to long-term UVC irradiation of
human skin, it is also known to have potential carcinogenicity [13]. When UVC irradiation
is applied to treat localized infections, one must consider the possible side-effects of UVC
delivered at effective antimicrobial fluences on normal mammalian cells and tissue. The
safety issue of UVC germicidal treatment requires that the pathogenic microbe is selectively
eradicated while the normal tissue cells are spared.
Sosnin et al. convinced the UVC fluence that led to necrosis in Chinese hamster ovary cells,
one type of fibroblasts, was more than ten-times higher than that used for killing of E. coli in
vitro [14]. Dai et al. investigated UVC inactivated nearly all bacteria (99%) in suspensions
over keratinocytes in confluent monolayer cultures, while it only reduced approximately 6%

viability of keratinocytes [15]. Another study demonstrated that UVC selectively inactivated
2 logs (99%) of C. albicans, while only approximately 0.77 log10 (18.9%) of keratinocytes
was killed at the same UVC fluence [11]. Dean et al. found that no significant adverse
effects were induced in human primary corneal epithelial cells when the cells were exposed
to 1.93mJ/cm2 UVC (265nm), which induced 100% inhibition of growth of all the bacterial
species cultured on agar plates [7]. Mohr et al. used UVC to reduce pathogen contamination
of platelet concentrates [16]. The results showed UVC inactivated more than 4log10 Gram-
positive S. aureus, Bacillus cereus and S. epidermidis, and Gram-negative E. coli, P.
aeruginosa and Klebsiella pneumoniae. However, UVC irradiation caused mild damage to
platelet and leaded to more enhanced platelet metabolism, including enhancing glucose
consumption, lactate accumulation and a stronger decrease in pH during storage.
Yin et al. Page 4
In a study using a murine model, Dai et al. demonstrated that the mouse skin tolerated the
UVC irradiation at the effective fungicidal fluence. Although UVC caused mild wrinkling of
the skin at 24h after light exposure, it showed full recovery in the following days [11]. In
order to evaluate the carcinogenicity of UVC, one animal study using hairless mice
determined that UVC was less carcinogenic than UVB [17].
Most of the experimental results mentioned above suggest that UVC at appropriate fluences
does not cause significant damages to host cells and tissues. However, UVC irradiation still
has potential to induce nonspecific damage. Studies demonstrated that the DNA of
mammalian cells could indeed be damaged by UVC at its effective antimicrobial fluences.
Fortunately however, at the same time, the DNA repairing enzymes of the host cells could
rapidly repair the damaged DNA [3]. In addition, to minimize the UVC-induced non-
specific damage, the intact skin around the area to be treated could be shielded from UVC
illumination. On the other hand, application of UVC is limited in some special locations due
to its detrimental effects such as infections of the eyes [18].
2.4 The clinical application of UVC irradiation for infectious diseases

A few clinical studies have demonstrated the antimicrobial effect of UVC irradiation,
including the disinfection of cutaneous wounds, promotion of wound healing in non-healing
infected leg ulcers by increasing granulation tissue [19,20]. A study presented by Taylor et
al., reported that the mean bacterial CFU in joint arthroplasty surgical wounds was reduced
by 87% with 0.1 mW/cm2 (P < 0.001) and 92% with 0.3 mW/cm2 (P < 0.001) of UVC [21].
Thai et al. used UVC irradiation to treat cutaneous ulcers infected with MRSA [22]. In all
three patients, UVC treatment reduced the bacterial burden in wounds and promoted wound
healing. Two patients had complete wound closure following 1 week of UVC treatment.
Another trial was carried out by the same investigators in 22 patients with chronic ulcers
manifesting at least two signs of infection and critically colonized with bacteria. The
patients received a single UVC treatment and demonstrated significantly reductions of the
bacterial burden [23]. Boker et al. enrolled thirty patients with mild-to-moderate toenail
onychomycosis to treat with UVC. Improvement by at least 1 measurement point was
achieved in 60% of patient at 16-week follow-up compared with baseline. There were some
unusual and slight side effects such as temporary mild eythema of the treated toe [24]. In
addition to the inactivation of microbial cells in the cutaneous wound, UVC exposure is
beneficial for wound healing by promoting the expression of basic fibroblast growth factor
(bFGF) and transforming growth factor-β [25], although the exact mechanisms of UVC for
wound healing is still unclear. Shimomura et al. investigated the prophylactic efficacies of
UVC irradiation in 18 cases of catheter exit-site infections [26]. Although five cases
remained unchanged, ten cases (55%) became culture negative and a further three cases
showed a microbial decrease.
In summary, it has been known during the past one-hundred years that UVC irradiation is
highly bactericidal; however, using UVC illumination for the prophylaxis and treatment of
localized infections is still at very early stages of development. Most of the studies are
limited to in vitro and ex vivo levels, while in vivo animal studies and clinical studies are
much rarer. A major advantage of using UVC over antibiotics is that UVC can eradicate
resistant and pathogenic microorganisms much more rapidly without any systemic side-
effects. UVC may also be much more cost effective than the commonly used antibiotics.
3 Photodynamic therapy as an anti-infective

3.1 Introduction and mechanism of photodynamic therapy
Another prominent member of the light-based therapies is known as antimicrobial
photodynamic inactivation (PDI) or photodynamic therapy (PDT). PDT can direct injury to
Yin et al. Page 5
the target cells and tissues by producing reactive oxygen species (ROS) induced by the
appropriate wavelength of visible light interacting with pre-applied photosensitizer (PS)
drugs. The PS can be specifically activated by light of certain wavelength to its excited state.
Interaction of the long-lived excited triplet state of the drug or dye with the ground state of
molecular oxygen (also a triplet) results in development of ROS such as singlet oxygen
(1O2) through energy transfer and hydroxyl radicals (HO•) through electron transfer. The
formation of ROS and 1O2 can chemically attack a very wide range of biomolecules [27–30]
(see Fig 2). The high selectivity of PDT for rapidly growing and hyperproliferating cells
suggested it could be useful for selective inactivation of microorganisms which possess very
fast growth rates [31]. Recently, PDT has been applied as a discovery and treatment
alternative approach for localized infections, which represents an emerging new field [32].
This development has been motivated by the relentless growth in antibiotic resistance now
found in all classes of microbial cells and in all countries of the world. The broad-spectrum
and rapid killing effect of PDT is particularly appealing in this regard [33].
3.2 Antibacterial efficacy of photodynamic therapy

Bacterial cells can be divided into two groups called Gram-positive and Gram-negative
bacteria, characterized by large differences in the cellular structure and organization (Fig 3A
and B).
Gram-positive and Gram-negative bacteria have diversity in their three-dimensional

architecture and outer cell wall. In Gram-positive bacteria the outer wall (15–80nm thick)
demonstrates a relatively high degree of porosity which allows the most commonly used PSs
to readily diffuse into the inner plasma membrane [34]. On the contrary, in Gram-negative
bacteria, the outer wall possesses a highly organized compact structure which acts as a
permeability barrier and prevents diffusion to the inner plasma membrane for most high
molecular weight compounds [35]. The different permeability barriers between Gram-
positive and Gram-negative bacteria are responsible for the observed sensitivity differences
when treated with many antimicrobial agents. In principle, PDT has been shown to be more
effective against Gram-positive than Gram-negative, especially when neutral or anionic PSs
were used [31]. However, non-cationic PSs only bind to the outer membrane of Gram-
negative bacteria cells, and do not inactivate them after illumination since the ROS is not
produced in sensitive regions. The molecular structures of a range of PSs used for
antimicrobial applications are shown in Fig 4.
A number of PSs, including cationic substituted Zn(II)-phthalocyanines [36] (Fig 4A), poly-
S-lysine-porphyrin conjugates [37] (Fig 4K), meso-tetrahydroporphyrin,
tetrahydroporphyrin-tetratosylat (THPTS) [38,39] and cationic water-soluble gallium(III)
phthalocyanines (GaPcs) [40] (Fig 4B), can substantially reduce MRSA populations (4–5
log10) after illumination. Moreover, it has been shown that a PS conjugate of

polyethylenimine and chlorin(e6) (pEI-ce6) (Fig 4I) irradiated with red light is capable of
reducing MRSA CFU by 2.7 log10 in a murine skin abrasion model [41]. Methylene blue
(MB) mediated PDT inactivated VRE [42], and vancomycin-porphyrin conjugates were able
to inactive in vitro vancomycin-sensitive and VRE [43]. Cationic PSs such as pL-ce6 and
MB could destroy the protective effects of extracellular slime and stationary bacterial
growth phase when PDI was used to inactive isogenic pairs of wild-type and mutant S.
epidermidis and S. aureus [44].
Multidrug-resistant (MDR) and pandrug-resistant (PDR) Gram-negative bacteria are less

prevalent than MRSA but bring about an equally grave threat of truly intractable infections
[45,46]. In a model study, PDT also showed high efficiency to anti-infection of Gram-
negative bacteria. Sixty MDR P. aeruginosa isolates were inactivated by toluidine blue O
(TBO) (Fig 4C) mediated PDT with up to 6–7log10 reductions in CFU [47]. This study also
Yin et al. Page 6
demonstrated that antibiotic-resistant P. aeruginosa was just as sensitive to PDI as antibiotic-

susceptible strains. Another report showed that MDR strains of Aeromonas hydrophila were
inactivated by PDT mediated by cationic phthalocyanines [48]. Using 5-phenyl-10,15,20-
tris(N-methyl-4pyridyl)-porphine trichloride (also known as Sylsens B or Tri-P(4)) (Fig 4E)

as the PS, Trannoy et al. obtained a PDI reduction of Yersinia enterolitica CFU by 5 log10
[49].
MDR and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis (MDR-

TB and XDR-TB) are a rising threat in the developing world which requires long term
treatment. Infections have serious adverse effects, are difficult to cure and even fatal [50].
PDT studies on TB have been focused on the homologous system M. bovis Bacille Calmette
Guerin (BCG) both in vitro applying phenothiazinium salts and in murine models of
localized mycobacterial induced granulomatous formation [51,52]. In a similar fashion, the
antimicrobial effects of PDT have been assessed on infections caused by M. marinum [53]
and on rapidly growing nontuberculous mycobacteria keratitis [54].
Recently, some novel designed and synthesized PSs have been explored for anti-infective
therapy with PDT such as the closed all-carbon cages called fullerenes. C60(>ME1N6 +C3)
and C60(>ME3N6 +C3) (Fig 4L), which are two analogous pentacationic [60] fullerenyl
monoadducts with variation of the methoxy-ethyleneglycol length, exhibited high activity
for targeting and PDI Gram-positive and Gram-negative bacteria [55]. Polycationic
chitosan-conjugated rose bengal (CSRB) as PS showed anti-biofilm ability on Enterococcus

faecalis (Gram positive) using PDT [56]. Temoporfin liposomes conjugated with a specific
lectin (wheat germ agglutinin, WGA) on the liposomal surface eradicated all MRSA by PDT
compared with non-modified liposomes [57]. A new PS integrated a potent second
generation photosensitizer (temoporfin) and a novel antimicrobial peptide (WLBU2) into
liposomes that killed all MRSA upon illumination with 652 nm laser (1 W/cm2, 100 s, 100
J/cm2) [58]. Tsai et al. showed that 0.25 μM micellar hematoporphyrin (Hp) completely
eradicated S. aureus under a light fluence of 50 J/cm2 [59]. The potentiating bactericidal
effect of chitosan on Hp- or TBO-mediated PDI was also found with other Gram-positive
bacteria (S. pyogenes and S. epidermidis) and two Gram-negative bacteria (A. baumannii
and P. aeruginosa) while causing less damage to human normal tissues [60]. Tetracationic
meso-arylsubstituted porphyrin (RM24) induced PDI can completely eradicate cultures of
108 CFU/mL P. aeruginosa PAO1 in stationary phase [61].
Moreover, PDT demonstrated significant killing effects on some bacteria which are very
difficult to inactivate by antibiotics. For example, Hypocrellin A (HA)-mediated PDT
significantly reduced the survival rate of Bacillus subtilis to less than 0.1% (P<0.05) [6].
Walther et al. reported that protochlorophyllide mediated PDT inactivated Listeria
monocytogenes and Bacillus subtilis, while Yersinia pseudotuberculosis were found to be

insensitive to protochlorophyllide treatment [62]. However, the two bacteria were showed
susceptibility to eradication by protochlorophyllide (10 mg/L) combined with polymyxin B
nonapeptide at 50 and 20 mg/L, respectively.
In addition to in vitro studies, several in vivo studies have been carried out to investigate and
improve the efficacy of antimicrobial PDT in animal models that mimic clinical human
infections. Different kinds of wounds such as burns, abrasions, soft tissue disruption, and
excisional wounds have been investigated. These wounds are easy to infect and frequently
involve Gram-negative as well as Gram-positive bacteria. Hamblin et al. first investigated
the effect of PDT in vivo using mouse excisional wound models infected with P. aeruginosa
and E. coli, respectively [63,64]. A light dose dependent response was observed in bacteria
killing. Moreover, only the animals treated with PDT survived in P. aeruginosa infected
wounds. PDT using TBO significantly inactivated Vibrio vulnificus that could otherwise
Yin et al. Page 7
lead to fatal sepsis. By using a mouse model of burn infected with Acinetobacter baumannii,
Dai et al. found that, PEI-Ce6 mediated PDT produced 1.7–3 log10 reduction of bacteria
while it did not inhibit the wound healing [65]. In a soft tissue model developed by
intramuscular injection of bacterial suspension into the mouse thigh muscle, Berthiaume et
al. studied the effect of PDT against P. aeruginosa using bacteria mixed in vitro with the tin
(IV) chlorin e6—monoclonal antibody conjugate and shining the light after injection [66].
The significant decrease of bacteria was demonstrated. Hypocrellin B: lanthanum (HB:La+3)
mediated PDT was able to reduce multidrug-resistant P. aeruginosa burden in burn wounds,
inactive bacterial levels 2–3log in blood and delay bacteremia compared with untreated
control group. In addition, Hashimoto et al. reported the in vitro reduction of P. aeruginosa
[67]. Park and coworkers investigated chlorin(e6)-mediated PDT antimicrobial effects in a
mouse model in which skin wounds were infected by bioluminescent S. aureus Xen29 [68].
MB-induced PDT produced maximal bacterial killing and minimized killing of host
neutrophils in murine MRSA arthritis models [69]. PDT with Na-pheophorbide significantly
prevented leg swelling, inhibited bone destruction on osteomyelitis models in rats infected
by methicillin-sensitive S. aureus (MSSA) [70].
PDT has been widely applied in oral and dental infections where Gram-negative bacteria are
really common and are thought to be responsible for the most damaging infections. The use
of PDT in different models of peri-implantitis demonstrated a decrease of bacterial burden
with different bacterial strains. The viabilities of A. actinomycetemcomitans, P. gingivalis,
and Prevotella intermedia bacteria were reduced significantly after TBO mediated PDT [71].
PDT was able to reduce Fusobacterium sp and Prevotella sp counts [72,73]. Significant
results were also obtained in non-surgical treatment of periodontitis in in vivo models. PDT
using TBO caused a significant reduction of P. gingivalis CFU in a rat model [74]. The
inactivation of P. gingivalis was achieved in infected sites using PDT with chlorin(e6) in a
beagle dog model [75]. Bottura et al. used a model of immunosuppressed rats to demonstrate
the effect of PDT on reducing bone loss resulted in experimental periodontitis [76]. Several
more studies were in agreement with the previous literature, and further manifested the
effectiveness of PDT in treatment of periodontal disease [77–79]. In order to evaluate the
effect of PDT on inflammatory cells in periodontitis, Seguier et al. tested two different drug
delivery systems [80]. The results demonstrated that PDT suppressed the gingival
inflammatory reaction during chronic periodontitis and resulted in a specific decrease of
antigen-presenting cell populations based on which drug delivery system was used.
3.3 Antifungal efficacy of photodynamic therapy

The clinical location and visible appearance of fungal infection varies widely, from
superficial, subcutaneous to severe systemic infectious disease. Fungal pathogens are
variable in their cell envelopes, possessing outer wall formed from mixtures of mannan,
chitin polymers, and beta-glucans (Fig 3C). This feature causes them to be inherently more
permeable to external substances when compared to Gram-negative bacteria.
The hypothesis that cationic PSs would be more efficient in PDI has been tested in Candida
species, which are proposed to be the most common fungal pathogens, by several in vitro
and in vivo studies [81–83]. C. albicans were inactivated by PDT using PSs including RB,
Photofrin (Porfimer sodium) and Al (III)-tetrasulfonated phthalocyanine [84–86]. In another
study, C. albicans suspensions were irradiated with helium–neon or gallium-aluminum-
arsenide lasers using five different PSs including methylene blue (MB), thionin, crystal
violet, toluidine blue O (TBO) and phthalocyanine. Except for phthalocyanine, all other PSs
tested resulted in a significant reduction in C. albicans CFU following PDT [81]. PDT
mediated by Photogem, a porphyrin PS, against four different species of Candida, C.
tropicalis, C. albicans and C. dubliniensis were completely eliminated, except C. krusei [87].
MB-mediated PDT achieved a drug-dependent photo-killing of oral candidosis infected by
Yin et al. Page 8
C. albicans in a murine model [88]. Junqueira et al. demonstrated that the rats treated by
MB-mediated PDT had less candidiasis lesions compared with the untreated control group
[83]. On the other hand, Giroldo et al. confirmed that MB-induced PDT increased the
permeability of cell membrane of C. albicans, which in turn could increase the sensitivity of
this microorganism to other antifungal agents [89]. Effective killing of azole-resistant strains
of C. albicans [88,90,91] and C. glabrata, [87,91] by PDT was also achieved with the use of
TBO [88], MB [90], cationic porphyrin [91] and Photogem® [87] as PSs. However,
investigators reported that PDT produced less substantial killing of azole-resistant strains in
Candida [87,90]. Dovigo et al. investigated the PDI of oral candidiasis infected by C.
albicans in a murine model using curcumin as the PS [92]. Results showed that exposure to
curcumin (Fig 4H) combined with light-emitting diode (LED) light caused a significant
reduction in C. albicans viability following PDT in a PS concentration dependent manner
without harming the host tissue of mice. Mima et al. investigated that Photogem®
illuminated by 455 or 630 nm LED light (305 J/cm2) inactivated C. albicans in a murine
model of oral candidosis. The results showed a significant reduction in C. albicans recovered
from the tongue (P < 0.001) compared control group [93]. Mima et al. did another in vitro
study showed the effectiveness of Photogem® induced PDT for the inactivation of different
five species of Candida on maxillary complete dentures, including C. albicans, C. tropicalis,
C. glabrata, C. krusei and C. dubliniensis. The results showed PDT reduced 3.99 log10 CFU
from dentures which were infected all species of Candida [94].
T. rubrum is a common pathogenic fungus genus of dermatophytes. PDT was suggested as a

potential treatment modality to kill T. rubrum with the use of the broad band white light and
PSs, including porphyrins deuteroporphyrin mono-methylester (DP-mme) and 5,10,15-
tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) [95]. PDT
inactivated T. rubrum in both suspension culture and its isolated microconidia [96]. Red
light showed the higher penetration was suggested to be more effective especially for nail
infections by PDT. Results of the study demonstrated that Sylsens B was the better PS
against both hyphae and the microconidia of T. rubrum which completely inactivated the
fungal spores and destroyed the fungal hyphae.
Aspergillus fumigatus can cause opportunistic infections in the lung, sinuses and brain of
patients with predisposing conditions [97]. Aspergillosis caused by this fungus is frequently
fatal, increasing in frequency, and difficult to treat. The treatment is even more complicated
by the increasing incidence of drug-resistant strains. Significant fungicidal effects of PDT on
A. fumigatus was demonstrated in vitro, implying that PDT may be a treatment approach for
certain localized fungal infections that form granulomas and can adapt themselves to
invasive growth [98,99]. However, in a study performed by Gonzales et al. comparing the
conidial phototoxicity of Metarhizium anisopliae and A. nidulans with TBO and MB under
various incubation times and light doses, the investigators observed that even though
conidial killing rates reached up to 99.7%, and germination of the surviving conidia was
delayed, none of the treatments achieved a 100% fungicidal effect [100].
3.4 Anti-bacterial/fungi biofilm efficacy of photodynamic therapy

The formation of biofilm is most commonly associated with chronic infections which are
typical of microorganisms adhering both to each other and/or to surfaces or interfaces and
embedded in an extracellular polymeric matrix. Once biofilm is established, these
communities form a protective niche that facilitates growth and survival of bacteria in a
hostile environment [101]. The protected environment and the density of the film, as well as
the significantly different cellular properties from those in free-floating bacteria of the same
species, have been implicated in about 1,000-fold resistance to antiseptics, detergents and
antibiotics [102]. Owing to the prevalence of biofilms as a cause of many diseases and the
fact that they significantly increase the bacterial resistance to conventional antibiotic
Yin et al. Page 9
therapies, it is necessary to develop new modalities to treat microbial biofilms. PDI is a

possible alternative to inactivate biofilms. Moreover, PDI is a simple non-mechanical
therapy and it can be also used for the treatment of children, handicapped people and
medically compromised individuals.
S. epidermidis and S. aureus are often the pathogens of persistent infections occurring with
implanted medical devices that are hard to eradicate [103]. Sharma et al. studied the effects
of TBO-induced PDT on the structure and the viability of biofilms of S. epidermidis and of a
MRSA strain [104]. They found PDT significantly reduced staphylococcal biofilms, and the
combination of tetrasodium EDTA with PDT enhanced the phototoxic efficacy in S.
epidermidis biofilms, but not in S. aureus biofilms. Zanin et al. found that TBO (0.1 mg/ml)
induced PDI reduced 2.10–3.11 log 10 CFU of S. mutans composed in biofilms treated in a
dose-dependent manner [105,106]. The average reduction in CFU varied between 1.00 to
2.44 log 10 for the biofilms formed by two and three species, and it was verified that TBO-
based PDT has a substantial effect on staphylococcal biofilms which can decrease 5 log10
CFU irradiated with red light, disrupt the architecture of biofilm and damage the membranes
of bacterial cells. PDT also has an influence on S. mutans biofilms in different maturity
stages (inactivated 4 log10 CFU), as well as mature S. sanguinis and S. sobrinus biofilms.
MB-PDT induced an average reduction of 2.81 log 10 of S. mutans biofilms, as well as an

average CFU reduction of 3.29 log 10 of S. aureus biofilms [107]. Merocyanine 540 (Fig 4J)
mediated PDT showed a significant inactivation effect on the survival of S. aureus biofilms.
However, the light fluences required to inactivate biofilms were much higher than those
used to photoinactivate planktonic cultures, due to hindrance of the penetration of light and/
or PS within the biofilms [108]. PDI with the cationic porphyrin, tetra-substituted N-methyl-
pyridylporphine (TMP) was effective in biofilm adhering to medical implant surfaces when
combined with vancomycin and the host defenses [109]. Tri-meso (N-methyl-pyridyl), meso
(N-tetradecyl-pyridyl) porphine (C14) (Fig 4M) presented a significantly greater PDI effect
on the eradication of S. epidermidis biofilms when compared with the parental tetra-
substituted N-methyl-pyridyl-porphine (C1) [110]. Erythrosine (ER) was found to be able to
inactivate S. mutans biofilms better than protoporphyrin and MB with the inactivation effect
enhanced by 2 log10 [111]. Erythrosine induced PDT was also more potent than MB, RB
and TBO against Aggregatibacter actinomycetemcomitans biofilms [112]. PDI mediated by
both 5-ALA and TMP at different concentrations could eliminate P. aeruginosa biofilms
[113,114]. Recently, Hegge et al. investigated the phototoxicity of curcumin in S.
epidermidis biofilm and suspension cells in two models in vitro [115]. The phototoxicity of
curcumin towards planktonic S. epidermidis was dependent on the presence and type of
nanocarrier (PEG 400, HPcCD and F 127). A different situation was observed in a biofilm,
where the phototoxicity of curcumin was less sensitive to the selected nanocarrier.
Therefore, curcumin should be optimally formulated to target both biofilms and planktonic
bacteria in order to obtain maximum photoinactive effect.
Peri-implantitis involves the bacterial colonization, typically in the form of biofilms on the
implant surfaces and may lead to system infection and destroy the implant surface.
Approximately 20 % of the oral bacteria are streptococci, which are correlative of the dental
plaque formation and the development of oral biofilms. Streptococcus mutans and
Streptococcus sanguinis normally exist as regular members of the mature dental biofilm
community, and causes caries and periodontal diseases. Pereira et al. examined the effect of
PDT using ER and Rose Bengal (RB) as the PSs, respectively, and LED on S. mutans and S.
sanguinis biofilms [116]. PDT induced significant decreases in the viability of both
microorganisms, especially S. sanguinis.
Yin et al. Page 10
Biofilm formation is also a key event in the development of fungal disease. Similar with the
previously mentioned bacterial biofilms, fungal biofilms cause treatment resistance and high
mortality, particularly in immunocompromised patients [117]. Candidiasis, caused most
commonly by C. albicans and to a lesser extent by C. tropicalis, C. parapsilosis or C.

glabrata, is often associated with biofilm formation on the surface of medical devices and
tissues [118]. Candida biofilm is one of the most common causes of clinical complications
found with implanted biomaterials such as intravascular catheters, prostheses, pacemakers,
stents, urinary catheters, shunts and orthopedic implants and unfortunately treatment with
current antifungal agents are challenging [119]. Candida biofilms resist most of the present
antifungal agents, except echinocandins. However, an increase in resistance to
echinocandins is now frequently reported [120].
It was revealed that C. albcans biofilms were susceptible to photofrin induced PDT [121],
and C. albicans and C. dubliniensis were sensitive to erythrosine-mediated PDT, however
the biofilms of both Candida spp. were more resistant than their planktonic counter parts
[122]. Another study also showed the effects of photofrin (10μg/ml) mediated PDT against
C. albicans biofilms and its germ tubes [121]. Biofilms treated with PDT showed significant
reduction of metabolic activity compared to the biofilms treated with amphotericin B (10μg/
ml). The same authors also investigated the effect of PDT against biofilms of C. albicans
and C. dubliniensis using erythrosine (400 mM) illuminated by green LED light (532±10
nm, 237 mW/cm2, 42.63J/cm2) [123]. Scan electronic images demonstrated a decrease in the
amounts of hyphae and conidia in the biofilm following PDT. However biofilms of both
Candida species showed more resistance than their planktonic counterparts. The
combination of erythrosine and RB-mediated PDT demonstrated some effect on biofilms,
and was also effective in destroying and reducing the blastoconidia and hyphae of C.
albicans [122]. MB-PDT combined with 660nm laser exhibited a modest reduction in
biofilm C. albicans species [107]. Mentareva et al. demonstrated that the best phototoxic
effects on C. albicans biofilms were obtained by Zn (II) and Ga (III) phtalocyanines, such as
ZnPcMe (Fig 4A) and GaPc2 induced PDT, compared with other diverse PSs [40]. Coleman
et al. demonstrated that through coupling of saponins with PSs (RB (Fig 4F) and
chlorin[e6]), incubating with the yeasts for 30 min and irradiating with visible light, it was
possible to inhibit the formation of C. albicans biofilm and dramatically potentiate PDI in
vitro [124]. In one in vitro study, Donnelly et al. developed a mucoadhesive patch that
contained TBO as a PS delivery system for PDT of oropharyngeal candidiasis [125]. This
patch was able to resist dissolution when it interacted with artificial saliva and prevented
staining of lips, teeth and buccal mucosa. When TBO was released directly into an aqueous
sink upon illumination (635nm, 100mW/cm2, 200J/cm2), patches were capable of killing C.
albicans biofilm. Recently, Ribeiro et al. evaluated that the effectiveness of
chloroaluminum-phthalocyanine (ClAlPc) induced PDT in the treatment of C. albicans
planktonic cultures and biofilm with different drug delivery system [126]. Cationic
nanoemulsions –ClAlPc and free ClAlPc caused significant damage to the fungal cells
membrane (P < 0.05); furthermore, cationic nanoemulsions –ClAlPc reduced significantly
both cell metabolism and fungal colony counts and also showed better killing rates for
Candida biofilms (P < 0.05).
PDT eradication of microbial biofilms could lead to a variety of applications in clinical

conditions. For examples, PDT has been used to target to: 1) superficial infections, including
elimination of dental plaque, periodonitis, gingivitis, endodontics and oral candidiasis; 2)
subcutaneous infections including sinusitis, and osteomyetitis; 3) internal organs infection
including endocarditis and the infection of cystic fibrosis; 4) various catheter infections and
5) some temporary or permanent indwelling devices such as joint prostheses, heart valves
and other implants, and so on [32]
Yin et al. Page 11
In conclusion, there is a wealth of literature describing PDT-based anti-biofilm strategies

that focus mostly on the use of different PSs against a variety of microbial species. In
contrast there are only a limited number of studies exploring the effects of PDT on
phenotypic biofilm elements (e.g.,adhesins and extracellular polysaccharide ) [127,128].

Moreover, there is lack of standard reproducible models for assessing PDT efficacy against
biofilms. The majority of published researches used methodologies where biofilms were
grown in/on plastic or silicon microtiter plates and surfaces. These bioassays have been
repeatedly criticized for lack of stability and clinical relevance and occasionally yield
inconsistent results. For fungal biofilm, it involves a heterogeneous mixture that is
composed of hyphae, pseudohyphae and blastoconidia embedded in extracellular polymeric
substances which form pores and channels and display diverse phenotypic features
compared to planktonic yeasts [129]. The extracellular matrix is composed of proteins,
polysaccharides, hexosamine, uronic acid and DNA which promote biofilm formation and
adhesion, protecting the cells from phagocytosis [129,130]. This in turn maintains and limits
the diffusion of substances, such as antifungal agents, and also restricts penetration of PS
and light during PDT [129,130]. Other reasons for increased resistance against both
antifungal agents and PDT are the presence of cell wall thickening, up-regulation of drug-
efflux pumps, and higher density and larger size of cells in yeast biofilms, decreased number
of targets for singlet oxygen per unit of cell volume, existence of multiple species (bacteria
and yeasts together), enzymatic alteration of active agent, presence of persisters which
represent a subpopulation of cells spontaneously going into a dormant, non-dividing state
and in turn remaining alive even after antimicrobial treatments [119,122,129,131,132].

Through modification of the treatment conditions such as incubation time, fluence rate, type
of PS and PS’ concentration, the efficacy of photo-inactivation can be enhanced especially
for resistant cases.
3.5 The efficacy of PDT on other pathogens

Several protozoa are quite dangerous and even deadly human pathogens and most of them
are immune to the presently used antimicrobial therapeutic modalities. Typical examples are
represented by Acanthamoeba, Leishmania spp, Entamoeba histolytica, Plasmodium spp and
Giardia intestinalis.
Species of Acanthamoeba are facultative parasites, capable of living within the tissues of
vertebrates and are the causative agents of granulomatous amoebic encephalitis and amoebic
keratitis [133]. The life-cycle of Acanthamoeba species are divided into two stages: a
dormant cyst and trophozoite, and both stages are infectious. The trophozoitic stage is
delimited by a plasma membrane that mainly consists of phospholipids, while the mature
cysts’ wall is composed of an inner (endocyst) and outer (exocyst) layer separated by a
space with a tightly organized structure. The special osmotically inextensible wall,
especially endocyst layer, makes the cyst highly resistant to various chemical agents [134].
Limited reports have shown that cationic porphyrins and phthalocyanines, excited by visible
light, caused an efficient photosensitized killing of parasitic protozoa. More specifically,
tetracationic phthalocyanine (RLP068)-mediated PDT showed the photo-toxicity effect on
Acanthamoeba palestinensis which almost induced complete inhibition of the viability of A.
palestinensis in the trophozoite stage [135]. Hypocrellins B (HB) induced PDT manifested
effectiveness on Acanthamoeba cysts and trophozoites in vitro. The results showed HB-PDT
induced a total inhibition of the trophozoites and cysts in a dose-dependent manner and a
better phototoxic inactivation on the trophozoites than the cysts. However, HB-PDT also
manifested phototoxicity on the corneal epithelial cells and stromal cells in the rabbit model
[136]. Another study showed that MB-mediated PDT reduced the respiratory activity of
Acanthamoeba castellanii (ATCC 50370) trophozoites in an MB-concentration dependent
manner [137]. Recently, Siddiqui et al. used Sn (IV) porphyrin (a synthetic
Yin et al. Page 12
metalloporphyrin) as PSs illuminated by visible light to treat A. castellanii [138]. The results
demonstrated that PDT suppressed A. castellanii growth, the ability of cytopathogenicity
and decrease the adhesion to human corneal epithelial cells in a concentration-dependent
manner. However, PDT had no effect on its viability.
Leishmania is an intracellular parasite that parasitizes the phagosomal compartment of

macrophages in the vertebrate host and is transmitted to human body via the bite of infected
sandflies. Leishmaniasis causes substantial disfigurement and sometimes mortality in the
developing countries. The clinical manifestations of Leishmaniasis in humans include
cutaneous, mucocutaneous and visceral forms with a wide range of geographic distribution
[139]. Recent therapeutic modalities include systemic and topical treatments [140].
However, none of these treatments have been evaluated to be completely satisfactory, and
only a few of the touted agents have been proved adequately in clinical trials. Up to now,
multiple investigations performed have shown the effects of PDT on killing Leishmania in
vivo and in vitro. Some PSs manifested phototoxicity to inactive Leishmania strains,
including MB [141], (3,7-bis(N,N-dibutylamino) phenothiazinium bromide (PPA904) (Fig
4D), δ-aminolevulinic acid-derived protoporphyrin IX (ALA) [142–144], chloroaluminum
phthalocyanine encapsulated ultra-deformable liposomes (UDL-ClAlPc) [145], dimethyl
and diethyl carbaporphyrin ketals (CKOMe and CKOEt) [146]. PDT caused obvious
inactivation on several Leishmania strains, such as L. panamensis, L. chagasi [145], L.
tarentolae [146], regardless whether their life-stages were promastigotes, axenic or
intracellular amastigotes. In some clinical studies, MB-, ALA- or MAL-mediated PDT

showed effectiveness to treat cutaneous leishmaniasis. Cosmetic results were excellent and
nearly no recurrence occurred after PDT [140,147–149]. Topical PDT showed safety as well
as effectiveness, and can be applied as an alternative approach for cutaneous leishmaniasis.
Plasmodium spp are the main causative pathogens of mosquito-borne diseases transmitted
from one individual to another resulting in transfusion-transmitted malaria (TTM). There are
several infective members of the Plasmodium genus, including P. falciparum, P. ovale, P.
vivax and P. malariae [150]. As a result of increased international travel, migration, and the
spread of drug-resistant parasites, malaria is a growing issue in malaria-endemic areas and
industrialized nations. Phthalocyanines (one of the most effective, HOSiPcOSi(CH,),
(CH,),N(CH,), (Pc 4) ), illuminated by red light, manifested the photoinactivation of P.
falciparum parasitized in red cells in a dose-dependent manner and could be used to make
blood components safe [151]. Another study demonstrated that ALA-induced inactivation of
plasmodial heme-cycle intermediates, by exposure to white light, reduced P. falciparum in
culture to levels such that they were not detectable by light microscopy nor by lactate
dehydrogenase assay [152].
Human African trypanosomiasis, caused by Trypanosoma brucei rhodesiense or

Trypanosoma brucei gambiense is a disease contracted after biting by the tsetse fly, and is
usually a disease involving the nervous system, also known as sleeping sickness.
Conversely, American trypanosomiasis, or Chagas’ disease, is commonly produced by
infection by T. cruzi [150]. None of the diseases have an obvious cutaneous appearance
(except the puncture wound caused by the biting insect disease vector), but the protozoa
exist in the bloodstream of infected persons. Recent studies showed silicon phthalocyanine
Pc4, thiazole orange and gentian violet induced PDT manifested photoinaction for T. cruzi
[153]. Especially, gentian violet -induced PDT has already been used to disinfect T. cruzi in
donated blood, and this also offers a strong potential lead for conventional drug discovery
against T. cruzi [154]. In one in vitro study, Do Campo et al. demonstrated the a 1% solution
of MB or 0.001% solution of MB illuminated by white light (100W) immobilized T. brucei
in citrated guinea pig blood without any subsequent disease when administered to mice
[155]. Another study showed that the ultraviolet-absorbing compound Amotosalen (4′-
Yin et al. Page 13
aminomethyl-4,5′,8-trimethylpsoralen) when illuminated by UV completely inactivated the

infective form of T. cruzi both in platelet concentrates and in fresh-frozen plasma [156].
Gottlieb et al. inactivated T. cruzi trypomastigote forms in the composition of blood by PDT
with three phthalocyanine dyes, including HOSiPcOSi(CH3)2(CH2)3N(CH3)2(Pc4),

HOSiPcOSi(CH3)2(CH2)3N+(CH3)3I-(Pc5) and aluminum tetrasulfophthalocyanine
hydroxide (AlOHPcS4) [157]. The compound Pc 4 was shown to be highly effective in
inactivating T. cruzi, Pc 5 was less effective and AlOHPcS4 was ineffective. Ultrastructural
analysis showed the parasites’ mitochondria were a primary target of phthalocyanine dyes-
induced PDT.
3.6 The efficacy of PDT on viruses

It has been reported that PDT can also be used to inactivate enveloped viruses. Pristine C60
fullerenes induced photo-inactivation of vesicular stomatitis virus (VSV) (Rhabdoviridae) or
Semliki forest virus (Togaviridae), which produced up to 7-log10 of loss of infectivity
[158,159]. Hirayama et al. demonstrated that methoxy-polyethylene glycol conjugated
fullerene induced PDT, illuminated by white light (120 J/cm2), and damaged more than 5
log10 of plaque forming units of VSV [160]. Lin et al. compared two region-isomers of
carboxyfullerene, illuminated with or without light, inactivation dengue-2 and other
enveloped viruses [161]. The asymmetric isomer showed greater dark activity, albeit at
much higher concentrations than required for its PDT effect. The authors concluded that this
difference was attributed to the interaction with the lipid envelope of the virus.
In general, polyomaviruses are considered to be highly resistant to PDI. The degree of

resistance is dependent upon the structure of the PS; TBO, acridine orange and MB are
effective PSs for PDI of polyomaviruses. Higher doses of PS and light may be required in
order to inactivate the oncogenic properties of these viruses than those required to inactivate
infectivity [162]. Human papillomavirus (HPV) infection is very common in many
populations and manifests several clinical features, including genital warts, verruca vulgaris,
verruca plantaris, verruca plana and so on. The clinical applications of PDT have been well
reviewed by Lee et al. in terms of efficacy, safety and cosmetic outcome [163]. In most
clinical cases ALA and methyl aminolevulinic acid (MAL) are used as PSs, even if they are
off-label treatments.
3.7 The efficacy of PDT on vectors

Insects act as the crucial roles to disseminate tropical disease via the blood of bitten
individuals. Owing to deforestation, insect vectors can now invade cities and are thus
considered responsible for developing new endemic regions of disease. Many of the affected
countries spend enormous resources to control insect vectors, usually entailing the use of
chemical insecticides. However, chemical insecticides are considered to possibly cause side
effects on humans and are detrimental to the environment. Other strategies have been
developed including biological control and photo-insecticides.
Photo-activated insecticides were demonstrated to be a novel compelling alternative to

chemical insecticides. The ideal photo-activated insecticides possess the following
characters. First of all they are non-toxic in the dark and second they are not permanently
accumulated in the environment, because they are usually bleached and chemically degraded
by light. The third is that usually, they employ light energy to produce their phototoxicity
and are intrinsically less harmful to the environment because they are used at very low
concentrations. The concept of using PDT inactivation of pest populations was realized as
early as in the 1930’s using xanthene derivatives against Anopheles and Aedes larvae and
the developments in this field have been reviewed by Ben Amor and Jori [164]. Among the
limited reports in this area, porphyrins are particularly interesting as photo-insecticides
Yin et al. Page 14
because they exhibit several absorption bands in the UV/visible spectrum, being therefore
efficiently excited by the solar spectrum [165]. Under this concept, Lucantoni et al. tested
the phototoxicity of a synthetic meso-substituted porphyrin meso-tri(N-methylpyridyl),
meso-mono(N-tetradecylpyridil) porphine (C14) [166]. The photo-activable porphyrin

showed phototoxicity on the dengue vector Aedes aegypti in laboratory conditions. In
artificial pool tests, greatest population reductions were achieved using dosages on the
countryside of 20 and 40 g/ha. Shao et al. synthesized a series of tetraethynyl silanes (TETS)
as photo-activated insecticides to test their killing effects on 4th-instar larvae of A.
albopictus (Skuse) [167]. After 3h incubation in a dark room, A. albopictus were treated by
ultraviolet irradiation (300–400 nm, maximum at 365 nm) for 1.5h at the fluence rate of
2.074 wW/cm2. Among them, the compound with four thiophene groups attached on the
same silicon atom with the triple bond exhibited excellent photo-activated insecticidal
activity. Relationship analysis between structure and activity showed that the presence of
thiophene ring was critical for the overall activity [167].
3.8 Blood product disinfection by PDT

Transfusion-related infections and consequent fatalities continue to be reported in quite a
few publications [168,169], and blood is currently not tested for many potentially dangerous
known pathogens. In order to guarantee the safety of blood products from pathogenic
microbes, PDT is an alternative approach with the main advantage of being potentially
efficient against viruses, bacteria, fungi, and parasites. However, the main challenge in
blood disinfection is to kill or inactivate the target microorganism without disruption of

blood cells or serum proteins and moreover the PS should be without toxicity. MB is not an
ideal PS for this application due to its ineffectiveness against intracellular viruses and
tendency to cause collateral damage to blood components, typically to factor VIII and
fibrinogen [170]. Thionin, the demethylated derivative of MB, mediating PDT has been
shown to be efficient without damaging red blood cells and platelets [171].
Dimethylmethylene blue (DMMB), one type of phenothiazinium dye, illuminated by visible
light showed good effects on the inactivation of DNA and RNA model viruses, including
vesicular stomatitis virus and duck HBV (DHBV, a model for HBV; single-and double-
stranded DNA virus), and leukocytes in RBCs. However, clinical studies have not yet been
started with DMMB [172–174].
The naturally occurring vitamin B2, riboflavin (Fig 4G), illuminated by UVA or visible
light, has also been developed as a nucleic acid-binding agent to be used for pathogen
inactivation which results in photoinactivation of nucleic acid-containing pathogens in
plasma, platelets, and RBCs. Preclinical studies have revealed the effectiveness of reduction
in infectivity of some viruses, including HIV-1, bovine viral diarrhea virus (BVDV), a
model for HCV; single-stranded RNA virus), and pseudorabies virus, as well as bacteria in
blood products [175–178].
3.9 Aquaculture disinfection by PDT

Aquaculture comprises all types of culture of fish and other aquatic animals as well as
culture of plants in fresh, brackish and marine environments. These enterprises are
susceptible to water-borne disease caused by bacteria, viruses, helminths, protists and
oomycetes, to a lesser extent, fungi. Among them, bacterial infection is one of the major
concerns in the aquaculture industry [179]. Application of immobilized PS could mediate
PDT to inactivate fish pathogenic microorganisms and if successful would manifests
obvious advantage without low risk neither to the fishes nor to the environment. Only a few
studies have been explored in this field, but preliminary results were gained at both
laboratory level and at pilot stations. Porphyrin derivatives, as the PSs, have also a great
potential for the disinfection of fish farming plant water, including Gram-positive bacteria
Yin et al. Page 15
(e.g. MRSA), Gram-negative bacteria (e.g. E. coli), fungi (e.g. C. albicans) and fungal-like
pathogens (e.g. Saprolegnia spp.) and parasitic protozoa (e.g. Acanthamoeba palestinensis)
[179]. Microbial population was decreased 5–6 log10 CFU after 10 min of irradiation with a
low fluence rate (ca. 50 mW/cm2) in the presence of micromolar PS concentration [180].
Another study showed that a micromolar concentration of a porphyrinic PS eliminated the
naturally or artificially induced infection of saprolegniosis in trout farming pools
(inactivation of 6–7 log10) without collateral damage of the fish. The same low
concentrations demonstrated also higher phototoxicity activity over more traditional
pathogens MRSA and E. coli (up to 7 log10 decrease in bacterial CFU) [181]. The
bactericidal effects of PDT were also exhibited for V. vulnificus that frequently infects fish
farming water [182]. Similarly, cationic porphyrins-induced PDT inactivated up to 7 log10
of ten bacterial species (i.e. V. anguilarum, V. parahaemolyticus, Photobacterium damselae
subsp. piscicida, Photobacterium damselae subsp. damselae, E. coli, Enterobacter, A.
salmonicida, S. aureus, Pseudomonas sp, E. faecalis,) isolated from fish farming plant
waters in vitro [179].
3.10 Waste water disinfection by PDT

At present, antimicrobial PDT is considered to have potential for use in the environmental
area, including water disinfection. Effects were observed on the eradication of faecal
bacteria [183–186], viruses [187] and helminth eggs [188] in environmental water. Bonnett
et al. used a phthalocyanine immobilized on a polymeric membrane of chitosan in a model
reactor of water disinfection [189]. Two mono-hydroxyl zinc-porphyrin derivatives with

anionic and cationic net charge, respectively, were immobilized on poly(methyl
methacrylate). The cationic derivative immobilized on poly(methyl methacrylate) polymer
showed more effective inhibitory effect on the photo-inactivation of Deinococcus
radiodurans [179].
3.11 Food disinfection by PDT

Numerous outbreaks of foodborne illness have been attributed to the contamination of
produce due to inadequate sanitation of food contact surfaces [190]. Recent research has
indicated that pathogens can acquire resistance to commonly used sanitizers or disinfectants
and, as a result of such adaptation, cross-resistance of pathogens to antibiotics and
production of multi-antibiotic resistant pathogens have been observed, which is a risk to
animal health status and the safety of food products [191]. Brovko et al. investigated the
photodynamic bactericidal effect of acriflavine neutral, phloxine B, RB and malachite green
(oxalate salt) against two Gram-negative strains (E. coli LJH 128 and Salmonella
Typhimurium C1058), two Gram-positive strains (Bacillus sp. C578 and Listeria
monocytogenes LJH 375), and yeast (Saccharomyces cerevisiae C1172) [191]. The results
showed that acriflavine neutral-induced PDT significantly reduced the viabilities of all
pathogens tested. RB caused a significant reduction of bacterial viability incubated both in
the dark and under illumination. Malachite green induced PDT was gave more effective
inactivation of Gram-positive bacteria than Gram-negative bacteria and yeasts. Phloxine B
demonstrated a significant photoinactivation on Gram-positive bacteria, whether in the dark
or under illumination; but Gram-negative bacteria and yeasts were unaffected. Conjugation
of RB and phloxine B with poly(vinylamine) resulted in an enhanced bactericidal effect
during both dark and light incubation. No killing effect was observed for yeasts incubated
with dye conjugates. The presented data suggest that a photodynamic approach for
constructing “self-decontaminating” materials has potential in the food industry.
3.12 Effects of PDT on host cells and tissues

As mentioned above, PDT is an alternative approach to inactivate pathogenic
microorganisms. The ideal outcome of antimicrobial PDT is that the target pathogenic
Yin et al. Page 16
microbes are selectively inactivated while host cells and tissues are preserved. Therefore,
one of the criteria to evaluate the effectiveness of a PS is that it should demonstrate
selectivity of microbial cells over host tissue and cells [192]. A recent study indicates that
using tetracationic phthalocyanine (RLP068) as a PS leads to an about 5 log10 decrease in

the CFU of microbial pathogens with no collateral damage at the same doses to typical
human cells, such as fibroblasts and keratinocytes [36]. Soukos et al. found that poly-L-
lysine (pL)-chlorin e6 (ce6) conjugates could efficiently target photo-destruction towards
Gram-negative (P. gingivalis) and Gram-positive (Actinomyces viscosus) oral bacterial
species while it spared the oral epithelial cell line (HCPC-1) [193]. Tegos et al. investigated
the broad-spectrum antimicrobial photodynamic activities induced by two series of
functionalized C60; one series had one, two, or three polar diserinol groups (BF1–3), another
series had one, two, or three quarternary pyrrolidinium groups (BF4–6). The bis- and tris-
cationic fullerenes showed highly germicidal effects on all tested microbes (4–6 log10),
including Gram-positive bacteria, Gram-negative bacteria, and fungi, without any harm to
mammalian cells (fibroblasts) [192]. One in vivo research, carried out by Trindade et al.
evaluated the toxicity of Photogem® mediated PDT on rat palatal mucosa. Their data
demonstrated Photogem®-induced PDT was not toxic to the rat palatal mucosa detected by
macroscopic analysis and thermal mapping [194]. An in vitro study was carried out by Zeina
et al. to investigate the effect of PDT on keratinocytes [195]. They found a therapeutic/
dosing protocol whereby PDT inactivated microorganisms effectively without damaging
adjacent keratinocytes. The researchers used a combination of MB and visible light
previously used for microbial killing. The kill rates for keratinocytes were 200-fold lower
than for bacteria and 18-fold lower than for Candida. Using different PSs, Tanaka et al.
demonstrated the cytocidal effects of PDT on neutrophils [196]. They studied the
morphological alteration and the viability of murine peripheral-blood neutrophils after PDT
performed in vitro by using each PS at a concentration that achieved a maximum germicidal
effect on MRSA. Most neutrophils survived (>80%) after PDT using TBO or MB, however,
other PSs used for PDT (such as erythrosine B, RB, Photofrin, crystal violet, NMB and
Laserphyrin) caused morphological change and viabilities reduction (<70%) to neutrophils.
Shrestha et al. evaluated the potential of a PS-conjugated chitosan as a matrix-reinforcing
agent on dentin collagen [197]. Rose Bengal-conjugated chitosan (CSRB) was synthesized
by using a chemical cross-linking technique and it was characterized for its photobiological,
photophysical, and cytotoxicity properties. Dentin collagen was applied for the CSRB cross-
linking experiments and later on evaluated for mechanical properties, chemical changes, and
resistance to enzymatic degradation. The CSRB-cross-linked dentin collagen demonstrated
higher resistance to collagenase degradation and superior mechanical properties.
In conclusion, in vitro researches have shown that, under the same dosimetric parameters of
the photodynamic process, host cells are less sensitive to PDI than bacteria, suggesting a
safety margin between bacterial inactivation and host cell damage if PDT is used in vivo.
This selectivity might be due to the protection that the plasma and nuclear membrane afford
in mammalian cells that may act as an additional barrier for the PS or the PS high-energy
reaction products (ROS) formed after light. Differences in sensitivity may also reflect
differences in cell volume/size which increase the amount of ROS needed to kill the cell.
Mammalian cells are approximately 25–50 times larger than the bacterial test species and
may therefore they are likely harder to be killed [195]. The selectivity can also be explained
by the selective uptake of cationic PSs by bacteria compared to that by mammalian cells
[193].
3.13 Clinical applications of anti-infective PDT

Although PDT was discovered in the antimicrobial field over 100 years ago, the first clinical
trials in humans did not take place until 1970s. The clinical applications of anti-infective
Yin et al. Page 17
PDT have been also evaluated and performed in vivo. Numerous studies indicate that PDT
appears to be very efficient for the treatment of superficial and localized infections,
including dental disease, cutaneous superficial and subcutaneous infection, gastric
infections, and so on.
Qin et al. found an about 21% killing rate of microorganism in biofilms collected from
patients with periodontitis, using 1 mg/ml TBO irradiated by 12 J/cm2 red laser [198].
Dörtbudak et al. reported that TBO-PDT successfully decontaminated implants with
bacterial colonization of 15 patients, reducing about 2log10 bacterial CFU [199]. Soukos et
al. used MB (25 μg/ml) PDT, illuminated by 665 nm red light (30 J/cm2), to fully inactivate
all bacterial species except E. faecalis (53% killed) in infected root canals of extracted teeth
[200]. A 97% elimination of bacteria biofilm infected by E. faecalis in root canals were
achieved by PDT with the same concentration of MB and increased fluence (222 J/cm2) of
red light. Fontana et al. reported that MB -PDT eliminated 63% subgingival species in the
planktonic phase and 31% in the biofilm derived from patients’ dental plague with chronic
periodontitis [201]. Another study, presented by William et al. showed that TBO-PDT could
reduce 10-fold S. mutans which was present in a collagen matrix similar to mimicking
carious dentin or appearing in decayed teeth [202]. Mima et al. used Photogem-PDT to treat
five cases of denture stomatitis infected by Candida spp. The results showed four patients
achieved clinical resolution and one patient demonstrated reduction in palatal inflammation.
Two patients occurred recurrence of denture stomatitis during the follow-up period [203]. In
one randomized clinical trial, Photogem®-induced PDT significantly reduced the CFU/ml of
Candida species and showed clinical success rates 45% as effective as topical nystatin in the
treatment of denture stomatitis [204].
Lee et al. used MAL-mediated PDT to treat six patients with refractory Malassezia
folliculitis infected by Malassezia yeasts [205]. The results showed that a great improvement
was achieved in four patients following continuous three sessions of PDT and a moderate
improvement was achieved in one patient. Piracinini et al. treated a patient with recalcitrant
onychomycosis infected by T. rubrum who did not respond to the topical and systemic
antifungal agents’ treatment for 18 months. Following three sessions of ALA-PDT in a 15-
days interval, remission of the infection was released and without recurrence during a 24-
months’ follow-up period [206]. In another contrary study, thirty patients received ALA-
PDT and achieved a 43.3% cure rate 12 months after treatment, however, cure rate dropped
down to 36.6% after 18 months [207]. Nine patients with interdigital mycoses of the feet
received 20% ALA-PDT treatment and achieved good therapeutic effects in most patients,
however recurrences were seen quickly [208].
Another compelling application of PDT for superficial cutaneous infection is the treatment
of acne vulgaris. Hongcharu et al. first revealed the efficacy of topical ALA-PDT to treat
acne vulgaris on the back in a small but well administrated study in adults [209]. Another
study was carried out by Itoh et al. using topical 20% ALA irradiated by lower fluence red
light (13J/cm2) in Japanese patients. The acne lesions were improved and lasted at least 6
months [210,211]. Gold et al. evaluated the efficacy of ALA-PDT illuminated by intense
pulsed light (IPL) and other light sources on moderate to severe inflammatory acne vulgaris
in a clinical trial. Twenty patients were enrolled. Among them, fifteen completed the whole
trial and twelve patients responded to the treatment. Among respondents, 50.1% patients
reduced the active inflammatory acne lesions at the end of the four-week treatment, 68.5%
was improved four weeks after the last treatment, and 71.8% was improved twelve weeks
after the last treatment [212]. A large sample, randomized, split-face, placebo-controlled
study was carried out by Yin et al. in China in 2010 [213]. These authors recruited one
hundred and eighty patients and compared different topical ALA concentrations vs. placebo
for PDT in patients with moderate to severe acne. Every patient was treated once at intervals
Yin et al. Page 18
of 10-days for four sessions. After 24 weeks, the sides treated by ALA-PDT demonstrated
clinical improvement compared with the control sides treated only by red light (P < 0.01)
(Fig 5). The beneficial outcomes were dose-dependent; however, the authors pointed out
that the occurrence of side-effects also showed a dose-dependent increase.
There have been further studies reporting beneficial effects of PDT on various bacterial
infections, acne vulgaris, leishmaniasis, viral infections, gastric infections, etc. Table 2
summarizes the representative clinical applications of antimicrobial PDT. Although the
overall clinical outcomes in all the studies were considered to be positive, discrepancies
were found in the therapeutic efficacies in different trials. It was likely based on the use of
different light sources, different PSs, concentration and incubation time between PS and
infected tissue.
By and large, PDT has been investigated in the laboratory and the clinic, and was found to
be able to inactivate numerous pathogenic microorganisms efficiently. However, the
application of PDT as an anti-infective in the clinic is still at a relatively early stage and
faces some limitations that need to be overcome before it can assume its future application
as a therapeutic approach for deep infections. PDT will not replace antimicrobial
chemotherapy, but may improve the treatment of localized infections, promote healing and
reduce the cost of the treatment with low side-effects. It would be prudent to say that there is
not any convincing evidence to support s that PDT has superior advantages over the
traditional modalities at present. Furthermore, meta-analyses and randomized long term

clinical studies are necessary to confirm the beneficial effect of antimicrobial PDT
according to the tenets of evidence-based medicine. Further studies on the exploration of
new PSs and more efficient light delivery systems are required to establish optimal treatment
parameters. Antimicrobial PDT may hold promise as a substitute for currently available
antibiotic therapy in the treatment of recalcitrant, recurrent, and multi-drug resistant
bacterial and fungal infections especially in poorly vascularized tissues where antibiotics
have difficulty reaching.
4 Blue light for infectious disease

4.1 Introduction
Another novel light-based approach, the use of blue light (400–500nm) as a mono-therapy,
is gaining increasing attention owing to its potential antimicrobial effect without addition of
exogenous PS. Moreover, it is accepted that blue light is much less harmful to mammalian
cells than ultraviolet irradiation [214]. In addition to safety benefits, prolonged exposure of
materials to 405-nm visible light would not induce the problematic levels of
photodegradation that are associated with similar periods of material exposure to UV light,
particularly in the UV-C region of around 254 to 260 nm. The biological response of blue
light was firstly reported in 1881 by Darwin who reported phototropic response in plants to
blue light. Up to now, the phototropic response of blue light has been identified in all three
biological domains, including bacteria, eukaryotes, and archaea [215]. Recently, new
exciting discoveries have been made concerning the genomes of many photosynthetic and
chemotropic bacteria and fungi, which can encode photoreceptor proteins. For examples,
blue light receptors have been studied in Neurospora crassa and other fungi, which can be
divided into two general classes: the cryptochromes and the phototropins. The two major
blue light photoreceptors are, (i) white collar (WC)-1 and (WC)-2, which control dark to
light transition and (ii) VIVID, a protein important in the second light signaling system.
Together, they control responses to daily changes in light intensity and also are involved in
the modulation of the circadian clock [216,217]. Other photoreceptors have been studied in
E.coli [218] and A. baumannii [219] and are functional coded for production of a BLUF
protein (blue light sensing using flavin), encoded by the blue-light-sensing A (bslA) gene.
Yin et al. Page 19
The discoveries of those photoreceptors have triggered the new concept that not only
phototrophic bacteria but also photosynthetic and chemotrophic microorganisms can sense
light [220]. So far, it has been found that some bacteria and fungi can chose their lifestyle
between the motile single-cellular planktonic state and the multicellular surface-attached
community state (biofilms) by photo-regulated pathways, including the second messenger
cyclic di-GMP system, and direct interactions of photoreceptors with transcription factors
[221,222]. Blue light has been verified as a crucial factor that can affect all these
physiological responses and decide the bacterial lifestyle [223,224]. Blue light not only can
regulate bacterial motility, suppress biofilm formation, but also subsequently potentiate light
inactivation of bacteria. On the other hand, the presence of blue light may also activate or
increase bacterial virulence [219].
The lethality of blue light for bacteria has been demonstrated both in vitro and in vivo which
can produce a broad-spectrum bactericidal effect on both Gram-negative and Gram-positive
bacteria. While the wavelength range of 402–420 nm has been reported to be the most
effective antimicrobial spectral range, both 455 nm and 470 nm wavelengths have also been
found to be of antimicrobial potential for some bacterial species (e.g., S. aureus) [219]. The
precise mechanism of the antimicrobial effect of blue light is not fully elaborated yet. The
generally accepted hypothesis is that blue light excites endogenous intracellular porphyrins
to behave as PSs, and this photon absorption sequentially leads to energy transfer and
ultimately, the production of highly cytotoxic ROS – most notably 1O2 in a similar manner
to PDT [225]. However, different bacteria demonstrate variable susceptibilities to blue light.
Studies have reported that Gram-positive species, in general, were more sensitive to 405 nm
light inactivation than Gram-negative species, which is generally consistent with the results
obtained in a recent study [226,227]. It has also been theorized that the differences in
inactivation kinetics may be due to organism-specific differences in porphyrin levels,
individual porphyrin sub-types (i.e., porphyrin wavelength absorption maxima), or as a
consequence of the relatively short distance that 1O2 molecules can diffuse (~20 nm in
solution) within cell structures [33,226]. In addition, it has been speculated that less oxygen-
tolerant bacterial species may be particularly susceptible to the effects of ROS as
microorganisms, such as some microaerophilic species, have been found to possess fewer
key antioxidant defenses than most aerobes [228,229]. For example, studies have
demonstrated that blue light is able to inactivate the anaerobic oral pathogens Prevotella,
Porphyromonas, and Fusobacterium as well as microaerophilic pathogens such as P. acnes
and H. pylori [33,230]. However, inactivation results achieved with anaerobic/
microaerophilic bacteria have not provided conclusive evidence as to whether oxygen-
sensitive anaerobic bacteria are any more susceptible to blue light than aerobes as many of
these bacteria are also known to accumulate high levels of porphyrins. Possible explanations
for this difference in inactivation susceptibility could be that different characteristics of
bacteria, the unusual cell envelope, confers some resistance to 405 nm light penetration and/
or that bacterial cell envelopes provide greater innate resistance to ROS given that bacteria
species are capable of evading phagocytic oxidative burst damage [227].
4.2 Blue light inactivation of bacteria and fungi

Blue light is lethal to many species of bacteria and fungi, even including filamentous fungi,
although the effectiveness is dependent on the light wavelength, energy levels and microbial
genus and species [216]. Blue non-coherent light sources, such as LED, the halogen lamp,
and the plasma-arc curing (PAC) light, are often used in vivo and in vitro studies. The
bactericidal efficacy in vitro of blue light for Escherichia coli, MRSA and P. aeruginosa,
even Streptococcus mutans in biofilms has been reported [231].
Maclean et al. studied the bactericidal effect of 405-nm blue light emitted from a LED array
on some bacterial species, including Gram-positive S. epidermidis, S. aureus, Enterococcus
Yin et al. Page 20
faecalis, Streptococcus pyogenes, Clostridium perfringens, Gram-negative E. coli,

Acinetobacter baumannii, P. aeruginosa, Proteus vulgaris and K. pneumonia which are all
associated with hospital acquired infection [226]. The results demonstrated that most of
Gram-positive and Gram-negative species were successfully eliminated, except E. faecalis

which was the least sensitive to inactivation by 405-nm blue light.
Murdoch et al. reported that bactericidal effect of high-intensity 405nm light, delivered by
an array of LED, on taxonomically diverse bacterial pathogens from the genera Salmonella,
Shigella, Escherichia, Listeria, and Mycobacterium [227]. L. monocytogenes was most
readily inactivated in suspension, whereas S. enterica was most resistant. In surface
exposure tests, L. monocytogenes was more susceptible than Gram-negative enteric bacteria
to 405nm light when exposed on an agar surface but interestingly less susceptible than S.
enterica after drying onto PVC and acrylic surfaces. Mycobacterium terrae, which was
chosen as a safe, comparative, surrogate microorganism for the highly pathogenic M.
tuberculosis, was inactivated by 4–5 log10 CFU/ml between a dose of 144 and 288 J/cm2 by
405nm light.
Blue light photoinactivation—as evaluated for other bacteria such as Helicobacter pylori,
Propionibacterium acnes, Porphyromonas gingivalis and some black-pigmented bacteria
[225,232–234] —has been attributed to the photo-excitation of endogenous intracellular
porphyrins by blue light in the wavelength region from 400 nm to 500 nm and, more
particularly, 400 nm to 420 nm in the cases of H. pylori and P. acnes [225,232]. The studies
on the in vitro antimicrobial effect of blue light are summarized in Table 3
However, the types and the amount of porphyrins can lead to different photo-inactivation
rates in different bacteria. For example, Gram-positive S. epidermidis and S. aureus
produced coproporphyrin as the predominant porphyrin species, and these species produce
2–3 times higher coproporphyrin than in Gram-negative strains (i.e. E. coli, Aeromonas
strains and Acinetobacter) whereas there was no primary porphyrin produced in some Gram-
negative bacteria [235]. Owing to fact that different bacteria produce different predominant
porphyrins and their peak absorption wavelengths of these porphyrins may be at variance.
As a consequence, different wavelengths may be required for their optimal photo-
stimulation. All the aforementioned factors clearly induced the diversity of bactericidal
efficacy by blue light. Other factor that must be taken into account are that the bacteria may
produce different levels of porphyrins when cultured in laboratory cultivation conditions
than they do in their natural physiological states, such as when they are infecting a
mammalian host [236].
Fungi are able to sense light in a broad spectrum range, from UV to far-red light because
they have blue-light receptors. Flavins or flavoproteins are considered to act as blue light
receptors and can absorb blue light [237]. This observation makes them able to serve as a
cue’ in the asexual development of fungal spores, and affecting growth, metabolism,
pigment formation, and tropism [238,239]. This observation makes them able to serve as a
cue’ in the asexual development of fungal spores, and affecting growth, metabolism,
pigment formation, and tropism [237], or could photoexcite endogenous porphyrins in a
similar manner to PDT [240]. However, there have been few reports demonstrating the
fungicidal efficacy of blue light. Only recently, De Lucca et al. found that blue light
(470nm) alone significantly reduced Fusarium graminearum (FG) germinating conidial
viability by approximately 36, 42, and 47% respectively at fluences of 40, 80 and 100 J/cm2
[216]. However, no significant viability loss was observed for the FG non-germinated
conidia treated with blue light (20–100 J/cm2).
Yin et al. Page 21
4.3 Effects of blue light on host cells and tissues

In order to use blue light for the treatment of infectious diseases, it is very important to
assess the possible harm that the blue light could cause against host cells and tissues so that
they can avoid non-specific damage. However, only a few studies have been conducted so
far in this area.
In one in vitro study, Hockberguer et al. showed that some cells, such as mouse 3T3
fibroblasts, foreskin keratinocytes from human, kidney epithelial cells from monkey were
irradiated with blue light (450nm at 6.3 W/cm2) for 20 min respectively, produced hydrogen
peroxide (H2O2), an important ROS which can lead cellular damage and could cause
pathological changes associated with exposure to blue light [241].
Wataha et al. treated 3T3 mouse fibroblasts with three dental blue (400nm–500nm) light
sources, and detected succinate dehydrogenase (SDH) activity of mitochondria after a range
of fluences (1.3 to 60 J/cm2) [242]. Results showed that fluences ranging from 5 J/cm2 to 15
J/cm2 irreversibly completely suppressed SDH activity compared to dark controls up to 72 h
post-exposure.
Godley et al. investigated the interaction of human primary retinal epithelial (PRE) cells
with blue light (390–550 nm at 2.8 mW/cm2) and reported significant damage to the
mitochondrial DNA after 3 h of light exposure. At the same time, release of ROS (1O2,
O2−•, HO•) increased in isolated mitochondria after irradiation. Hence, it has been
confirmed that blue light can damage cells via ROS release and subsequent DNA damage
[243].
Shnitkind et al. studied the effects of blue light illumination on two immortalized
keratinocyte cell lines, HaCaT and hTERT1. The cells were irradiated by 420nm blue light
at fluences of 54 or 134 mJ/cm2. The results showed that blue light inhibited IL-1α release,
which proved that blue light has anti-inflammatory potential on keratinocytes [244].
Conversely, a recent clinical research performed by Kleinpenning et al. did not demonstrate
significant side effects on human skin [214]. Eight healthy volunteers were illuminated with
a blue light source (390–460 nm) with a peak emission of 420 nm over 5 consecutive days at
20 J/cm2. To analyze the irradiation effects, skin biopsies were analyzed for p53 expression,
vacuolization, sunburn cells, elastosis, matrix metalloproteinase-1 (MMP-1), and melan-A
expression. As a result, neither inflammatory nor sunburn cells were observed before or after
irradiation. No significant changes in p53 and MMP-1 expressions or sign of elastosis were
observed after blue light exposure. Keratinocytes demonstrated a significant incremental
perinuclear vacuolization during treatment (P=0.02) with a tendency towards significance
after cessation of treatment (P=0.09). Irradiated skin demonstrated minor hyperpigmentation

and a significant increase in melan-A-positive cells (P=0.03) histologically. Therefore, the
investigators concluded that short-term use of blue light in cutaneous treatment could be
considered safe.
In conclusion, blue light has been occasionally reported to be phototoxic to mammalian cells
at certain spectrum ranges in a wavelength dependent manner. The photocytotoxic effect
was assumed to be similar to that which occurred in bacteria, such as photo-excitation of
intracellular chromophores and subsequent cytotoxic ROS formation. Thus, an ideal blue
light therapy should choose an optimal wavelength to selectively excite the chromophores in
pathogenic bacteria with minimized effect on chromophores in mammalian cells [219].
Yin et al. Page 22
4.4 The clinical application of blue light for infectious diseases

One of the major application of blue light for infectious disease in the clinic is the treatment
on acne vulgaris which is an important dermatologic disorder infected mainly by P. acnes.
Several studies have reported the benefits of blue light on acne vulgaris. Table 4 summarizes
the clinical studies of blue light therapy for acne vulgaris.
Blue light also can inactivate H. pylori, which is the primary pathogen causing atrophic
gastritis and peptic ulcers and is increasingly resistant to antibiotics [245]. Ganz et al. treated
10 patients with blue light (405 nm, 40.5J/cm2) via an optical fiber inserted in the endoscope
channel that illuminated a 1-cm diameter area of the gastric mucosa [246]. Significant
decreases (91%) in H. pylori colonies per gram of tissue in treated sites was observed by
comparing biopsies from treated and adjacent untreated sites. Some patients showed
reductions of bacterial burden approaching 99%. Another pilot trial was carried out by
Lembo et al. using a blue (408 nm) laser source with multi-segmented balloon that allowed
whole stomach illumination to treat 18 patients with H. pylori infection [247]. A generalized
reduction in bacterial load was observed in the stomach. Significant reduction in bacterial
colonization was found in the antrum (>97%), followed by the body (>95%) and the fundus
(>86%) with no apparent dose-response correlation. However, repeated breath tests showed
H. pylori repopulated in days following illumination.
In conclusion, blue light can treat inflammatory and non-inflammatory acne lesions caused
by P. acnes and digestive disorders caused by H. pylori. Blue light therapy has been
demonstrated in clinical studies to be feasible and safe, and may represent a novel
alternative approach to the eradication of P. acnes and H. pylori, especially in patients who
have failed standard antibiotic treatment. However further studies are necessary to establish
appropriate protocols for each clinical condition.
4.5 Summary
In summary, blue light, at certain wavelengths (range 402–420 nm), has been found to be
effective in the inactivation of pathogens in both laboratory and clinic. The overall outcomes
were demonstrated to be positive in all the studies. This is a new compelling approach that
can eliminate a wide range of medically important pathogenic bacteria, including MRSA, P.
aeruginosa, A. baumannii and E. faecalis. The absence of a requirement for pretreatment
with a PS increases its likelihood for widespread applications, although discrepancies have
been seen in the antibacterial efficacy in different studies. It was likely that the differences
were owing to the different light delivery regimen used in the studies, differences in the
spectra of light sources, differences in light fluences, variable microbial genera, different
patient populations, etc. In common, the clinical application of blue light for infectious
diseases is still at the early stage of development. The sample sizes of all published clinical
studies are still very limited and the target bacteria are mainly P. acnes and H. pylori.
Further studies with larger sample sizes are warranted to optimize the parameters of blue
light therapy, and to investigate the use of blue light for infections caused by a wider range
of pathogens. Further development of blue light inactivation approaches could lead to
potential applications as decontamination systems for air, contact surfaces, and medical
instruments within the clinical environment [248,249]. Light with wavelengths centered at
405-nm has shown a significantly broad antibacterial spectrum which offer potential
application in numerous fields including food sterilization, prevention of contamination,
sewage disposal and other disinfection industries [236].
Yin et al. Page 23
5. Other light-based anti-infectives

5.1. Anti-infective effect of femtosecond pulsed lasers
It has been proposed that femtosecond lasers, or lasers that have a pulse duration in the
region 10−15s, are able to break down transparent or semitransparent biological tissues due
to nonlinear absorption of laser energy with minimal thermal and mechanical effects [250].
As a result of the adverse thermal damage possible with other laser systems, femtosecond
lasers have been hypothesized to be a new approach as anti-infectives. The basic idea is that
the entire energy of a pulse (which could be quite small) is delivered in a very short time, so
if the average power is 10 mW and the pulse duration is 100 fs then the peak power is 100
GW which is sufficient to produce efficient 2-photon absorption. Recently, a series of
studies have demonstrated the efficacy of a visible femtosecond laser or a near-infrared
subpicosecond fiber laser on elimination of several kinds of viral species, including M13
bacteriophage, tobacco mosaic virus, HPV, and human immunodeficiency virus (HIV)
[251–258]
In 2007, Tsen et al. found that a very low power visible femtosecond laser (as low as 0.5 nJ/
pulse, 425 nm wavelength, 100 fs pulse width, peak power density greater than or equal to
50 MW/cm2) irradiated M13 phages efficiently [254]. Their further research demonstrated
that one kind of visible femtosecond diode-pumped continuous-wave (CW) mode-locked Ti-
sapphire laser (λ = 425 nm) could inactivate M13 bacteriophage, encephalomyocarditis virus
and Salmonella typhimurium. The laser emitted a continuous array of 60 fs pulses at a

repetition rate of 80 MHz, average power of about 50 mW. It has a pulse width of full-width
at half maximum (FWHM) 100fs. The laser inactivated nearly the entire aforementioned
microorganism very efficiently, especial for Salmonella typhimurium [258]. Another recent
study reported the inactivation of murine cytomegalovirus (MCMV), an envelope, double-
stranded DNA virus, illuminated by visible femtosecond laser ( λ = 425 nm) which was
reduced 5 log 10 titer and the virus demonstrated selective aggregation of viral capsid and
tegument proteins [259]. However, the global structure of MCMV virions including
membrane and capsid, was not changed significantly evaluated by electron microscopy;
meanwhile the double-chain of MCMV genomic DNA did not show any break or
crosslinking.
Manevitch and colleagues showed that the application of the femtosecond laser at a fluence
of 7×1031 photons m−2 s−1 effectively and selectively inactivated T. rubrum from nail
clippings obtained from infected toenails [260]. The laser wavelength used was 800nm
which wavelength could effectively traverse the nail to eliminate dermatophytes at any level.
Most importantly, it killed fungal cells without any collateral damage to the nail structure as
confirmed by canning electron microscopy. This is the only study assessing the fungicidal
efficacy of this laser system for onychomycosis in vitro at present. In fact, it is difficult to
selectively delivery laser energy to microorganisms embedded in the nail plate without
causing collateral damage to the nail structure overlying the fungi. Femtosecond infrared
titanium sapphire lasers can theoretically circumvent this problem owing to the nonlinear
interactions of femtosecond lasers with biological media and the ability to selectively deliver
energy at the laser focus [261]. This study further demonstrated femtosecond infrared laser
was a new alternative approach for the infection of fungus.
It has been proposed that use of a near-infrared (NIR) femtosecond laser via impulsive
stimulated raman scattering (ISRS) to produce damage (e.g. to the protein coat of a virus) is
a method for selectively inactivating microorganisms [254]. There were different
mechanisms of inactivation of different microorganism by femtosecond laser. Inactivation of
viruses involving the breaking of hydrogen/hydrophobic bonds or the separation of the weak
protein links in the protein shell of a viral particle. On the contrary, inactivation of bacteria
Yin et al. Page 24
is related to the damage of their DNA due to 2-photon absorption of a visible femtosecond
laser [258].
When a NIR femtosecond laser induced the inaction of virus and bacteria, its safety to the
mammalian cells was considered. The relative research demonstrated that if the wavelength
and pulse width of the femtosecond laser were appropriately selected, there was a window in
power density that enabled them to achieve selective inactivation of target viruses and
bacteria without causing cytotoxicity to mammalian cells. It was suggested that this strategy
targeted the mechanical (vibrational) properties of micro-organisms, and thus its
antimicrobial efficacy was likely to be affected by genetic differences in the micro-
organisms [255].
Advantages of such a new laser technology over the presently prevailed disinfection
methods have been proposed to be: 1. it is a noninvasive disinfection technology, because no
extraneous materials are needed in the disinfection process; 2.it is a harmless environmental
disinfection method since no chemicals are used in the pathogen inactivation process; 3. it is
a general method for selective disinfection of pathogens with potential minimal side effects
[258].
5.2. Anti-infective effect of dual wavelength (870/930 nm) laser

Recently by using the wavelengths that had previously demonstrated cellular light mediated
damage effects in optical traps, Bornstein et al. developed a dual wavelength laser system
(with the wavelengths of 870 nm and 930 nm) for the inactivation of bacteria and fungi
[262]. During in vitro studies, by using 1.5cm diameter flat-top light spots (fluence rate of
5.66 W/cm2, at physiological temperatures), the investigators successfully inactivated S.
aureus, E. coli, C. albicans and T. rubrum. Using non-lethal light fluence, the authors
observed a decrease in transmembrane potentials and an increase in ROS generation in
pathogenic microorganisms (MRSA and C. albicans) and human embryonic kidney cells. It
was also postulated that the dual wavelengths caused an optically mediated —
mechanotransduction of cellular redox pathways, decreased transmembrane potentials, and
increased ROS generation. The cellular energetics of prokaryotic, fungal pathogens, as well
as host cells, was affected in a similar manner when treated with the dual wavelengths at the
fluence used. After thermal tolerance experiments in live porcine skin, human pilot studies
were performed, examining the photo-inactivation of MRSA in the nose, and of fungi
present in onychomycosis. The investigators did not observe non-specific damages either to
the nares or the nail matrix, yet found successful photo-inactivation of pathogens.
Krespi et al. performed a prospective clinical study using a dual wavelength laser (870/930
nm) for nasal decolonization of S. aureus and MRSA [263]. Twenty-five patients colonized
with S. aureus or MRSA were enrolled and non-randomly divided into 4 treatment groups:
dual-wavelength laser with low-power alone (GR1); dual wavelength laser with low-power
in combination with erythromycin (E-mycin) cream (GR2); dual wavelength laser with low-
power in combination with peroxide irrigation (GR3); and single wavelength 940-nm laser
with high-power alone (GR4). Laser illumination was delivered by using a fiber diffuser,
with the fluences of 200 to 600 J/cm2 for each naris circumferentially. Nasal decolonization
efficacy for the groups of GR1, GR2, GR3, and GR4 was respectively 1 out of 3, 13 out of
14, 2 out of 4, and 4 out of 4. No side effects or discomfort were observed. The authors
concluded that dual wavelength laser therapy could eradicate S. aureus and MRSA. It also
could potentially re-sensitize bacteria which was drug-resistant to the common antibiotic,
erythromycin.
A possible mechanism of the resensitization of bacteria to antibiotics using dual wavelength

laser was that, the laser illumination interacted with the respiratory processes of the bacterial
Yin et al. Page 25
plasma membranes, lowered the membrane potential, and subsequently inactivated the
bacterial resistance mechanism, which depended on the membrane drug efflux pump [263].
6. Can pathogenic microorganisms develop resistance to light based anti-

infectives?
Before light-based antimicrobials become widely used, one question that must be addressed
is: can pathogenic microorganisms develop resistance to light-based anti-infectives? If this is
indeed possible, the mechanism of resistance development is likely to be different for each
light-based modality.
6.1 Can pathogenic microorganisms develop resistance to PDT?

An early study investigating the development of resistance by bacteria after PDI was carried
out by Lauro et al. [264]. Peptostreptococcus micros and Actinobacillus
actinomycetemcomitans were subject to 10 cycles of repeated sub-lethal PDI and regrowth
using visible light combined with the PS consisting of porphycene-polylysine conjugates
(2,7,12,17-tetrakis(2-methoxyethyl)-9-glutaramidoporphycene and 2,7,12,17-tetrakis(2-
methoxyethyl)-9-p-carboxybenzyloxyporphycene). The investigators found that the repeated
PDI of P. micros and A. actinomycetemcomitans by both PSs did not induce the
development of resistance by bacteria to PDI in partially photo-inactivated bacteria.
Taveres et al. assessed the development of bacterial resistance to PDI mediated by white
light and Tri-Py+-Me-PF (5,10,15-tris(1-Methylpyridinium-4-yl)-20-(pentafluorophenyl)-
porphyrin triiodide) [265] Vibrio fischeri and recombinant E. coli were the studied bacteria.
The results showed that after 10 consecutive cycles of sub-lethal PDI followed by regrowth,
E. coli did not develop appreciable resistance to PDI. In another study, Giuliani et al.
investigated the potential of P. aeruginosa, S. aureus and C. albicans strains to develop
resistance to PDI after beingexposed to RLP068/Cl, a tetracationic Zn(II) phthalocyanine
derivative [266]. The authors reported that 20 consecutive cycles of sub-lethal PDI with
RLP068/Cl did not generate any resistant mutants.
6.2 Can pathogenic microorganisms develop resistance to UVC irradiation?

Dai et al. investigated the potential development of resistance to UVC (254 nm) inactivation
by T. rubrum by performing five consecutive cycles of sub-lethal UVC inactivation
followed by fungal regrowth in vitro [9]. The investigators did not observe significant
difference in fungal inactivation rate among the five cycles of fungal inactivation and
regrowth when 120 mJ/cm2 UVC energy was delivered. The investigators concluded that the
development of resistance to UVC inactivation is not rapidly acquired by T. rubrum that are
subject to repeated sub-lethal UVC inactivation.
Alcantara-Diaz et al. evaluated the divergent adaptation of E. coli to repeated cycles of sub-
lethal UVC inactivation at 254 nm [267]. Five cultures of wild type E. coli PQ30 were
exposed to 80 consecutive bacterial inactivation by UVC and regrowth. The initial fluence
of UVC was 1mJ/cm2 for each cycle, and the fluence was increased 2-fold every 10
inactivation-regrowth cycles. Quantitative dose response curves were obtained by exposing
the bacteria in exponentially growing phase to several UVC fluences. The investigators
reported that all cultures were observed to develop different degrees resistance to UVC
inactivation after 80 consecutive cycles of sub-lethal bacterial inactivation and regrowth.
The adaptation of bacteria to cyclic UVC inactivation was believed to be a consequence of
selecting advantageous mutations in those genes related to DNA repair and replication.
Yin et al. Page 26
6.3 Can pathogenic microorganisms develop resistance to blue light?

To the best of our knowledge, this question has not yet been experimentally addressed. As
the mechanism of blue light inactivation of microorganisms is suggested to be similar to that
of PDI [219,225,268], it is expected that the potential of resistance development by

pathogenic microorganisms to blue light inactivation is less than that of antibiotics. In a
recent study carried out in our lab (data not published), we found that, after 10 consecutive
cycles of sub-lethal bacterial (A. baumannii) inactivation by blue light at 415 nm and
bacterial regrowth, there was a significant decrease (rather than an increase) in bacterial
resistance to blue light inactivation between the first cycle and the 10th cycle (P <0.05).
However, a contradictory finding was reported by Guffey et al. [261]. In that study, the
investigators found that the inactivation of S. aureus by blue light (405 nm) was increasingly
effective through the first 4 cycles of sub-lethal inactivation-regrowth (P<0.05). From the 5th
cycle, the increase of bacterial resistance to blue light inactivation was observed (P<0.05).
6.4 Summary
There have been no reported cases of PDT induced resistance of microorganisms despite
numerous attempts to induce resistance by repeated cycles of sub-lethal PDI and microbial
re-growth. It is believed that PDT acts at multiple sites within microorganism cells [269],
and, subsequently, offers less potential for the development of resistance by microorganisms
to PDI than antibiotics, which usually act on a single-target. Development of resistance by
microorganisms to UVC inactivation is possible after excessive repetition of UVC

inactivation. This is because one primary function of UVC inactivation is to damage
microbial DNA. However, for treatment of wound or other localized infections using UVC,
only a limited number of repetitions of therapy are expected to be required. Variable results
have been reported regarding the development of resistance by microorganisms to blue light
inactivation. Further studies are warranted to elucidate the intrinsic mechanism of blue light
inactivation of microorganisms.
7. Conclusion
Light based anti-infective therapies have become a rapidly growing area of interest in recent
years. The reasons for this rise in interest are manifold; multi antibiotic resistance is the
predominant growth-driving factor, but other relevant considerations may be the public
distrust of big-pharmaceutical companies and their products, the desire to use —natural
therapies and blue light is considered somewhat similar to sunlight; and the rapid microbial
killing typically seen in phototherapy; so when the light is switched off the killing is done.
Since many of the applications of phototherapy can in fact be performed by midday sunlight
that has been suitable concentrated and/or filtered by cheap disposable devices,
antimicrobial phototherapy may in future be transitioned into a therapy suitable for the
developing world, where it is accepted that infectious diseases represent a much bigger
burden to public health than they do in developed nations. As always in anti-infective
therapies, selectivity towards microbial cells compared to host mammalian cells is also
critical. Fortunately this appears to be the rule rather than exception for the phototherapies
discussed above.
Acknowledgments
Research in the Hamblin laboratory is supported by US NIH grant R01AI050875. Rui Yin was supported in part by
National Natural Science Foundation of China (No.81172495) in this work. Tianhong Dai was supported by an
Airlift Research Foundation Extremity Trauma research grant (No. 109421). Ana Jorge and Wanessa de Melo were
supported by CAPES (Coordination for the Improvement of Higher Level Personnel) from Brazil. Asheesh Gupta
was supported by BOYSCAST Fellowship 2010–11, Department of Science and Technology, Government of India.
Yin et al. Page 27
Abbreviations
ALA 5-aminolevulinic acid methyl ester

BCG M. bovis Bacille Calmette Guerin

bFGF basic fibroblast growth factor
BVDV bovine viral diarrhea virus
CFU colony forming units
ClAlPC chloroaluminum-phthalocyanine
CPD cyclobutane pyrimidine dimer
CSRB chitosan-conjugated rose bengal
DMMB dimethylmethylene blue
DP-mme deuteroporphyrin mono-methylester
EDTA ethyelenediamine tetraacetic acid
ER erythrosine
GaPc cationic gallium phthalocyanine
HA(B) hypocrellin A(B)

Hp hematoporphyrin
HPV human papillomavirus
LED light-emitting diode
MAL 5-aminolevulinic acid
MB methylene blue
MDR multi-drug resistant
MRSA methicillin resistant Staphylococcus aureus
MSSA methicillin sensitive Staphylococcus aureus
PDI photodynamic inactivation
PDR pan-drug resistant
PDT photodynamic therapy
PEI-ce6 polyethylenimine chlorin(e6) conjugate
PS photosensitizer
RB rose Bengal
RBC red blood cell
ROS reactive oxygen species
TB Mycobacterium tuberculosis
TBO toluidine blue O
THPTS meso-tetrahydroporphyrin-tetratosylate
TMP tetra-substituted N-methyl-pyridylporphine
Tri-P(4) 5-phenyl-10,15,20-tris(N-methyl-4pyridyl)-porphine trichloride
Yin et al. Page 28
UVC ultraviolet c
VRE vancomycin resistant Enterococcus

VSV vesicular stomatitis virus
WGA wheat germ agglutinin
XDR extensively-drug resistant
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Highlights (for review)

Light-based anti-infectives work against biofilms and localized infections.
Ultraviolet C light will kill all pathogens without killing host mammalian cells.
Photodynamic inactivation produces reactive oxygen species that kill pathogens.
Blue light can kill many pathogens due to accumulation of porphyrins.
Femtosecond infrared laser pulses can selectively kill pathogens.
Yin et al. Page 46
Figure 1.
Spectrum of ultraviolet irradiation.
Yin et al. Page 47
Figure 2.
Schematic illustration of photodynamic action. The PS initially absorbs a photon that excites
it to the first excited singlet state and this can relax to the more long-lived triplet state. The
triplet PS can interact with molecular oxygen in two pathways, Type I and Type II, leading
to the formation of reactive oxygen species (ROS) and singlet oxygen 1O2, respectively. The
formation of ROS and 1O2 can chemically attack a very wide range of biomolecules.
Yin et al. Page 48
Figure 3.
Schematic illustration of cell wall structures of microbial pathogens. A: Gram-negative
bacteria. B: Gram-positive bacteria. C: Fungal cells.
Yin et al. Page 49
Figure 4.
Chemical structures of PSs described in this review. (A) Cationic phthalocyanine, Zn-PC-
Me; (B) Hydroxygallium(III) 2,3,9,10,16,17,23,24-octakis-[3-(N-methyl) -pyridyloxy]-
phthalocyanine octaiodide; (C) Toluidine blue O; (D) 3,7-Bis(N,N-dibutylamino)
phenothiazinium bromide, PPA904; (E) 5-Phenyl-10,15,20-tris(N-methyl-4-pyridyl)-
porphine trichloride, Sylsens B; (F) Rose Bengal; (G) Riboflavin; (H) Curcumin; (I)
Polyethylenimine chlorin(e6), PEI-ce6; (J) Merocyanine 540; (K) Poly-S-lysine porphyrin
conjugate, pL-TMPP; (L) Functionalized fullerene, C60(>ME1N6+C3); (M) Tri-meso (N-
methyl-pyridyl), meso (N-tetradecyl-pyridyl) porphine, C14.
Yin et al. Page 50
Figure 5.
Improvements of severe inflammatory acne treated by 15% ALA-PDT (a c) and control
(only red light alone) (d f). (a, d) before treatment; (b, e) 2 weeks after four consecutive
treatments; (c, f) at follow-up visit after 24 weeks [213].
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Table 1
Studies of UVC irradiation for inactivation of bacteria/fungi in vitro
Yin et al.
Light Source Radiant Exposure Bacterial/Fungi species/strains Inactivation efficacy Reference
254nm UVC 15.54 mW/cm2 MRSA, VRE antibiotic-susceptible strains of S. aureus and E. Illuminated 5 seconds, 99.9% MRSA and VRE inactivation; illuminated 9 5
faecalis seconds, 100% MRSA inactivation; illuminated 45 seconds, 100% VRE
inactivation
254nm UVC 5mW/cm2 MRSA, Streptococcus pyogenes Illuminated 5 seconds, methicillin-resistant, coagulase-negative 6
Staphylococcus and Streptococcus pyogenes inactivation; Illuminated 15
seconds, methicillin-susceptible S. aureus and Enterococci species

inactivation
265nm UVC 1.93 mJ/cm2 S. aureus, E. coli, Pseudomonas aeruginosa, S. pyogenes Illuminated 1 seconds, 100% inhibition for all strains 7
254nm UVC 1500mJ/cm2 catheter biofilms of E. coli, coagulase-negative Staphylococcus, Mean killing rates of the bacteria in catheter biofilms were 89.6% (11.8 mJ/ 8
E. faecalis, Streptococcus, P. aeruginosa, Coryneforms cm2), 98% (47 mJ/cm2) and 99% (1500 mJ/cm2)
254nm UVC 120mJ/cm2 Trichophyton rubrum, T. mentagrophytes, Epidermophyton 3–5 log10 of fungal inactivation 9
floccosum, Microsporum canis.
UVC 15.54 mW/cm2 bacteria (P. aeruginosa and Mycobacterium abscessus) and fungi Illuminated 3–5 seconds, 99% bacteria inactivation; Illuminated 15–30 10
(Candida albicans, Aspergillus fumigatus) seconds, 99% fungi inactivation
Page 51
Table 2
Summary of representative clinical applications of antimicrobial PDT
Yin et al.
Disease Causative microorganism Site of infection Photosensitizer No. of patients Treatment outcome Ref.
Leg ulcer, diabetic foot Up to 2-log reduction in bacterial load was
Gram (+)/Gram (−) bacteria PPA904 10 270
lesions achieved after a single PDT.
Leg ulcer, diabetic foot Bacterial counts were significantly lower after
MRSA PPA904 32 270,271
lesions PDT. No side effects were experienced.
Localized bacterial infection
PDT was used once a week for 12 weeks.

Interim data on 24 patients showed that 4 of 12
Leg ulcer, diabetic foot
Gram (+)/Gram (−) bacteria PPA904 48 ulcers in the PDT group closed completely, 270,271
lesions
compared with 0 of 12 ulcers in the placebo
group.
11 patients (32 Completely healing was seen after one or two

L. major Skin ALA 140
lesions) PDT sessions.
Leishmaniasis
20 patients (31 3 months after PDT, 29 lesions were
L. major Skin ALA 149
lesions) completely healed.
Numbers of both inflammatory and non-

inflammatory acne lesions were significantly
Acne vulgaris Skin/sebaceous gland Indole-3-acetic acid (IAA) 25 272
decreased. No adverse effects were observed
except for transient pruritus in one patient.
Acne 22% of patients showed excellent improvement

after one PDT session and another 34% showed
excellent improvement after two PDT sessions.
Acne vulgaris Skin/sebaceous gland ALA 78 273
The rest (44%) required three PDT sessions to
further reduce the number and size of residual
lesions. Adverse effects were minimal.
No significant acute side effects were noted and

Herpes simplex virus Skin MB 4 274
the lesions healed rapidly.
The overall complete remission rate of 1–4

Viral infection sessions of PDT was 98.2% and the
corresponding HPV clearance rate was 83.9%.
Papillomavirus Cervical condylomata ALA 56 275
Ten cases showed complete removal of cervical
lesions and HPV infection after a single PDT.
Adverse effects were minimal.
A maximum eradication effect was achieved at

4 h post-irradiation when 85% of biopsies in
Gastric infection H. pylori Stomach ALA 13 the monochromatic and 66% in the white light 276
exposed zones, and 58 and 33% in the
respective control zones were HP-negative.
PDT was effective in reducing numbers of A.

Dental pockets and
Dental infection A. actinomycetemcomitans Phenothiazine chloride 10 actinomycetemcomitans than scaling and root 277
gingival tissue
planning.
Page 52
Disease Causative microorganism Site of infection Photosensitizer No. of patients Treatment outcome Ref.
After one year, 13 patients (43.3%) were cured.
Yin et al.
The remaining patients had changes compatible
Onychomycosis T. rubrum Tonail ALA 30 207
with dermatophyte infection covering more
than 10% of the nail plate.
Page 53
Table 3
Studies on the in vitro antimicrobial effect of blue light
Yin et al.
Light Source Radiant exposure Bacterial species/strains Inactivation efficacy Ref
405-nm diode laser 20 J/cm2 H. pylori >99.9% 225
405 nm light-emitting diode 15 J/cm2 at lamp aperture P. gingivalis >75% 233
380–520 nm broadband light 4.2–42 J/cm2 P. gingivalis, P. intermedia, P. nigrescens, P. intermedia and P. nigrescens: >5 log10 at 4.2 J/cm2; P. melaninogenica: >5 278
P. elaninogenica, S. constellatus
log10 at 21 J/cm2; P. gingivalis:1.83 log10 at 42 J/cm2
400–500 nm blue lamps 260 and 1300 mW/cm2 for up P. gingivalis, F. nucleatum, S. mutans, E. The minimal inhibitory dose for P. gingivalis and F. nucleatum was 16–62 J/cm2, 279
to 3 min faecalis for S. mutans and S. faecalis was 159–212 J/cm2
405 and 470 nm light 15 J/cm2 S. aureus, P. aeruginosa S. aureus: 90% at 405 nm, 62% at 470 nm; P. aeruginosa: 95.1% at 405 nm, 280
96.5% at 470 nm
407–420 nm five P. acnes strains decreased by 15.7% immediately and 24.4% at 60 min after the irradiation. 231
407–420 nm 75J/cm2 P. acnes less than 2-log10 units (99%) illuminated once; decreased by 4-log10 units 281
(99.99%) after two illuminations and by 5-log10 units (99.999%) after three
illuminations
Page 54
Table 4
Studies on blue light therapy for acne vulgaris
Yin et al.
Light source Clinical trial type Acne type Treatment regimen No. of Total follow-up time outcome Side-effects Ref
patients/acne
location
407–420 nm metal halide open clinical trial Mild to twice a week up to 5 30 during the trial period (0, Acne lesions were Two patients 281
lamp moderate acne weeks 1, 3 and 5 weeks) and at 1 reduced by 64%. experienced
month after the final dryness.
treatment
Handle 412nm LED prospective, Mild-to- 2J/cm2/day full-face 32/facial acne eight weeks 100 percent of Three adverse 282
single-center, moderate, treatment or 29J/cm2/ subjects considered events: minimal
open-label study including day localized spot their overall transient skin
inflammatory or treatment, twice daily appearance was dryness and
non- at home improved, clarity minimal transient
inflammatory (97%), radiance hyperpigmentation
(73%), tone (80%),
texture (80%), and
smoothness (83%)
home use Blue-light LED IRB approved inflammatory Four Treatments were 30/facial acne followed-up till resolution significant reduction NA 283
randomized self- conducted twice daily in lesion size and
control study in the clinic lesion erythema as
well as the
improvement in the
overall skin condition
controlled with the
placebo
hand-held, Blue-light LED open clinical trial mild-to-mode a daily dose of ~29 J/ 33/facial acne eight weeks 90% improvements NA 284
rate cm2, twice daily
inflammatory
acne
407 to 420 nm LED A prospective, inflammatory underwent 8 sessions 60/facial acne 4 weeks The improvement 23.3% patients 285
randomized, open acne lesions of light therapy for 15 achieved by the blue with mild
and comparative grades II or III minutes each, twice a light was the same as desquamation and/
study week, within the one with benzoyl or dryness
minimum intervals of peroxide
48h
415nm LED open-lab el study 48 J/cm2, receiving 45 2, 4 and 8 weeks after mean improvement 286
two treatments of 20 treatment score was 3.14 at 4
minutes per week for weeks and 2.90 at 8
a period of 4–8 weeks weeks
blue LED, peak open-label study mild to moderat Over 4 weeks, patients 30 at weeks 5, 8 and 12 An overall effect on 287
wavelength inflammatory
409–419 nm e acne received eight 10- or counts was observed

20-minute light at week 5, and a
treatments statistically
Page 55
significant decrease
in inflamed counts
Light source Clinical trial type Acne type Treatment regimen No. of Total follow-up time outcome Side-effects Ref
patients/acne
location
Yin et al.
was detected at the
week 8 assessments,
which continued to
week 12.
Page 56

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Curr Opin Pharmacol. 2013 October ; 13(5): . doi:10.1016/j.coph.2013.08.009.

Light based anti-infectives: ultraviolet C irradiation,

2Department of Dermatology, Harvard Medical School, Boston, MA, USA

5Programa de Pós-Graduação Interunidades Bioengenharia - EESC/FMRP/IQSC and Centro de

© 2013 Elsevier Ltd. All rights reserved.

Prominent among novel non-antibiotic approaches is likely to be the group of light-based

In this review, we will provide an overview of recent advances in exploration and

2. Antimicrobial efficacy of UVC irradiation

2.2 UVC irradiation for bacterial/fungi infections in vivo

antibiotic-susceptible strains of E. faecalis and S. aureus [5], Enterococci species,

including coagulase-negative Staphylococcus, Streptococcus, E. coli, E. faecalis, P.

UVC also inactivated 3–5 log10 of dermatophyte suspensions in vitro, including

2.3 Effects of UVC irradiation on host cells and tissues

over keratinocytes in confluent monolayer cultures, while it only reduced approximately 6%

2.4 The clinical application of UVC irradiation for infectious diseases

3 Photodynamic therapy as an anti-infective

3.2 Antibacterial efficacy of photodynamic therapy

Gram-positive and Gram-negative bacteria have diversity in their three-dimensional

log10) after illumination. Moreover, it has been shown that a PS conjugate of

Multidrug-resistant (MDR) and pandrug-resistant (PDR) Gram-negative bacteria are less

demonstrated that antibiotic-resistant P. aeruginosa was just as sensitive to PDI as antibiotic-

tris(N-methyl-4pyridyl)-porphine trichloride (also known as Sylsens B or Tri-P(4)) (Fig 4E)

MDR and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis (MDR-

chitosan-conjugated rose bengal (CSRB) as PS showed anti-biofilm ability on Enterococcus

monocytogenes and Bacillus subtilis, while Yersinia pseudotuberculosis were found to be

3.3 Antifungal efficacy of photodynamic therapy

T. rubrum is a common pathogenic fungus genus of dermatophytes. PDT was suggested as a

3.4 Anti-bacterial/fungi biofilm efficacy of photodynamic therapy

therapies, it is necessary to develop new modalities to treat microbial biofilms. PDI is a

medically compromised individuals.

MB-PDT induced an average reduction of 2.81 log 10 of S. mutans biofilms, as well as an

commonly by C. albicans and to a lesser extent by C. tropicalis, C. parapsilosis or C.

PDT eradication of microbial biofilms could lead to a variety of applications in clinical

In conclusion, there is a wealth of literature describing PDT-based anti-biofilm strategies

phenotypic biofilm elements (e.g.,adhesins and extracellular polysaccharide ) [127,128].

and in turn remaining alive even after antimicrobial treatments [119,122,129,131,132].

3.5 The efficacy of PDT on other pathogens

manner. However, PDT had no effect on its viability.

Leishmania is an intracellular parasite that parasitizes the phagosomal compartment of

intracellular amastigotes. In some clinical studies, MB-, ALA- or MAL-mediated PDT

Human African trypanosomiasis, caused by Trypanosoma brucei rhodesiense or

aminomethyl-4,5′,8-trimethylpsoralen) when illuminated by UV completely inactivated the

with three phthalocyanine dyes, including HOSiPcOSi(CH3)2(CH2)3N(CH3)2(Pc4),

3.6 The efficacy of PDT on viruses

In general, polyomaviruses are considered to be highly resistant to PDI. The degree of

3.7 The efficacy of PDT on vectors

Photo-activated insecticides were demonstrated to be a novel compelling alternative to

meso-mono(N-tetradecylpyridil) porphine (C14) [166]. The photo-activable porphyrin

3.8 Blood product disinfection by PDT

blood disinfection is to kill or inactivate the target microorganism without disruption of

blood products [175–178].

3.9 Aquaculture disinfection by PDT

3.10 Waste water disinfection by PDT

reactor of water disinfection [189]. Two mono-hydroxyl zinc-porphyrin derivatives with

3.11 Food disinfection by PDT

3.12 Effects of PDT on host cells and tissues

using tetracationic phthalocyanine (RLP068) as a PS leads to an about 5 log10 decrease in

3.13 Clinical applications of anti-infective PDT

infections, and so on.