Homogeneous, flexible light sources with pure, deep red color and sufficiently high power densities are necessary for more effective and widely used photodynamic therapy (PDT), but have been difficult to achieve with lasers or LED arrays at reasonably low cost. Quantum dot light emitting diodes (QLEDs) have outstanding wavelength tunability, ideal color purity, sufficient power density and unique form factors as thin, flexible, light weight and uniformly large area light sources, which will meet the pressing needs for PDT.
Here we report QLEDs fabricated with emission wavelengths precisely tuned to match the absorption peaks of several FDA approved photosensitizers. Preliminary in-vitro studies with rigid on-glass QLEDs as photosensitizer activators demonstrate they can kill cancerous A431 cells or Methicillin-resistant Staphylococcus aureus (MRSA) with efficiency comparable or better than control LED sources, indicating their potential for PDT treatments of cancers or infections. Computer simulation of light propagation in a tissue mimicking phantom suggests that about 50% of the QLED power can be delivered to a depth of about 4 mm from the treated surface. Recent progress on the fabrication of low-cost flexible QLEDs will be presented. Finally, the perspectives of using these devices for PDT to address medical conditions such as cancer treatment, wound repair or cosmetic dermatology will be discussed.
Catheter–associated urinary tract infections (CAUTIs) cause millions of infections in the US every year, with direct medical costs exceeding billions of dollars and resulting in > 1 million ER visits and hospitalizations, resulting in thousands of deaths. CAUTI is thought to be a major reservoir both containing and creating highly drug-resistant infections, due to the chronicity of infection, biofilm formation, and the setting of institutionalization with chronic exposure to antibiotics, thus enabling resistance.
To reduce antibiotic resistance developing in this setting, we are attempting to apply both antimicrobial photodynamic therapy (aPDT), antimicrobial blue light (aBL), and combinations of both with minimal use of antibiotics. To this end, we established a rat model of UTI. In this model, we catheterized female rats, infected them with a standard uropathogenic E.Coli (UPEC; UTI89), infused the bladder with methylene blue (MB) and potassium Iodide (KI), and illuminated the bladder once with a diffusing fiber connected to a 1W 660nm laser. Multilog killing was observed, but given the transient nature of ROS generation, regrowth of UPEC was seen 24 hours later. To this end, we are experimenting with the combination of illumination with antibiotics. When tetracyclines are illuminated by aBL, we have found > 6 log(10) steps of microbial killing in vitro, and significant drops in the antibiotic MIC effected by the combination of light and drug. Multiple treatments with aBL and aPDT both with and without limited intravesical application of antimicrobials may light the way to solving this problem.
We report a novel class of highly water-soluble decacationic methano[60]fullerene decaiodides C60[>M(C3N6+C3)2]-(I−)10 [1-(I−)10] capable of co-producing singlet oxygen (Type-II) and highly reactive hydroxyl radicals, formed from superoxide radicals in Type-I photosensitizing reactions, upon illumination at both UVA and white light wavelengths. The O2‒·-production efficiency of 1-(I−)10 was confirmed by using a O2‒·-reactive bis(2,4-dinitrobenzenesulfonyl)tetrafluorofluorescein probe and correlated to the photoinduced electron-transfer event going from iodide anions to 3C60*[>M(C3N6+C3)2] leading to C60‒·[>M(C3N6+C3)2]. Incorporation of a defined number (ten) of quaternary ammonium cationic charges per C60 in 1 was aimed to enhance its ability to target pathogenic Gram-positive and Gram-negative bacterial cells. We used the well-characterized malonato[60]fullerene diester monoadduct C60[>M(t-Bu)2] as the starting fullerene derivative to provide a better synthetic route to C60[>M(C3N6+C3)2] via transesterification reaction under trifluoroacetic acid catalyzed conditions. These compounds may be used as effective photosensitizers and nano-PDT drugs for photoinactivation of pathogens.
Antimicrobial photodynamic inactivation (aPDI) is a new approach to killing microbial cells involving the excitation of photosensitizers (PS) by the correct wavelength of light to produce microbicidal reactive oxygen species. aPDI is independent of the antibiotic resistance status of the target cells, and is thought unlikely to produce resistance itself. Among a wide range of antimicrobial PS that have so far been investigated, tetracyclines occupy a unique niche. They are potentially dual-action compounds that can both kill bacteria under illumination, and prevent bacterial regrowth by inhibiting ribosomes. Demeclocycline (DMCT) can be efficiently activated by blue light (405 nm), while doxycycline (DOTC) is excited best by UVA light (360 nm). Both compounds were able to eradicate Gram-positive and Gram-negative bacteria at concentrations up to (100µM) and fluences up to 10J/cm2. The addition of potassium iodide (400mM) potentiates bacteria killing by up to six extra logs with little amount of DMCT and DOTC (5µM). In contrast to methylene blue (MB), tetracyclines can photoinactivate bacteria in rich growth medium. Bacteria regrowth inhibition and even further bacterial killing were observed when bacteria were partially killed with photosensitized DOTC or DMCT, while MB allowed complete regrowth. MIC studies were carried out either in the dark or exposed to continuous blue light (0.5mW/cm2). Up to 3 extra steps (8-fold) of antibiotic activity was found in the light compared to dark, while 5 extra steps (32-fold) of that was observed with 200mM KI. The mechanism is proposed to be singlet oxygen addition to iodine anion to form peroxyiodine which can decomposes into hydrogen peroxide and molecular iodine.
Rose Bengal (RB) is a halogenated xanthene dye that has been used to mediate antimicrobial photodynamic inactivation. While highly active against Gram-positive bacteria, RB is largely inactive in killing Gram-negative bacteria. We have discovered that addition of the non-toxic salt potassium iodide (100mM) potentiates green light (540nm)-mediated killing by up to six extra logs with Gramnegative bacteria Escherichia coli and Pseudomonas aeruginosa,Gram-positive methicillin resistant Staphylococcus aureus, and fungal yeast Candida albicans. The mechanism is proposed to be singlet oxygen addition to iodide anion to form peroxyiodide, which decomposes into radicals, finally forms hydrogen peroxide and molecular iodine. The effects of these different bactericidal species can be teased apart by comparing killing in three different scenarios: (1) cells+RB+KI are mixed together then illuminated with green light; (2) cells+RB are centrifuged then KI added then green light; (3) RB+KI+green light then cells added after light. We showed that KI could potentiate RBPDT in a mouse model of skin abrasions infected with bioluminescent P.aeruginosa.
Low-level light therapy (or photobiomodulation therapy) is a rapidly growing approach to treating a wide range of diseases and disorders that afflict humanity. This Tutorial Text covers the basic molecular and cellular mechanisms of action, applications for treating diseases in animal models, and its use in clinical trials and therapeutic practice in patients. Other topics include the two basic chromophores and how they trigger the signaling pathways, activation of transcription factors, and mobilization of stem cells; how the light-source design and the relevant energy parameters can affect the outcome of therapy; and the physics and tissue-optics principles that concern LLLT.
We have previously shown that transcranial low level light therapy (LLLT) can ameliorate brain damage in mice subjected to traumatic brain injury and improve neurological function. We used a 810-nm laser and delivered 18 J/cm2 at an irradiance 25 mW/cm2. LLLT was either delivered once at 4 hours after controlled cortical impact TBI, once a day for 3 days, or once a day for 14 days. One and 3 applications of LLLT had beneficial effects on the mice, with 3 being better than 1, but 14 applications had no beneficial effect. We now report immunofluorescence studies in mouse brain sections that offer some explanation for this intriguing finding. Mice were injected with BrdU for 1 week before sacrifice (a marker for proliferating cells) and antibodies to double cortin (DCX-1,a marker of migrating neurons), Tuj-1 ( a marker of neuroprogenitor cells), BDNF (brain derived neurotrophic factor) and synapsin-1 ( a marker for newly formed synaptic connections between existing neurons). We found increased BrdU incorporation indicating proliferating cells in the dentate gyrus of the hippocampus, the subventricular layer of the lateral ventricle, as well as the brain tissue surrounding the cortical lesion. Interestingly these cells were more abundant at 7 days than at 28 days post TBI. Co-labeling of BrdU with Neu-N was performed indicating that the proliferating cells were in fact neuronal in nature. Mice with 3 laser treatments had much more BrdU incorporation than mice with 14. Upregulation of BDNF was seen at 7 days, and increased expression of DCX-1 and Tuj-1 was seen at 28 days in the lesion region, indication that neuroprogenitor cells may have migrated there from sites of neurogenesis. Increased syapsin-1 was seen in the cortex at 28 days indicating that neural plasticity may be stimulated by LLLT. Taken together these data suggest that transcranial LLLT may have applications beyond TBI in areas such as neurodegenerative disease and psychiatric disorders.
It is accepted that the mechanisms of low level laser therapy (LLLT) involves photons that are absorbed in the
mitochondria of cells and lead to increase of mitochondrial metabolism resulting in more electron transport, increase
of mitochondrial membrane potential, and more ATP production. Intracellular calcium changes are seen that
correlate with mitochondrial stimulation. The situation with two other intermediates is more complex however:
reactive oxygen species (ROS) and nitric oxide (NO). Evidence exists that low levels of ROS are produced by LLLT
in normal cells that can be beneficial by (for instance) activating NF-kB. However high fluences of light can
produce large amounts of ROS that can damage the cells. In oxidatively stressed cells the situation may be different.
We exposed primary cultured cortical neurons to hydrogen peroxide (H2O2) or cobalt chloride (CoCl2) oxidative
insults in the presence or absence of LLLT (810-nm laser at 0.3 or 3 J/cm2). Cell viability of cortical neurons was
determined by lactate dehydrogenase assay. ROS in neurons was detected using an ROS probe, MitoRox with
confocal microscopy. Results showed that LLLT dose-dependently reversed ROS production and protected cortical
neurons against H2O2 or CoCl2 induced oxidative injury in cultured cortical neurons. Conclusion: LLLT can protect
cortical neurons against oxidative stress by reversing the levels of ROS.
Low-level laser (or light) therapy (LLLT) is attracting growing interest to treat both stroke and traumatic brain
injury (TBI). The fact that near-infrared light can penetrate into the brain allows non-invasive treatment to be carried
out with a low likelihood of treatment-related adverse events. It is proposed that red and NIR light is absorbed by
chromophores in the mitochondria of cells leading to changes in gene transcription and upregulation of proteins
involved in cell survival, antioxidant production, collagen synthesis, reduction of chronic inflammation and cell
migration and proliferation. We developed a mouse model of controlled cortical impact (CCI) TBI and examined the
effect of 0, 1, 3, and 14 daily 810-nm CW laser treatments in the CCI model as measured by neurological severity
score and wire grip and motion test. 1 laser Tx gave a significant improvement while 3 laser Tx was even better.
Surprisingly 14 laser Tx was no better than no treatment. Histological studies at necropsy suggested that the
neurodegeneration was reduced at 14 days and that the cortical lesion was repaired by BrdU+ve neural progenitor
(stem) cells at 28 days. Transcranial laser therapy is a promising treatment for acute (and chronic TBI) and the lack
of side-effects and paucity of alternative treatments encourages early clinical trials.
In the past four decades numerous studies have reported the efficacy of low level light (laser) therapy (LLLT) as a
treatment for diverse diseases and injuries. Recent studies have shown that LLLT can biomodulate processes in the
central nervous system and has been extensively studied as a stroke treatment. However there is still a lack of
knowledge on the effects of LLLT at the cellular level in neurons. The present study aimed to study the effect of 810
nm laser on several cellular processes in primary cortical neurons cultured from mouse embryonic brains. Neurons
were irradiated with light dose of 0.03, 0.3, 3, 10 and 30 J/cm2 and intracellular levels of reactive oxygen species,
nitric oxide and calcium were measured. The changes in mitochondrial function in response to light were studied in
terms of adenosine triphosphate (ATP) and mitochondrial membrane potential (MMP). Light induced a significant
increase in calcium, ATP and MMP at lower fluences and a decrease at higher fluence. ROS was induced
significantly by light at all light doses. Nitric oxide levels also showed an increase on treatment with light. The
results of the present study suggest that LLLT at lower fluences is capable of inducing mediators of cell signaling
process which in turn may be responsible for the biomodulatory effects of the low level laser. At higher fluences
beneficial mediators are reduced but potentially harmful mediators are increased thus offering an explanation for the
biphasic dose response.
There are many reports showing that low-level light/laser therapy (LLLT) can enhance wound healing,
upregulate cell proliferation and has anti-apoptotic effects by activating intracellular protective genes. In
the field of immune response study, it is not known with any certainty whether light/laser is proinflammatory
or anti-inflammatory. Increasingly in recent times dendritic cells have been found to play
an important role in inflammation and the immunological response. In this study, we try to look at the
impact of low level near infrared light (810-nm) on murine bone-marrow derived dendritic cells. Changes
in surface markers, including MHC II, CD80 and CD11c and the secretion of interleukins induced by light
may provide additional evidence to reveal the mystery of how light affects the maturation of dendritic
cells as well how these light-induced mature dendritic cells would affect the activation of adaptive
immune response.
Discoveries are rapidly being made in multiple laboratories that shed "light" on the fundamental molecular and
cellular mechanisms underlying the use of low level light therapy (LLLT) in vitro, in animal models and in clinical
practice. Increases in cellular levels of respiration, in cytochrome c oxidase activity, in ATP levels and in cyclic
AMP have been found. Increased expression of reactive oxygen species and release of nitric oxide have also been
shown. In order for these molecular changes to have a major effect on cell behavior, it is likely that various
transcription factors will be activated, possibly via different signal transduction pathways. In this report we compare
and contrast the effects of LLLT in vitro on murine embryonic fibroblasts, primary cortical neurons, cardiomyocytes
and bone-marrow derived dendritic cells. We also examined two human cell lines, HeLa cancer cells and HaCaT
keratinocytes. The effects of 810-nm near-infra-red light delivered at low and high fluences were addressed.
Reactive oxygen species generation, transcription factor activation and ATP increases are reported. The data has led
to the hypothesis that cells with a high level of mitochondrial activity (mitochondrial membrane potential) have a
higher response to light than cells with low mitochondrial activity.
Low level laser (or light) therapy (LLLT) has been clinically applied for many indications in medicine that require
the following processes: protection from cell and tissue death, stimulation of healing and repair of injuries, and
reduction of pain, swelling and inflammation. One area that is attracting growing interest is the use of transcranial
LLLT to treat stroke and traumatic brain injury (TBI). The fact that near-infrared light can penetrate into the brain
would allow non-invasive treatment to be carried out with a low likelihood of treatment-related adverse events.
LLLT may have beneficial effects in the acute treatment of brain damage injury by increasing respiration in the
mitochondria, causing activation of transcription factors, reducing key inflammatory mediators, and inhibiting
apoptosis. We tested LLLT in a mouse model of TBI produced by a controlled weight drop onto the skull. Mice
received a single treatment with 660-nm, 810-nm or 980-nm laser (36 J/cm2) four hours post-injury and were
followed up by neurological performance testing for 4 weeks. Mice with moderate to severe TBI treated with 660-
nm and 810-nm laser had a significant improvement in neurological score over the course of the follow-up and
histological examination of the brains at sacrifice revealed less lesion area compared to untreated controls. Further
studies are underway.
Anti-tumor immunity is stimulated after PDT for cancer due to the acute inflammatory response, exposure and
presentation of tumor-specific antigens, and induction of heat-shock proteins and other danger signals. Nevertheless
effective, powerful tumor-specific immune response in both animal models and also in patients treated with PDT for
cancer, is the exception rather than the rule. Research in our laboratory and also in others is geared towards identifying
reasons for this sub-optimal immune response and discovering ways of maximizing it. Reasons why the immune
response after PDT is less than optimal include the fact that tumor-antigens are considered to be self-like and poorly
immunogenic, the tumor-mediated induction of CD4+CD25+foxP3+ regulatory T-cells (T-regs), that are able to inhibit
both the priming and the effector phases of the cytotoxic CD8 T-cell anti-tumor response and the defects in dendritic cell
maturation, activation and antigen-presentation that may also occur. Alternatively-activated macrophages (M2) have also
been implicated. Strategies to overcome these immune escape mechanisms employed by different tumors include
combination regimens using PDT and immunostimulating treatments such as products obtained from pathogenic
microorganisms against which mammals have evolved recognition systems such as PAMPs and toll-like receptors
(TLR). This paper will cover the use of CpG oligonucleotides (a TLR9 agonist found in bacterial DNA) to reverse
dendritic cell dysfunction and methods to remove the immune suppressor effects of T-regs that are under active study.
KEYWORDS: Melanoma, Photodynamic therapy, Picosecond phenomena, Tumors, Luminescence, Absorption, In vitro testing, Control systems, Near infrared, Skin cancer
Photodynamic therapy (PDT) has been successfully used to treat many malignancies, and has afforded highly
encouraging results in skin cancers such as basal cell carcinoma. However, pigmented melanoma remains a notable
exception from the range of tumors treated by PDT largely due to the fact that melanin has high absorption of light in
wavelength regions where most clinically approved photosensitizers (PS) absorb light (600-690 nm). Moreover,
melanoma cells sequester exogenous molecules including photosensitizers inside melanosomes. The aforementioned
drawbacks of the clinically used PS have motivated us to search for new classes of PS with improved spectral properties,
such as bacteriochlorins (BC) to be used in PDT of melanoma. To overcome the PDT-resistance mechanisms of
melanoma, particularly the high optical absorption of melanin, three near-infrared (NIR) absorbing synthetic stable BC
were used in PDT treatment of melanoma. Dose and fluence dependent cell killing, intracellular localization (particularly
in melanosomes), and correlation between the melanin level and cell death were examined. Intracellular melanosomes
are ruptured after illumination as shown by electron microscopy. The best in vitro performing BC were tested upon
delivery in micellar nanoparticles against a mouse pigmented melanoma. Two of the BC were effective at significantly
lower concentrations (<0.5 μM) than common photosensitizers in present use.
Despite over forty years of investigation on low-level light therapy (LLLT), the fundamental mechanisms underlying
photobiomodulation remain unclear. In this study, we isolated murine embryonic fibroblasts (MEF) from transgenic
NF-kB luciferase reporter mice and studied their response to 810-nm laser radiation. Significant activation of NFkB
was observed for fluences higher than 0.003 J/cm2. NF-kB activation by laser was detectable at 1-hour time
point. Moreover, we demonstrated that laser phosphorylated both IKK α/β and NF-kB 15 minutes after irradiation,
which implied that laser activates NF-kB via phosphorylation of IKK α/β. Suspecting mitochondria as the source of
NF-kB activation signaling pathway, we demonstrated that laser increased both intracellular reactive oxygen species
(ROS) by fluorescence microscopy with dichlorodihydrofluorescein and ATP synthesis by luciferase assay.
Mitochondrial inhibitors, such as antimycin A, rotenone and paraquat increased ROS and NF-kB activation but had
no effect on ATP. The ROS quenchers N-acetyl-L-cysteine and ascorbic acid abrogated laser-induced NF-kB and
ROS but not ATP. These results suggested that ROS might play an important role in the signaling pathway of laser
induced NF-kB activation. However, the western blot showed that antimycin A, a mitochondrial inhibitor, did not
activate NF-kB via serine phosphorylation of IKK α/β as the laser did. On the other hand, LLLT, unlike
mitochondrial inhibitors, induced increased cellular ATP levels, which indicates that light also upregulates
mitochondrial respiration. ATP upregulation reached a maximum at 0.3 J/cm2 or higher. We conclude that LLLT not
only enhances mitochondrial respiration, but also activates the redox-sensitive transcription factor NF-kB by
generating ROS as signaling molecules.
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