Photochemistry and Photobiology, 2009, 85: 1364–1374
Near-infrared Photoinactivation of Bacteria and Fungi at
Physiologic Temperatures
Eric Bornstein*1, William Hermans2, Scott Gridley2 and Jeffrey Manni3
1
Nomir Medical Technologies, Waltham, MA
Blue Sky Biotech, Inc., Worcester, MA
3
JGM Associates, Inc., Burlington, MA
2
Received 13 March 2009, accepted 27 June 2009, DOI: 10.1111 ⁄ j.1751-1097.2009.00615.x
ABSTRACT
We examined a laser system (870 and 930 nm), employing
wavelengths that have exhibited cellular photodamage properties
in optical traps. In vitro, with 1.5 cm diameter flat-top projections (power density of 5.66 W cm)2), at physiologic temperatures, we achieved photoinactivation of Staphylococcus aureus,
Escherichia coli, Candida albicans and Trichophyton rubrum.
Using nonlethal dosimetry, we measured a decrease in transmembrane potentials (DWmt and DWp) and an increase in
reactive oxygen species (ROS) generation in methicillin-resistant
S. aureus (MRSA), C. albicans and human embryonic kidney
cells. We postulate that these multiplexed wavelengths cause an
optically mediated mechano-transduction of cellular redox
pathways, decreasing DW and increasing ROS. The cellular
energetics of prokaryotic and fungal pathogens, along with
mammalian cells, are affected in a similar manner when treated
with these multiplexed wavelengths at the power densities
employed. Following live porcine thermal tolerance skin experiments, we then performed human pilot studies, examining
photodamage to MRSA in the nose and fungi in onychomycosis.
No observable damage to the nares or the nail matrix was
observed, yet photodamage to the pathogens was achieved at
physiologic temperatures. The selective aspect of this nearinfrared photodamage presents the possibility for its future
utilization in human cutaneous antimicrobial therapy.
INTRODUCTION
Photodamage of bacterial and fungal pathogens without the
need of a photosensitizer has generally been the province of UV
wavelengths of light. As early as 1903, Bernard and Morgan
established that arc lamps had a tremendous bactericidal action
between 226 and 328 nm (UVC + UVB) (1). A century later,
researchers are still using these energies in human studies, and
since 2002, at least three publications have reported the
successful germicidal activity of UV light on human prokaryotic
and eukaryotic pathogens in vivo (2–4). Notwithstanding these
reported human studies and suggested therapies, it is an
unfortunate truth that UV light, while being genotoxic to the
organisms, is also photocarcinogenic to human cells (5–8).
*Corresponding author email: [email protected]
(Eric S. Bornstein)
! 2009 The Authors. Journal Compilation. The American Society of Photobiology 0031-8655/09
These data suggest that a higher level of safety must be
researched to explore the possible phototherapy of human
infectious diseases that would eliminate the dangers associated
with UV therapy. A photoinactivating near-infrared (IR)
therapy for prokaryotic and eukaryotic pathogens, if possible,
would fit such a safety profile.
Two decades ago, the science of optical traps improved
greatly (9) showing significantly decreased photodamage
during optical trapping of Escherichia coli with near-IR
wavelengths. More recently, it has been demonstrated by
Liang et al. (10) and Neuman et al. (11) that there are two
distinctive near-IR wavelengths (870 and 930 nm) that are
capable of causing photodamage in both E. coli (prokaryotic)
and Chinese hamster ovary (CHO) cells (eukaryotic) in vitro,
during confocal laser microscopy (10,11). In Neuman’s experiments, he hypothesized that the high cell death curves were the
result of the generation of a singlet oxygen reaction, and that
this oxygen species was created independently by the absorption of 870 and 930 nm selectively, in unknown intracellular
endogenous chromophores of both the prokaryotic and
eukaryotic species.
Neuman determined that this reactive oxygen species (ROS)
disturbed the proton-motive force of the E. coli, and thus
caused the cells’ flagella rotation to slow and finally cease. The
proton-motive force (Dp) refers to the storing of energy as
a combination of a proton and voltage gradient across a
membrane that drives the synthesis of ATP. The two components of Dp are DY (transmembrane potential) and DpH
(chemical gradient of H+). Also termed ‘‘energy transduction,’’ this entire process refers to proton transfer through
the respiratory complexes embedded in a membrane, utilizing
electron transfer reactions to pump protons across the
membrane and create an electrochemical potential. This
potential energy is stored in the form of an electrochemical
gradient (plasma membranes in prokaryotes and mitochondrial membranes in eukaryotes), is created during normal
cellular respiration, and is an excellent measure of the
robustness of cellular bioenergetics (12,13).
Neuman postulated that the unidentified endogenous
absorptive chromophores were ubiquitous in both the prokaryotic and eukaryotic cells, as the increase in kill rate
affected both species. This in effect demonstrated a distinctive
measure of near-IR cellular photodamage in the absence of
any exogenous drug or chemical that had heretofore not been
observed with other near-IR wavelengths (10,11). Neuman
1364
Photochemistry and Photobiology, 2009, 85
also reported that oxygen is critical in this unique photodamage pathway, as aerobic and anaerobic experiments were
performed with virtually no damage occurring in the anaerobic
system (11). In further support of these data, that the
chromophores were ubiquitous across prokaryotic and eukaryotic species, Sacconi et al. (14) demonstrated similar results by
observing photodamage effects within minutes, in eukaryotic
yeast cells at 880 nm but no photodamage at 830 nm (14).
With these data (10–12,14) we hypothesized that a multiplexed beam of the 870 and 930 nm wavelengths, blending the
individual photodamage phenomena caused by each at physiologically tolerable power densities, could potentially cause DY
and Dp alterations in the plasma membranes of prokaryotic
pathogens, and the mitochondrial membranes of eukaryotic
pathogens.
If this type of selective photodamage to pathogens were
possible, it could create safe adjunctive therapies for cutaneous
infectious disease treatments, such as targeting dangerous
pathogens like methicillin-resistant Staphylococcus aureus
(MRSA) (15), without deleterious photothermal or photocarcinogenic effects. To begin testing these hypotheses we took
the first step of reviewing the available spectroscopy literature,
to prospectively discern the nature of these ubiquitous
chromophores. With this information, we could then postulate
a mechanism of action for the creation of ROS, and the
alteration of DY and Dp in cells.
870 nm Absorption in cytochrome c
It has been observed (16) in CHO air-dried monolayers that
there is a unique spectral absorption band in the oxidized
cytochrome c oxidase complex at 870 nm. The molecular
components giving rise to the 870 nm absorption band were
postulated to be copper (Cu) ions outside of the heme (Fe)
redox pathway. This absorption peak was also recorded in vivo
(17) and it was proposed that the absorption was occurring in
Cu-A and Cu-B metallo-protein reaction centers of the
cytochrome. J}
obsis-vander Vliet also showed that small
structural or conformational changes in the binuclear site of
cytochrome c oxidase can modify both the rates of the
molecular oxygen reduction and the rates of internal electron
and proton transfer reactions in the enzymatic cytochrome
complex (18). Additionally, it has been reported that metalloproteins in the cytochrome chains exist as chromophores, and
that they can be exploited by different laser systems (16,19–22).
930 nm Absorption in cell membranes
It has been observed (23) that porcine fat (long chain
hydrocarbons) showed a strong absorption peak at exactly
930 nm, during infrared spectroscopy studies. Conway’s data
were corroborated when a high absorption band was found at
930 nm in porcine fat (24). Irie postulated that this unique
absorption band was, in all probability, caused by the carbon–
hydrogen (C–H) covalent atomic bonds in the oil structure of
the fat.
Despite the many fundamental differences between cells of
eukaryotic and prokaryotic species, cells of virtually every type
of living organism share in common the structural features of
lipid carbon–hydrogen (C–H) bonds in the form of a phospholipid bilayer membrane. Phospholipid bilayers define the
1365
periphery of cells and of mitochondria and are compiled of
vast numbers of lipid (fat) and protein molecules that are held
together chiefly through noncovalent hydrophobic molecular
interactions. These membranes act as semipermeable barriers
to organic polar compounds, and inorganic ions that are
necessary for normal cellular function and redox reactions.
This is known as the fluid mosaic model of the plasma
membrane (25–27). Finally, there are two interesting reports in
the literature of 930 nm absorption in the specific electron
transport molecules of Rhodobacter capsulatus mutants lacking
bacteriopheophytin in its photosynthetic reaction center, and
in a synthetic proton pump gate (with Cu-B complexes),
fabricated to mimic the Cu-B metallo-protein reaction centers
of cytochrome c oxidase (28,29).
Postulate for the generation of ROS
The implication of cytochrome c (870 nm) and plasma and
mitochondrial membranes (930 nm), as the identified absorptive chromophores in Neuman’s findings suggest: (1) the
possible generation of minute optically mediated mechanical
forces on plasma and mitochondrial membranes that can
regulate a cell’s biochemical activity in a manner that is equally
as potent as chemical or pharmacological signals (30,31); (2)
the possibility that these energies are capable of eliciting a
molecular response in cells that can influence biochemical
signal transduction (32–34); and (3) the possibility that
cytochrome c, cytochrome c oxidase and lipid bilayers are
the ubiquitous highly conserved chromophores postulated by
Neuman (35,36).
Hence, if the C–H covalent bonds in the long chain fatty
acids of the lipid bilayers actively absorb targeted photon
energy at 930 nm, while cytochrome c simultaneously absorbs
photon energy at 870 nm (and possibly at 930 nm), there
would be increased and targeted kinetic interactions in the
redox chains of irradiated cells. These targeted kinetic interactions could alter the fluid mosaic nature of the mitochondrial
and plasma membranes and cytochromes of the cells, thereby
creating a dynamic disorder in redox pathways. Also, a
simultaneous local kinetic energy increase in cytochrome c
(870 nm) could create a different orientation of the metalloprotein reaction centers in the cytochrome. This could then
produce functional oxidation-reduction changes near Cucontaining chromophores in redox chains. Cytochrome c
oxidase is also referred to as ‘‘respiratory complex IV’’ and
is the terminal enzyme in the electron transport chain that
receives an electron from each of four cytochrome c molecules,
and transfers them to one oxygen molecule, creating two
molecules of water from molecular oxygen. The probable
target chromophores that are interacting with these two
distinct wavelengths are in effect at the last stage of the energy
transduction and oxygen reduction processes of cellular
respiration (12).
The creation of a dynamic disorder in redox chains with
near-IR radiation a priori would be expected to alter the
thermodynamics of vital redox membrane-bound respiratory
proteins in mitochondrial and plasma membranes (such as
cytochrome c), thereby altering DY and Dp. If this photodamage signal transduction effect could be accomplished at
power densities and energy densities only producing tolerable
human physiologic temperatures, we would term such an
1366 Eric Bornstein et al.
interaction optically mediated mechano-transduction of cellular redox pathways (12,22).
To demonstrate selective photodamage in vivo, we must also
analyze the known literature for potential negative IR effects on
the mammalian mitochondria. As such, Frank et al. (37) showed
that one of the effects of IR on mitochondria is a downward
regulation in the mitochondrial transmembrane potentials
(DYmt) in human fibroblasts. This lowering of DYmt has also
been shown to be an early marker in the cellular apoptotic
process (38), but can also occur when cells are not absolutely
committed to apoptosis (39). Therefore, in light of our in vitro
data, we decided to further titrate physiologically tolerable
energy densities in a live porcine model, that are approximately
1 log less (J cm)2) in energy than the in vitro (saline) model
because of the greater absorption properties of skin to near-IR
energy (34). After isolating such a dosimetry in Yorkshire pigs
(40), we then ran two human pilot studies against bacterial and
fungal pathogens, to test for potential negative sequelae in
delicate human tissue, the nasal mucosa and nail matrix.
In the first human pilot (MRSA in the nares) we performed
a study to observe whether the effect of lowering Dp and DY
in the resistant prokaryotic plasma membrane would possibly
hamper the functionality of multidrug transport systems in
staphylococcus, that rely on endogenous cellular energy for
their functionality (41–46). In the second human pilot, we
attempted to photoinactivate different fungal species in
patients with diagnosed and cultured onychomycosis.
MATERIALS AND METHODS
Optics. A multifunctional custom-fabricated Class IV CDRH compliant turnkey laser system, 20 W CW dual wavelength (10 W at 870 nm,
10 W at 930 nm) laser (Lasertel, Tucson, AZ) was enabled (Fig. 1).
The laser energy was shaped through a custom infrared microlens. This
was fabricated and used with a uniformly illuminated spot (PhotoGlow, Dennis, MA). This assured a uniform energy density (J cm)2)
across the diameter of the illumination spot (Fig. 2). The fiber size was
600 lm core NA 0.37, with spot uniformity less than ±15%. Spot size
could be varied from 1 to 4 cm diameter based on the distance of the
lens from the irradiation site.
Figure 1. Experimental indexing fixture.
Figure 2. Flat-top spot uniformity profile of microlens.
Indexing fixture. We employed a custom indexing fixture with a
breadboard table (12¢ · 18¢ · 0.5¢). Two linear translation stages were
stacked at a 90" angle to give 50 mm of travel in the x and y
directions. The upper and lower indexing tables were 6¢ · 6¢ · 0.5¢
plates.
Calibration of in vitro power and temperature. To assess time–
temperature interactions for these experiments, 2 mL of PBS was
aliquoted into selected wells of the 24-well tissue culture plates, placed
on the indexing fixture and tested at 20 different dosimetry parameters
with a scientific thermometer. This was accomplished with a uniform
1.5 cm spot of the blended 870 and 930 nm energies. After multiple
temperature trials, the theoretical threshold dosimetry for photoinactivation experiments was chosen as 12 min (720 s) with blended
10 W (5 W of each wavelength) producing an energy density of
4074 J cm)2 with a flat-top projection. This uniform energy dose
consistently produced a temperature steady-state limit of 42.5"C in
PBS, in the last 5 min of the irradiation cycle; this was measured with a
scientific thermometer placed in the 2 mL of PBS to the bottom of the
well of the 24-well plates (approximately 2 cm depth). This temperature limit was chosen with examination of data from the literature,
describing normal growth for the species we tested at the experimental
time ⁄ temp parameters:
(1) 42.5"C is within the thermal tolerance maxima for seven different
species of keratinophilic fungi (Trichophyton mentagrophytes,
Epidermophyton floccosum, Microsporum canis, T. interdigitale
and T. rubrum [47]);
(2) 42.5"C for 5 min will have minimal effect on log growth curves of
S. aureus (48);
(3) 42.5"C is well below the observed tissue damage threshold for
time–temperature tolerance on human skin (49).
All culture dilutions for the experiments were performed using PBS,
and cells were suspended in PBS. This was done to allow for negligible
absorption of laser energy by trace amounts of the various culture
media that the cells were grown in, and to idealize the experiments to
the tested in vitro temperature curves for PBS. A temperature pretest
with live cultures was also performed in triplicate on E. coli, at an
energy density of 4074 J cm)2 with a flat-top projection, and the DT
began at 21"C and finished at 41"C.
Cell culture. All microbial and mammalian cell cultures, cell
counting, assay production and data analysis were completed by
BlueSky Biotech (Worcester, MA). For all manual colony counts,
digital photographs of each plate were taken, and the examiner was
shielded from any knowledge of the amount of energy used to irradiate
each sample that was tested.
Trichophyton rubrum. T. rubrum ATCC 52022 liquid cultures were
grown in Peptone-Dextrose (PD) medium (10 g L)1 peptone, 10 g L)1
dextrose) at 37"C with no agitation. Culture dilutions were performed
using PBS. When the OD 300 nm was measured to be 0.210, 2 mL of
this suspension was aliquoted into selected wells of 24-well tissue
culture plates and presented for laser irradiation experiments. Following laser treatments, 3·100 lL aliquots were removed from each well
and spread onto separate plates and incubated at 37"C for approximately 91 h for CFU counts.
Photochemistry and Photobiology, 2009, 85
Candida albicans. C. albicans ATCC 14053 liquid cultures were
grown in Difco yeast mold broth (21 g L)1) (BD Diagnostic Systems,
Sparks, MD). On the day prior to laser experimentation, a 50 mL culture
was inoculated using a glycerol stock of C. albicans (stored at 80"C), and
allowed to grow approximately 16 h. It was then subcultured, and
growth was monitored by measuring the optical density at 600 nm until
the culture reached an OD of approximately 1. An aliquot of 1 mL was
removed from the subculture and serially diluted to 1:500 in PBS. This
dilution was allowed to incubate at room temperature for approximately
2 h. Two milliliters of this suspension was aliquoted into selected wells of
24-well tissue culture plates and presented for laser irradiation experiments. Following photoinactivation laser treatments, 3·100 lL aliquots were removed from each well and serially diluted to 1:1000 resulting
in a final dilution of 1:5 · 105 of initial culture and spread onto separate
plates and incubated at 37"C for approximately 16–20 h for CFU
counts. Following sublethal dosimetry of laser treatments, membrane
potential and glutathione assays were performed on control and
treatment cultures and the examiner was shielded from any knowledge
of the amount of energy used to irradiate each sample that was tested.
Escherichia coli. E. coli K12 liquid cultures were grown in Luria
Bertani (LB) medium (25 g L)1). Culture dilutions were performed
using PBS. Drawing from a seed culture, multiple 50 mL LB cultures
were inoculated and grown at 37"C overnight. The next morning, the
healthiest culture was chosen and used to inoculate 5% into 50 mL LB
at 37"C and the OD 600 nm was monitored over time taking
measurements every 30–45 min until the culture was in stationary
phase. An aliquot of 100 lL was removed from the subculture and
serially diluted to 1:1200 in PBS. This dilution was allowed to incubate
at room temperature approximately 2 h or until no further increase in
OD 600 nm was observed. Two milliliters of this suspension was
aliquoted into selected wells of 24-well tissue culture plates and
presented for laser irradiation experiments. Following laser treatments,
100 lL was removed from each well and serially diluted to 1:1000
resulting in a final dilution of 1:12·105 of initial K12 culture. Each final
dilution of 3· 200 lL was spread onto separate plates. The plates were
then incubated at 37"C for approximately 16 h for CFU counts.
Staphylococcus aureus. Staphylococcus aureus (ATCC 12600) liquid
cultures were grown in LB medium (25 g L)1). Culture dilutions were
performed using PBS, pH 7.4. On the day prior to laser experimentation,
a 50 mL culture was inoculated using a glycerol stock of S. aureus
(stored at 80"C), and allowed to grow for approximately 16 h. It was
then subcultured, and growth was monitored by measuring the optical
density at 600 nm until the culture reached an OD of approximately 1.
An aliquot of 1 mL was removed from the subculture and serially diluted
to 1:300 in PBS. This dilution was allowed to incubate at room
temperature for approximately 2 h. Two milliliters of this suspension
was aliquoted into selected wells of 24-well tissue culture plates and
presented for laser irradiation experiments. Following laser treatments,
100 lL was removed from each well and serially diluted to 1:1000
resulting in a final dilution of 1:3·105 of initial culture. Each final
dilution of 3· 100 lL was spread onto separate plates. The plates were
then incubated at 37"C for approximately 16–20 h for CFU counts.
Methicillin-resistant S. aureus (MRSA). MRSA (ATCC BAA-43)
liquid cultures were grown in tryptic soy broth medium (30 g L)1) with
overnight cultures containing 40 lg mL)1 methicillin. Culture dilutions were performed using PBS, pH 7.4. On the day prior to laser
experimentation, a 50 mL culture was inoculated using a glycerol stock
of MRSA (stored at 80"C), and allowed to grow for approximately
16 h. It was then subcultured, and growth was monitored by
measuring the optical density at 600 nm until the culture reached an
OD of approximately 1. An aliquot of 1 mL was removed from the
subculture and serially diluted to 1:300 in PBS. This dilution was
allowed to incubate at room temperature for approximately 2 h. Two
milliliters of this suspension was aliquoted into selected wells of 24-well
tissue culture plates and presented for laser irradiation experiments.
Following laser treatments, membrane potential and glutathione
assays were performed on control and treatment cultures and the
examiner was shielded from any knowledge of the amount of energy
used to irradiate each sample that was tested.
NIH 3T3 mouse fibroblasts. Five T75 flasks were cultured for 72 h
until reaching approximately 70% confluence. The flasks were rinsed
with 10 mL of 0.25% trypsin, and then treated with 3 mL of 0.25%
trypsin and placed in a 37"C incubator until cells had become
dislodged. The trypsin was then quenched with 8 mL of growth media
1367
containing 10% fetal bovine serum (FBS), and the cells were counted
on Cedex (Innovatis, Bielefeld, Germany). Cells were then centrifuged
for 10 min at 1778 g and washed with treatment media (clear growth
media without phenol red and without FBS), then centrifuged for
10 min at 1778 g. Cells were resuspended in treatment media at
roughly 1.00 · 105 cells per mL and plated with control in 2 mL of
treatment media (clear growth media without phenol red and without
FBS). Cells were allowed to attach to plate for 3 h in a 37"C incubator
and then removed from well via the trypsinization treatment described
above, and again counted on Cedex.
Human embryonic kidney (HEK-293) cells. A suspension HEK 293
cell line was cultured in shake flasks at 37"C and 8% CO2 supplementation using standard laboratory practices. Cells were counted on
a Cedex to determine cell density and viability. Cells from a log phase
culture were aliquoted at 4 million cells per well in 2 mL of Freestyle
293 growth media (Invitrogen, Carlsbad, CA) on a 24-well plate.
Live animal dosimetry test. An IACUC-approved study was conducted on two Yorkshire pigs at an AALAC-accredited facility
(Toxikon Corp., Bedford, MA). Each animal had thirty-three 1 cm
sites identified on its back and were subjected to lasing at a range of
dosimetry parameters. Needle thermocouples were placed at both 2 and
20 mm depths to determine surface and subsurface temperature
increases at different dosimetries. The lased areas were clinically
observed daily for 72 h, and 2.5 cm biopsies were taken from the lased
sites at day 1, day 2 and day 3, for examination microscopically (against
control specimens) for evidence of adverse thermal changes resulting
from irradiation. This study was undertaken to further the understanding of live dermal thermal tolerances for different dosimetric parameters
in vivo. The pigs were sacrificed at the end of the study.
Human onychomycosis pilot study. To calibrate dosimetry for
human toenail studies, clear and mycotic toenails were extracted and
obtained from cadavers donated for research, following Massachusetts
public health guidelines in a laboratory accredited for research activity.
These nails were then placed between the laser and a National Institute
of Standards and Technology traceable power meter to test for power
attenuation and throughput values. The data were then coupled to the
live porcine thermal ⁄ dosimetry data previously titrated to prevent
thermal injury to skin, and a toenail therapeutic dose was created.
Following IRB approval, the great toe nails of seven patients with
positive fungal cultures (Phaeoannellomyces, Trichophyton and
Rhodotorula species) were topically irradiated four times (days 1, 7,
14 and 60), utilizing a flat-top microlens identical to that employed in
the in vitro and Yorkshire pig tests. Each patient underwent four
exposures with 870 nm ⁄ 930 nm for 240 s (energy density – 408 J cm)2
with 1.5 cm spot) to each infected great toenail, followed by 930 nm
for 120 s (energy density – 204 J cm)2). Nail temperatures were
recorded with an infrared thermometer. After the second irradiation
(day 7), a commercial, over-the-counter tinea pedis spray was
employed only between the toes two times a day, to inhibit nail
reinoculation from adjacent tinea pedis. Debridement of thickened
nails was accomplished if necessary by a dermatologist.
Human MRSA pilot study in the nose (nares). Positive MRSA and
methicillin-sensitive S. aureus (MSSA) anterior nares cultures were
obtained with nasal swabs, diluted in 2 mL of PBS and vortexed for
120 s before plating on chromogenic agar in triplicate. This was
accomplished in six patients (12 nostrils) in a Human IRB pilot study
(49). One patient tested positive for MRSA only, three tested positive for
MSSA only, and two tested positive for both MRSA and MSSA. All
MRSA and MSSA cultures were also tested and shown to be completely
uninhibited on premade (4 lg mL)1) erythromycin TBS agar plates,
showing robust growth. Utilizing a prototype 10 cm flat-top diffuser,
each patient underwent exposure with 870 nm ⁄ 930 nm for 240 s (energy
density – 110 J cm)2) to each anterior nostril followed by 930 nm for
180 s (energy density – 83 J cm)2) on day 1 and on day 3. Temperatures
of the nares were recorded every 30 s with an IR thermometer. Generic
erythromycin paste (2%) was then applied three times a day for 5 days.
Quantitative cultures from each nostril were obtained and plated in
triplicate on chromogenic agar before and 20 min after exposure on day
1 and day 3. A final culture was taken and plated in triplicate on day 5.
Commercial membrane potential kits. BacLight Bacterial Membrane
Potential Kit (B34950; Invitrogen) provides carbocyanine dye
DiOC2(3) (3,3¢-diethyloxacarbocyanine iodide, Component A) and
CCCP (carbonyl cyanide 3-chlorophenylhydrazone, Component B),
both in dimethyl sulfoxide, and a 1· PBS solution (Component C).
1368 Eric Bornstein et al.
DiOC2(3) exhibits green fluorescence in all bacterial cells, but the
fluorescence shifts toward red emission as the dye molecules self
associate at the higher cytosolic concentrations caused by larger
membrane potentials. Proton ionophores such as CCCP destroy
membrane potential by eliminating the proton gradient, hence causing
higher green fluorescence.
The APO LOGIX JC-1 Assay Kit (Peninsula Laboratories, Inc.,
San Carlos, CA) measures the mitochondrial membrane potential in
cells. In nonapoptotic cells, JC-1 (5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢-tetraethylbenzimidazolylcarbocyanine iodide) exists as a monomer in the
cytosol (green) and also accumulates as aggregates in the mitochondria
which stain red. In healthy cells with normal mitochondrial membrane
potentials, the JC-1 aggregates will have an intense red fluorescence. If
the cell is treated so that the mitochondrial DY is diminished, the JC-1
aggregates will exit the mitochondria, distribute in the cytoplasm and
fluoresce green in their monomeric form. The intensity of the red
fluorescent signal versus the green can be used to measure mitochondrial DY versus control. Fluorescence was measured using a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA).
Commercial total glutathione quantification kit (Dojindo Laboratories, Kumamoto, Japan). Glutathione is almost always found in its
reduced form, as the enzyme which reverts it from its oxidized form
(GSSG), glutathione reductase, is constitutively active and easily
inducible upon an oxidative cellular challenge or oxidative stress. The
ratio of reduced to oxidized glutathione within cells is used scientifically as a measure of oxidative stress in cells. After laser treatment,
cells were recovered by centrifugation and lysed in approximately
1 mL of assay buffer by gentle sonication on ice. Absorbance was
measured using a plate-ready SpectraMax M5.
forming units counted at 20 h incubation versus control
(Table 1).
In vitro E. coli photoinactivation experiments
Two equivalent samples of E. coli cells were exposed to
blended laser energy for 12 min irradiation cycles. The first
was exposed to 3259 J cm)2 and plated in triplicate, and the
second was exposed to 4074 J cm)2 and plated in triplicate.
The 3259 J cm)2 exposure produced photoinactivation of
16.5% of the cells after 20 h incubation. The 4074 J cm)2
exposure produced photoinactivation of 97% of the colony
forming units counted at 16 h incubation versus control
(Table 2).
In vitro T. rubrum photoinactivation experiments
Two equivalent samples of T. rubrum cells were exposed to
blended laser energy for 12 min irradiation cycles. The first
was exposed to 4074 J cm)2 and plated in triplicate, and the
second was exposed to 4500 J cm)2 and plated in triplicate.
Both exposures to T. rubrum produced photoinactivation
of 100% of the colony forming units counted at 91 h
incubation versus control. It is probable that a lower energy
density would accomplish photoinactivation of these fungi
in vitro, and warrant further experiments (Table 3).
RESULTS
In vitro S. aureus photoinactivation experiments
In vitro C. albicans photoinactivation experiments
Two equivalent samples of S. aureus cells were exposed to
blended laser energy for 12 min irradiation cycles. The first
was exposed to 3667 J cm)2 and plated in triplicate, and the
second was exposed to 4074 J cm)2 and plated in triplicate.
The 3667 J cm)2 exposure produced photoinactivation of
25% of the cells after 20 h incubation. The 4074 J cm)2
exposure produced photoinactivation of 98% of the colony
Two equivalent samples of C. albicans cells were exposed to
blended laser energy for 12 min irradiation cycles. The first
was exposed to 4074 J cm)2 and plated in triplicate, and the
second was exposed to 4500 J cm)2 and plated in triplicate.
Both of the exposures to C. albicans produced photoinactivation of 100% of the colony forming units counted at 20 h
incubation versus control. It is probable that a lower energy
Table 1. Staphylococcus aureus photoinactivation experiment results.
870 nm + 930 nm
output power (W)
9.0
9.0
9.0
10.0
10.0
10.0
Plate
Beam spot
1.5 cm2
diameter
Treatment
time
(s)
Total
energy
(J)
Energy
density
(J cm)2)
Power
density
(W cm)2)
Control
CFUs
Postlaser
CFUs
Kill
percentage
1
2
3
4
5
6
1.76
1.76
1.76
1.76
1.76
1.76
720
720
720
720
720
720
6480
6480
6480
7200
7200
7200
3667
3667
3667
4074
4074
4074
5.09
5.09
5.09
5.66
5.66
5.66
430
400
390
413
378
363
320
280
316
6
5
11
25.6
30.0
19.0
98.5
98.7
97.0
Table 2. Escherichia coli photoinactivation experiment results.
870 nm + 930 nm
output power (W)
8.0
8.0
8.0
10.0
10.0
10.0
Plate
Beam spot
1.5 cm2
diameter
Treatment
time
(s)
Total
energy
(J)
Energy
density
(J cm)2)
Power
density
(W cm)2)
Control
CFUs
Postlaser
CFUs
Kill
percentage
1
2
3
4
5
6
1.76
1.76
1.76
1.76
1.76
1.76
720
720
720
720
720
720
5760
5760
5760
7200
7200
7200
3259
3259
3259
4074
4074
4074
4.53
4.53
4.53
5.66
5.66
5.66
95
91
81
79
64
91
80
68
74
5
1
1
15.8
25.3
8.6
93.7
98.4
98.9
Photochemistry and Photobiology, 2009, 85
1369
Table 3. Trichophyton rubrum photoinactivation experiment results.
870 nm + 930 nm
output power (W)
10.0
10.0
10.0
11.0
11.0
11.0
Plate
Beam spot
1.5 cm2
diameter
Treatment
time
(s)
Total
energy
(J)
Energy
density
(J cm)2)
Power
density
(W cm)2)
Control
CFUs
Postlaser
CFUs
Kill
percentage
1
2
3
4
5
6
1.76
1.76
1.76
1.76
1.76
1.76
720
720
720
720
720
720
7200
7200
7200
7920
7920
7920
4074
4074
4074
4500
4500
4500
5.68
5.68
5.68
6.25
6.25
6.25
13
16
15
27
17
24
0
0
0
0
0
0
100
100
100
100
100
100
Table 4. Candida albicans photoinactivation experiment results.
870 nm + 930 nm
output power (W)
10.0
10.0
10.0
11.0
11.0
11.0
Plate
Beam spot
1.5 cm2
diameter
Treatment
time
(s)
Total
energy
(J)
Energy
density
(J cm)2)
Power
density
(W cm)2)
Control
CFUs
Postlaser
CFUs
Kill
percentage
1
2
3
4
5
6
1.76
1.76
1.76
1.76
1.76
1.76
720
720
720
720
720
720
7200
7200
7200
7920
7920
7920
4074
4074
4074
4500
4500
4500
5.68
5.68
5.68
6.25
6.25
6.25
41
37
36
37
50
46
0
0
0
0
0
0
100
100
100
100
100
100
density would accomplish photoinactivation of these fungi
in vitro, and warrant further experiments (Table 4).
In vitro mouse 3T3 fibroblast photoinactivation experiments
Two equivalent samples were taken from the aggregate yield of
five T75 flasks of fibroblasts. The treatment sample was
exposed to 4686 J cm)2 in 12 min. Both samples were then
allowed to reattach to plate for 3 h in a 37"C incubator, then
removed from well via trypsinization and counted on Cedex.
There was only minimal viability loss (15%) in the treatment
sample at approximately 13% higher energy density and power
density than what proved 97–100% lethal for bacterial and
fungal samples in vitro (Tables 5 and 6). As the 3T3 fibroblasts
were only observed for 3 h postexposure, further data as to the
robustness of mammalian cells were gathered in vivo in
Yorkshire pigs with histological analysis and human studies.
These data are presented below.
Table 5. Mouse 3T3 fibroblast photoinactivation dosimetry.
Beam spot Treatment Total Energy Power
870 nm + 930 nm 1.5 cm2
time
energy density density
output power (W) diameter
(s)
(J) (J cm)2) (W cm)2)
11.5
1.76
720
8280
4686
6.51
Table 6. Mouse 3T3 fibroblast photoinactivation data.
Viability following 3 h
incubation postlasing
Treatment plate
Control plate
33.70%
39.60%
Statistical significance of photoinactivation in vitro results
Fisher’s exact test (comparing two binomial proportions) was
used to examine the success rate (defined as 100% photoinactivation of microbes) between those experiments conducted
below the threshold energy density value for 100% photoinactivation and those conducted at or above the threshold. Of
the six experiments conducted below the threshold energy
density value, none achieved 100% kill. In contrast, of the 18
experiments conducted at or above the threshold, 12 achieved
100% kill. The P-value associated with Fisher’s exact test
comparing the two success rates is 0.014. Of the six experiments
conducted at or above the threshold ED value that failed to
achieve 100% kill, none had a kill rate lower than 93.7%. In
contrast, of the six experiments conducted below the threshold
ED value, none had a kill rate higher than 30.0%.
In vitro sublethal dosimetry experiments for DY analysis
There are selected fluorescent dyes that can be taken up by
intact cells and accumulate within these cells within 15–30 min
without appreciable staining of other protoplasmic constituents. These dye indicators of membrane potential have been
available for many years and have been employed to study cell
physiology. The fluorescence intensity of these dyes can be
easily monitored, as their spectral fluorescent properties are
responsive to changes in the value of the transmembrane
potentials DYmt and DYp. We irradiated MRSA, C. albicans
and HEK cell cultures with sublethal dosimetry from the laser
system and subjected treatment and control cultures to the
commercial membrane potential assay described in the Materials and Methods section.
The MRSA (Fig. 3) data show that the red fluorescence
is dissipated versus control by pretreatment with the laser
and is dose dependent (4.53–4.81 W cm)2 for 26 min
and 4.53 W cm)2 for 30 min). This indicates that the laser
1370 Eric Bornstein et al.
exposure interacted sufficiently with respiratory processes of
the plasma membranes of the cells to alter and lower the
membrane potential DYp.
The HEK-293 data (Fig. 4) show that the ratio of green
fluorescence to red is increased versus control (i.e. red
fluorescence is dissipated) by pretreatment with the laser and
is dose dependent (4.53–4.81 W cm)2 for 26 min). This indicates that the laser exposure interacted sufficiently with
respiratory processes of the mitochondrial membranes of the
cells to alter and lower the mitochondrial membrane potential
DYmt. Decreased mitochondrial membrane potential values
were also seen with the C. albicans cell cultures.
and subjected tested and control samples to the commercial
glutathione assays described in the Materials and Methods
section. The data show that there was an increase in ROS
generated by the laser exposure in the C. albicans and HEK293 cells, and that the phenomenon is dose dependent (i.e.
reduced glutathione [GSH] % of total glutathione, as
described in Schroeder et al. [50]). This indicates that the laser
exposure interacted sufficiently with respiratory processes of
In vitro sublethal dosimetry experiments for glutathione analysis
We irradiated MRSA, C. albicans (Fig. 5) and HEK-293
(Fig. 6) cell cultures with sublethal dosimetry from the laser
Figure 5. Candida albicans. Reduced glutathione (GSH) % of total
glutathione in C. albicans after increasing dosage of nonlethal
870 nm ⁄ 930 nm laser energy.
Figure 3. DY MRSA data. Lowering effect on DYp in MRSA after
increasing dosage of nonlethal 870 nm ⁄ 930 nm laser energy. Red
fluorescence is dissipated versus control by pretreatment with the laser
and is dose dependent.
Figure 4. DY HEK-293 Data. Lowering effect on DYmt in HEK-293
cells after increasing dosage of nonlethal 870 nm ⁄ 930 nm laser energy.
Ratio of green fluorescence to red is increased versus control by
pretreatment with the laser and is dose dependent.
Figure 6. HEK-293 cells. Reduced glutathione (GSH) % of total
glutathione in HEK-293 cells after increasing dosage of nonlethal
870 nm ⁄ 930 nm laser energy.
Photochemistry and Photobiology, 2009, 85
1371
the cells to increase ROS. Similar values were also seen with
the MRSA cell cultures and the Glutathione assay.
In vivo Yorkshire pig dosimetry and histology thermal studies
Dosimetry tests were performed on the shaved backs of
Yorkshire pigs to measure histologic thermal effects of the
laser energies (40). Utilizing a 2.0 cm flat-top projection with a
blended 2.3 W output of combined (870 nm ⁄ 930 nm) energy
density (552 J cm)2) followed by 930 nm alone at 2.3 W
output at energy density (276 J cm)2), there were positive
thermal histological changes seen at 48 h. These changes
included slight subepidermal erythema, mild necrosis, mild
perivascular mononuclear cell infiltrate and mild collagen
degeneration. When measured again histologically at 72 h,
there was no discernable histological difference from the
control sites. The temperatures at this threshold energy density
(276 + 572 J cm)2 = 828 J cm)2) at a depth of 2 mm were
39.1"C after 450 s exposure, and 39.4"C after 570 s. The
temperatures observed at a depth of 20 mm were 40"C after
450 s exposure, and 41.6"C after 570 s. This threshold energy
density combination (828 J cm)2) was then defined by our
team as the maximum upper limit of possible dosimetry
parameters, to be applied to human skin, when measured up to
the published Individual Maximum Safe Radiant Exposure
(IMSRE) levels for human phototherapy and the ‘‘thermal
thresholds for tissue damage’’ data from previous studies
(49,51).
Figure 8. Human great toenail with onychomycosis. Representative
great toenail (baseline on left, day 63 on right) that received 612 J cm)2
of laser energy with 1.5 cm flat-top projection on day 1, day 10 and
day 40 of fungal photoinactivation study. Clear nail is beginning to
grow out from the nail matrix (growth center) of the nail by day 63,
before final scheduled dose of energy.
Human onychomycosis pilot study
All seven patients reached a mycological negative through nail
biopsy and culture at 60 days. The experimental temperatures
did not exceed 38"C, well within the (IMSRE) levels for human
phototherapy and thermal tissue damage thresholds (49,51,52).
No negative sequelae, adverse events or inhibited growth
(matrix damage) were recorded with observation from
a dermatologist of clear nail growth linearly from the matrix
in four subjects (Fig. 8) (53–55).
Human MRSA pilot study in the nose (nares)
Erythromycin-resistant MRSA was completely cleared by
triplicate culture in all three carriers after the second laser
treatment on day 3. All three cultures stayed clear through day
5 (Fig. 7). Erythromycin-resistant MSSA was completely
cleared by triplicate culture in four of the five carriers after
the second laser treatment on day 3 and remained clear
through day 5. In one patient the erythromycin-resistant
MSSA (baseline count >1000 CFUs) showed a 3 log reduction in MSSA on the day 5 culture. No negative sequelae or
adverse events were observed with an intranasal camera and
concurrent examination by a board-certified otolaryngologist.
The average maximum temperature of the nares reached in all
patients was 37.2"C (51) and was well within the (IMSRE)
levels for human phototherapy and thermal tissue damage
thresholds (49,51).
Figure 7. Erythromycin resistant MRSA. All erythromycin MRSA are
cleared from the human nares (shown by culture), after 1 laser
exposure with flat-top optical diffuser (193 J cm)2) followed by 2%
erythromycin paste.
DISCUSSION
An essential tenet of potential therapies for infectious disease
has always been that such therapies must be nontoxic to the
mammalian tissues that are infected to have any intrinsic
safety and value. Furthermore, it has generally been a fact that
antimicrobials affect either bacterial or fungal cellular processes that are not common to the mammalian host, giving them
their range of safety, and therapeutic value (56,57).
UV light has a DNA-damaging effect that is also common
to the mammalian tissues, and is therefore teratogenic to
humans (5,6,8). Hence, the question with the experiments we
have undertaken is: If within the path of the beam of blended
870 nm ⁄ 930 nm energy, the DY and Dp of cells (mammalian,
fungal, bacterial) are universally modified and lowered, and
ROS is universally generated, are there safe power densities
and energy densities available, with flat-top projections, where
these wavelengths could be used to treat cutaneous pathogens
in diverse human disease states?
All of our in vitro experimental data support a universal
lowering of DY and Dp among all seven cell types tested across
three major species and suggests that C. albicans and T. rubrum
will undergo photoinactivation with lower energy densities than
were employed in this series of experiments. The data lead to
the conclusion that not only the electromechanical, but also the
electrodynamical aspects of all cell membranes (mitochondrial
and plasma), have no differing properties that can adequately
be separated by the unique action spectrum of these infrared
wavelengths. This means that all cells in the path of the beam are
affected with a lowering of DY and Dp, and a generation of ROS,
not only the microbial cells. For the sublethal ROS and DY
1372 Eric Bornstein et al.
experiments on all cell types, we lowered the power density to
a level that failed to photoinactivate E. coli, and extended the
irradiation exposure times.
Our data agree with Schroeder et al. (50,58) in two
important areas. First, Schroeder used a broadband near-IR
source in human fibroblasts to show that the equilibrium of
reduced and oxidized glutathione shifted to the oxidized form
(i.e. GSH % of total glutathione) under near-IR irradiation,
suggesting ROS generation. Our data show similar results with
870 nm ⁄ 930 nm near-IR energy. Second, Schroeder showed
that a near-IR response involves generation of ROS that are
derived from the mitochondria in human fibroblasts. They
further proposed that the likely chromophores absorbing the
near-IR energy are the components of the electron transport
system (16,17,20). Therefore, when analyzing our data, it
stands to reason that similar components of the mammalian
electron transport systems (e.g. highly conserved cytochrome c
and lipid bilayers), that are present in prokaryotic and
eukaryotic cell types, would be affected in comparable
manners across the spectrum of species (12,35,36).
Additionally, our in vitro and human experimental results
support the supposition that the universal membrane depolarization caused by these wavelengths is more graded and
transient to mammalian cells in the path of the beam, than to
the bacteria and fungi at physiologic temperatures. This
suggests that the measures of temporal and energetic robustness of the mammalian cells appear to be greater in the face of
optical depolarization and subsequent ROS generation, than is
seen in the bacterial or fungal cells. This idea is implied in our
human pilot data via the photoinactivation of fungal pathogens in onychomycosis patients and the apparent resensitization of erythromycin-insensitive MRSA and MSSA to topical
2% erythromycin at physiologic temperatures.
One possible explanation for the MRSA pilot results is
that the optically mediated lowering of Dp and DY in the
resistant prokaryotic plasma membrane could inhibit the
functionality of efflux pumps to first-generation macrolides in
staphylococcus, as they rely on endogenous cellular energy
from a stable Dp and DY (45,46). This observation with
erythromycin is supported by research that has shown the
following: (1) Erythromycin is effluxed in S. aureus with an
ATP-coupled efflux pump (MRSA) that ceases to function in
the presence of the ATP synthesis inhibitor arsenate, and is
also sensitive to the uncoupler dinitrophenol (45). (2)
Another erythromycin efflux pump (MdeA) is dependent on
the proton motive force and an energized membrane (DY),
and has been shown to be sensitive to the uncouplers CCCP
and reserpine (46).
Further, the lack of observable negative sequelae in the
tested human tissues (nasal mucosa and nail matrix) is also
supported by Frank et al. (37), who found that the transient
decrease in the mitochondrial membrane potential (DYmt) in
mammalian mitochondria is a component of the ‘‘process’’ in
which IR radiation affects mammalian cells, yet that apoptosis
is not a forgone conclusion with transient membrane depolarization in human fibroblasts. These data are also in agreement
with the work of Davies (59), who suggested that researchers
had, heretofore, almost certainly underestimated the in vivo
(mammalian) cellular capacity to cope with oxidative stress.
Therefore these pilot data offer a provisional hope for further
therapy possibilities, as nasal colonization with MRSA, and
mycotic nails, are two important dermatologic infectious
disease states where patient outcomes would potentially
improve with alternative selective phototherapies (60–62).
In contrast, many researchers have reported that excessive
exposure to near-IR light will cause an oxidative stress
response in human skin that leads to premature photoaging
effects. This stress response is believed to originate from the
mitochondrial electron transport chain which causes a significant decrease in the antioxidant content of human skin.
Continued and repeated exposure to IR light has also been
shown to reduce the expression of Type I procollagen
(33,50,58,63). Near-IR energy can also have a detrimental
effect on the local immune system in mouse and human skin
(64–66). However, our human data suggest that a near-IR
photodamage effect can be produced against bacterial and
fungal pathogens in human subjects, while meeting the
published criteria for calculating the IMSRE for human
phototherapy, as no negative interactions were reported in
either the onychomycosis or MRSA human studies (51).
Finally, in further substantiation of the original work of
Neuman and Liu, we have recently determined that the
action spectrum of these wavelengths in vitro is unique in the
near IR at the dosimetries that we employed. In published action
spectrum photodamage experiments in vitro, with a threshold
energy density of 4482 J cm)2, with identical protocols to the
in vitro experiments described herein, we only produced an
average of 27% photoinactivation against E. coli with the
five different wavelength combinations of 830 nm ⁄ 930 nm,
810 nm ⁄ 930 nm, 885 nm ⁄ 930 nm, 810 nm ⁄ 870 nm and
870 nm ⁄ 885 nm (67).
Our a priori concept of optically mediated mechanotransduction, leading to universal membrane depolarization
can claim validity only insofar as it stands in clear relation to
the conservation of all normal cellular thermodynamic processes and does not violate the existing laws of photobiology
(68–71). Thus by reaffirming what the photobiology and
cellular energetics data of our experiments have already
revealed (i.e. that bacterial, fungal and mammalian redox
pathways are affected in the same manner with multiplexed
870 and 930 nm energies), we believe that this novel universal
optical depolarizing effect can be independently exploited in
cutaneous pathogens, at safe power densities, energy densities
and at physiologic temperatures. This technology hence
warrants further studies for human therapy.
Conflicts of interest—Eric Bornstein DMD is the Chief Science Officer
of Nomir Medical Technologies, the inventor of the Noveon#
phototherapy device. Dr. Bornstein is employed by Nomir and retains
stock in the company.
William Hermans and Scott Gridley are employees of Blue Sky
Biotech, an independent CRO contracted by Nomir Medical Technologies to complete parts of this research. Jeffrey Manni is an
employee of JGM Associates, Inc., an independent CRO contracted by
Nomir Medical Technologies to complete parts of this research.
Acknowledgements—None.
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