U.S. patent application number 17/687904 was filed with the patent office on 2022-06-23 for photoeradication of microorganisms with pulsed purple or blue light.
The applicant listed for this patent is CAREWEAR CORP., SAN DIEGO STATE UNIVERSITY RESEARCH FOUNDATION. Invention is credited to JOHN C. CASTEL, CHUKUKA S. ENWEMEKA.
Application Number | 20220193445 17/687904 |
Document ID | / |
Family ID | |
Filed Date | 2022-06-23 |
United States Patent
Application |
20220193445 |
Kind Code |
A1 |
ENWEMEKA; CHUKUKA S. ; et
al. |
June 23, 2022 |
PHOTOERADICATION OF MICROORGANISMS WITH PULSED PURPLE OR BLUE
LIGHT
Abstract
The present invention is directed to a system and method for
photoeradication of microorganisms from a target. The method
includes the step of obtaining test data for a plurality of
experiments each of which comprises irradiating test microorganisms
with a plurality of light pulses having a wavelength that ranges
from 380 nm to 500 nm. The light pulses have a plurality of pulse
parameters (peak irradiance, pulse duration, and off time between
adjacent light pulses) and are provided at a radiant exposure that
ranges from 0.5 J/cm.sup.2 to 60 J/cm.sup.2 during each of a
plurality of irradiation sessions. The test data comprises a
survival rate for the test microorganisms after irradiation with
the light pulses. The method also includes the step of analyzing
the test data to identify the pulse parameters for the light pulses
and the radiant exposure for each of the irradiation sessions that
result in a desired survival rate for the test microorganisms. The
method further includes the step of irradiating the microorganisms
of the target with light pulses having the identified pulse
parameters at the identified radiant exposure for each of the
irradiation sessions so as to photoeradicate all or a portion of
the microorganisms.
Inventors: |
ENWEMEKA; CHUKUKA S.; (SAN
DIEGO, CA) ; CASTEL; JOHN C.; (RENO, NV) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SAN DIEGO STATE UNIVERSITY RESEARCH FOUNDATION
CAREWEAR CORP. |
SAN DIEGO
RENO |
CA
NV |
US
US |
|
|
Appl. No.: |
17/687904 |
Filed: |
March 7, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16826229 |
Mar 21, 2020 |
11266855 |
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17687904 |
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16205182 |
Nov 29, 2018 |
10981017 |
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16826229 |
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15955773 |
Apr 18, 2018 |
10639498 |
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16205182 |
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PCT/US2017/034396 |
May 25, 2017 |
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15955773 |
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62341691 |
May 26, 2016 |
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International
Class: |
A61N 5/06 20060101
A61N005/06; A61L 2/24 20060101 A61L002/24; A61L 2/28 20060101
A61L002/28; A61L 2/00 20060101 A61L002/00; A61K 41/00 20060101
A61K041/00 |
Claims
1. A lighting system for sanitization of a surface located within
an environment contaminated with microorganisms, comprising: a
light source configured to generate purple or blue light having a
wavelength that ranges from 380 nm to 500 nm; and an electronic
circuit configured to control the light source so that the pulsed
purple or blue light is applied in a pulsed mode of irradiation to
provide a plurality of light pulses in which (a) the light pulses
have a peak irradiance and a pulse duration sufficient to optically
excite a photoactive molecule capable of photoeradication of the
microorganisms and (b) the light pulses are separated by an off
time sufficient to allow the photoactive molecule to return to a
ground state creating an oxidation reaction that produces free
radicals which destroy a cellular structure of the microorganisms
to thereby cause photoeradication of all or a portion of the
microorganisms on the surface.
2. The lighting system of claim 1, wherein the light source is
configured to directly illuminate the surface with the purple or
blue light.
3. The lighting system of claim 1, further comprising an optical
element configured to redirect the purple or blue light from the
light source to the surface.
4. The lighting system of claim 3, wherein the optical element
comprises a diffuser configured to evenly distribute the purple or
blue light across the surface.
5. The lighting system of claim 1, wherein the light source
comprises one of lasers, light emitting diodes (LEDs), organic
light emitting diodes (OLEDs), printed light emitting diodes
(printed LEDs), polymer light emitting diodes (PLEDs), quantum dot
light emitting diodes (QDLEDs), and fluorescent tubes.
6. The lighting system of claim 1, wherein the peak irradiance of
the light pulses at the surface ranges from 0.3 mW/cm.sup.2 to 60
mW/cm.sup.2, the pulse duration of the light pulses ranges from 5
microseconds to 1,000 microseconds, and the off time between the
light pulses ranges from 10 microseconds to 1 second.
7. The lighting system of claim 1, wherein the light pulses are
provided at a duty factor that ranges from 20% to 33% and a pulse
repetition rate that ranges from 33 kHz to 40 kHz.
8. The lighting system of claim 1, wherein the light source is
further configured to generate white light, and wherein the
electronic circuit is configured to control the light source so
that the white light is applied in a continuous wave mode of
irradiation to thereby provide lighting within the environment.
9. The lighting system of claim 8, wherein the electronic circuit
is configured to control the light source so that the white light
has a color temperature in the range of 1700 kelvins (K) to 9500
kelvins (K).
10. The lighting system of claim 8, wherein the electronic circuit
is configured to control the light source so that the white light
is applied either simultaneously or sequentially with the pulsed
purple or blue light.
11. The lighting system of claim 1, wherein the microorganisms
comprise an aerobic or anaerobic bacteria (Gram positive and Gram
negative).
12. The lighting system of claim 11, wherein the microorganisms
comprise one of methicillin-resistant Staphylococcus aureus (MRSA),
Salmonella spp., E. coli, and Listeria spp.
13. The lighting system of claim 1, wherein the microorganisms
comprise a virus.
14. The lighting system of claim 13, wherein the microorganisms
comprise one of SARS-CoV, 2019-nCoV, human coronavirus 229E, bat
coronavirus HKU9-1, H1N1, norovirus (NoV), feline calicivirus,
influenza virus type A, HCoV-OC43, and HCoV-NL63.
15. The lighting system of claim 1, wherein the microorganisms
comprise a fungus.
16. The lighting system of claim 1, wherein the environment
comprises one of a locker room, a restroom, an airplane, a school,
a beach, a playground, a playing field, a hospital, and a clinical
environment.
17. A lighting system for photoeradication of microorganisms from a
surface, comprising: a light source configured to generate purple
or blue light having a wavelength that ranges from 380 nm to 500
nm; and an electronic circuit configured to control the light
source so that the purple or blue light is applied in a pulsed mode
of irradiation to thereby cause photoeradication of all or a
portion of the microorganisms on the surface.
18. The lighting system of claim 17, wherein the light source is
configured to directly illuminate the surface with the purple or
blue light.
19. The lighting system of claim 17, further comprising an optical
element configured to redirect the purple or blue light from the
light source to the surface.
20. The lighting system of claim 19, wherein the optical element
comprises a diffuser configured to evenly distribute the purple or
blue light across the surface.
21. The lighting system of claim 17, wherein the light source
comprises one of lasers, light emitting diodes (LEDs), organic
light emitting diodes (OLEDs), printed light emitting diodes
(printed LEDs), polymer light emitting diodes (PLEDs), quantum dot
light emitting diodes (QDLEDs), and fluorescent tubes.
22. The lighting system of claim 17, wherein the purple or blue
light comprises a plurality of light pulses in which (a) the light
pulses have a peak irradiance and a pulse duration sufficient to
optically excite a photoactive molecule capable of photoeradication
of the microorganisms and (b) the light pulses are separated by an
off time sufficient to allow the photoactive molecule to return to
a ground state creating an oxidation reaction that produces free
radicals which destroy a cellular structure of the microorganisms
to thereby cause photoeradication of all or a portion of the
microorganisms.
23. The lighting system of claim 17, wherein the purple or blue
light comprises a plurality of light pulses that destroy the RNA or
DNA genomes of all or a portion of the microorganisms.
24. The lighting system of claim 17, wherein the purple or blue
light comprises a plurality of light pulses in which the peak
irradiance of the light pulses at the surface ranges from 0.3
mW/cm.sup.2 to 60 mW/cm.sup.2, the pulse duration of the light
pulses ranges from 5 microseconds to 1,000 microseconds, and the
off time between the light pulses ranges from 10 microseconds to 1
second.
25. The lighting system of claim 17, wherein the purple or blue
light comprises a plurality of light pulses provided at a duty
factor that ranges from 20% to 33% and a pulse repetition rate that
ranges from 33 kHz to 40 kHz.
26. The lighting system of claim 17, wherein the light source is
further configured to generate white light, and wherein the
electronic circuit is configured to control the light source so
that the white light is applied in a continuous wave mode of
irradiation.
27. The lighting system of claim 26, wherein the electronic circuit
is configured to control the light source so that the white light
has a color temperature in the range of 1700 kelvins (K) to 9500
kelvins (K).
28. The lighting system of claim 26, wherein the electronic circuit
is configured to control the light source so that the white light
is applied either simultaneously or sequentially with the pulsed
purple or blue light.
29. The lighting system of claim 17, wherein the microorganisms
comprise an aerobic or anaerobic bacteria (Gram positive and Gram
negative).
30. The lighting system of claim 29, wherein the microorganisms
comprise one of methicillin-resistant Staphylococcus aureus (MRSA),
Salmonella spp., E. coli, and Listeria spp.
31. The lighting system of claim 17, wherein the microorganisms
comprise a virus.
32. The lighting system of claim 31, wherein the microorganisms
comprise one of SARS-CoV, 2019-nCoV, human coronavirus 229E, bat
coronavirus HKU9-1, H1N1, norovirus (NoV), feline calicivirus,
influenza virus type A, HCoV-OC43, and HCoV-NL63.
33. The lighting system of claim 17, wherein the microorganisms
comprise a fungus.
34. The lighting system of claim 17, wherein the surface is located
within an environment contaminated with the microorganisms.
35. The lighting system of claim 34, wherein the environment
comprises one of a locker room, a restroom, an airplane, a school,
a beach, a playground, a playing field, a hospital, and a clinical
environment.
36. Alighting system for photoeradication of microorganisms from a
surface located within an environment, comprising: a light source
configured to generate purple or blue light and white light; and an
electronic circuit configured to control the light source so that
(a) the purple or blue light is applied in a pulsed mode of
irradiation to thereby cause photoeradication of all or a portion
of the microorganisms on the surface and (b) the white light is
applied in a continuous wave mode of irradiation to thereby
providing lighting within the environment.
37. The lighting system of claim 36, wherein the electronic circuit
is configured to control the light source so that the white light
has a color temperature in the range of 1700 kelvins (K) to 9500
kelvins (K).
38. The lighting system of claim 36, wherein the electronic circuit
is configured to control the light source so that the white light
is applied either simultaneously or sequentially with the pulsed
purple or blue light.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of and claims priority to
U.S. patent application Ser. No. 16/826,229, filed on Mar. 21,
2020, which is a continuation-in-part of and claims priority to
U.S. patent application Ser. No. 16/205,182, filed on Nov. 29,
2018, which is a continuation-in-part of and claims priority to
U.S. patent application Ser. No. 15/955,773, filed on Apr. 18,
2018, which is a continuation of and claims priority to PCT Patent
Application Serial No. PCT/US2017/034396, filed on May 25, 2017,
which is based on and claims priority to U.S. Provisional
Application Ser. No. 62/341,691, filed on May 26, 2016, each of
which is incorporated herein by reference in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not applicable.
STATEMENT REGARDING JOINT RESEARCH AGREEMENT
[0003] Not applicable.
BACKGROUND OF THE INVENTION
[0004] Various devices for delivering light to a region of skin for
therapeutic or cosmetic purposes are known in the art. The use of
phototherapy, in particular blue light, as an armamentarium for
antimicrobial activity has been of great interest, particularly
since Enwemeka et al., first reported in 2007 that 405 nm and 470
nm light inactivate the methicillin-resistant Staphylococcus aureus
(MRSA) bacteria. Current research indicates that bacteria kill rate
is tied to the intensity and total amount of irradiation energy;
that is, the higher the intensity used and the higher the total
energy of irradiation, the better the bacteria kill rate. For
example, conventional light emitting diodes (LEDs) operating in a
continuous wave (CW) mode of irradiation have been used to deliver
light at high irradiances and radiant exposures to increase
bacteria kill rates. However, there is a risk that these high
irradiances and radiant exposures may damage other tissues in the
region under treatment through thermal or photochemical effects or
may provide a significant optical hazard to the subject undergoing
treatment.
BRIEF SUMMARY OF THE INVENTION
[0005] The present invention is directed to a system and method for
photoeradication of microorganisms from a target using pulsed
purple or blue light. In some embodiments, the target includes an
endogenous or exogenous photoactive molecule in which (a) the light
pulses have a peak irradiance and a pulse duration sufficient to
optically excite the photoactive molecule and (b) the light pulses
are separated by an off time sufficient to allow the photoactive
molecule to return to a ground state creating an oxidation reaction
that produces free radicals which destroy a cellular structure of
all or a portion of the microorganisms. In other embodiments, the
target is irradiated with pulsed purple or blue light so as to
destroy the RNA or DNA genomes of all or a portion of the
microorganisms.
[0006] In a first embodiment, the target comprises a region of
skin, tissue, a wound, or cells of a human or animal body that are
infected by various types of microorganisms, such as bacteria,
viruses or fungi. The infected skin, tissue, wound or cells are
treated by irradiating the target with pulsed purple or blue light.
Preferably, the pulsed purple or blue light is provided at
specified pulse parameters, dosages and time intervals so as to
inactivate all or a portion of the microorganisms at low
irradiances and radiant exposures compared to those of a continuous
wave (CW) mode of irradiation. As a result, it is believed that the
microorganisms can be inactivated without damage to other tissues
or cells in the region under treatment. In some cases, the target
is a host cell subject to viral invasion that is destroyed without
disrupting non-invaded cells.
[0007] In a second embodiment, the target comprises an environment
contaminated with various types of microorganisms, such as
methicillin-resistant Staphylococcus aureus (MRSA) or viruses such
as SARS-CoV, 2019-nCoV, human coronavirus 229E, bat coronavirus
HKU9-1, H1N1, norovirus (NoV) and feline calicivirus (which are
part of the Caliciviridae family), influenza virus type A,
HCoV-OC43, and HCoV-NL63. The contaminated environment may
comprise, for example, a locker room, a public or private restroom,
an airplane, a school, a beach, a playground, a playing field, a
hospital or a clinical environment. The contaminated environment is
irradiated with pulsed purple or blue light as discussed above so
as to photoeradicate the microorganisms and thereby sanitize the
area.
[0008] In a third embodiment, the target comprises food under
storage or transport conditions that is contaminated with
microorganisms, such as Salmonella spp., E. coli, Listeria spp,
norovirus (NoV) (which is part of the Caliciviridae family), or
hepatitis A. The food may be contained within, for example, a
refrigeration system, a food display system, a food storage area,
or a food processing system. The food is irradiated with pulsed
purple or blue light as discussed above so as to photoeradicate the
microorganisms and thereby enhance shelf-life and reduce the
potential for significant infection transmission to human and
animal populations.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] Various exemplary embodiments of the present invention are
described in detail below with reference to the attached drawing
figures, as described below.
[0010] FIG. 1 is a graph depicting P. acnes survival when
irradiated with 450 nm light at radiant exposures of 20 and 60
J/cm.sup.2; irradiance of 4.5 mW/cm.sup.2; continuous wave (CW)
mode of irradiation; and single irradiation at 0 hours.
[0011] FIG. 2 is a graph depicting P. acnes survival when
irradiated with 450 nm light at a radiant exposure of 20
J/cm.sup.2; irradiance of 4.5 mW/cm.sup.2; continuous wave (CW)
mode of irradiation; and multiple irradiation at 0, 24 and 48
hours.
[0012] FIG. 3 is a graph depicting P. acnes survival when
irradiated with 450 nm light at a radiant exposure of 20
J/cm.sup.2; irradiance of 4.5 mW/cm.sup.2; continuous wave (CW)
mode of irradiation; and multiple irradiation at 0, 4, 24 and 48
hours.
[0013] FIG. 4 is a graph depicting P. acnes survival when
irradiated with 450 nm light at radiant exposures of 30, 40, 45 and
60 J/cm.sup.2; irradiance of 4.5 mW/cm.sup.2; continuous wave (CW)
mode of irradiation; and multiple irradiation at 0 and 4 hours.
[0014] FIG. 5 is a graph depicting P. acnes survival when
irradiated with 450 nm light at a radiant exposure of 60
J/cm.sup.2; average irradiance of 5 mW/cm.sup.2; continuous wave
(CW) mode of irradiation and pulsed mode of irradiation (33% duty
factor); and multiple irradiation at 0 and 4 hours.
[0015] FIG. 6 is a graph depicting P. acnes survival when
irradiated with 450 nm light at a radiant exposure of 20
J/cm.sup.2; average irradiance of 5 mW/cm.sup.2; continuous wave
(CW) mode of irradiation and pulsed mode of irradiation (33% and
20% duty factors); and multiple irradiation at 0 and 4 hours.
[0016] FIG. 7 is a graph depicting P. acnes survival when
irradiated with 450 nm light at radiant exposures of 5, 10 and 20
J/cm.sup.2; average irradiance of 2 mW/cm.sup.2; pulsed mode of
irradiation (33% duty factor); and multiple irradiation at 0, 4, 24
and 48 hours.
[0017] FIG. 8 is a graph depicting P. acnes survival when
irradiated with 450 nm light at radiant exposures of 5, 10 and 20
J/cm.sup.2; average irradiance of 3.5 mW/cm.sup.2; pulsed mode of
irradiation (33% duty factor); and multiple irradiation at 0, 4, 24
and 48 hours.
[0018] FIG. 9 is a graph depicting P. acnes survival when
irradiated with 450 nm light at radiant exposures of 5, 10 and 20
J/cm.sup.2; irradiance of 5 mW/cm.sup.2; continuous wave (CW) mode
of irradiation; and multiple irradiation at 0, 4, 24 and 48
hours.
[0019] FIG. 10 is a graph depicting P. acnes survival when
irradiated with 450 nm light at radiant exposures of 5, 10 and 20
J/cm.sup.2; average irradiance of 3 mW/cm.sup.2; pulsed mode of
irradiation (33% duty factor); and multiple irradiation at 0, 4, 24
and 48 hours.
[0020] FIG. 11 is a graph depicting P. acnes survival when
irradiated with 450 nm light at radiant exposures of 5, 10 and 20
J/cm.sup.2; average irradiance of 3 mW/cm.sup.2; pulsed mode of
irradiation (33% duty factor); and multiple irradiation at 0, 3, 6
and 24 hours.
[0021] FIG. 12 is a graph depicting P. acnes survival when
irradiated with 450 nm light at radiant exposures of 5, 10 and 20
J/cm.sup.2; average irradiance of 3 mW/cm.sup.2; pulsed mode of
irradiation (33% duty factor); and multiple irradiation at 0, 3 and
6 hours.
[0022] FIG. 13 is a graph depicting P. acnes survival when
irradiated with 450 nm light at a radiant exposure of 5 J/cm.sup.2;
average irradiance of 3 mW/cm.sup.2; pulsed mode of irradiation
(33% duty factor); and multiple irradiation at 0, 3, 6, 24, 27, 30,
48, 51 and 54 hours.
[0023] FIG. 14 is a graph depicting P. acnes survival when
irradiated with 450 nm light at a radiant exposure of 5 J/cm.sup.2;
average irradiance of 3 mW/cm.sup.2; pulsed mode of irradiation
(33% duty factor); and multiple irradiation at 0, 3, 6, 24, 27, 30,
48, 51, 54, 72, 75 and 78 hours.
[0024] FIG. 15 is a graph depicting P. acnes survival when
irradiated with 450 nm light at a radiant exposure of 5 J/cm.sup.2;
average irradiance of 2 mW/cm.sup.2; pulsed mode of irradiation
(33% duty factor); and multiple irradiation at 0, 3, 6, 24, 27, 30,
48, 51, 54, 72, 75 and 78 hours.
[0025] FIG. 16 is a graph depicting P. acnes survival when
irradiated with 450 nm light at a radiant exposure of 5 J/cm.sup.2;
average irradiance of 2 mW/cm.sup.2; pulsed mode of irradiation
(33% duty factor); and multiple irradiation at 0, 3, 6, 24, 27, 30,
48, 51 and 54 hours.
[0026] FIG. 17 is a graph depicting P. acnes survival when
irradiated with 450 nm light at a radiant exposure of 3.6
J/cm.sup.2; average irradiance of 2 mW/cm.sup.2; pulsed mode of
irradiation (33% duty factor); and multiple irradiation at 0, 3,
24, 27, 48, 51, 72 and 75 hours.
[0027] FIG. 18 is a graph depicting P. acnes survival when
irradiated with 450 nm light at a radiant exposure of 3.6
J/cm.sup.2; average irradiance of 2 mW/cm.sup.2; pulsed mode of
irradiation (33% duty factor); and multiple irradiation at 0, 3,
24, 27, 48, 51, 72, 75, 96 and 99 hours.
[0028] FIG. 19 is a graph depicting P. acnes survival when
irradiated with 450 nm light at a radiant exposure of 3.6
J/cm.sup.2; average irradiance of 2 mW/cm.sup.2; pulsed mode of
irradiation (33% duty factor); and multiple irradiation at 0, 3, 6,
24, 27, 30, 48, 51, 54, 72, 75 and 78 hours.
[0029] FIG. 20 is a graph depicting P. acnes survival when
irradiated with 450 nm light at a radiant exposure of 5 J/cm.sup.2;
average irradiance of 2 mW/cm.sup.2; pulsed mode of irradiation
(33% duty factor); and multiple irradiation at 0, 3, 6, 24, 27, 30,
48, 51 and 54 hours.
[0030] FIG. 21 is a graph depicting P. acnes survival when
irradiated with 450 nm light at a radiant exposure of 3.6
J/cm.sup.2; average irradiance of 2 mW/cm.sup.2; pulsed mode of
irradiation (33% duty factor); and multiple irradiation at 0, 3, 6,
24, 27, 30, 48, 51 and 54 hours.
[0031] FIG. 22 illustrates a plurality of light emitting diodes
printed on a flexible film (referred to herein as a printed LED
flexible lamp or a lighted substrate) that is suitable for use in
accordance with the present invention;
[0032] FIG. 23 is a schematic illustration depicting an
experimental set-up used in bacteria irradiation with a 458 nm
laser light.
[0033] FIG. 24A is a graph depicting porphyrin emission extracted
from P. acnes excited with 458 nm light.
[0034] FIG. 24B is a graph depicting porphyrin fluorescence
emission extracted from P. acnes excited with 450 nm light from the
printed LED flexible lamp shown in FIG. 22 at an average irradiance
of 3 mW/cm.sup.2; pulsed mode of irradiation (33% duty factor); and
multiple irradiation at 0, 10, 20 and 30 minutes.
[0035] FIG. 24C is a graph depicting porphyrin emission extracted
from a sample of Protoporphyrin IX excited with 450 nm light from
the printed LED flexible lamp shown in FIG. 22.
[0036] FIG. 25 illustrates the general structure of a light source
in the form of a very thin layered structure that is suitable for
delivering pulsed light in accordance with the present
invention.
[0037] FIGS. 26A and 26B illustrate various exemplary shapes of
light sources that are suitable for use in the light source of FIG.
25.
[0038] FIG. 27 illustrates an exemplary OLED structure for use in
the light source of FIG. 25.
[0039] FIG. 28 illustrates an exemplary printable LED structure for
use in the light source of FIG. 25.
[0040] FIG. 29 illustrates a light source with a bottom light
emitting configuration that is suitable for delivering pulsed light
in accordance with the present invention.
[0041] FIG. 30 illustrates a light source with a top light emitting
configuration that is suitable for delivering pulsed light in
accordance with the present invention.
[0042] FIG. 31 is a block diagram of an electronic circuit for
controlling a light source in accordance with the present
invention, wherein the electronic circuit includes a drive circuit
and microcontroller that is preprogrammed to provide a fixed dose
of light.
[0043] FIG. 32 is a block diagram of an electronic circuit for
controlling a light source in accordance with the present
invention, wherein the electronic circuit includes sensors that
operate in a closed loop to provide feedback to a microcontroller
so as to dynamically control the light source.
[0044] FIGS. 33 and 34 are graphs depicting GBS survival for
different combinations of 0.002, 0.02 or 0.05 mg/mL PPIX
supplementation and/or irradiation with 450 nm light at a radiant
exposure of 7.56 J/cm.sup.2; average irradiance of 3 mW/cm.sup.2;
pulsed mode of irradiation (33% duty factor); and multiple
irradiation at 0, 72 and 144 hours.
[0045] FIG. 35 is a graph depicting GBS survival for different
combinations of 0.002 or 0.05 mg/mL CP III supplementation and/or
irradiation with 450 nm light at a radiant exposure of 7.56
J/cm.sup.2; average irradiance of 3 mW/cm.sup.2; pulsed mode of
irradiation (33% duty factor); and multiple irradiation at 0, 72
and 144 hours.
[0046] FIG. 36 is a graph depicting GBS survival for different
combinations of 0.002 or 0.02 mg/mL CP III supplementation and/or
irradiation with 450 nm light at a radiant exposure of 7.56
J/cm.sup.2; average irradiance of 3 mW/cm.sup.2; pulsed mode of
irradiation (33% duty factor); and multiple irradiation at 0, 72
and 144 hours.
[0047] FIGS. 37 and 38 are graphs depicting GBS survival for
different combinations of 0.02 mg/mL PPIX supplementation (with
optional incubation and wash after supplementation) and/or
irradiation with 450 nm light at a radiant exposure of 7.56
J/cm.sup.2; average irradiance of 3 mW/cm.sup.2; pulsed mode of
irradiation (33% duty factor); and multiple irradiation at 0, 72
and 144 hours.
[0048] FIGS. 39, 40 and 41 are graphs depicting GBS survival for
different combinations of 0.05 mg/mL PPIX supplementation (with
optional incubation and wash after supplementation) and/or
irradiation with 450 nm light at a radiant exposure of 7.56
J/cm.sup.2; average irradiance of 3 mW/cm.sup.2; pulsed mode of
irradiation (33% duty factor); and multiple irradiation at 0, 72
and 144 hours.
[0049] FIG. 42 is a graph depicting GBS survival for different
combinations of 0.05 mg/mL CP III supplementation (with optional
incubation and wash after supplementation) and/or irradiation with
450 nm light at a radiant exposure of 7.56 J/cm.sup.2; average
irradiance of 3 mW/cm.sup.2; pulsed mode of irradiation (33% duty
factor); and multiple irradiation at 0, 72 and 144 hours.
[0050] FIGS. 43 and 44 are graphs depicting GBS survival for
different combinations of 0.08 mg/mL PPIX supplementation (with
optional incubation and wash after supplementation) and/or
irradiation with 450 nm light at a radiant exposure of 7.56
J/cm.sup.2; average irradiance of 3 mW/cm.sup.2; pulsed mode of
irradiation (33% duty factor); and multiple irradiation at 0, 72
and 144 hours.
[0051] FIGS. 45, 46 and 47 are graphs depicting GBS survival for
different combinations of 0.2 mg/mL PPIX supplementation (with
optional incubation and wash after supplementation) and/or
irradiation with 450 nm light at a radiant exposure of 7.56
J/cm.sup.2; average irradiance of 3 mW/cm.sup.2; pulsed mode of
irradiation (33% duty factor); and multiple irradiation at 0, 72
and 144 hours.
[0052] FIGS. 48 and 49 are graphs depicting GBS survival for
different combinations of 0.2 mg/mL CP III supplementation (with
optional incubation and wash after supplementation) and/or
irradiation with 450 nm light at a radiant exposure of 7.56
J/cm.sup.2; average irradiance of 3 mW/cm.sup.2; pulsed mode of
irradiation (33% duty factor); and multiple irradiation at 0, 72
and 144 hours.
[0053] FIG. 50 is a graph depicting GBS survival for different
combinations of 0.08 mg/mL CP III supplementation (with optional
incubation and wash after supplementation) and/or irradiation with
450 nm light at a radiant exposure of 7.56 J/cm.sup.2; average
irradiance of 3 mW/cm.sup.2; pulsed mode of irradiation (33% duty
factor); and multiple irradiation at 0, 72 and 144 hours.
[0054] FIG. 51 illustrates a light device incorporating printed
light emitting diodes placed externally on a cheek region of a
human body for use in accordance with the present invention.
[0055] FIG. 52 illustrates a light device incorporating printed
light emitting diodes placed externally on an inner wrist region of
a human body for use in accordance with the present invention.
[0056] FIGS. 53A and 53B illustrate a nasal applicator
incorporating printed light emitting diodes that is suitable for
use in accordance with the present invention.
[0057] FIGS. 54A, 54B, 54C, 54D and 54E illustrate various
embodiments of a respirator mask incorporating printed light
emitting diodes that is suitable for use in accordance with the
present invention.
[0058] FIG. 55 is a schematic illustration depicting an
experimental set-up used in bacteria irradiation with pulsed 450 nm
blue light.
[0059] FIG. 56 are light micrographs showing non-irradiated control
MRSA (left panel) and irradiated MRSA (right panel).
[0060] FIG. 57 are transmission electron microscope images showing
normal cell division in non-irradiated control MRSA (A and C) and
disruption of normal cell division in irradiated MRSA (B and
D).
[0061] FIG. 58 are transmission electron microscope images showing
structural damage to irradiated MRSA (B) compared to non-irradiated
control MRSA (A), as well as normal cell division in non-irradiated
control MRSA (A) compared to irradiated MRSA (B).
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0062] The present invention is directed to methods and systems for
the photoeradication of microorganisms from a target using pulsed
purple or blue light (and optionally pulsed ultraviolet, red or
infrared light). While the invention will be described in detail
below with reference to various exemplary embodiments, it should be
understood that the invention is not limited to the specific
methodologies or device configurations of these embodiments. In
addition, although the exemplary embodiments are described as
embodying several different inventive features, one skilled in the
art will appreciate that any one of these features could be
implemented without the others in accordance with the present
invention.
I. General Overview
[0063] The methods and systems of the present invention may be used
to photoeradicate many different types of microorganisms with
pulsed purple or blue light (and optionally pulsed ultraviolet, red
or infrared light) provided at specified pulse parameters, dosages
and time intervals. In some embodiments, the pulsed light consists
of a sequence of light pulses each of which has a peak irradiance
and a pulse duration sufficient to optically excite photoactive
molecules capable of photoeradication of the microorganisms and
move them to an excited singlet state. The light pulses are
separated by an off time sufficient to allow the photoactive
molecules to return to their ground state, which transition creates
a reaction with triplet oxygen that reduces the oxygen molecule to
a highly reactive singlet state which includes singlet oxygen
(.sup.1O.sub.2), hydroxyl radicals (.OH) and superoxide
(O.sub.2.sup.-) ions. These are capable of destroying cellular
structure of the microorganisms and thereby photoeradicate all or a
portion of the microorganisms. This mechanism of microorganism
suppression or eradication is broad spectrum and believed to be
applicable to various types of microorganisms, including bacteria,
viruses and fungi. In contrast, when light is applied in a
continuous wave mode, the photoactive molecules are maintained in
an excited state and fewer return to a ground state (they do so
randomly as opposed to a cascade that follows the pulsing) and,
thus, the effect of the light is reduced.
[0064] In a first embodiment, the invention is used to provide
treatment for many different types of diseases, disorders, or
conditions. For example, a region of skin, tissue, or a wound
infected by bacteria may be irradiated with pulsed purple or blue
light for the treatment of a bacterial infection. The treatment can
be provided to subjects having or who are susceptible to or at
elevated risk for experiencing a bacterial skin, tissue or wound
infection. Subjects may be susceptible to or at elevated risk for
experiencing a bacterial infection due to family history, age,
environment, and/or lifestyle. As used herein, "susceptible" and
"at risk" refer to having little resistance to a certain disease,
disorder or condition, including being genetically predisposed,
having a family history of, and/or having symptoms of the disease,
disorder or condition.
[0065] The bacterial infection may comprise any infection caused by
aerobic and anaerobic bacteria (Gram positive and Gram negative).
Exemplary bacterial infections include acne, psoriasis, cellulitis,
erysipelas, erythrasma, folliculitis and skin abscesses,
hidradenitis suppurativa, impetigo and ecthyma, lymphadenitis,
lymphangitis, necrotizing skin infections, staphylococcal scalded
skin syndrome, as well as wound and tissue infections such as
osteomyelitis.
[0066] As another example, cells infected by various types of
viruses may be irradiated with pulsed purple or blue light for the
treatment of a viral infection. The treatment can be provided to
subjects having or who are susceptible to or at elevated risk for
experiencing a viral infection. Exemplary viral infections include
SARS-CoV, 2019-nCoV, human coronavirus 229E, bat coronavirus
HKU9-1, H1N1, norovirus (NoV) and feline calicivirus (which are
part of the Caliciviridae family), influenza virus type A,
HCoV-OC43, and HCoV-NL63.
[0067] In particular, the current emergence of the 2019-nCoV and
the previous emergence of the severe acute respiratory syndrome
(SARS), caused by a formerly un-known coronavirus, SARS-CoV,
exemplify the considerable zoonotic potential of coronaviruses and
their ability to seriously affect human health. The novel
coronavirus, 2019-nCoV, is a virus closely related to SARS-CoV and
human coronavirus 229E, and originated from Wuhan, China. Both
2019-nCoV and the SARS-CoV share a common ancestor that resembles
the bat coronavirus HKU9-1.
[0068] The human airway epithelium (HAE) represents the entry port
of many human respiratory viruses, including coronaviruses (CoVs),
which have very similar spike protein 3-D structures that are
considered to have strong binding affinity to the human cell
receptor, angiotensin-converting enzyme 2 (ACE2). Therefore, the
cells with ACE2 expression may act as target cells and thus are
susceptible to 2019-nCoV infection. Such cells include type II
alveolar cells (AT2) or respiratory epithelial cells of the lungs.
The symptoms of COVID-19, which is the disease caused by 2019-nCoV,
include fever, cough, shortness of breath, headache and sore
throat. There are presently no antivirals or vaccines, and the
current treatment of COVID-19 is supportive care (treatment of
symptoms).
[0069] As another example, cells infected by various types of fungi
may be irradiated with pulsed purple or blue light for the
treatment of a fungal infection. The treatment can be provided to
subjects having or who are susceptible to or at elevated risk for
experiencing a fungal infection. Exemplary fungal infections
include tinea capitis, tinea corporis, tinea cruris, tinea pedis,
and tinea unguium.
[0070] In a second embodiment, the invention is used to enable
photoeradication of microorganisms from contaminated environments.
The microorganisms may comprise any aerobic and anaerobic bacteria
(Gram positive and Gram negative) or any virus. For example,
community associated methicillin-resistant Staphylococcus aureus
(MRSA) strains or SARS-CoV, 2019-nCoV, human coronavirus 229E, bat
coronavirus HKU9-1, H1N1, norovirus (NoV) and feline calicivirus
(which are part of the Caliciviridae family), influenza virus type
A, HCoV-OC43, and HCoV-NL63 are often acquired from places that
people congregate, such as locker rooms, public or private
restrooms, airplanes, ambulances, fire trucks, emergency services
personnel vehicles, schools, beaches, playgrounds, playing fields,
etc. Hospital associated MRSA or SARS-CoV, 2019-nCoV, human
coronavirus 229E, bat coronavirus HKU9-1, H1N1, norovirus (NoV) and
feline calicivirus (which are part of the Caliciviridae family),
influenza virus type A, HCoV-OC43, and HCoV-NL63 are typically
contacted in hospitals and clinical environments. These
contaminated environments may be irradiated with pulsed purple or
blue light to provide a more effective way to sanitize such places.
The pulsed purple or blue light may be placed together with a white
light to act as a dual light source, one for lighting and one for
bactericidal or anti-viral effects. The pulse treatment sequences
could be applied daily to the environment or on any other periodic
or scheduled basis (e.g., the pulse treatment sequence could be
applied to the interior surfaces of an airplane between the various
successive flights).
[0071] In a third embodiment, the invention is used to enable
photoeradication of microorganisms from food. The bacteria may
comprise any aerobic and anaerobic bacteria (Gram positive and Gram
negative), norovirus (NoV) (which is part of the Caliciviridae
family), or hepatitis A. For example, foods may be contaminated
with Salmonella spp., E. coli, or Listeria spp. Salmonella spp.
contains CspH, a cold shock protein that protects the bacteria at
50.degree. C. As such, refrigeration at this temperature may not be
adequate to kill the bacteria. Refrigeration in retail may be at a
higher temperature further exacerbating the problem of bacterial
growth. Globally, there are more than 80.3 million cases annually
of Salmonella spp. with more than 115,000 deaths. Contaminated
foods may be irradiated with pulsed purple or blue light to provide
a more effective way to extend shelf life and prevent bacterial
transmission to humans and animals exposed to such foods. The
pulsed light may be built into commercial, residential or portable
refrigeration systems, food display systems, food storage areas and
systems for food processing. For example, the pulsed purple or blue
light may be activated whenever a door of a refrigeration system is
opened and closed. When the door is opened, the light would be
white and when closed the pulsed purple or blue light would be
applied at the correct dose and treatment sequence. The treatment
sequence would be applied daily and/or as new food items are added
to the food storage or processing system.
[0072] For all three embodiments discussed above, exemplary
bacteria include Propionibacterium acnes, Group B Streptococcus,
Propionibacterium spp., Staphylococcus spp. (including methicillin
resistant strains), Clostridium spp., Escherichia spp., Pseudomonas
spp., Campylobacter spp., Listeria spp., Leuconostoc spp., Bacillus
spp., Acinetobacter spp., Streptococcus spp., Brucella spp.,
Proteus spp., Klebsiella spp., Shigella spp., Helicobacter spp.,
Mycobacterium spp., Enterococcus spp., Salmonella spp., Chlamydia
spp., Porphyromonas spp., Stenotrophomonas spp., and
Elizabethkingia spp. Of course, other types of bacteria may also be
targeted in accordance with the present invention.
[0073] Also, for all three embodiments discussed above, exemplary
viruses include SARS-CoV, 2019-nCoV, human coronavirus 229E, bat
coronavirus HKU9-1, H1N1, norovirus (NoV) and feline calicivirus
(which are part of the Caliciviridae family), influenza virus type
A, HCoV-OC43, HCoV-NL63, and hepatitis A.
[0074] In some cases, the microorganism itself synthesizes a
photoactive molecule that functions as a photoreceptor for the
pulsed ultraviolet, purple or blue light--i.e., the photoactive
molecule is endogenous to the microorganisms. An example of this
type of microorganism is the Propionibacterium acnes (P. acnes)
bacteria, which synthesizes the photoactive molecule porphyrin. In
the case of viral microorganisms, the pulsed ultraviolet, purple or
blue light may be absorbed by the viral RNA or protein
photoreceptor leading to the destruction or destabilization of the
virus rendering it inactive.
[0075] In other cases, the microorganisms do not synthesize a
sufficient amount of a photoactive molecule, such as the Group B
Streptococcus (GBS) bacteria or different types of viruses, such as
the coronavirus. For example, while GBS contains chromophores such
as characteristic brick-red pigment ornithine rhamnopolyenic
(rhamnolipid), a .beta.-haemolysin/cytolysin, and granadaene, it is
believed that these chromophores are not implicated in the
suppression of bacteria growth upon irradiation with pulsed blue or
purple light. In these cases, the microorganisms are associated or
supplemented with a photoactive molecule that is exogenous to the
microorganisms and functions as a photoreceptor for the pulsed
purple or blue light, as discussed below.
[0076] In some embodiments, the microorganisms are suspended in a
medium that is endogenous to a human body (e.g., an
organically-rick media (ORM) such as human saliva) and the medium
contains the photoactive molecule--i.e., the photoactive molecule
is an extracellular endogenous photosensitizer. For example, human
saliva contains riboflavin, tyrosine, tryptophan, pyridoxine and
folic acid.
[0077] In other embodiments, the microorganisms are hosted within a
plurality of cells that are endogenous to a human body and the
cells themselves contain the photoactive molecule. For example, it
is believed that lung cells that have ACE2 expression, such as type
II alveolar cells (AT2) or respiratory epithelial cells, may be the
main target cells during 2019-nCoV infection. These cells may
contain the photoactive molecules porphyrin, NADH, and FAD. In the
case of red blood cells which are targets for viral invasion during
2019-nCoV infection, the blood cell contains the photoactive
molecule porphyrin.
[0078] In yet other embodiments, the photoactive molecule comprises
a photosensitizer that is administered to the target. Examples of
photosensitizers that may be applied topically to skin, tissue or a
wound include curcumin, aminolevulinic acid (ALA), protoporphyrin
IX (PPIX), coproporphyrin III (CP III), flavin mononucleotide
(FMN), or nicotinamide adenine dinucleotide (NAD). These
photosensitizers may be administered in various doses that are
typically in the range of 5 to 20 micromolar. Of course, these
doses may vary based on the photoactivation required and topical
and systemic toxicity profiles. Examples of photosensitizers that
may be ingested include curcumin, aminolevulinic acid (ALA), and
chloroquine and its derivatives. These photosensitizers may be
administered in various doses from as little as 1 milligram to 5000
milligrams per day using oral or intravenous administration. Of
course, these doses may vary based on the photoactivation required
and systemic toxicity profiles. It should be understood that these
photosensitizers may be used in combination with any of the
embodiments described above.
[0079] In accordance with the invention, bacteria, viruses or other
microorganisms are inactivated via the application of ultraviolet,
purple or blue light in a pulsed mode of irradiation. As used
herein, "ultraviolet light" refers to light having a wavelength
ranging from about 200 nm to about 380 nm (e.g., 200, 210, 220,
230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350,
360, 370, 380 nm or some value therebetween). Particularly suitable
wavelengths are about 260 nm and 370 nm. As used herein, "purple or
blue light" refers to light having a wavelength ranging from about
380 nm to about 560 nm (e.g., 380, 390, 400, 410, 420, 430, 440,
450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560 nm or
some value therebetween). Particularly suitable wavelength are
about 410 nm and 450 nm.
[0080] Also, pulsed red light may be used for its anti-inflammatory
effects to reduce the cytokine reaction response associated with
the high levels of Reactive Oxygen Species (ROS) in targeted cells
and tissues associated with microorganism destruction. As used
herein, "red light" refers to light having a wavelength ranging
from about 600 nm to about 830 nm (e.g., 600, 610, 620, 630, 640,
650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770,
780, 790, 800, 810, 820, 830 nm or some value therebetween).
Particularly suitable red wavelengths are about 640 nm and 645 nm.
In addition, pulsed infrared light may be used to kill
microorganisms through the production and release of nitric oxide
(NO) from the mitochondria. As used herein, "infrared light" refers
to light having a wavelength ranging from about 780 nm to about
1060 nm (e.g., 780, 790, 800, 810, 820, 830, 840, 850, 860, 870,
880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000,
1010, 1020, 1030, 1040, 1050, 1060 nm or some value therebetween).
Particularly suitable infrared wavelengths are about 904 nm and 940
nm. The pulses provided can be a square wave, rectified sinusoidal
waveform or any combination thereof, although other pulse shapes
may also be used in accordance with the present invention. A square
wave with a rise time of less than 1 microsecond is preferred.
[0081] The pulsed purple or blue light (and optionally pulsed
ultraviolet, red or infrared light) is provided at radiant
exposures that can range from about 0.5 J/cm.sup.2 to about 60
J/cm.sup.2, with a preferred range of about 3.6 J/cm.sup.2 to about
20 J/cm.sup.2, and a more preferred range of about 3.6 J/cm.sup.2
to about 5 J/cm.sup.2. The average irradiance can range from about
0.1 mW/cm.sup.2 to about 20 mW/cm.sup.2, with an average irradiance
of about 2 mW/cm.sup.2 to about 3 mW/cm.sup.2 being preferred at
the tissue surface, and may be higher for contaminated environment
or food surface.
[0082] It should be understood that the low levels of light used in
connection with the present invention (e.g., average irradiances of
about 2 mW/cm.sup.2 to about 3 mW/cm.sup.2 and radiant exposures of
about 3.6 J/cm.sup.2 to about 5 J/cm.sup.2) are very safe to the
eye and meet international blue light safety requirements,
including IEC TR 62778:2014 (Application of IEC 62471 for the
assessment of blue light hazard to light sources and luminaries),
IEC 62471:2006 (Photobiological safety of lamps and lamp systems),
IEC 60601-2-57:2011 (Medical Electrical equipment--Part 2-57:
Particular requirements for the basic safety and essential
performance of non-laser light source equipment intended for
therapeutic, diagnostic, monitoring and cosmetic/aesthetic use).
This provides a significant advantage over continuous wave (CW)
light sources that deliver light at high irradiances and radiant
exposures. These higher output light sources also require
significant cooling and heat sinking and, thus, are not ideal for
refrigeration applications.
[0083] The pulse parameters of the pulsed purple or blue light are
preferably selected to optimize the photochemical reaction of the
photoactive molecule used to photoeradicate the microorganisms, as
described above. In most embodiments, the peak irradiance of the
light pulses ranges from 0.3 mW/cm.sup.2 to 60 mW/cm.sup.2 with a
preferred range of about 6 mW/cm.sup.2 to about 15 mW/cm.sup.2 at a
duty factor of 33%. At other duty factors, the peak irradiance may
be higher or lower so that the average irradiance stays within the
preferred ranges of irradiance described herein. The duty factor is
typically in the range of about 20% to about 33%.
[0084] In most embodiments, the pulse duration of the light pulses
ranges from 5 microseconds to 1,000 microseconds, and the off time
between the light pulses ranges from 10 microseconds to 1 second. A
suitable combination may range from about 5 microseconds to about
30 microseconds for the pulse duration with off times ranging from
about 10 microseconds to about 100 microseconds. A particularly
suitable combination provides a pulse duration of about 10
microseconds and an off time of about 20 microseconds. The light
pulses are typically provided at a pulse repetition rate that
ranges from 33 kHz to 40 kHz.
[0085] It should be understood that the optimal pulse parameters
for destruction of microorganisms will vary between different types
of microorganisms. For example, the pulse intensity and on/off
states that result in the depletion and restoration/recovery of
porphyrins or other photoactive molecules involved in bacterial
photoeradication will vary between different types of bacteria.
[0086] The pulsed purple or blue light (and optionally pulsed
ultraviolet, red or infrared light) may be applied one time (i.e.,
single irradiation session) and is preferably provided multiple
times (i.e., multiple irradiation sessions) at the desired radiant
exposures (fluence) and power density (irradiance). One skilled in
the art will appreciate that the irradiation time for each
irradiation session is dependent on the dose, and is typically in
the range of about 20 minutes to about 45 minutes for the
irradiances and radiant exposures used in connection with the
present invention.
[0087] In some embodiments, the duration of exposure may be
controlled by the decay and recharge in fluorescence of the
photoactive molecule. In that configuration, the exposure would
stop when the fluorescence has depleted to a preset level
indicating that there is insufficient photo-activity to maintain
bacterial kill and then restart when the fluorescence has returned
to another preset level. Also, the irradiation sessions may be
timed to a replication cycle of the microorganisms so that the
irradiation sessions restart prior to replication of the
microorganisms.
[0088] Preferably, the pulsed purple or blue light (and optionally
pulsed ultraviolet, red or infrared light) is applied during a
plurality of irradiation sessions at pre-defined time intervals in
accordance with an irradiation schedule, as described below. The
time interval between irradiation sessions may range from about one
hour to about four hours (e.g., 1, 2, 3, 4 hours or some value
therebetween) with two or three (or more) irradiation sessions per
day. These timed irradiation sessions may also be repeated on two,
three, four or more days in certain embodiments, or in the case of
environmental or food irradiation on a daily basis. In some
embodiments, the pulsing has been found to be optimized when the
irradiation schedule includes the application of pulses three times
per day with a three hour time interval between applications, which
may be repeated on two or more days (e.g., when the pulses are
applied at 0, 3, 6, 24, 27, 30, 48, 51 and 54 hours). Of course,
other irradiation schedules that are suitable for a particular
application may also be used within the scope of the present
invention. The irradiation schedule is preferably chosen to provide
a survival rate for the microorganisms of less than 50% and
preferably 0% (i.e., a survival rate of 50%, 40%, 30%, 20%, 10%,
5%, 0% or some value therebetween).
[0089] In addition to the basic pulsing of the purple or blue light
as described above, the pulsed mode may be further modulated when
used for irradiation of human or animal tissue, i.e., gated on and
off with a low frequency signal timed to coincide with the heart
rate or at a rate similar to the heart rate (i.e., 0.5 to 2 Hz or a
multiple or harmonic thereof). This signal allows for recharge of
the free oxygen, porphyrins or other molecules responsible for
photochemical activation of cellular killing mechanisms during the
resting period of the photostimulation. Duty factors in the range
of 5% to 95% may be used for this modulation.
[0090] Any suitable light source may be used to generate the pulsed
purple or blue light (and optionally pulsed ultraviolet, red or
infrared light) in accordance with the present invention. Exemplary
light sources include various types of lasers, light emitting
diodes (LEDs), organic light emitting diodes (OLEDs), printed light
emitting diodes (printed LEDs), polymer light emitting diodes
(PLEDs) also known as light emitting polymers (LEPs), quantum dot
light emitting diodes (QDLEDs), and fluorescent tubes emitting
light in the purple or blue spectral region. Exemplary light
sources are described in greater detail below.
[0091] For the first embodiment directed to the treatment of
various diseases, disorders or conditions, devices comprised of
printed LEDs or OLEDs on flexible substrates are preferred due to
their ability to produce light with an intensity that is
substantially constant across the surface of the light source so as
to provide substantially uniform light emission when the light
source is in contact with or illuminates skin or tissue of a
subject. The devices are also able to conform to the skin or tissue
surface to maximize optical coupling. In addition, a layer of
hydrogel may be used as an adhesive to contact the flexible
substrate to the skin or tissue surface and thereby provide optical
light piping and coupling. The hydrogel layer may also contain
therapeutic substances used to enhanced bacterial inhibition or
viral suppression or kill.
[0092] Various exemplary light devices that may be used to deliver
pulsed light in accordance with the invention are shown in FIGS.
51-54.
[0093] FIG. 51 illustrates a light device comprising an
oblong-shaped light source 300 in the form of printed LEDs on a
flexible substrate that is controlled by a controller 310, as
described herein. In this embodiment, light source 300 is
configured to be placed externally on a cheek region of a human
body. The pulsed light emitted by this device may comprise pulsed
blue light, pulsed blue and red light, or pulsed blue and infrared
light and may be used, for example, to target microorganisms
suspended in human saliva within the oral cavity. A biocompatible
hydrogel may also be used. In other embodiments, the light source
is configured for internal placement within the oral cavity so as
to directly target the microorganisms suspended in human
saliva.
[0094] FIG. 52 illustrates a light device comprising a
butterfly-shaped light source 400 in the form of printed LEDs on a
flexible substrate that is controlled by a controller 410, as
described herein. In this embodiment, light source 400 is
configured to be placed externally on an inner wrist region of a
human body. The pulsed light emitted by this device may comprise
pulsed blue light, pulsed blue and red light, or pulsed blue and
infrared light, for transcutaneous application over the inner wrist
region (or other areas in close proximity to blood flow) in order
to target microorganisms in the blood, such as treatment of a blood
borne virus on platelets or immune cells. A biocompatible hydrogel
may also be used.
[0095] FIGS. 53A and 53B illustrate a nasal applicator comprising
headgear 500 with a pair of nasal inserts 510 and 520 that are
configured to be inserted into the nasal cavity, but allow
breathing during use. Each of nasal inserts 510 and 520
incorporates printed LEDs and may be used, for example, to target
microorganisms located within the nasal cavity. The pulsed light
emitted by this device may comprise pulsed blue light, pulsed blue
and red light, or pulsed blue and infrared light, or a combination
of all three wavelengths.
[0096] FIG. 54A illustrates a respirator mask that functions as
part of an air filtration system. The respirator mask includes an
outer mask 600 that supports a light patch and filter assembly 610
configured to filter airborne particles (e.g., a bacteria or virus
suspended in human saliva) from the air passed through various
filter layers, as well as photoeradicate all or a portion of the
microorganisms (e.g., a bacteria or virus) that become trapped
within those filter layers thereby preventing the microorganisms
from penetrating into the oral and nasal cavities and lungs of the
user.
[0097] Light patch and filter assembly 610 is shown in greater
detail in FIGS. 54B and 54C and, in general, comprises a plurality
of filter and light layers 612 sealed together by a sealing area
614. As best shown in FIG. 54C, the layers of light patch and
filter assembly 610 include an outer filter layer 620 (i.e., the
layer facing the environment), an outer folded filter layer 622, a
printed LED layer 624, an inner folded filter layer 626, and an
inner filter layer 628 (i.e., the layer facing the user).
Preferably, outer filter layer 620 and inner filter layer 628 are
each made of a material that is greater than 95% efficient at
filtering 0.3-.mu.m particles (i.e., an N95 material), although
other materials may also be used. Outer folded filter layer 622 and
inner folded filter layer 626 are each made of a filter material
(including, but not limited, to an N95 material) that is formed in
a folded configuration, as shown, in order to provide support for
printed LED layer 624 and to provide a cavity that enables air
circulation between outer and inner filter layers 620 and 628 and
printed LED layer 624, respectively. In other embodiments, suitable
spacers may be used in place of outer folded filter layer 622 and
inner folded filter layer 626.
[0098] Printed LED layer 624 comprises printed LEDs on a flexible
substrate, as described herein, which is connected via a wire 618
to a controller that includes a power source, such as a
rechargeable or non-rechargeable battery pack (not shown). In this
embodiment, printed LED layer 624 is configured to emit pulsed
light in a direction toward outer folded filter layer 622 and outer
filter layer 620. The pulsed light may comprise a single wavelength
or a combination of pulsed purple light, pulsed blue light or
pulsed infrared light. The pulsed light is able to photoeradicate
all of a portion of the microorganisms (e.g., bacteria or viruses)
that become trapped within outer folded filter layer 622 and outer
filter layer 620--i.e., the pulsed light effectively "cleans" those
filters. Preferably, outer folded filter layer 622 is made of a
material that is semi-transparent so as to permit the transmission
of pulsed light from printed LED layer 624 to outer filter layer
620. In some embodiments, pulsed or continuous wave (CW)
ultraviolet light (e.g., UVA, UVB or UVC) may also be used for its
bactericidal or anti-viral effects.
[0099] In this embodiment, printed LED layer 624 comprises a solid
layer (printed LEDs on a flexible substrate) that does not enable
airflow therethrough. Therefore, a pair of cutouts 616a and 616b
are provided on each side of printed LED layer 624 to permit
airflow around the sides of printed LED layer 624. As such, the air
flows transversely through outer filter layer 620 and outer folded
filter layer 622, laterally from outer folded filter layer 622
around the sides of printed LED layer 624 to inner folded filter
layer 626, and then transversely through inner folded filter layer
626 and inner filter layer 628. Non-breathable material 630a and
630b is provided on each side of inner filter layer 628 to direct
the air flow through this filter layer. It should be noted that the
filter layers are omitted in FIG. 54B in order to illustrate the
position of cutouts 616a and 616b.
[0100] In other embodiments, the printed LED layer includes one or
more openings that enable the direct flow of air from outer folded
filter layer 622 to inner folded filter layer 626. For example,
FIG. 54D illustrates a printed LED layer 642 and a sealing area 640
that are similar to those described in connection with FIG. 54B
(noting again that the filter layers are omitted in FIG. 54D), with
the exception that printed LED layer 642 includes a plurality of
vertical slots 644 to enable the flow of air therethrough. As
another example, FIG. 54E illustrates a printed LED layer 652 and a
sealing area 650 that are similar to those described in connection
with FIG. 54B (noting again that the filter layers are omitted in
FIG. 54E), with the exception that printed LED layer 652 includes a
plurality of holes 654 to enable the flow of air therethrough. Of
course, the number and shape of the openings in the printed LED
layer will vary between different embodiments. It should be
understood that cutouts 616a and 616b are not required in these
embodiments.
[0101] In some embodiments, printed LED layer 624 (or the printed
LED layers shown in FIGS. 54D and 54E) include LEDs printed on both
sides of the substrate so that pulsed light is also emitted in a
direction toward inner folded filter layer 626 and inner filter
layer 628. The pulsed light may comprise a single wavelength or a
combination of pulsed purple light, pulsed blue light or pulsed
infrared light. The pulsed light is able to photoeradicate all of a
portion of the microorganisms (e.g., bacteria or viruses) that
become trapped within inner folded filter layer 626 and inner
filter layer 628--i.e., the pulsed light effectively "cleans" those
filters. Preferably, inner folded filter layer 626 is made of a
material that is semi-transparent so as to permit the transmission
of pulsed light from printed LED layer 624 to inner filter layer
628. In some embodiments, pulsed or continuous wave (CW)
ultraviolet light (e.g., UVA, UVB or UVC) may also be used for its
bactericidal or anti-viral effects.
[0102] In yet other embodiments, a second printed LED layer (not
shown) is positioned between the user and inner filter layer 628
and configured to emit pulsed light in a direction toward inner
filter layer 628 and inner folded filter layer 626. The pulsed
light may comprise a single wavelength or a combination of pulsed
purple light, pulsed blue light or pulsed infrared light. The
pulsed light is able to photoeradicate all of a portion of the
microorganisms (e.g., bacteria or viruses) that become trapped
within inner filter layer 628 and inner folded filter layer
626--i.e., the pulsed light effectively "cleans" those filters.
Preferably, inner filter layer 628 is made of a material that is
semi-transparent so as to permit the transmission of pulsed light
from the second printed LED layer to inner folded filter layer 626.
In some embodiments, pulsed or continuous wave (CW) ultraviolet
light (e.g., UVA, UVB or UVC) may also be used for its bactericidal
or anti-viral effects.
[0103] It should be understood that the present invention is not
limited to the configuration of the respirator mask described
above, and that other mask designs may also be used within the
scope of the invention. For example, in some embodiments, the
printed LED layer is removeably secured within the respirator mask
in such a manner as to enable access to the printed LED layer. For
example, the printed LED layer may be positioned within a
compartment that is closeable using Velcro.RTM. or any other
suitable fastener, but which enables access to the compartment for
removal of the printed LED layer. This configuration enables the
printed LED layer to be inserted into a variety of different types
of respirator masks. Thus, while the respirator mask itself is
disposable, the printed LED layer is re-useable in different masks
following cleaning and decontamination. Also, other types of light
sources may also be used in accordance with the present
invention.
[0104] Any of the light devices described above may also be used in
combination with detectors to verify the kill rates or to filter
change points.
[0105] For the second and third embodiments directed to
photoeradication of microorganisms from contaminated environments
and food, respectively, it is possible to use strips or sheets of
printed LEDs or OLEDs, high power LEDs or even tube lamps emitting
modulated light in the UVA through blue spectral range. Certain of
these strips or sheets may have rows of blue lamps in parallel or
interspersed with white lamps or phosphor coated or Stokes
conversion quantum dots or the like to produce white light. As used
herein, "white light" means light having a color temperature in the
range of about 1700 kelvins (K) to about 9500 kelvins (K).
[0106] In some embodiments, the light source is controlled by an
electronic circuit with a drive circuit and microcontroller that
provides a preprogrammed sequence of light pulses at a fixed dose
during each irradiation session. In other embodiments, the light
source is controlled by an electronic circuit with one or more
sensors that operate in a closed loop to provide feedback to a
microcontroller so as to dynamically control the dose during a
treatment session. Exemplary electronic circuits for both
preprogrammed control and dynamic control of a light source are
described in greater detail below. In some embodiments, the
controller will receive input from a lighting system or the opening
or closing of a door in a refrigeration or storage unit to activate
the pulsed purple or blue lights and provide the appropriate daily
irradiation sequence.
II. In Vitro Testing of P. acnes Cultures to Determine Feasibility
and Reproducibility of Inactivation with Light Irradiation
[0107] Testing was performed to determine the feasibility and
reproducibility of 450 nm light to inactivate P. acnes using
printed LED flexible lamps operated in a continuous wave (CW)
irradiation mode, a pulsed irradiation mode with a 33% duty factor,
and a pulsed irradiation mode with a 20% duty factor (referred to
hereinafter as CW mode, 33% DF pulsed mode, and 20% DF pulsed
mode).
Culturing P. acnes
[0108] A vial of P. acnes (ATCC 6919) was obtained from the
American Type Culture Collection (ATCC) and cultures started
according to ATCC recommendations. Briefly, under anaerobic
conditions, the lyophilized pellet was rehydrated in 500 .mu.L
modified reinforced clostridial broth (pre-reduced) and transferred
into another tube containing 5 mL clostridial broth. Test tubes
were labelled as "Start Cultures" (SC) and 200 .mu.L transferred
onto a reinforced clostridial agar plate. The bacteria were
streaked on the plates for isolation of single colonies by the
"clock plate technique" to check colony morphology and purity.
These plates were called "start plates." Both tube/plate were
placed in an anaerobic chamber with BD Gas-Pak EZ anaerobic
container system sachets. The anaerobic chamber was then placed
inside a 37.degree. C. incubator for 72 hours. After 72 hours of
growth period, culture tubes were removed from the 37.degree. C.
incubator and tested for their susceptibility to irradiation with
450 nm blue light.
Illumination of Bacteria
[0109] A 5 mL liquid culture was grown for 3 days and 1 mL pipetted
into a sterile microcentrifuge tube and centrifuged at 13,300 rpm
for five minutes. The supernatant was removed and discarded. The
pellet was re-suspended in 1 mL saline and optical density adjusted
using McFarland standard to 0.8 to 1.0 at 625 nm for a
concentration of 10.sup.8 CFU/mL. The bacteria were diluted to a
concentration of 1.times.10.sup.6 CFU/mL and 2 .mu.L streaked onto
reduced clostridial agar plates (12 well plates were used, which
enabled triplicate irradiations with 3 patches simultaneously) and
irradiated in an anaerobic chamber with various protocols. A set of
non-irradiated plates served as controls. After irradiation, plates
were placed upside down in the anaerobic chamber, along with a
Gas-pak sachet and incubated at 37.degree. C. for 72 hours. The
colonies were then counted, percentage survival computed and
morphology checked. Illumination times and intervals were optimized
based on experimentation as to which dose and sequence provided the
most effective kill rates.
Quantification of Bacterial Colonies
[0110] Standardized digital images of P. acnes colonies were taken
72 hours after incubation, with the camera positioned 10 cm
perpendicularly above each plate to ensure consistency of colony
magnification. Colonies were counted, and percent survival
calculated, comparing irradiated groups and non-irradiated
controls.
Description of Test Protocols and Test Results
Example 1
[0111] A first series of experiments involved culturing P. acnes
and illuminating the bacteria with a 450 nm lighted substrate, an
example of which is shown in FIG. 22 (described below). The lighted
substrate was set to operate in CW mode and driven with a constant
current source. The peak and average irradiances were the same,
i.e., 4.5 mW/cm.sup.2, due to the continuous output. The bacteria
were irradiated one, three or four times with different radiant
exposures, as described below. Three substrates were provided for
the experiments and all were tested and calibrated.
[0112] The data is presented in FIGS. 1-4. As illustrated in FIG.
1, single irradiation of bacteria with radiant exposures of 20
J/cm.sup.2 and 60 J/cm.sup.2 produced statistically significant
decreases in percent survival when compared to the control, with
maximum reduction of 31% observed at 60 J/cm.sup.2. As illustrated
in FIG. 2, triple irradiation of bacteria with a radiant exposure
of 20 J/cm.sup.2 at 0, 24 and 48 hours reduced the percent survival
to 62%, which was significantly different from the control. As
illustrated in FIG. 3, when bacteria were irradiated four times
with a radiant exposure of 20 J/cm.sup.2 at 0, 4, 24 and 48 hours,
a significant decrease in percent survival of 31% was observed when
compared to the control. This percent survival was much lower than
the percent survival of 62% observed with triple irradiation at 0,
24 and 48 hours (FIG. 2). It was found that four exposures at 20
J/cm.sup.2 applied at 0, 4, 24 and 48 hours provided the highest
kill rates as compared to a single, double or triple exposure.
[0113] FIG. 4 illustrates the relationship between different
radiant exposures for a double exposure occurring at 0 and 4 hours.
Double irradiation of bacteria with radiant exposures of 30, 40, 45
and 60 J/cm.sup.2 at 0 and 4 hours produced a significant dose
dependent decrease in percent survival as the dose increased, with
complete inactivation observed at 60 J/cm.sup.2.
Example 2
[0114] A second series of experiments involved culturing P. acnes
and illuminating the bacteria with the 450 nm lighted substrate
shown in FIG. 22. The lighted substrate was set to operate in CW
mode, 33% DF pulsed mode, or 20% DF pulsed mode, as described
below, and driven with a constant current source. The average
irradiance was the same for all modes, i.e., 5 mW/cm.sup.2. The
peak irradiance was 15 mW/cm.sup.2 in the 33% DF pulsed mode (i.e.,
three times higher than CW mode) and 25 mW/cm.sup.2 in the 20% DF
pulsed mode (i.e., five times higher than CW mode). In the 33% DF
pulsed mode, the pulse duration was 10 microseconds with an off
time of 20 microseconds, and the pulse repetition rate was 33 kHz.
In the 20% DF pulsed mode, the pulse duration was 5 microseconds
with an off time of 20 microseconds, and the pulse repetition rate
was 40 kHz. The bacteria were irradiated twice at 0 and 4 hours
with different radiant exposures, as described below. Three
substrates were provided for the experiments and all were tested
and calibrated.
[0115] The data is presented in FIGS. 5 and 6. As illustrated in
FIG. 5, double irradiation of bacteria at 0 and 4 hours with a
radiant exposure of 60 J/cm.sup.2 in CW mode and 33% DF pulsed mode
revealed marked decreases in percent survival when compared to the
control, with maximum reduction to a percent survival of 2.2%
observed in the 33% DF pulsed mode. As illustrated in FIG. 6,
double irradiation of bacteria at 0 and 4 hours with a radiant
exposure of 20 J/cm.sup.2 in CW mode, 33% DF pulsed mode and 20% DF
pulsed mode again revealed that the 33% DF pulsed mode was more
efficacious in suppressing bacteria growth than the CW mode and the
20% DF pulsed mode, with a percent survival of 16% for the 33% DF
pulsed mode compared to percent survivals of 87% and 80% for the CW
mode and 20% DF pulsed mode, respectively.
Example 3
[0116] A third series of experiments involved further testing with
a change to the exposure schedule based on the findings described
in FIG. 3, i.e., that four irradiation exposures (0, 4, 24 and 48
hours) provided the best kill rate at a radiant exposure of 20
J/cm.sup.2. These experiments involved culturing P. acnes and
illuminating the bacteria with the 450 nm lighted substrate shown
in FIG. 22. The lighted substrate was set to operate in CW mode or
33% DF pulsed mode, as described below, and driven with a constant
current source. The average irradiance was selectable at 2 and 3.5
mW/cm.sup.2 in the 33% DF pulsed mode compared to an average
irradiance of 5 mW/cm.sup.2 in the CW mode. In the 33% DF pulsed
mode, the pulse duration was 10 microseconds with an off time of 20
microseconds, and a pulse repetition rate of 33 kHz. The bacteria
were irradiated four times at 0, 4, 24 and 48 hours with different
radiant exposures of 5, 10 and 20 J/cm.sup.2. Three substrates were
provided for the experiments and all were tested and
calibrated.
[0117] The data is presented in FIGS. 7-9. As illustrated in FIG.
7, irradiation of cultures four times (0, 4, 24 and 48 hours) with
radiant exposures of 5, 10 and 20 J/cm.sup.2 and an average
irradiance of 2 mW/cm.sup.2 in the 33% DF pulsed mode showed
significant decreases in percent survival when compared to the
control; however, no significant differences were observed between
the doses. These results indicated that no significant differences
in the percent survival existed with increases in radiant exposure
from 5 J/cm.sup.2 to 20 J/cm.sup.2 at an average irradiance of 2
mW/cm.sup.2.
[0118] As illustrated in FIG. 8, irradiation of cultures four times
(0, 4, 24 and 48 hours) with radiant exposures of 5, 10 and 20
J/cm.sup.2 and an average irradiance of 3.5 mW/cm.sup.2 in the 33%
DF pulsed mode showed significant decreases in percent survival
when compared to the control; however, no significant differences
were observed between the doses. These results indicate that no
significant differences in the percent survival existed with
increases in radiant exposure from 5 J/cm.sup.2 to 20 J/cm.sup.2 at
an average irradiance of 3.5 mW/cm.sup.2.
[0119] As illustrated in FIG. 9, irradiation of cultures four times
(0, 4, 24 and 48 hours) with radiant exposures 5, 10 and 20
J/cm.sup.2 and an irradiance of 5 mW/cm.sup.2 in the CW mode showed
significant decreases in percent survival when compared to the
control. However, unlike the results shown in FIGS. 7 and 8, the
decrease in percent bacterial survival was dose dependent with
significant decreases observed between the radiant exposures of 5
J/cm.sup.2 and 10 J/cm.sup.2 and between 10 J/cm.sup.2 and 20
J/cm.sup.2 at an irradiance of 5 mW/cm.sup.2.
[0120] Thus, it can be seen that the CW mode behaved in a linear
dose dependent fashion. However, a significant and unexpected
result was obtained with respect to the 33% DF pulsed mode, i.e.,
there was no significant radiant exposure dependency on kill rates
for different average irradiances of 2 and 3.5 mW/cm.sup.2. As a
result, a novel treatment was discovered using two to four doses in
sequence in the 33% DF pulsed mode at a radiant exposure of 5
J/cm.sup.2.
III. In Vitro Testing of P. acnes Cultures to Determine Optimal
Parameters of Inactivation with Light Irradiation
[0121] The preliminary results described above revealed that pulsed
blue light was more effective in suppressing bacteria growth than
continuous wave. Thus, further testing was performed to determine
the optimal parameters of 450 nm light to inactivate P. acnes using
printed LED flexible lamps operated in a pulsed irradiation mode
with a 33% duty factor.
[0122] A suspension of P. acnes bacteria diluted to a concentration
of 1.times.10.sup.6 CFU/mL, as discussed in Section II above, was
streaked onto reduced clostridial agar plates that were irradiated
or not (controls) in an anaerobic chamber. The tests were conducted
using three different irradiation substrates (with the same power
output) with each placed at the top of two wells on a 12 well
plate. This enabled results to be obtained in triplicates (six sets
of data for each experiment). After irradiation protocols were
completed, plates were placed upside down in the anaerobic chamber,
along with a Gas-pak sachet and incubated at 37.degree. C. for 72
hours. The colonies were then counted, percentage survival computed
and morphology checked.
Description of Test Protocols and Test Results
Example 1
[0123] A first experiment involved culturing P. acnes and
illuminating the bacteria with the 450 nm lighted substrate shown
in FIG. 22. The lighted substrate was set to operate in 33% DF
pulsed mode, as described below, and driven with a constant current
source. The average irradiance was 3 mW/cm.sup.2. The pulse
duration was 10 microseconds with an off time of 20 microseconds,
and a pulse repetition rate of 33 kHz. The bacteria were irradiated
four times at 0, 4, 24 and 48 hours with different radiant
exposures of 5, 10 and 20 J/cm.sup.2.
[0124] As illustrated in FIG. 10, irradiation of cultures four
times (0, 4, 24 and 48 hours) with radiant exposures of 5, 10 and
20 J/cm.sup.2 and an average irradiance of 3 mW/cm.sup.2 in the 33%
DF pulsed mode showed decreases in percent survival when compared
to the control, i.e., 89.1%, 34.3% and 0.5%, respectively. The
decrease in percent bacterial survival was dose dependent with
significant decreases observed between the radiant exposure of 5
and 10 J/cm.sup.2, between 5 and 20 J/cm.sup.2, and between 10 and
20 J/cm.sup.2.
Example 2
[0125] A second experiment involved culturing P. acnes and
illuminating the bacteria with the 450 nm lighted substrate shown
in FIG. 22. The lighted substrate was set to operate in 33% DF
pulsed mode, as described below, and driven with a constant current
source. The average irradiance was 3 mW/cm.sup.2. The pulse
duration was 10 microseconds with an off time of 20 microseconds,
and a pulse repetition rate of 33 kHz. The bacteria were irradiated
four times at 0, 3, 6 and 24 hours with different radiant exposures
of 5, 10 and 20 J/cm.sup.2.
[0126] As illustrated in FIG. 11, irradiation of cultures four
times (0, 3, 6 and 24 hours) with radiant exposures of 5, 10 and 20
J/cm.sup.2 and an average irradiance of 3 mW/cm.sup.2 in the 33% DF
pulsed mode showed decreases in percent survival when compared to
the control, i.e., 85.7%, 16.3% and 0.0%, respectively. The overall
decrease in percent survival was higher than that seen in the first
example. Again, the decrease in percent bacterial survival was dose
dependent with significant decreases observed between the radiant
exposure of 5 and 10 J/cm.sup.2, between 5 and 20 J/cm.sup.2, and
between 10 and 20 J/cm.sup.2.
Example 3
[0127] A third experiment involved culturing P. acnes and
illuminating the bacteria with the 450 nm lighted substrate shown
in FIG. 22. The lighted substrate was set to operate in 33% DF
pulsed mode, as described below, and driven with a constant current
source. The average irradiance was 3 mW/cm.sup.2. The pulse
duration was 10 microseconds with an off time of 20 microseconds,
and a pulse repetition rate of 33 kHz. The bacteria were irradiated
three times at 0, 3 and 6 hours with different radiant exposures of
5, 10 and 20 J/cm.sup.2.
[0128] As illustrated in FIG. 12, irradiation of cultures three
times using a shorter time interval (0, 3 and 6 hours) with radiant
exposures of 5, 10 and 20 J/cm.sup.2 and an average irradiance of 3
mW/cm.sup.2 in the 33% DF pulsed mode showed decreases in percent
survival when compared to the control, i.e., 60.9%, 11.7% and 0.2%,
respectively. The decrease in percent bacterial survival was dose
dependent with significant decreases observed between the radiant
exposure of 5 and 10 J/cm.sup.2, between 5 and 20 J/cm.sup.2, and
between 10 and 20 J/cm.sup.2.
Example 4
[0129] A fourth experiment involved culturing P. acnes and
illuminating the bacteria with the 450 nm lighted substrate shown
in FIG. 22. The lighted substrate was set to operate in 33% DF
pulsed mode, as described below, and driven with a constant current
source. The average irradiance was 3 mW/cm.sup.2. The pulse
duration was 10 microseconds with an off time of 20 microseconds,
and a pulse repetition rate of 33 kHz. The bacteria were irradiated
nine times at 0, 3, 6, 24, 27, 30, 48, 51 and 54 hours with a
radiant exposure of 5 J/cm.sup.2.
[0130] As illustrated in FIG. 13, a change in the irradiation
schedule to three times per day, every three hours over the course
of three days for a total of nine times (0, 3, 6, 24, 27, 30, 48,
51 and 54 hours) with a radiant exposure of 5 J/cm.sup.2 and an
average irradiance of 3 mW/cm.sup.2 in the 33% DF pulsed mode
showed a significant decrease in percent survival when compared to
the control, i.e., 2.57%.
Example 5
[0131] A fifth experiment involved culturing P. acnes and
illuminating the bacteria with the 450 nm lighted substrate shown
in FIG. 22. The lighted substrate was set to operate in 33% DF
pulsed mode, as described below, and driven with a constant current
source. The average irradiance was 3 mW/cm.sup.2. The pulse
duration was 10 microseconds with an off time of 20 microseconds,
and a pulse repetition rate of 33 kHz. The bacteria were irradiated
twelve times at 0, 3, 6, 24, 27, 30, 48, 51, 54, 72, 75 and 78
hours with a radiant exposure of 5 J/cm.sup.2.
[0132] As illustrated in FIG. 14, a change in the irradiation
schedule to three times per day, every three hours over the course
of four days for a total of twelve times (0, 3, 6, 24, 27, 30, 48,
51, 54, 72, 75 and 78 hours) with a radiant exposure of 5
J/cm.sup.2 and an average irradiance of 3 mW/cm.sup.2 in the 33% DF
pulsed mode showed a significant decrease in percent survival when
compared to the control, i.e., 1.13%. The overall decrease in
percent survival was higher than that seen in the fourth example
when bacteria were irradiated over the course of three days (with
all other parameters being the same).
Example 6
[0133] A sixth experiment involved culturing P. acnes and
illuminating the bacteria with the 450 nm lighted substrate shown
in FIG. 22. The lighted substrate was set to operate in 33% DF
pulsed mode, as described below, and driven with a constant current
source. The average irradiance was 2 mW/cm.sup.2. The pulse
duration was 10 microseconds with an off time of 20 microseconds,
and a pulse repetition rate of 33 kHz. The bacteria were irradiated
twelve times at 0, 3, 6, 24, 27, 30, 48, 51, 54, 72, 75 and 78
hours with a radiant exposure of 5 J/cm.sup.2.
[0134] As illustrated in FIG. 15, the irradiation of bacteria three
times per day, every three hours over the course of four days for a
total of twelve times (0, 3, 6, 24, 27, 30, 48, 51, 54, 72, 75 and
78 hours) with a radiant exposure of 5 J/cm.sup.2 and a change in
the average irradiance to 2 mW/cm.sup.2 in the 33% DF pulsed mode
showed a complete bacterial eradication when compared to the
control, i.e., 0.0%. The overall decrease in percent survival was
slightly higher than that seen in the fifth example when the
average irradiance was 3 mW/cm.sup.2 (with all other parameters
being the same).
Example 7
[0135] A seventh experiment involved culturing P. acnes and
illuminating the bacteria with the 450 nm lighted substrate shown
in FIG. 22. The lighted substrate was set to operate in 33% DF
pulsed mode, as described below, and driven with a constant current
source. The average irradiance was 2 mW/cm.sup.2. The pulse
duration was 10 microseconds with an off time of 20 microseconds,
and a pulse repetition rate of 33 kHz. The bacteria were irradiated
nine times at 0, 3, 6, 24, 27, 30, 48, 51 and 54 hours with a
radiant exposure of 5 J/cm.sup.2.
[0136] As illustrated in FIG. 16, the irradiation of bacteria three
times per day, every three hours over the course of three days for
a total of nine times (0, 3, 6, 24, 27, 30, 48, 51 and 54 hours)
with a radiant exposure of 5 J/cm.sup.2 and an average irradiance
of 2 mW/cm.sup.2 in the 33% DF pulsed mode showed a complete
bacterial eradication when compared to the control, i.e., 0.0%. The
overall decrease in percent survival was the same as that seen in
the sixth example when the irradiation was applied over the course
of four days for a total of twelve times (with all other parameters
being the same).
Example 8
[0137] An eighth experiment involved culturing P. acnes and
illuminating the bacteria with the 450 nm lighted substrate shown
in FIG. 22. The lighted substrate was set to operate in 33% DF
pulsed mode, as described below, and driven with a constant current
source. The average irradiance was 2 mW/cm.sup.2. The pulse
duration was 10 microseconds with an off time of 20 microseconds,
and a pulse repetition rate of 33 kHz. The bacteria were irradiated
eight times at 0, 3, 24, 27, 48, 51, 72 and 75 hours with a radiant
exposure of 3.6 J/cm.sup.2.
[0138] As illustrated in FIG. 17, the irradiation of bacteria two
times per day, every three hours over the course of four days for a
total of eight times (0, 3, 24, 27, 48, 51, 72 and 75 hours) with a
lower radiant exposure of 3.6 J/cm.sup.2 and an average irradiance
of 2 mW/cm.sup.2 in the 33% DF pulsed mode showed a slight decrease
in percent survival when compared to the control, i.e., 86.9%,
which is significantly higher than that seen in the seventh example
when the radiant exposure was 5 J/cm.sup.2 and the irradiation was
applied three times per day (with all other parameters being the
same).
Example 9
[0139] A ninth experiment involved culturing P. acnes and
illuminating the bacteria with the 450 nm lighted substrate shown
in FIG. 22. The lighted substrate was set to operate in 33% DF
pulsed mode, as described below, and driven with a constant current
source. The average irradiance was 2 mW/cm.sup.2. The pulse
duration was 10 microseconds with an off time of 20 microseconds,
and a pulse repetition rate of 33 kHz. The bacteria were irradiated
ten times at 0, 3, 24, 27, 48, 51, 72, 75, 96, and 99 hours with a
radiant exposure of 3.6 J/cm.sup.2.
[0140] As illustrated in FIG. 18, the irradiation of bacteria two
times per day, every three hours over the course of five days for a
total of ten times (0, 3, 24, 27, 48, 51, 72, 75, 96, and 99 hours)
with a radiant exposure of 3.6 J/cm.sup.2 and an average irradiance
of 2 mW/cm.sup.2 in the 33% DF pulsed mode showed a decrease in
percent survival when compared to the control, i.e., 78.9%, which
is just slightly lower than that seen in the eighth example when
the irradiation was applied over the course of four days (with all
other parameters being the same).
Example 10
[0141] A tenth experiment involved culturing P. acnes and
illuminating the bacteria with the 450 nm lighted substrate shown
in FIG. 22. The lighted substrate was set to operate in 33% DF
pulsed mode, as described below, and driven with a constant current
source. The average irradiance was 2 mW/cm.sup.2. The pulse
duration was 10 microseconds with an off time of 20 microseconds,
and a pulse repetition rate of 33 kHz. The bacteria were irradiated
twelve times at 0, 3, 6, 24, 27, 30, 48, 51, 54, 72, 75 and 78
hours with a radiant exposure of 3.6 J/cm.sup.2.
[0142] As illustrated in FIG. 19, the irradiation of bacteria three
times per day, every three hours over the course of four days for a
total of twelve times (0, 3, 6, 24, 27, 30, 48, 51, 54, 72, 75 and
78 hours) with a radiant exposure of 3.6 J/cm.sup.2 and an average
irradiance of 2 mW/cm.sup.2 in the 33% DF pulsed mode showed a
decrease in percent survival when compared to the control, i.e.,
32.6%, which is lower than that seen in the eighth and ninth
examples.
Example 11
[0143] An eleventh experiment involved culturing P. acnes and
illuminating the bacteria with the 450 nm lighted substrate shown
in FIG. 22. The lighted substrate was set to operate in 33% DF
pulsed mode, as described below, and driven with a constant current
source. The average irradiance was 2 mW/cm.sup.2. The pulse
duration was 10 microseconds with an off time of 20 microseconds,
and a pulse repetition rate of 33 kHz. The bacteria were irradiated
nine times at 0, 3, 6, 24, 27, 30, 48, 51 and 54 hours with a
radiant exposure of 5 J/cm.sup.2.
[0144] As illustrated in FIG. 20, the irradiation of bacteria three
times per day, every three hours over the course of three days for
a total of nine times (0, 3, 6, 24, 27, 30, 48, 51 and 54 hours)
with a radiant exposure of 5 J/cm.sup.2 and an average irradiance
of 2 mW/cm.sup.2 in the 33% DF pulsed mode showed a complete
bacterial eradication when compared to the control, i.e., 0.0%.
This experiment confirmed the data from the seventh experiment (see
Example 7).
Example 12
[0145] A twelfth experiment involved culturing P. acnes and
illuminating the bacteria with the 450 nm lighted substrate shown
in FIG. 22. The lighted substrate was set to operate in 33% DF
pulsed mode, as described below, and driven with a constant current
source. The average irradiance was 2 mW/cm.sup.2. The pulse
duration was 10 microseconds with an off time of 20 microseconds,
and a pulse repetition rate of 33 kHz. The bacteria were irradiated
nine times at 0, 3, 6, 24, 27, 30, 48, 51 and 54 hours with a
radiant exposure of 3.6 J/cm.sup.2.
[0146] As illustrated in FIG. 21, the irradiation of bacteria three
times per day, every three hours over the course of three days for
a total of nine times (0, 3, 6, 24, 27, 30, 48, 51 and 54 hours)
with a radiant exposure of 3.6 J/cm.sup.2 and an average irradiance
of 2 mW/cm.sup.2 in the 33% DF pulsed mode showed a significant
decrease in percent survival when compared to the control, i.e.,
11.7%. Although the percentage survival was low, it was still
higher than that seen in the eleventh example when the radiant
exposure was 5 J/cm.sup.2 (with all other parameters being the
same).
[0147] Summary
[0148] Further testing was performed using light patches that emit
pulsed blue light at a 33% duty cycle. The various protocols tested
showed effective reduction in bacteria growth. Testing using
irradiation intervals of 3 hours proved to be more efficient than
irradiation intervals of 4 hours. It is believed that such observed
efficiency may be due in part to targeting the bacteria at the
appropriate time during the replication cycle or due to porphyrin
depletion/replenishing mechanisms in the bacteria.
[0149] Irradiation protocols were optimized to yield 100% bacterial
suppression of 1.times.10.sup.6 CFU/mL P. acnes cultures. Optimal
bacterial suppression of 100% was attained (i) when cultures were
irradiated at 0, 3, 6 and 24 hours with a radiant exposure of 20
J/cm.sup.2 and an average irradiance of 3 mW/cm.sup.2 (see Example
2), (ii) when cultures were irradiated three times per day, every
three hours over the course of four days for a total of twelve
times (0, 3, 6, 24, 27, 30, 48, 51, 54, 72, 75 and 78 hours) with a
radiant exposure of 5 J/cm.sup.2 and an average irradiance to 2
mW/cm.sup.2 (see Example 6), and (iii) when cultures were
irradiated three times per day, every three hours over the course
of three days for a total of nine times (0, 3, 6, 24, 27, 30, 48,
51 and 54 hours) with a radiant exposure of 5 J/cm.sup.2 and an
average irradiance of 2 mW/cm.sup.2 (see Example 11).
[0150] One skilled in the art will appreciate that the present
invention allows for improved device performance with reduced
battery requirements, a substantial reduction in treatment time,
and improved patient safety due to a significant reduction in heat
and optical hazard.
IV. In Vitro Testing of GBS Cultures Supplemented with Porphyrins
to Obtain Bacterial Suppression with Light Irradiation
[0151] The results described above revealed that pulsed blue light
was effective in suppressing bacteria growth for P. acnes, which
synthesizes the photoactive molecule porphyrin. However, some
bacteria such as Group B Streptococcus (GBS) do not synthesize a
sufficient amount of porphyrin or other photoactive molecules, in
which case a photosensitizer is needed to function as a
photoreceptor for the pulsed blue light. Thus, further testing was
performed to assess GBS bacterial suppression when supplemented
with exogenous porphyrins (either protoporphyrin IX (PPIX) or
coproporphyrin III (CP III)) and irradiated with pulsed blue light
using printed LED flexible lamps.
Study 1: GBS Supplemented with Different Concentrations of
Porphyrins
[0152] GBS wild type strain COH1 was added to 3 mL of Todd Hewitt
broth and grown aerobically overnight at 37.degree. C. From the
overnight cultures, 500 uL of GBS was added to fresh Todd Hewitt
broth and grown logarithmically at 37.degree. C. until reaching a
concentration of 10.sup.8 CFU/mL. The culture was then centrifuged
at 1300 rpm for three minutes, the supernatant was removed and
discarded, and 1 mL of fresh Todd Hewitt broth was added. Serial
dilutions of NaCl were made to obtain a suspension of GBS diluted
to a concentration of 1.times.10.sup.4 CFU/mL. The GBS were
streaked onto Todd Hewitt agar plates both with and without the
addition of diluted porphyrins (different concentrations of PPIX or
CP III).
[0153] Some of the plates were irradiated in a chamber using the
450 nm lighted substrate shown in FIG. 22. The lighted substrate
was set to operate in 33% DF pulsed mode and driven with a constant
current source. The average irradiance was 3 mW/cm.sup.2. The pulse
duration was 10 microseconds with an off time of 20 microseconds,
and a pulse repetition rate of 33 kHz. The plates subject to light
treatment were irradiated three times at 0, 72 and 144 hours with a
radiant exposure of 7.56 J/cm.sup.2 during each irradiation session
(i.e., each irradiation session was 42 minutes with an incubation
period there between of 30 minutes).
[0154] After irradiation protocols were completed, the plates were
placed upside down in the anaerobic chamber and incubated at
37.degree. C. for 24 hours. The plates were photographed, colonies
counted and statistics of percent survival recorded.
Example 1
[0155] In a first experiment, the GBS were assigned to six
experimental groups: (1) GBS only (control); (2) GBS and light; (3)
GBS supplemented with 0.05 mg/mL PPIX; (4) GBS supplemented with
0.002 mg/mL PPIX and light; (5) GBS supplemented with 0.02 mg/mL
PPIX and light; and (6) GBS supplemented with 0.05 mg/mL PPIX and
light. The results are shown in FIG. 33.
[0156] As can be seen in FIG. 33, irradiation of GBS without the
addition of any PPIX showed a minimal decrease in percent GBS
survival when compared to the control, i.e., approximately 87%,
which demonstrates that GBS does not synthesize a sufficient amount
of porphyrin. Also, GBS supplemented with 0.05 mg/mL PPIX, but
without any irradiation, did not show much decrease in percent GBS
survival when compared to the control, i.e., approximately 85%. In
addition, irradiation of GBS supplemented with 0.002 mg/mL PPIX did
not show much decrease in percent GBS survival when compared to the
control, i.e., approximately 83%, which demonstrates that the
concentration of PPIX was too low. However, irradiation of GBS
supplemented with 0.02 mg/mL PPIX and 0.05 mg/mL PPIX showed
significant decreases in percent GBS survival when compared to the
control, i.e., 0% and 3%, respectively. Thus, the decrease in
percent GBS survival was dependent on the concentration of PPIX
added to the GBS prior to irradiation.
Example 2
[0157] In a second experiment conducted separately, and with
observed slight decrease in CFU/mL, the GBS were assigned to six
experimental groups: (1) GBS only (control); (2) GBS and light; (3)
GBS supplemented with 0.05 mg/mL PPIX; (4) GBS supplemented with
0.002 mg/mL PPIX and light; (5) GBS supplemented with 0.02 mg/mL
PPIX and light; and (6) GBS supplemented with 0.05 mg/mL PPIX and
light. The results are shown in FIG. 34.
[0158] As can be seen in FIG. 34, irradiation of GBS without the
addition of any PPIX showed a small decrease in percent GBS
survival when compared to the control, i.e., approximately 81%,
which again demonstrates that GBS does not synthesize a sufficient
amount of porphyrin. GBS supplemented with 0.05 mg/mL PPIX, but
without any irradiation, showed some decrease in percent GBS
survival when compared to the control, i.e., approximately 50%. In
addition, irradiation of GBS supplemented with 0.002 mg/mL PPIX
showed even more decrease in percent GBS survival when compared to
the control, i.e., approximately 37%. Further, irradiation of GBS
supplemented with 0.02 mg/mL PPIX and 0.05 mg/mL PPIX showed
complete bacterial kill, i.e., 0% GBS survival. Thus, the decrease
in percent GBS survival was dependent on the concentration of PPIX
added to the GBS prior to irradiation, and also on the number of
CFU/mL.
Example 3
[0159] In a third experiment, the GBS were assigned to five
experimental groups: (1) GBS only (control); (2) GBS and light; (3)
GBS supplemented with 0.05 mg/mL CP III; (4) GBS supplemented with
0.002 mg/mL CP III and light; and (5) GBS supplemented with 0.05
mg/mL CP III and light. Because the CP III was solubilized in
ethanol, two additional experimental groups were included to test
whether the quantity of ethanol used as a solvent had any
significant effect on bacterial growth suppression: (6) ethanol
only and (7) ethanol and light. The results are shown in FIG.
35.
[0160] As can be seen in FIG. 35, irradiation of GBS without the
addition of any CP III showed a minimal decrease in percent GBS
survival when compared to the control, i.e., approximately 98%,
which demonstrates that GBS does not synthesize a sufficient amount
of porphyrin. Also, GBS supplemented with 0.05 mg/mL CP III, but
without any irradiation, did not show much decrease in percent GBS
survival when compared to the control, i.e., approximately 84%. In
addition, irradiation of GBS supplemented with 0.002 mg/mL CP III
did not show much decrease in percent GBS survival when compared to
the control, i.e., approximately 78%, which demonstrates that the
concentration of CP III was too low. However, irradiation of GBS
supplemented with 0.05 mg/mL CP III showed complete bacterial kill,
i.e., 0% GBS survival. Thus, the decrease in percent GBS survival
was dependent on the concentration of CP III added to the GBS prior
to irradiation. It should also be noted that ethanol did not have
any significant contribution to the observed suppressive
effect.
Example 4
[0161] In a fourth experiment, the GBS were assigned to five
experimental groups: (1) GBS only (control); (2) GBS and light; (3)
GBS supplemented with 0.02 mg/mL CP III; (4) GBS supplemented with
0.002 mg/mL CP III and light; and (5) GBS supplemented with 0.05
mg/mL CP III and light. Because the CP III was solubilized in
ethanol, two additional experimental groups were included to test
whether the quantity of ethanol used as a solvent had any
significant effect on bacterial growth suppression: (6) ethanol
only and (7) ethanol and light. The results are shown in FIG.
36.
[0162] As can be seen in FIG. 36, irradiation of GBS without the
addition of any CP III showed a minimal decrease in percent GBS
survival when compared to the control, i.e., approximately 90%,
which again demonstrates that GBS does not synthesize a sufficient
amount of porphyrin. Also, GBS supplemented with 0.02 mg/mL CP III,
but without any irradiation, showed a small decrease in percent GBS
survival when compared to the control, i.e., approximately 74%. In
addition, irradiation of GBS supplemented with 0.002 mg/mL CP III
showed a significant decrease in percent GBS survival when compared
to the control, i.e., approximately 17%. Further, irradiation of
GBS supplemented with 0.05 mg/mL CP III showed complete bacterial
kill, i.e., 0% GBS survival. Thus, the decrease in percent GBS
survival was dependent on the concentration of CP III added to the
GBS prior to irradiation, and also on the number of CFU/mL. It
should also be noted that ethanol did not have any significant
contribution to the observed suppressive effect.
Study 2: GBS Supplemented with Porphyrins with Incubation after
Supplementation
[0163] GBS wild type strain COH1 was added to 3 mL of Todd Hewitt
broth and grown aerobically overnight at 37.degree. C. From the
overnight cultures, 500 uL of GBS was added to fresh Todd Hewitt
broth and grown logarithmically at 37.degree. C. until reaching a
concentration of 108 CFU/mL. The culture was then centrifuged at
1300 rpm for three minutes, the supernatant was removed and
discarded, and 1 mL of fresh Todd Hewitt broth was added.
[0164] The culture was then split into three Eppendorf tubes--a
first tube for GBS and second and third tubes for GBS with diluted
porphyrins used for incubation. Diluted porphyrins (PPIX or CP III)
were added into the second and third tubes and incubated for 30
minutes at 37.degree. C. The third tube was then washed three times
with phosphate-buffered saline (PBS) and centrifuged at 1300 rpm
for three minutes, and the supernatant was removed and discarded.
Serial dilutions of NaCl were made to obtain a suspension of GBS
diluted to a concentration of 1.times.10.sup.4 CFU/mL. The GBS were
streaked onto Todd Hewitt agar plates.
[0165] Some of the plates were irradiated in a chamber using the
450 nm lighted substrate shown in FIG. 22. The lighted substrate
was set to operate in 33% DF pulsed mode and driven with a constant
current source. The average irradiance was 3 mW/cm.sup.2. The pulse
duration was 10 microseconds with an off time of 20 microseconds,
and a pulse repetition rate of 33 kHz. The plates subject to light
treatment were irradiated three times at 0, 72 and 144 hours with a
radiant exposure of 7.56 J/cm.sup.2 during each irradiation session
(i.e., each irradiation session was 42 minutes with an incubation
period therebetween of 30 minutes).
[0166] After irradiation protocols were completed, the plates were
placed upside down in the anaerobic chamber and incubated at
37.degree. C. for 24 hours. The plates were photographed, colonies
counted and statistics of percent survival recorded.
Example 5
[0167] In a fifth experiment, the GBS were assigned to six
experimental groups: (1) GBS only (control); (2) GBS and light; (3)
GBS supplemented with 0.02 mg/mL PPIX (incubated); (4) GBS
supplemented with 0.02 mg/mL PPIX (incubated) and light; (5) GBS
supplemented with 0.02 mg/mL PPIX (incubated and washed); and (6)
GBS supplemented with 0.02 mg/mL PPIX (incubated and washed) and
light. The results are shown in FIG. 37.
[0168] As can be seen in FIG. 37, irradiation of GBS without the
addition of any PPIX showed a minimal decrease in percent GBS
survival when compared to the control, i.e., approximately 97%,
which demonstrates that GBS does not synthesize a sufficient amount
of porphyrin. Also, GBS supplemented with 0.02 mg/mL PPIX
(incubated), but without any irradiation, did not show much
decrease in percent GBS survival when compared to the control,
i.e., approximately 92%. In addition, irradiation of GBS
supplemented with 0.02 mg/mL PPIX (incubated) showed a small
decrease in percent GBS survival when compared to the control,
i.e., approximately 68%. This result is different than those seen
in FIGS. 33 and 34 because the bacteria was incubated at 37.degree.
C. for approximately thirty minutes before irradiation, which gives
them more time to grow, hence starting with an overall increased
CFU/mL compared to Examples 1 and 2 where irradiation was commenced
immediately after dilutions were made. This is reflected in the
higher percent of remnant colonies obtained in this experiment. GBS
supplemented with 0.02 mg/mL PPIX (incubated and washed) showed
some decrease in percent GBS survival when compared to the control,
i.e., approximately 92%. It is believed that there is a slight
decrease in percent GBS survival because PPIX may have some minimal
suppressive effects that may be concentration dependent. Further,
irradiation of GBS supplemented with 0.02 mg/mL PPIX (incubated and
washed) showed a significant decrease in percent GBS survival when
compared to the control, i.e., approximately 18%.
[0169] Overall, the decrease in percent GBS survival was greatest
for GBS supplemented with PPIX that was incubated and washed prior
to irradiation, which suggests that a sufficient quantity of PPIX
was internalized into the GBS during the incubation period. It
should be noted that the percent GBS survival for GBS supplemented
with PPIX that was incubated prior to irradiation was greater than
the percent GBS survival for GBS supplemented with PPIX that was
incubated and washed prior to irradiation. This difference is
believed to be caused by the fact that incubation with porphyrin
has some minimal effect on bacterial survival and, thus, it is
likely that more dead bacteria will be washed during the washing
step of the incubated sample resulting in a reduced starting
CFU/mL.
Example 6
[0170] In a sixth experiment, the GBS were assigned to six
experimental groups: (1) GBS only (control); (2) GBS and light; (3)
GBS supplemented with 0.02 mg/mL PPIX (incubated); (4) GBS
supplemented with 0.02 mg/mL PPIX (incubated) and light; (5) GBS
supplemented with 0.02 mg/mL PPIX (incubated and washed); and (6)
GBS supplemented with 0.02 mg/mL PPIX (incubated and washed) and
light. The results are shown in FIG. 38. It should be noted that
the results for Example 6 are similar to those of Example 5, which
is indicative of the reproducibility of the data.
Example 7
[0171] In a seventh experiment, the GBS were assigned to six
experimental groups: (1) GBS only (control); (2) GBS and light; (3)
GBS supplemented with 0.05 mg/mL PPIX (incubated); (4) GBS
supplemented with 0.05 mg/mL PPIX (incubated) and light; (5) GBS
supplemented with 0.05 mg/mL PPIX (incubated and washed); and (6)
GBS supplemented with 0.05 mg/mL PPIX (incubated and washed)) and
light. The results are shown in FIG. 39.
[0172] As can be seen in FIG. 39, irradiation of GBS without the
addition of any PPIX showed a minimal decrease in percent GBS
survival when compared to the control, i.e., approximately 80%,
which demonstrates that GBS does not synthesize a sufficient amount
of porphyrin. Also, GBS supplemented with 0.05 mg/mL PPIX
(incubated), but without any irradiation, did not show much
decrease in percent GBS survival when compared to the control,
i.e., approximately 72%. In addition, irradiation of GBS
supplemented with 0.05 mg/mL PPIX (incubated) showed a small
decrease in percent GBS survival when compared to the control,
i.e., approximately 60%. As explained above, this result is
different than those seen in FIGS. 33 and 34 because the bacteria
was incubated at 37.degree. C. for approximately thirty minutes
before irradiation, which gives them more time to grow, hence
starting with an overall increased CFU/mL compared to Examples 1
and 2 where irradiation was commenced immediately after dilutions
were made. This is reflected in the higher percent of remnant
colonies obtained in this experiment. GBS supplemented with 0.05
mg/mL PPIX (incubated and washed) showed some decrease in percent
GBS survival when compared to the control, i.e., approximately 92%.
Again, it is believed that there is a slight decrease in percent
GBS survival because PPIX may have some minimal suppressive effects
that may be concentration dependent. Further, irradiation of GBS
supplemented with 0.05 mg/mL PPIX (incubated and washed) showed a
significant decrease in percent GBS survival when compared to the
control, i.e., approximately 18%.
[0173] Overall, the decrease in percent GBS survival was greatest
for GBS supplemented with PPIX that was incubated and washed prior
to irradiation, which suggests that the PPIX was internalized into
the GBS during the incubation period. It should be noted that the
percent GBS survival for GBS supplemented with PPIX that was
incubated prior to irradiation was greater than the percent GBS
survival for GBS supplemented with PPIX that was incubated and
washed prior to irradiation. This difference is believed to be
caused by the fact that incubation with porphyrin has some minimal
effect on bacterial survival and, thus, it is likely that more dead
bacteria will be washed during the washing step of the incubated
sample resulting in a reduced starting CFU/mL.
Example 8
[0174] In an eighth experiment, the GBS were assigned to six
experimental groups: (1) GBS only (control); (2) GBS and light; (3)
GBS supplemented with 0.05 mg/mL PPIX (incubated); (4) GBS
supplemented with 0.05 mg/mL PPIX (incubated) and light; (5) GBS
supplemented with 0.05 mg/mL PPIX (incubated and washed); and (6)
GBS supplemented with 0.05 mg/mL PPIX (incubated and washed) and
light. The results are shown in FIG. 40. It should be noted that
the results for Example 8 are similar to those of Example 7, which
is indicative of the reproducibility of the data.
Example 9
[0175] In a ninth experiment, the GBS were assigned to six
experimental groups: (1) GBS only (control); (2) GBS and light; (3)
GBS supplemented with 0.05 mg/mL PPIX (incubated); (4) GBS
supplemented with 0.05 mg/mL PPIX (incubated) and light; (5) GBS
supplemented with 0.05 mg/mL PPIX (incubated and washed); and (6)
GBS supplemented with 0.05 mg/mL PPIX (incubated and washed) and
light. The results are shown in FIG. 41.
[0176] As can be seen in FIG. 41, irradiation of GBS without the
addition of any PPIX showed a minimal decrease in percent GBS
survival when compared to the control, i.e., approximately 94%,
which demonstrates that GBS does not synthesize a sufficient amount
of porphyrin. Also, GBS supplemented with 0.05 mg/mL PPIX
(incubated), but without any irradiation, did not show much
decrease in percent GBS survival when compared to the control,
i.e., approximately 90%. In addition, irradiation of GBS
supplemented with 0.05 mg/mL PPIX (incubated) showed a large
decrease in percent GBS survival when compared to the control,
i.e., approximately 20%. GBS supplemented with 0.05 mg/mL PPIX
(incubated and washed) also showed some decrease in percent GBS
survival when compared to the control, i.e., approximately 54%.
Again, it is believed that there is a slight decrease in percent
GBS survival because PPIX may have some minimal suppressive effects
that may be concentration dependent. Further, irradiation of GBS
supplemented with 0.05 mg/mL PPIX (incubated and washed) showed a
significant decrease in percent GBS survival when compared to the
control, i.e., approximately 6%, which suggests that the PPIX was
internalized into the GBS during the incubation period.
Example 10
[0177] In a tenth experiment, the GBS were assigned to six
experimental groups: (1) GBS only (control); (2) GBS and light; (3)
GBS supplemented with 0.05 mg/mL CP III (incubated); (4) GBS
supplemented with 0.05 mg/mL CP III (incubated) and light; (5) GBS
supplemented with 0.05 mg/mL CP III (incubated and washed); and (6)
GBS supplemented with 0.05 mg/mL CP III (incubated and washed) and
light. Because the CP III was solubilized in ethanol, two
additional experimental groups were included to test whether the
quantity of ethanol used as a solvent had any significant effect on
bacterial growth suppression: (7) ethanol only and (8) ethanol and
light. The results are shown in FIG. 42.
[0178] As can be seen in FIG. 42, irradiation of GBS without the
addition of any CP III showed a small decrease in percent GBS
survival when compared to the control, i.e., approximately 82%,
which again demonstrates that GBS does not synthesize a sufficient
amount of porphyrin. Also, GBS supplemented with 0.05 mg/mL CP III
(incubated), but without any irradiation, showed a small decrease
in percent GBS survival when compared to the control, i.e.,
approximately 90%. In addition, irradiation of GBS supplemented
with 0.05 mg/mL CP III (incubated) showed complete bacterial kill,
i.e., 0% GBS survival. GBS supplemented with 0.05 mg/mL CP III
(incubated and washed) showed minimal decrease in percent GBS
survival when compared to the control, i.e., approximately 72%.
Further, irradiation of GBS supplemented with 0.05 mg/mL CP III
(incubated and washed) showed complete bacterial kill, i.e., 0% GBS
survival, which suggests that the CP III was internalized into the
GBS during the incubation period. It should also be noted that
ethanol did not have any significant contribution to the observed
suppressive effect.
Example 11
[0179] In an eleventh experiment, the GBS were assigned to six
experimental groups: (1) GBS only (control); (2) GBS and light; (3)
GBS supplemented with 0.08 mg/mL PPIX (incubated); (4) GBS
supplemented with 0.08 mg/mL PPIX (incubated) and light; (5) GBS
supplemented with 0.08 mg/mL PPIX (incubated and washed); and (6)
GBS supplemented with 0.08 mg/mL PPIX (incubated and washed) and
light. The results are shown in FIG. 43.
[0180] As can be seen in FIG. 43, irradiation of GBS without the
addition of any PPIX showed a minimal decrease in percent GBS
survival when compared to the control, i.e., approximately 98%,
which demonstrates that GBS does not synthesize a sufficient amount
of porphyrin. Also, GBS supplemented with 0.08 mg/mL PPIX
(incubated), but without any irradiation, did not show much
decrease in percent GBS survival when compared to the control,
i.e., approximately 84%. In addition, irradiation of GBS
supplemented with 0.08 mg/mL PPIX (incubated) showed some decrease
in percent GBS survival when compared to the control, i.e.,
approximately 52%. GBS supplemented with 0.08 mg/mL PPIX (incubated
and washed) showed a small decrease in percent GBS survival when
compared to the control, i.e., approximately 76%. Again, it is
believed that there is a slight decrease in percent GBS survival
because PPIX may have some minimal suppressive effects that may be
concentration dependent. Further, irradiation of GBS supplemented
with 0.08 mg/mL PPIX (incubated and washed) showed some decrease in
percent GBS survival when compared to the control, i.e.,
approximately 40%, which suggests that at least some of the PPIX
was internalized into the GBS during the incubation period.
Example 12
[0181] In a twelfth experiment, the GBS were assigned to six
experimental groups: (1) GBS only (control); (2) GBS and light; (3)
GBS supplemented with 0.08 mg/mL PPIX (incubated); (4) GBS
supplemented with 0.08 mg/mL PPIX (incubated) and light; (5) GBS
supplemented with 0.08 mg/mL PPIX (incubated and washed); and (6)
GBS supplemented with 0.08 mg/mL PPIX (incubated and washed) and
light. The results are shown in FIG. 44. It should be noted that
the results for Example 12 are similar to those of Example 11,
which is indicative of the reproducibility of the data.
Example 13
[0182] In a thirteenth experiment, the GBS were assigned to six
experimental groups: (1) GBS only (control); (2) GBS and light; (3)
GBS supplemented with 0.2 mg/mL PPIX (incubated); (4) GBS
supplemented with 0.2 mg/mL PPIX (incubated) and light; (5) GBS
supplemented with 0.2 mg/mL PPIX (incubated and washed); and (6)
GBS supplemented with 0.2 mg/mL PPIX (incubated and washed) and
light. The results are shown in FIG. 45.
[0183] As can be seen in FIG. 45, irradiation of GBS without the
addition of any PPIX showed a minimal decrease in percent GBS
survival when compared to the control, i.e., approximately 95%,
which demonstrates that GBS does not synthesize a sufficient amount
of porphyrin. Also, GBS supplemented with 0.2 mg/mL PPIX
(incubated), but without any irradiation, did not show much
decrease in percent GBS survival when compared to the control,
i.e., approximately 80%. In addition, irradiation of GBS
supplemented with 0.2 mg/mL PPIX (incubated) showed some decrease
in percent GBS survival when compared to the control, i.e.,
approximately 50%. GBS supplemented with 0.2 mg/mL PPIX (incubated
and washed) showed a small decrease in percent GBS survival when
compared to the control, i.e., approximately 78%. Again, it is
believed that there is a slight decrease in percent GBS survival
because PPIX may have some minimal suppressive effects that may be
concentration dependent. Further, irradiation of GBS supplemented
with 0.2 mg/mL PPIX (incubated and washed) showed a significant
decrease in percent GBS survival when compared to the control,
i.e., approximately 10%, which suggests that the PPIX was
internalized into the GBS during the incubation period.
Example 14
[0184] In a fourteenth experiment, the GBS were assigned to six
experimental groups: (1) GBS only (control); (2) GBS and light; (3)
GBS supplemented with 0.2 mg/mL PPIX (incubated); (4) GBS
supplemented with 0.2 mg/mL PPIX (incubated) and light; (5) GBS
supplemented with 0.2 mg/mL PPIX (incubated and washed); and (6)
GBS supplemented with 0.2 mg/mL PPIX (incubated and washed) and
light. The results are shown in FIG. 46. It should be noted that
the results for Example 14 are similar to those of Example 13,
which is indicative of the reproducibility of the data.
Example 15
[0185] In a fifteenth experiment, the GBS were assigned to six
experimental groups: (1) GBS only (control); (2) GBS and light; (3)
GBS supplemented with 0.2 mg/mL PPIX (incubated); (4) GBS
supplemented with 0.2 mg/mL PPIX (incubated) and light; (5) GBS
supplemented with 0.2 mg/mL PPIX (incubated and washed); and (6)
GBS supplemented with 0.2 mg/mL PPIX (incubated and washed) and
light. The results are shown in FIG. 47. It should be noted that
the results for Example 15 are similar to those of Examples 13 and
14, which is again indicative of the reproducibility of the
data.
Example 16
[0186] In a sixteenth experiment, the GBS were assigned to six
experimental groups: (1) GBS only (control); (2) GBS and light; (3)
GBS supplemented with 0.2 mg/mL CP III (incubated); (4) GBS
supplemented with 0.2 mg/mL CP III (incubated) and light; (5) GBS
supplemented with 0.2 mg/mL CP III (incubated and washed); and (6)
GBS supplemented with 0.2 mg/mL CP III (incubated and washed) and
light. Because the CP III was solubilized in ethanol, two
additional experimental groups were included to test whether the
quantity of ethanol used as a solvent had any significant effect on
bacterial growth suppression: (7) ethanol only and (8) ethanol and
light. The results are shown in FIG. 48.
[0187] As can be seen in FIG. 48, irradiation of GBS without the
addition of any CP III showed a small decrease in percent GBS
survival when compared to the control, i.e., approximately 96%,
which again demonstrates that GBS does not synthesize a sufficient
amount of porphyrin. Also, GBS supplemented with 0.2 mg/mL CP III
(incubated), but without any irradiation, showed a small decrease
in percent GBS survival when compared to the control, i.e.,
approximately 82%. In addition, irradiation of GBS supplemented
with 0.2 mg/mL CP III (incubated) showed complete bacterial kill,
i.e., 0% GBS survival. GBS supplemented with 0.2 mg/mL CP III
(incubated and washed) showed minimal decrease in percent GBS
survival when compared to the control, i.e., approximately 80%.
Further, irradiation of GBS supplemented with 0.2 mg/mL CP III
(incubated washed) showed complete bacterial kill, i.e., 0% GBS
survival, which suggests that the CP III was internalized into the
GBS during the incubation period. It should also be noted that
ethanol did not have any significant contribution to the observed
suppressive effect.
Example 17
[0188] In a seventeenth experiment, the GBS were assigned to six
experimental groups: (1) GBS only (control); (2) GBS and light; (3)
GBS supplemented with 0.2 mg/mL CP III (incubated); (4) GBS
supplemented with 0.2 mg/mL CP III (incubated) and light; (5) GBS
supplemented with 0.2 mg/mL CP III (incubated and washed); and (6)
GBS supplemented with 0.2 mg/mL CP III (incubated and washed) and
light. Because the CP III was solubilized in ethanol, two
additional experimental groups were included to test whether the
quantity of ethanol used as a solvent had any significant effect on
bacterial growth suppression: (7) ethanol only and (8) ethanol and
light. The results are shown in FIG. 49.
[0189] As can be seen in FIG. 49, irradiation of GBS without the
addition of any CP III showed a small decrease in percent GBS
survival when compared to the control, i.e., approximately 98%,
which again demonstrates that GBS does not synthesize a sufficient
amount of porphyrin. Also, GBS supplemented with 0.2 mg/mL CP III
(incubated), but without any irradiation, showed a small decrease
in percent GBS survival when compared to the control, i.e.,
approximately 82%. In addition, irradiation of GBS supplemented
with 0.2 mg/mL CP III (incubated) showed complete bacterial kill,
i.e., 0% GBS survival. GBS supplemented with 0.2 mg/mL CP III
(incubated and washed) showed a small decrease in percent GBS
survival when compared to the control, i.e., approximately 72%.
Further, irradiation of GBS supplemented with 0.2 mg/mL CP III
(incubated and washed) showed some decrease in percent GBS survival
when compared to the control, i.e., approximately 42%. It should
also be noted that ethanol did not have any significant
contribution to the observed suppressive effect.
Example 18
[0190] In an eighteenth experiment, the GBS were assigned to six
experimental groups: (1) GBS only (control); (2) GBS and light; (3)
GBS supplemented with 0.2 mg/mL CP III (incubated); (4) GBS
supplemented with 0.2 mg/mL CP III (incubated) and light; (5) GBS
supplemented with 0.2 mg/mL CP III (incubated and washed); and (6)
GBS supplemented with 0.2 mg/mL CP III (incubated and washed) and
light. Because the CP III was solubilized in ethanol, two
additional experimental groups were included to test whether the
quantity of ethanol used as a solvent had any significant effect on
bacterial growth suppression: (7) ethanol only and (8) ethanol and
light. The results are shown in FIG. 50. It should be noted that
the results for Example 18 are similar to those of Example 17,
which is indicative of the reproducibility of the data.
V. Electron Microscopic Evidence of Disruption and Damage to Cell
Membrane of MRSA after Irradiation with Pulsed Blue Light
[0191] Further testing was performed using an electron microscope
to determine the impact on the cell membrane of MRSA when
irradiated with pulsed blue light using printed LED flexible
lamps.
Methods
[0192] MRSA was cultured in Tryptic soy broth (TSB) and divided
into two groups of 1.times.10.sup.8 CFU/mL. Each group was
separately plated on Tryptic soy agar (TSA). One group was
irradiated with a sub-lethal dose of 2.5 J/cm.sup.2 of 33% pulsed
450 nm blue light, while the other group served as non-irradiated
control. A sub-lethal dose was used to reduce the antimicrobial
effect of blue light; otherwise, it would have photo-eradicated the
entire bacterial culture. Irradiated and non-irradiated MRSA
cultures were then placed in a 37.degree. C. incubator for 24
hours. MRSA colonies obtained from both groups were resuspended in
TSB, fixed and then processed for Transmission Electron Microscopy
(TEM) and light microscopy using standard procedures, as shown in
FIG. 55. Samples were then sectioned and studied using a Zeiss
light microscope and a Technai TEM.
Results
[0193] Light microscopy of control and irradiated (i.e., treated)
MRSA showed that the samples were appropriately processed for TEM
imaging. FIG. 56 are light micrographs showing non-irradiated
control MRSA (left panel) and irradiated MRSA (right panel).
[0194] TEM of irradiated MRSA clearly revealed structural and
morphological changes in the treated group compared to the control
group. While the cell walls of non-irradiated control MRSA appeared
normal, smooth and stable, irregularities were seen in the cell
walls of irradiated MRSA, which appeared disrupted and fractured,
with breaks in portions of the cell membranes. FIG. 57 provides
evidence showing normal cell division in non-irradiated control
MRSA (A and C) and disruption of normal cell division in MRSA
irradiated with pulsed 450 nm blue light (B and D). Of note is the
structural modification of the nuclear material of irradiated MRSA
[Magnification=110,000] (B and D).
[0195] Also observed were cytoplasmic evidence of metabolic
irregularities in the irradiated (i.e., treated) group. FIG. 58
provides evidence showing structural damage to irradiated MRSA (B)
compared to non-irradiated control MRSA (A). FIG. 58 also shows
normal cell division in non-irradiated control MRSA (A) compared to
irradiated MRSA (B) [Magnification=67,000].
[0196] Control cells evinced high levels of metabolic activities in
their cytoplasm and the nuclei, and this is evidenced by the fine
distribution of intracellular content throughout the cell, as shown
in FIG. 57 (A, C) and FIG. 58 (A). Contrary to this observation,
the treated cells shown in FIG. 57 (B, D) and FIG. 58 (B) have very
dense clumps of nuclear material, suggestive of oxidative stress
and low metabolism. Furthermore, the non-irradiated control MRSA of
FIG. 57 (A, C) and FIG. 58 (A) can be seen undergoing normal cell
division as expected of a healthy rapidly replicating bacterium,
while cell division can be seen to be irregular and structurally
disrupted in the irradiated MRSA of FIG. 57 (B, D) and FIG. 58 (B).
As shown in FIG. 57 (A), early in the cell replication cycle,
non-irradiated control MRSA can be seen with indentations of the
membrane (arrowed), an indication that the cell is in the early
stage of dividing or replicating. Later in the cell replication
cycle, non-irradiated control MRSA were seen with clear-cut
cleavage furrows, as shown in FIG. 57 (C) and FIG. 58 (A). In
comparison, the cell membrane of irradiated MRSA appeared disrupted
and irregular with no evidence of cell replication, as shown in
FIG. 57 (B and D) and FIG. 58 (B).
Conclusion
[0197] These findings indicate that disruption of cell replication,
and structural damage of bacterial cell walls, are two other
mechanisms accounting for the antimicrobial effect of pulsed blue
light on MRSA. It is believed that these findings would also apply
to other types of microorganisms.
VI. Detection of Emission Spectra of Light Absorbing Pigment
"Porphyrins" in P. acnes
[0198] P. acnes gives off a florescence in the red spectrum of
600-700 nm (typically 620 nm to 640 nm) when illuminated with
pulsed purple or blue light. Without being bound by any one theory,
it is believed that porphyrins are excited (i.e., optically pumped)
with each light pulse and, upon return to their ground state,
create an oxidation reaction that produces free radicals which
subsequently destroy mitochondrial membranes, DNA or other cellular
structures, as described above. Thus, if a photo sensor is used to
detect red or other wavelengths given off by the cells undergoing
irradiation, then the feedback can be used to control the dose of
light, e.g., irradiation would take place until the porphyrin level
had reached a minimum point and would not begin until porphyrin
recovery took place. This may take place as part of the replication
cycle of the microorganism.
[0199] Testing was performed to detect the emission spectra of
light absorbing pigment porphyrins in P. acnes.
[0200] A 72 hour culture of P. acnes was centrifuged at 13,300 rpm
for five minutes, supernatant discarded and re-suspended in 1 mL
saline. It was then washed again by centrifuging at 13,300 rpm for
5 minutes with 1 mL saline (washing ensures that no other
factors--aside from the innate porphyrins found in the P. acnes
cells--would contribute to the detection). The optical density was
adjusted using McFarland standard to 0.8 to 1.0 at 625 nm for a
concentration of 108 CFU/mL. A volume of 10 .mu.l of this solution
was placed on concanavalin A coated dishes and excited as described
below.
[0201] As illustrated in FIG. 23, the set-up used for irradiating
the bacteria employed a 458 nm Ar-Ion laser assembly (Edmund
optics, USA) delivering continuous wave (CW) light with spectral
bandwidth of less than 1 nm and a total power of 150 mW. The
original, divergent laser beam was collimated using a lens (focal
length 35 mm) placed about 22 mm from the laser aperture. The final
beam diameter was 15 mm with a cross-sectional area of 3 cm.sup.2,
which approximately matched the area of the dish coated with
concanavalin A. The sample was placed on a translation stage
positioned about 25 cm from the collimating lens.
[0202] The sample was placed onto an inverted microscope (Axio
Observer--Z1, Carl Zeiss, Oberkochen, Germany) and illuminated
through the back port of the microscope using a continuous wave
Ar-Ion laser (Edmund Optics) with a 458 nm wavelength. The
excitation light (0.5 mW) was focused by a 63.times. water
immersion objective with numerical aperture (NA) of 1.2. The
microscope scanning head consisted of a computer-controlled OptiMiS
TruLine (Aurora Spectral Technologies, Milwaukee, Wis., USA). The
detection was done using a modified OptiMiS detection module (a
commercial prototype of OptiMiS d-Lux), which included a
galvanometric scanner used to descan the fluorescence beam which
then falls on an electron multiplying charged coupled device
(iXon3, Andor, Belfast, UK). This descanning concept allowed the
use of OptiMiS as a confocal (rather than a two-photon) microscope.
This in turn reduced the laser-induced photo-bleaching of the
cellular fluorescence, which was critical in these experiments
given the low intensity of the signal detected. In order to capture
full spectrum images, the sample was scanned line by line. The line
images were further reconstructed resulting in an image for each
wavelength channel (total of 300 channels).
[0203] The extraction of the porphyrin spectrum was performed by
first measuring the spectrum of the emission from the bacteria and
the spectrum of the media outside of the bacteria. Next, the
spectrum of the media was subtracted from the bacterial emission
spectrum while considering the emission ratios between the bacteria
and the outside media. Finally, the resulting spectrum was fitted
with two Gaussians as the emission has two humps.
[0204] FIG. 24A depicts the porphyrin emission spectrum extracted
from P. acnes excited with 458 nm light and detected using OptiMiS
TruLine (Aurora Spectral Technologies, Milwaukee, Wis.). The peak
wavelengths of the resulting emission were 619 nm and 670 nm for
the two apparent humps. This emission is characteristic of
cytochrome C which most likely is present in the acne bacteria.
However, it may still contain some emission from different
porphyrins such as coproporphyrins which may participate in the
bacterial eradication mechanism. It is proposed that the free
radical production by peroxides generated from porphyrins exposed
to purple or blue light may alter DNA structure and lead to
destruction of the bacteria during its replicative phase. Thus, the
timing of light exposures during the replicative phase may have
significant impact on the kill rates.
[0205] FIG. 24B demonstrates the fluorescence spectra of P. acnes
at various times during irradiation from zero to 30 minutes
(measured at T=1, 10, 20 and 30 minutes). It can be readily seen
that the fluorescence gradually depletes over time and thus can act
as an indicator of porphyrin depletion at the cellular level. The
porphyrin depletion follows the dose parameters (approximately 30
minutes of irradiation at 3 mW/cm.sup.2) which were found to be
optimal for photoeradication of bacteria. These experiments were
performed in vitro, in a petri dish in agar, illuminated with 450
nm printed LEDs on a flexible substrate. The output was pulsed at a
duty factor of 33% (10 microseconds on and 20 microseconds off) at
an irradiance of 3 mW/cm.sup.2. A calibrated ocean optics
OCEAN-FX-VIS-NIR-ES spectrometer was used to record the
fluorescence. The dish was held in a light tight housing. The blue
450 nm excitation light was filtered to eliminate all wavelengths
beyond 420 and 480 nm. A filter was used below the dish to
eliminate any wavelengths below 510 nm. The extraction of the
porphyrin spectrum was performed by first measuring the spectrum of
the emission from the bacteria and the spectrum of the media
outside of the bacteria. Next, the spectrum of the media was
subtracted from the bacterial emission spectrum while considering
the emission ratios between the bacteria and the outside media. The
spectra were then plotted.
[0206] FIG. 24C depicts the porphyrin emission spectrum extracted
from a sample of Protoporphyrin IX excited with 450 nm printed LEDs
on PET substrate and detected using an ocean optics
OCEAN-FX-VIS-NIR-ES spectrometer in the same test set-up as FIG.
24B. The peak wavelengths of the resulting emission were 620 nm and
670 nm and 750 nm for the three apparent humps. This emission is
similar to the initial porphyrin emission spectrum extracted from
P. acnes excited with 458 nm light (FIG. 24A); however, it has a
broader emission in the near infrared range.
[0207] Dosage can thus be optimized by detecting the emission
spectrum of porphyrins or other emissions from the targeted
bacteria, measuring fluorescence depletion or recovery, determining
the optimal excitation periods and recovery between treatment
sessions, and calculating ideal irradiation parameters, including
dosage, irradiance, fluence and treatment schedule. It is also
possible that killing of different strains of bacteria can be
optimized using this method. Ideal wavelengths to measure porphyrin
depletion are shown to be in the 620-640 nm range and the 670 nm
range from the data presented, which correlates well with our data
shown in FIGS. 24A, 24B and 24C. Ideal wavelengths to measure other
types of emissions can be determined by one skilled in the art.
VII. Exemplary Light Sources
[0208] Various types of light sources may be used to provide light
energy in accordance with the present invention. For example, the
light source may comprise various types of lasers, LEDs,
PLEDs/LEPs), QDLEDs, or fluorescent tubes emitting light in the
purple or blue spectral region (and optionally the red or infrared
spectral region). The light source may directly illuminate the
skin, tissue or other surface to be irradiated, such as a
contaminated environment or food to be exposed to pulsed light. The
light source may also be coupled by fiber optics or directly
mounted to the edge of a lens-diffuser that redirects the light to
the surface to be irradiated. These diffusers may contain numerous
optical elements designed to diffuse and distribute the light
evenly across the surface to be irradiated. These may include
Fresnel lenses, various light pipes, irregularities and additional
lens-like structures designed to pipe the light source and
distribute it evenly over the surface of the lens-diffuser
assembly. In order to improve the extraction or out-coupling of
light, the device may include an internal scattering layer of high
index particles such as TiOx in a transparent photoresist or a
micro-lens array (MLA) layer. The light sources may also be placed
directly behind the diffuser or Fresnel lens at a sufficient
distance to allow the production of a substantially uniform beam to
be applied to the surface to be irradiated. The light source may be
a combination of white light and purple or blue light, whereupon
the purple or blue light is pulsed and the white light is
continuous wave (CW). These different wavelengths can be applied
simultaneously or sequentially to irradiate an infected environment
or food.
[0209] A preferred light source is provided in the form of a very
thin layered structure. An example of this type of light source is
shown generally as reference numeral 10 in FIG. 25. Light source 10
is comprised of a plurality of layers 12 including a flexible light
emitter 40 located between an anode 50 and a cathode 60. A suitable
power source is connected to light source 10. Preferably, direct
current (DC) or pulsed DC is used to power light source 10. Light
source 10 is substantially planar in its form, although it is
preferably flexible and is more preferably conformable such that it
may conform to the contours of the body, surfaces within a
contaminated environment, or surfaces inside of a refrigeration or
food display system. The overall thickness of light source 10 is
typically about 10 mm or less (e.g., about 10, 9, 8, 7, 6, 5, 4, 3,
2, 1, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05 mm or less).
[0210] Light source 10 produces light with an intensity that is
substantially constant across the surface of the device so as to
provide substantially uniform light emission. As described in more
detail below, the light source may comprise, for example, organic
light emitting diodes (OLEDs) or printed light emitting diodes
(printed LEDs) (organic or inorganic) commonly referred to as LED
ink. It can be appreciated that the light source is capable of
decreasing hot spots on the surface of a subject's skin or tissue
to provide a safer delivery of light to a subject. Also, a
substantially uniform dose of light across the surface of the
device ensures that all of the skin, tissue, surface or food is
effectively treated with the same dose of light.
[0211] FIG. 22 shows an example of a light source that includes a
plurality of light emitting diodes 42 printed on a flexible PET
(polyethylene terephthalate)-ITO coated film 44 (referred to herein
as a printed LED flexible lamp or a lighted substrate). In this
example, the light emitting diodes 42 are printed in hexagon-shaped
LED clusters at a density of 2.5 LEDS/mm.sup.2. Of course, in other
examples, the LED clusters could have other shapes, the LEDS could
be printed across the entire surface of the film, and/or the LEDs
could be printed in other densities in accordance with the present
invention.
[0212] In one embodiment, flexible light emitter 40 comprises a
printed LED film that is laminated to a quantum dot film. In this
embodiment, all of the light emitting diodes of the printed LED
film (such as light emitting diodes 42 shown in FIG. 22) are
configured to emit purple or blue light. The quantum dot film
comprises a slurry of quantum dots (including cadmium-free quantum
dots (CFQDs)) coated on a flexible film and then another flexible
film is placed thereon in order to seal the quantum dots between
the two films. Alternatively, the CFQDs could be coated on the
printed LED substrate with a barrier coating or film applied to the
outer side in order to prevent oxidation of the CFQDs. The quantum
dots are configured to convert purple or blue light to red or
infrared light upon receipt of the pulsed purple or blue light from
the printed LED film so as to maintain the same pulse
characteristics for both the red or infrared light and the purple
or blue light. Various densities of quantum dots can be used to
control the percentage of light converted from purple or blue light
to red or infrared light. In one embodiment, the percentages of
purple or blue and red or infrared light range from 20% purple or
blue light/80% red or infrared light to 80% purple or blue
light/20% red or infrared light. Of course, other percentages of
purple or blue and red or infrared light could also be used as
desired for a certain application. Preferably, the purple or blue
light emitting diodes and the quantum dots are evenly distributed
across the surface of the device so as to provide a substantially
uniform emission of purple or blue and red or infrared light.
[0213] In another embodiment, flexible light emitter 40 comprises
only a printed LED film (i.e., a quantum dot film is not used in
this embodiment). In this embodiment, the light emitting diodes of
the printed LED film (such as light emitting diodes 42 shown in
FIG. 22) comprise a combination of purple or blue and red or
infrared light emitting diodes that are printed on a flexible film
(such as flexible film 44 shown in FIG. 22) in a checkerboard or
grid-like pattern. The respective number of purple or blue and red
or infrared light emitting diodes can be chosen to achieve the
desired levels of purple or blue and red or infrared light. In one
embodiment, the percentages of purple or blue and red or infrared
light preferably range from 20% purple or blue light/80% red or
infrared light to 80% purple or blue light/20% red or infrared
light. Of course, other percentages of purple or blue and red or
infrared light could also be used as desired for a certain
application. Preferably, the purple or blue and red or infrared
light emitting diodes are evenly distributed across the surface of
the device so as to provide a substantially uniform emission of
purple or blue and red or infrared light.
[0214] It should be understood that the irradiance and radiant
exposure of each of the purple or blue light and red or infrared
light will be a percentage of the total irradiance and radiant
exposure discussed above, as determined by the density of the
quantum dots (for the first embodiment discussed above) or the
respective numbers of purple or blue and red or infrared light
emitting diodes (for the second embodiment discussed above), or the
drive current applied respectively to the purple or blue and red or
infrared light emitting diodes by the driving device. As such, the
desired levels of purple or blue light and red or infrared light
may be selectively achieved for a particular light treatment.
[0215] In another embodiment, flexible light emitter 40 comprises
OLEDs laminated to a quantum dot film, wherein the OLEDs emit
purple or blue light and the quantum dot film converts a portion of
the purple or blue light to red or infrared light. In yet another
embodiment, flexible light emitter 40 comprises OLEDS that emit
both purple or blue and red or infrared light. It can be
appreciated that these embodiments are similar to the two
embodiments described above, with the exception that the printed
LED film is replaced with the OLEDs. In yet other embodiments,
flexible light emitter 40 comprises printed LED film or OLEDS that
emit only purple or blue light.
[0216] The light source may be in the form of an array, patch, pad,
mask, wrap, fiber, bandage or cylinder, for example. The light
source may have a variety of shapes and sizes. For example, the
light source may be square, rectangular, circular,
butterfly-shaped, elliptical, clover-shaped, oblong,
crescent/moon-shaped, or any other shape that is suitable for a
particular application. Examples of suitable light sources are
generally illustrated in FIGS. 26A and 26B, as well as the light
devices shown in FIGS. 51 and 52. The overall surface area of one
side of the light source may range from, for example, 1 cm.sup.2 to
1 m.sup.2, or in panels placed together to cover larger areas,
although typically the surface area is about 1 to 2000 cm.sup.2
(e.g., about 1, 4, 9, 16, 25, 36, 49, 64, 81, 100, 121, 144, 169,
196, 225, 289, 324, 361, 400, 441, 484, 529, 576, 625, 676, 729,
784, 841, 900, 961, 1024, 1089, 1156, 122, 1296, 1369, 1444, 1521,
1600, 1681, 1764, 1849, 1936 or 2000 cm.sup.2 or some range
therebetween). The light source is thus well adapted to be applied
to various areas of the subject's body, for example, the face,
forehead, cheek, back, wrist and the like. The light source may
also be applied other types of surfaces, such as surfaces within a
contaminated environment or surfaces inside of a refrigeration or
food display system.
[0217] The various elements/layers of the light source will now be
described in greater detail.
Substrate
[0218] In some embodiments, the light source includes a substrate.
The substrate may be any substance capable of supporting the
various layers of the light source. The substrate is preferably
flexible and/or conformable to a surface in which the light source
will be used, such as the contours of a subject's body, surfaces
within a contaminated environment, or surfaces inside of a
refrigeration, food display or processing system. The substrate can
comprise, for example, an inorganic material, an organic material,
or a combination of inorganic and organic materials. The substrate
may be, for example, made from metals, plastics or glass. The
substrate may be any shape to support the other components of the
light source, for example, the substrate may be substantially flat
or planar, curved, or have portions that are substantially flat
portions and curved portions. Most preferably, the substrate is
transparent, flexible, and conformable in nature. Ideally, the
material is a latex-free, non-toxic, non-allergenic material, which
is resistant to UV, sunlight and most infection control
products.
[0219] As used herein, the term "transparent" generally means
transparency for light and includes both clear transparency as well
as translucency. Generally, a material is considered transparent if
at least about 50%, preferably about 60%, more preferably about
70%, more preferably about 80% and still more preferably about 90%
of the light illuminating the material can pass through the
material. In contrast, the term "opaque" generally refers to a
material in which the light is substantially absorbed or reflected,
e.g., at least 90% of the light is absorbed or reflected, and
typically at least 95% of the light is absorbed or reflected.
[0220] In some embodiments, the substrate may be comprised of a
silicon-based material, rubber, thermoplastic elastomers (TTP), or
other polymeric material, such as polyester, polyethylene
terephthalate (PET), polyethylene naphthalate (PEN), polycarbonate,
polystyrene, polyacryl, polyether sulfone (PES), etc. Transparent
substrates may include, for example, polyethylene, ethylene-vinyl
acetate copolymers, polyimide (PI), polyetherimide (PEI),
ethylene-vinyl alcohol copolymers, polypropylene, polystyrene,
polymethyl methacrylate, PVC, polyvinyl alcohol, polyvinylbutyral,
polyether ether ketone, polysulfone, polyether sulfone, as well as
fluoropolymers, such as, fluorinated ethylene-propylene (FEP),
tetrafluoroethylene-perfluoroalkyl vinyl ether copolymers,
polyvinyl fluoride, tetrafluoroethylene-ethylene copolymers,
tetrafluoroethylene-hexafluoropropyl ene copolymers,
polychlorotrifluoroethylene, polyvinylidene fluoride, polyester,
polycarbonate, polyurethanes, polyimide or polyether imide.
[0221] In another embodiment, the transparent substrate is a
polyester film, such as Mylar. In another aspect, the substrate
comprises a poly-theretherketone film commercially available from
Victrex under the name APTIV. In still another aspect, the
substrate is a thin film sold under the name Flexent by Konica
Minolta or flexible glass such as Willow Glass by Dow Corning.
Ideally, substrates in direct or indirect contact with organic
layers will have exceptional barrier capabilities that withstand
heat, offer flexibility, have sustained reliability and can be mass
produced,
Conductive Layers (Electrodes)
[0222] The light source comprises a plurality of conductive layers
(i.e., electrodes), namely, a cathode and an anode. The anode may
comprise, for example, a transparent conductive oxide (TCO), such
as, but not limited to, indium tin oxide (ITO), zinc oxide (ZnO),
and the like. The cathode may also comprise, for example, a thin
metal film or fibers such as aluminum, copper, gold, molybdenum,
iridium, magnesium, silver, lithium fluoride and alloys thereof, or
a non-metal conductive layer.
[0223] Because the light source must emit light through one or both
electrodes, at least one of the electrodes must be transparent. The
transparent electrode is positioned on the side of the light source
designed to be facing the surface to be irradiated. For a light
source intended to emit light only through the bottom electrode
(i.e., the surface-facing electrode), the top electrode (i.e., the
electrode facing away from the surface) does not need to be
transparent. The top electrode may thus comprise an opaque or
light-reflective metal layer having a high electrical conductivity.
Where a top electrode does not need to be transparent, using a
thicker layer may provide better conductivity, and using a
reflective electrode may increase the amount of light emitted
through the transparent electrode by reflecting light back towards
the transparent electrode. Fully transparent light sources may also
be fabricated, where both electrodes are transparent.
[0224] The thickness of each electrode is typically about 200 nm or
less (e.g., about 200, 180, 160, 140, 120, 100, 90, 80, 70, 60, 50,
40, 30 nm or less). Preferably, the thickness of each electrode is
less than 10 nm (e.g., about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.8,
0.6, 0.4, 0.2 nm or less or some range therebetween).
[0225] The electrodes are preferably flexible in nature. In some
embodiments, the conductive materials of one or both of the
electrodes may include, but are not limited to, transparent
conductive polymer materials, such as indium tin oxide (ITO),
fluorine-doped tin oxide (FTO), ZnO--Ga2O3, ZnO--Al.sub.2O.sub.3,
SnO2-Sb2O3, and polythiophene. In addition, the electrodes may be
comprised of silver or copper grids or bushbars plated on a
transparent substrate or silver nanowires or nanoparticles
deposited on a substrate with a poly(3,4-ethylenedioxythiophene)
poly(styrenesulfonate) (PEDOT:PSS) coating. Additional conductive
polymer layers may be added to improve conductivity.
[0226] In one aspect, the transparent conductive electrode may be
carbon-based, for example, carbon nanotubes, carbon nanowires, or
graphene, and the like. One preferred electrode (typically for
infrared) comprises graphene. While one or two layers of graphene
are preferred, the electrode may comprise about 1 to 20 layers of
graphene (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20 layers or some range therebetween). The graphene
electrode(s) also have the effect of protecting the photoactive
layer sandwiched between them from oxidation. Therefore,
environmental stability of the light source can be improved. The
graphene electrode may optionally have a plurality of plasmonic
nanostructures, which may have various morphologies (spherical,
rods, discs, prisms, etc.). Exemplary nanostructures include those
made of gold, silver, copper, nickel, and other transition metals,
for example gold nanoparticles, silver nanoparticles, copper
nanoparticles, nickel nanoparticles, and other transition metal
nanoparticles. In general, any electrically conductive materials,
such as oxides and nitrides, of surface plasmonic resonance
frequencies in the visible spectrum can be made into plasmonic
nanostructures for the same purpose. In some embodiments, the
plasmonic particles have the size of about 1 nm to about 300 nm
(e.g., about 10, 50, 100, 150, 200, 250, 300 nm, or some range
therebetween).
Light Emitter Layer
[0227] The light source includes a thin light emitter that may
comprise, for example, OLEDs or printed LEDs (organic or
inorganic). The thickness of the light emitter is preferably about
2 mm or less (e.g., about 2, 1.8, 1.6, 1.4, 1.2, 1, 0.9, 0.8, 0.7,
0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04,
0.03, 0.02, 0.01 mm or less). Most preferably, the flexible light
emitter is about 10 to 200 nm in thickness (e.g., about 200, 180,
160, 140, 120, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 nm, or some
range therebetween). The light emitter is preferably flexible and
emits light in response to an electric current applied to the anode
and cathode.
[0228] OLEDs
[0229] In some embodiments, the light source may comprise OLEDs in
which the flexible light emitter is a thin organic film. As used
herein, the term "organic" with respect to OLEDs encompasses
polymeric materials as well as small molecule organic materials
that may be used to fabricate organic opto-electronic devices. Such
materials are well known in the art. "Small molecule" refers to any
organic material that is not a polymer, and it will be appreciated
that "small molecules" may actually be quite large. "Small
molecules" may include repeat units in some circumstances. For
example, using a long chain alkyl group as a substituent does not
remove a molecule from the "small molecule" class. "Small
molecules" may also be incorporated into polymers, for example as a
pendent group on a polymer backbone or as a part of the backbone.
"Small molecules" may also serve as the core moiety of a dendrimer,
which consists of a series of chemical shells built on the core
moiety. The core moiety of a dendrimer may be a fluorescent or
phosphorescent small molecule emitter. A dendrimer may be a "small
molecule," and it is believed that all dendrimers currently used in
the field of OLEDs are small molecules. In general, a "small
molecule" has a well-defined chemical formula with a single
molecular weight, whereas a polymer has a chemical formula and a
molecular weight that may vary from molecule to molecule.
[0230] Generally speaking, in the flexible light emitter, electrons
and holes recombine to radiate photons. The radiative photon energy
emitted from the flexible light emitter corresponds to the energy
difference between the lowest unoccupied molecular orbital (LUMO)
level and the highest occupied molecular orbital (HOMO) level of
the organic material. Photons of lower energy/longer wavelength may
be generated by higher-energy photons through fluorescent or
phosphorescent processes.
[0231] As described below, the flexible light emitter may
optionally include one or more of a hole injection material (HIM),
a hole transport material (HTM), a hole blocking material (HBM), an
electron injection material (EIM), an electron transport material
(ETM), an electron blocking material (EBM), and/or an exciton
blocking material (ExBM).
[0232] In one aspect, the emissive electroluminescent layer may
include a hole injection material (HIM). A HIM refers to a material
or unit capable of facilitating holes (i.e., positive charges)
injected from an anode into an organic layer. Typically, a HIM has
a HOMO level comparable to or higher than the work function of the
anode, i.e., -5.3 eV or higher.
[0233] In another aspect, the emissive electroluminescent layer may
include a hole transport material (HTM). A HTM has the
characteristic of a material or unit capable of transporting holes
(i.e., positive charges) injected from a hole injecting material or
an anode. A HTM has usually high HOMO, typically higher than -5.4
eV. In many cases, HIM can also function as HTM, depending on the
adjacent layer.
[0234] In another aspect, the emissive electroluminescent layer may
include a hole blocking material (HBM). A HBM generally refers to a
material that, if deposited adjacent to an emitting layer or a hole
transporting layer in a multilayer structure, prevents the holes
from flowing through. Usually it has a lower HOMO as compared to
the HOMO level of the HTM in the adjacent layer. Hole-blocking
layers are frequently inserted between the light-emitting layer and
the electron-transport layer.
[0235] In another aspect, the emissive electroluminescent layer may
include an electron injection material (EIM). An EIM generally
refers to a material capable of facilitating electrons (i.e.,
negative charges) injected from a cathode into an organic layer.
The EIM usually has a LUMO level comparable to or lower than the
working function of the cathode. Typically, the EIM has a LUMO
lower than -2.6 eV.
[0236] In another aspect, the emissive electroluminescent layer may
include an electron transport material (ETM). An ETM generally
refers to a material capable of transporting electrons (i.e.,
negative charges) injected from an EIM or a cathode. The ETM has
usually a low LUMO, typically lower than -2.7 eV. In many cases, an
EIM can serve as an ETM as well, depending on the adjacent
layer.
[0237] In another aspect, the emissive electroluminescent layer may
include an electron blocking material (EBM). An EBM generally
refers to a material which, if deposited adjacent to an emissive or
electron transporting layer in a multilayer structure, prevents the
electron from flowing through. Usually it has a higher LUMO as
compared to the LUMO of the ETM in the adjacent layer.
[0238] In another aspect, the emissive electroluminescent layer may
include an exciton blocking material (ExBM). An ExBM generally
refers to a material which, if deposited adjacent to an emitting
layer in a multilayer structure, prevents the excitons from
diffusing through. ExBM should have either a higher triplet level
or singlet level as compared to the emitting layer or other
adjacent layer.
[0239] Exemplary OLED materials are described in Hammond et al.,
U.S. Published Patent Application No. 2010/0179469; Pan et al.,
U.S. Published Patent Application No. 2013/0006119; Buchholz et
al., PCT Published Patent Application No. WO 2012/010238; and
Adamovich et al., U.S. Published Patent Application No.
2007/0247061.
[0240] Referring to FIG. 27, a typical sequence of materials found
in the flexible light emitter between the anode and the cathode of
the OLED is HIM, HTM, emission layer, HBM, and ETM. Another typical
sequence of materials is HTM, emission layer, and ETM. Of course,
other sequences of materials are also possible. Further, the OLED
may comprise one or more interlayers.
[0241] In one aspect, the flexible light emitter comprises a single
layer. The flexible light emitter may comprise, for example, a
conjugated polymer which is luminescent, a hole-transporting
polymer doped with electron transport molecules and a luminescent
material, or an inert polymer doped with hole transporting
molecules and a luminescent material. The flexible light emitter
may also comprise an amorphous film of luminescent small organic
molecules which can be doped with other luminescent molecules.
[0242] In another aspect, the flexible light emitter may comprise
one or more different emissive materials in either the same
emission layer or in different emission layers. For example, the
flexible light emitter may comprise 5, 4, 3, 2, or 1 radiation
emitting materials. The various different emissive materials may be
selected from the emissive materials described in the references
set forth above, but any other suitable emissive material can be
employed. If two emissive materials are used in one emission layer,
the absorption spectrum of one of the two emissive materials
preferably overlaps with the emission spectrum of the other
emissive material. The emissive materials may be arranged in
stacked layers or side-by-side configurations. The emissive layer
may comprise a continuous region forming a single emitter or a
plurality of light emitters. The plurality of light emitters may
emit light with substantially different wavelengths. The plurality
of light emitters may be vertically stacked within the emissive
layer or they may form a mixture. In some embodiments, a dopant is
dispersed within an organic host matrix. In one embodiment, a layer
of quantum dots is sandwiched between two organic thin films.
[0243] In another aspect, the flexible light emitter may comprise a
plurality of layers sharing a common anode and/or cathode. In this
case, individual layers are stacked one on top of another. The
stacked configuration may generally include intermediate electrodes
disposed between adjacent layers such that successive layers share
an intermediate electrode, i.e., a top electrode of one layer is
the bottom electrode of another in the stack. The stacked layers
may be formed of different materials, and therefore, different
emissions spectra.
[0244] The flexible light emitter may be substantially transparent.
When mostly transparent layers are used, a plurality of emissive
layers may be vertically stacked without substantially blocking
light emission from individual layers. The flexible light emitter
may comprise a single or multiple layers, for example, a
combination of p- and n-type materials. The p- and n-type materials
may be bonded to each other in the layer. The bonding may be ionic
or covalent bonding, for example. The multiple layers of the
flexible light emitter may form heterostructures therebetween.
[0245] Printed LEDs
[0246] In some embodiments, the light source may comprise printed
LEDs (organic or inorganic), i.e., LED ink. With LED ink, each
light source is very small which enables the LEDs to be positioned
in very close proximity to each other. During fabrication, the LEDs
may be printed in a uniform manner whereby each LED operates as a
point source in which the beams from the individual LEDs are
substantially parallel to each other to provide substantially
uniform light across the surface of the device. Unlike conventional
LEDs, printed LEDs do not need to be positioned a sufficient
distance from the surface to be irradiated in order to deliver a
substantially uniform dose of light. There are several known
methods for printing such LEDs, as described below.
[0247] In one method, a plurality of individual LEDs are suspended
and dispersed in a liquid or gel comprising one or more solvents
and a viscosity modifier so as to form a diode ink that is capable
of being printed on a flexible substrate (e.g., through screen
printing, flexographic printing and the like). In one aspect, the
average surface area concentration of LEDs is from about 25 to
50,000 LEDs per square centimeter. In general, each LED includes a
light emitting region, a first metal terminal located on a first
side of the light emitting region, and a second metal terminal
located on a second side of the light emitting region. The first
and second metal terminals of each LED may be electrically coupled
to conductive layers (i.e., electrodes) to enable the light
emitting region to emit light when energized.
[0248] An exemplary light source is shown generally in FIG. 28,
wherein only five LEDs are provided in order to simplify the
description. As can be seen, this device includes a plurality of
conductors 80a-80e deposited on a flexible substrate 82. A
plurality of LEDs 84a-84e are deposited on the conductors 80a-80e
such that the first metal terminals of the LEDs 84a-84e are
electrically coupled to the conductors 80a-80e. One skilled in the
art will appreciate that the LEDs 84a-84e may be formed of various
shapes. Preferably, the LEDs 84a-84e settle into a position over
conductors 80a-80e such that they maintain their polarity based on
the shape of the LEDs. Next, a plurality of dielectric layers
86a-86e are deposited over the LEDs 84a-84e and the conductors
80a-80e, as shown. Another conductor 88 is then deposited over the
LEDs 84a-84e and dielectric layers 86a-86e such that the second
metal terminals of the LEDs 84a-84e are coupled to the conductor
88. One skilled in the art will appreciate that the substrate 82
and conductors 80a-80e may be transparent so that light is emitted
from the bottom of the device and/or conductor 82 may be
transparent so that light is emitted from the top of the device.
Various configurations of printed LEDs that may be manufactured in
accordance with the above method are described in Lowenthal et al.,
U.S. Pat. No. 8,415,879.
[0249] In another method, the light source comprises LEDs that are
created through a printing process. In this method, a substrate is
provided that includes a plurality of spaced-apart channels. A
plurality of first conductors are formed on the substrate such that
each first conductor is positioned in one of the channels. Next, a
plurality of substantially spherical substrate particles are
coupled to the first conductors and, then the substantially
spherical substrate particles are converted into a plurality of
substantially spherical diodes. The substantially spherical diodes
may comprise, for example, semiconductor LEDs, organic LEDs
encapsulated organic LEDs, or polymer LEDs. A plurality of second
conductors are then formed on the substantially spherical diodes.
Finally, a plurality of substantially spherical lenses suspended in
a polymer (wherein the lenses and suspending polymer have different
indices of refraction) are deposited over the substantially
spherical diodes and the second conductors. Thus, in this method,
the LED's are built up on the substrate as opposed to being mounted
on the substrate. Various configurations of printable LEDs that may
be manufactured in accordance with the above method are described
in Ray et al., U.S. Pat. No. 8,384,630.
[0250] In an exemplary embodiment, the light source is comprised of
printed LEDs having a thickness of 12 .mu.m, silver electrodes each
of which has a thickness of 5-10 .mu.m with a transparent silver
fiber having a thickness of 0.05-5 .mu.m, and a PET substrate
having a thickness of 125 .mu.m.
Micro-Lens Array
[0251] The light source may optionally include a light dispersion
layer, such as a micro-lens array. It has been found that one of
the key factors that limits the efficiency of OLED devices is the
inefficiency in extracting the photons generated by the
electron-hole recombination out of the OLED devices. Due to the
high optical indices of the organic materials used, most of the
photons generated by the recombination process are actually trapped
in the devices due to total internal reflection. These trapped
photons never leave the OLED devices and make no contribution to
the light output from these devices. In order to improve the
extraction or out-coupling of light from OLEDs, the device may
include an internal scattering layer of high index particles such
as TiOx in a transparent photoresist or a micro-lens array (MLA)
layer. Exemplary MLAs and methods for forming the same are
described in Gardner et al., U.S. Published Patent Application No.
2004/01217702; Chari et al. U.S. Pat. No. 7,777,416; Xu et al.,
U.S. Pat. No. 8,373,341; Yamae et al., High-Efficiency White OLEDs
with Built-up Outcoupling Substrate, SID Symposium Digest of
Technical Papers, 43 694 (2012); and Komoda et al., High Efficiency
Light OLEDS for Lighting, J. Photopolymer Science and Technology,
Vol. 25, No. 3 321-326 (2012). Barrier Layer
[0252] The light source may optionally include one or more
encapsulation or barrier layers that isolate the light emitter (or
other layers) from an ambient environment. The encapsulation or
barrier layer is preferably substantially impermeable to moisture
and oxygen. In general, the moisture and oxygen sensitive
components should be enclosed by materials having gas permeation
properties. The barrier preferably achieves low water vapor
permeation rates of 10.sup.-4 g/m.sup.2/day or less, 10.sup.-5
g/m.sup.2/day or less, and even more preferably about 10.sup.-6
g/m.sup.2/day or less.
[0253] The encapsulation or barrier layer may be glass or a
plastic, for example. Exemplary materials include a
polyetheretherketone film commercially available from Victrex under
the name APTIV. In still another aspect, the substrate is a thin
film sold under the name Flexent by Konica Minolta or flexible
glass such as Willow Glass by Dow Corning. Ideally, substrates in
direct contact with organic layers will have exceptional barrier
capabilities that withstand heat, offer flexibility, have sustained
reliability and can be mass produced.
[0254] The light source may be further covered with a transparent
or semi-transparent covering. The covering may provide comfort for
a subject using the light source. The covering may provide
protection to the light source, keeping dirt and fluid off of the
device and providing a cushion to protect the device from
impact.
Quantum Dot Layer
[0255] In some embodiments, a layer of quantum dots is used between
the light emitting surface and the surface to be irradiated in
order to convert all or a portion of the light emission into a
different wavelength. The wavelength is typically down converted to
a longer wavelength (Stokes conversion). For example, purple or
blue light may be converted into red light at 630 nm. Quantum dots
may contain cadmium or may be organic and cadmium free. Wavelength
conversion may occur with many wavelengths thus providing an
additional or multiple wavelengths from a fixed wavelength source.
Quantum dots may also be embedded into the hydrogel used to connect
the light source to the surface to be irradiated, such as a skin or
tissue surface. This may consist of a film or encapsulated quantum
dots layered on the surface of the hydrogel or the light emitting
surface of the lamp. Various densities of quantum dots can be used
to control the percentage of light converted from one wavelength to
another. For example, testing was performed using 50% and 75% blue
light and 50% and 25% red light in bacterial kill experiments. This
combination had an effect on the killing of P. acnes and may be
added to enhance the anti-inflammatory effects of the light due to
the addition of red light. The quantum dot converts light as it
receives the incident light so the continuous or pulse
characteristic of the light is maintained.
Bottom and Top Light Emitting Configurations
[0256] In some embodiments, the light source has a "bottom" light
emitting configuration or a "top" light emitting configuration.
[0257] FIG. 29 illustrates an exemplary embodiment of a light
source 110 with a "bottom" light emitting configuration. Light
source 110 comprises a flexible light emitter 140 located between
an anode 150 and a cathode 160, all of which are formed on a
transparent substrate 120. A power source (not shown) is provided
so that DC or pulsed DC is used to power the light source. Further,
a transparent barrier layer 180 protects the flexible light emitter
140 from moisture and oxygen.
[0258] In this embodiment, the flexible light emitter 140 comprises
an OLED or printed LEDs. Both the substrate 120 and the anode 150
are transparent. The substrate is comprised of a transparent
silicon rubber. The anode 150 is comprised of ITO. Light generated
from the flexible light emitter 140 is emitted through the
transparent anode 150 and substrate 120 such that the device has a
"bottom" light emitting configuration. The cathode 160 is comprised
of a conductive metal such as silver. The barrier layer is
comprised of Flexent film (Konica Minolta).
[0259] FIG. 30 illustrates an exemplary embodiment of a light
source 210 with a "top" light emitting configuration. Light source
210 comprises a flexible light emitter 240 located between an anode
250 and a cathode 260, all of which are formed on a bottom surface
222 of a substrate 220 (i.e., the surface facing towards a surface
to be irradiated). A power source (not shown) is provided so that
DC or pulsed DC is used to power the light source. Further, a
transparent barrier layer 280 protects the flexible light emitter
240 from moisture and oxygen.
[0260] In this embodiment, the flexible light emitter 240 comprises
an OLED or printed LEDs. The substrate 220 comprises a Mylar film
and a silver nanolayer is coated on its bottom side to form the
cathode 260 of the light source. The silver nanolayer is highly
reflective to the light generated by the flexible light emitter 240
such that the light is directed towards the surface to be
irradiated. Both the barrier layer 280 and the anode 250 are
transparent. The barrier layer 280 is comprised of Willow
transparent flexible glass (Dow Corning). The anode 250 is
comprised of ITO. Light generated from the flexible light emitter
240 is emitted through the transparent anode 250 and barrier layer
280 such that the device has a "top" light emitting
configuration.
Hydrogel or Adhesive Layer
[0261] In some embodiments, a transparent hydrogel, silicone
membrane or adhesive layer such as Dow silicone PSA or double-sided
adhesive tape layer is applied between the light emitting surface
of the lamp to attach it to the surface to be irradiated, such as a
skin or tissue surface. This layer may be a single use disposable
in cases where certain microorganisms are able to penetrate the
hydrogel, silicone or adhesive layer. As such, the hydrogel,
silicone or adhesive layer is not suitable for re-use due to the
infection control requirements and non-transmittal of infection
back to the user or subsequent users of the device. Alternatively,
this layer may be used multiple times in cases where there is no
microorganism penetration. For example, studies have shown that
certain bacteria do not grow on the surface of a hydrogel or
silicone layer. The hydrogel, silicone or adhesive layers may also
contain additional anti-bacterials and substances suitable to
enhance tissue regeneration for use in wound healing. The
double-sided transparent adhesive tape may be, for example, 3M 9964
Clear Polyester Diagnostic Microfluidic Medical Tape (3M.com).
Various hydrogels and silicones are available based on the adhesion
requirements for various skin types and wound dressings.
VIII. Control of Light Source
[0262] The light source is driven and controlled by an electronic
circuit that includes, for example, a power supply, drive circuit
and control module. For flexible light sources, the electronic
circuit may be provided in a separate housing electrically
connected to the light source or may be built into the flexible
material that mounts the light source.
[0263] The power supply may be any power supply capable of
supplying sufficient power to activate the light source. The power
supply may comprise a disposable or rechargeable battery, solar
cell, fuel cell, an adapter, or may be powered by the power grid.
The battery may be a printed battery, flexible lithium-ion primary
or secondary cells, carbon nanotube, electrochemical inks, or other
flexible organic or inorganic primary or secondary cells using
non-toxic or limited toxicity materials. In some embodiments, the
battery is roll-to-roll printed on the lamp substrate. The light
source is preferably driven by DC or pulsed DC. One skilled in the
art will understand that the output voltages and current levels of
the DC or pulsed DC control the peak output of each layer of the
device, which in combination with the treatment time control the
dose.
[0264] FIG. 31 is a block diagram of an exemplary system in which
the light source and light source drive circuit are controlled by a
microcontroller (discussed below) in accordance with a
preprogrammed treatment cycle that provides a sequence of light at
a fixed dose or wavelength based on the patch configuration. For
example, the microcontroller may be preprogrammed for treatment of
a specific disorder, such as acne. The microcontroller may control
the light source by adjusting the activation and deactivation of
the light source, voltage, current, light wavelength, pulse width,
duty factor, and light treatment time. One skilled in the art will
appreciate that other operating parameters may also be controlled
by the microcontroller in accordance with the present
invention.
[0265] The microcontroller is also connected to one or more I/O
devices, such as an LED that provides an indication of whether the
light source is on/off or an audio buzzer that alerts the user upon
completion of a particular treatment. An on/off switch may also be
provided to power the light source.
[0266] The system may be used in a number of treatment
(irradiation) sessions that together result in an overall treatment
time. For such cases, the microcontroller may include at least one
timer configured to measure session time and overall treatment time
or both. The timer may be used simply to monitor the session time
or overall treatment time or may be used to deactivate the light
source after completion of a session or overall treatment. The
timer may also be used to provide sequential treatments for
automatic dosing in a wound care dressing, for example. A real time
clock associated with the system can monitor the treatments and
track/manage treatment sequences and dosing.
[0267] FIG. 32 is a block diagram of an exemplary system in which
the light source is controlled by an electronic circuit with one or
more sensors that operate in a closed loop to provide feedback to a
microcontroller so as to dynamically control the light source
during a treatment (irradiation) session. As can be seen, the
electronic circuit is similar to that shown and described in
connection with FIG. 31, with the addition of one or more sensors
that operate in a closed loop to provide feedback to the
microcontroller. For example, in applications involving the
photoeradication of P. acnes and other microorganisms, a photo
sensor could be used to detect porphyrins or other spectra related
to the inactivation of the bacteria that emit light within the red
or other wavelength portion of the fluorescent spectrum, as
discussed above. An exemplary sensor is a photo sensor used to
detect porphyrins that emit light within the red or other portion
of the fluorescent spectrum in applications involving the
photoeradication of P. acnes with pulsed purple or blue light or
other spectra related to the inactivation of the bacteria. The
photo sensor may comprise, for example, a photo detector with a
sharp purple or blue notch filter and a bandpass filter that is
used to detect the red or other wavelength portion of the
fluorescent spectrum. This would be monitored during treatment and
as it decayed to a preset threshold the pulsed purple or blue light
stimulation would be turned off. From time to time, the system
would turn on to see if the porphyrin levels would return to a high
enough level to induce microorganism kill upon exposure to pulsed
purple or blue light. Of course, other types of sensors may also be
used in accordance with the invention. Certain sensors may be built
into the layers of the device, while other sensors may be applied
to the skin or other surface to be irradiated or may be built into
the illuminator.
[0268] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the disclosure pertains. Also,
as will be understood by one skilled in the art, for any and all
purposes, all ranges disclosed herein also encompass any and all
possible subranges and combinations of subranges thereof. Any
listed range can be easily recognized as sufficiently describing
and enabling the same range being broken down into at least equal
halves, thirds, quarters, fifths, tenths, etc. As will also be
understood by one skilled in the art, a range includes each
individual member.
[0269] While the present invention has been described and
illustrated hereinabove with reference to several exemplary
embodiments, it should be understood that various modifications
could be made to these embodiments without departing from the scope
of the invention. Therefore, the present invention is not to be
limited to the specific methodologies or device configurations of
the exemplary embodiments, except insofar as such limitations are
included in the following claims.
* * * * *