Note: Descriptions are shown in the official language in which they were submitted.
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PERMEABILIZING BIOFILMS
FIELD OF THE INVENTION
This invention relates to methods of permeabilizing biofilms, e.g., to
deliver compounds such as antibiotics, antiseptics or photosensitizing agents
into
or through biofilms using stress or pressure waves.
BACKGROUND OF THE INVENTION
Most microbial infections in the body are caused by bacteria growing
in biofilms composed of single or multiple bacterial species. Biofilms are
typically matrix-enclosed microbial aggregates associated with each other and
a
solid surface. Bacteria within biofilms have an increased resistance to
antimicrobial agents relative to that of planktonic cells of the same species.
The
relative impermeability of biofilms to compounds such as antimicrobial agents
may be one reason why microbial infections associated with biofilms are
difficult
to treat.
SUMMARY OF THE INVENTION
The invention is based on the discovery that stress or pressure waves
can be used to permeabilize biofilms, and thus can be used to transport
compounds into or through biofilms. The new methods can be used to deliver
compounds of a wide range of sizes and net charges into and through biofilms.
In general, the invention features a method of delivering a compound
into or through a biofilm. The method includes contacting the biofilm with the
compound and propagating a sufficient number of stress waves into the biofilm
to
increase the permeability of the biofilm, thereby enabling the compound to
pass
into the biofilm. Because the permeability is enhanced for up to several
minutes,
the stress wave can be applied in a first step, and the compound can be
applied to
the permeabilized biofilm in a later step. The later step can be separated by
several seconds or minutes from the first step. Alternatively, the stress
waves can
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be used to help drive the compound into the biofilm.
Stress or pressure waves (or impulse transients) are broad-band
compressive waves having a rise time of at least 500 ps to 100 us, e.g., 1 ns
to 1
us, 10 ns to 100 ns, 10 ns to 10 us, or 100 to 300 ns. Stress waves have no
measurable tensile component. The stress waves have a peak pressure of at
least
50 bar, e.g., 200-700, 300-500, or 550-650 bar. Preferably, the stress waves
have
a peak pressure of no more than 800, 1000, or 2000 bar. The stress waves have
a
quick rise time of 10 us or less, and have a duration of 100 ns to 1 us, e.g.,
200-
600 ns. Between 1 and 50 or more pulses (i.e., individual stress waves) can be
applied in one treatment, e.g., l, 2, 3, 4, or 5-10 pulses.
In some embodiments, the stress waves are generated by directing a
pulsed laser beam to a target material that is coupled to the biofilm. The
laser
beam typically has a wavelength of between 140 nm and about 12 um, e.g., 250,
400, 450, 500, 550, 625, 675, 725, 800, 900, 1000, or 1100 nm. The target
1 S material includes a material that absorbs laser energy, and laser-induced
rapid
heating of the absorbing material generates the stress wave. The target
material
can be, e.g., a polymer, such as polystyrene. The target material can also be
a
metal foil. Metals suitable for use in the metal foil include, e.g., aluminum
or
copper.
In certain embodiments, a material that is transparent to light, e.g., a
quartz or glass plate, is bonded to a surface of the target material to
confine the
plasma generated by a laser beam, thereby increasing the efficiency of
conversion
of laser energy into the mechanical energy of the stress wave.
For some applications, the compound is provided in a reservoir
containing a coupling medium suitable for mixing with the compound. The
reservoir is arranged to enable the coupling medium to directly contact a
surface
of the biofilm. The coupling medium may also contain a surfactant, such as
sodium lauryl sulfate.
The biofilm can be associated with an enamel surface of a mammal,
e.g., on a tooth surface. The biofilm can also be associated with a
periodontal
pocket in a mammal. In other embodiments, the biofilm is associated with a
tracheal or lung surface.
The compound can be a bioactive agent, e.g., a therapeutic agent such
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as an antimicrobial agent. Examples of antimicrobial agents include
antibiotics,
e.g., metronidazole and minocycline; antiseptics, e.g., chlorhexidine and
triclosan;
photosensitizing agents, e.g., the benzoporpherene derivative monoacid ring A
(BPD-MA).
S Biofilms that can be permeabilized with the methods of the invention
include those produced by a bacteria or a product of a bacteria, e.g., a
capsular
polysaccharide produced by a bacteria. The biofilm may contain one or several
different bacterial species. In some applications the bacterial species will
include
P. gingivalis or Actinomycete spp., e.g., A. viscosus. The biofilms may also
include one or more fungal or protozoan species, or products thereof.
The invention also includes a method of delivering a compound, such
as an antimicrobial agent, into or through a biofilm. The method includes
contacting the biofilm with the antimicrobial agent and exposing a target
material
coupled to the biofilm to a pulsed laser beam. One or more stress waves are
then
propagated through the biofilm contacting the antimicrobial agent, thereby
causing the antimicrobial agent to pass into or through the biofilm.
Also included in the invention is a method of permeabilizing a biofilm
by exposing the biofilm to stress waves, thereby permeabilizing the biofilm.
Thereafter, compounds can pass through the biofilm, e.g., by diffusion or an
applied force.
The invention further includes a method of treating disorders
associated with a biofilm by exposing the biofilm to a stress wave. In some
embodiments, the stress waves permeabilize the biofilm to allow entry by a
therapeutic agent. The stress waves can also permeabilize the biofilm to
enable
entry of other agents, e.g., atmospheric oxygen.
The methods described herein can be applied to biofilms in any animal
or human subject, e.g., a mammal such as a human, dog, horse, cow, or cat. The
methods are particularly suitable for permeabilizing biofilms associated with
diseases of the oral cavity, e.g., chronic destructive periodontitis. The
methods
can also used to permeabilize biofilms adhering to other solid surfaces not
found
in an animal, e.g., solid surfaces on instrumentalities used in food
processing and
in medical applications.
Permeabilizing a biofilm with stress waves allows compounds, such as
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bioactive agents and therapeutic agents, to be administered with highly
localized
effects to areas of diseased cells, thus sparing other tissues of the body. In
this
way, healthy tissues and organs are spared from adverse effects of a
systemically
administered drug.
Unless otherwise defined, all technical and scientific terms used herein
have the same meaning as commonly understood by one of ordinary skill in the
art
to which this invention pertains. Although methods and materials similar or
equivalent to those described herein can be used in the practice or testing of
the
present invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references mentioned
herein
are incorporated by reference in their entirety. In case of conflict, the
present
document, including definitions, will control. Unless otherwise indicated,
materials, methods, and examples described herein are illustrative only and
not
intended to be limiting.
Various features and advantages of the invention will be apparent from
the following detailed description and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a schematic drawing of a device for generating a stress wave
by laser ablation of a target coupled to a biofilm.
Fig. 2 is a graph illustrating the waveform of a stress wave generated
by the ablation of a black polystyrene target material with a single 23 ns Q-
switched ruby laser pulse.
Fig. 3 is a graph illustrating the phototoxicity of A. viscosus in biofilms
after incubation with 50 g.g/ml methylene blue (MB) for 1 minute (white bar)
and
5 minutes (hatched bar) followed by the application of a single pressure wave
and
red light (L+MB+PW) or light only (L+MB).
DETAILED DESCRIPTION
The invention provides new methods of delivering compounds, e.g.,
antimicrobial agents such as antibiotics, antiseptics, and photosensitizers,
into or
through biofilms using stress or pressure waves. The stress waves induce a
transient increase in the permeability of the biofilm, thereby allowing
delivery of
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the compound into the biofilm at the time the stress waves are applied, or
shortly
thereafter. The method is thus useful for delivering compounds to treat
conditions
associated with the presence of biofilms.
Biofilms are matrix-enclosed microbial aggregates associated with
each other and a solid surface. While a biofilm may contain microbial cells,
it
may also contain extracellular substances, such as proteins or
polysaccharides,
e.g., capsular polysaccharides expressed by the microbe.
Biofilms can be found in association with both low and high nutrient
sources. Examples of biofilms are disclosed in Table 1 of Wimpenny, Adv. Dent.
Res., 11:150-159, 1997, which is incorporated by reference herein in its
entirety.
Biofilms include those found in association with high nutrient sources such as
plant surfaces, e.g., rhizospheres, but also on inert surfaces such as contact
lenses,
prostheses, catheters, metal plates, joints, heart valves, and stems, Other
sources
of biofilms include animal surfaces, e.g., oral surfaces, including, e.g., the
cheek,
tongue, palate, epithelium, tooth surfaces, epithelia, such as the gut, rumen
and
vagina, and the surfaces of internal organs such as the lung and heart, and on
heart
valves.
When present in the oral cavity as subgingival biofilms, they are
referred to as dental plaque. Dental plaque is involved in the development of
conditions such as caries, periodontitis, dental implant failures, denture
stomatitis,
and oral yeast infections such as candidiasis.
Examples of biofilms associated with specific microbial infections
include those formed in supragingival deposits by gram positive Actinomycete
spp., especially A. viscosus. Gram-negative bacteria can also be found in
association with supragingival biofilms. These bacteria include P. gingivalis,
F.
nucleaturn, as well as coccobacilli and Capnocytophage spp.
Pathogens associated with non-oral biofilms include P. aeroginosa,
which is found in the trachea of patients suffering from cystic fibrosis
disease; E.
coli biofilms found in infections associated with urinary tract and intestinal
infections, and biofilms formed by Staphylococcus spp. in eye infections. The
latter infections are frequently associated with contact lenses.
Biofilms can also be found in association with eukaryotic cells, e.g.,
cells specialized for secretion of extracellular matrix such as those
described in
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Alberts et al., Molecular Biology of the Cell, 3rd Ed., 1994, at p. 1189.
These
cells include epithelial cells, e.g., ameloblasts, which secrete tooth enamel,
the
proteoglycan-secreting planum semilunatum cells of the vestibular apparatus of
the ear, and the interdental cell of the organ of Corti, which secretes the
tectorial
membrane-covering hair cells of the organ of Corti. Other cells include
nonepithelial cells such as fibroblasts, pericytes, nucleus pulposus cells of
invertebral disc, cementoblast/odontocytes, chondrocytes of hyaline cartilage,
fibrocartilage, or elastic cartilage, osteoblasts/osteocytes, and
osteoprogenitor
cells, which are the stem cells of osteoblasts. Other extracellular matrix-
secreting
cells include the hyalocytes of the vitreous body of the eye, and the stellate
cell of
the perilymphatic space of the ear.
The methods of the invention can be used to increase the permeability
of these naturally occurring biofilms to allow compounds to pass into the
biofilms
and into the underlying cells and tissues.
Methods of Generating Stress Waves
The properties of stress waves are described generally in W098/23325,
which is incorporated herein by reference in its entirety.
Stress waves can be generated by various energy sources. For
example, stress waves can be generated by ablation or thermoelastic expansion
of
an appropriate target material by a high energy optical source such as a laser
(Doukas et al., Physical Characteristics and Biological Effects ofLaser-
Induced
Stress Waves, Ultrasound in Med. & Biol., 22:151-164, 1996). When stress waves
are generated by laser, they can be referred to as laser stress waves.
The laser beam can be generated by standard optical modulation
techniques known in the art, e.g., Q-switched or mode-locked lasers using, for
example, electro or acousto-optic devices. Standard commercially available
lasers
that can operate in a pulsed mode in the infrared, visible, and/or near
infrared
spectrum include Nd:YAG, Nd:YLF, COZ, excimer, dye, Tiaapphire, diode,
holmium (and other rare-earth materials), and metal-vapor lasers. The pulse
widths of these light sources are adjustable, and can vary from several tens
of
femtoseconds (fs) to several hundred microseconds. For use in the new methods,
the optical pulse width can vary from 100 fs to about 10 us and is preferably
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between about 500 ps and 40 ns.
Stress waves can also be generated by extracorporeal lithotripters (one
example is described in Coleman et al., Ultrasound Med. Biol., 15:213-227,
1989). These stress waves have rise times of 30 to 450 ns, which is longer
than
laser-generated stress waves.
The type of lithotripter used is not critical, as long as it is capable of
generating stress waves. Thus, either electrohydraulic, electromagnetic, or
piezoelectric lithotripters can be used.
Stress waves can also be generated using transducers, such as
piezoelectric transducers. Preferably, the transducer is in direct contact
with the
coupling medium, and undergoes rapid displacement following application of an
optical, thermal, or electric field to generate the stress wave.
For example, dielectric breakdown can be used, and is typically
induced by a high-voltage spark (similar to those used in certain
extracorporeal
lithotripters; see e.g., Coleman et al., Ultrasound Med. Biol., 15:213-227,
1989).
In the case of a piezoelectric transducer, the transducer undergoes rapid
expansion
following application of an electrical field to cause a rapid displacement in
the
coupling medium.
Stress waves can alternatively be generated by inducing explosive
reactions in energetic materials such as those described in Kodama et al.,
Ultrasound Med. Biol. 24:1459 (1998). Useful energetic materials include
nitrocellulose (NC), glacidy azide polymer (GAP), bis-azidomethyloxetane
polymer (BAMO), azidomethyl methyloxetane polymer (AMMO), and silver
azide.
For some applications it is desirable to generate stress waves with the
aid of fiber optics. Fiber optic delivery systems are particularly
maneuverable and
can be used to irradiate target materials located adjacent to biofilms to
generate
stress waves in remote, otherwise inaccessible locations. These types of
delivery
systems, when optically coupled to lasers, for example, are preferred as they
can
be integrated into catheters and related flexible devices, and used to
irradiate most
organs in the human body. In addition, to launch a stress wave having the
desired
rise times and peak stress, the wavelength of the optical source can be easily
tailored to generate the appropriate absorption in a particular target
material,
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which then emits the stress waves.
When the stress wave is generated by irradiation of a target material,
the absorbing target material acts as an optically triggered transducer.
Following
absorption of light, the target material undergoes rapid thermal expansion, or
is
ablated, to launch a stress wave. Typically, metal and polymer films have high
absorption coefficients in the visible and ultraviolet spectral regions.
Many types of materials can be used as the target material in
conjunction with a laser beam, provided they fully absorb light at the
wavelength
of the laser used. The target material may be present as part of a container
and
can be composed of a metal such as aluminum or copper; a plastic, such as
polystyrene, e.g., black polystyrene; a ceramic; or a highly concentrated dye
solution. The target material must have dimensions larger than the cross-
sectional
area of the applied laser energy. In addition, the target material must be
thicker
than the optical penetration depth of the laser into the target so that no
light passes
through to strike the surface of the biofilm or underlying tissue. When the
target
material is present as part of a container, it must also be sufficiently thick
to
provide mechanical support. When the target material is made of a metal, the
typical thickness will be 1/32 to 1/16 inch, i.e., a metal foil. For plastic
target
materials, the thickness will be generally 1/16 to 1/8 inch.
The target material must be coupled to the biofilm by a coupling
medium. A coupling medium is a liquid, gel, or cream medium in which the
stress waves are propagated. The coupling medium enables a direct contact of
the
stress wave to the surface of the biofilm layer and minimizes acoustic
reflections.
In many applications the solution or gel in which the compound to be delivered
is
dissolved or suspended may itself act as the coupling medium. Alternatively, a
coupling medium can be used during application of the stress waves, and then,
if
desired, removed to apply the compound to the now permeabilized biofilm. Thus,
a solution containing the compound to be delivered, e.g., water, oil, such as
castor
oil, an isotonic medium such as phosphate buffered saline (PBS), or a gel such
as
a collagenous gel, can all be used as the coupling medium.
When using an extracorporeal lithotripter, a stress wave of the
appropriate rise time can be generated by propagating in a non-linear coupling
medium (e.g., water) for a distance determined as described in W098/23325.
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The compound to be delivered is thoroughly dispersed in, and is preferably
dissolved in, the coupling medium. Thus, hydrophilic compounds can be mixed
with an aqueous coupling medium (e.g., water, solutions of surfactants, such
as
sodium lauryl sulfate (SLS), benzalkonium chloride (BAC), cocoamidopropyl
betaine (CAPB)), and hydrophobic compounds can be mixed with an oil-based
coupling medium (such as castor oil). These agents also enhance the coupling
ability of the medium.
The coupling medium can in addition include a surfactant that
enhances transport of the compound to be delivered, e.g., by prolonging the
period
of time in which the biofilm remains permeable to the compound following the
generation of a stress wave. The surfactant can be, e.g., an ionic or nonionic
detergent and thus can include, e.g., sodium lauryl sulfate, cetyl trimethyl
ammonium bromide, and lauryl dimethyl amine oxide.
Stress wave characteristics can be measured using methods standard in
the art. For example, peak stress or pressure, and rise time can be measured
using
a polyvinylidene fluoride (PVDF) transducer method as described in Doukas et
al., Ultrasound Med. Biol., 21:961 (1995).
A useful parameter by which to assess the efficiency of generation of
the stress wave is the coupling coefficient (Cn,), which is defined as the
total
momentum transfer to the target material during ablation divided by the pulse
energy. The efficiency of conversion of laser energy to mechanical energy of
the
stress wave is given by the coupling coefficient of the target material.
The permeabilizing effects of stress waves can be enhanced using
confined ablation. In confined ablation, a laser beam-transparent material,
such as
a quartz optical window, or glass, is placed in close contact with the target
material. Confinement of the plasma created by ablating the target material by
using the transparent material increases the coupling coefficient by an order
of
magnitude (Fabro et al., J. Appl. Phys., 68:775, 1990). The transparent
material
can be quartz, glass, or transparent plastic. Since voids between the target
material and the confining transparent material allow the plasma to expand,
thereby decreasing the momentum imparted to the target, the transparent
material
is preferably bonded to the target material using an initially liquid
adhesive, such
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as carbon-containing epoxies, to prevent such voids.
Since the effects induced by the stress waves last for several minutes,
the transport rate of a drug diffusing passively through the biofilm along its
concentration gradient can be increased by applying hydrostatic pressure on
the
surface of the biofilm following application of the stress wave. The
hydrostatic
medium can be any liquid, such as water or phosphate buffered saline.
Compounds
Because stress waves exert physical forces to increase the permeability
of the biofilm, they can be used to transport many different types of
compounds.
Thus, the compounds can be bioactive agents such as photosensitizing agents,
e.g.,
benzoporpherene derivative monoacid ring A (BPD-MA); antibiotics, such as
metronidazole and minocycline, and antiseptics, such as chlorhexidine and
triclosan.
Additional bioactive compounds which can be used include therapeutic
agents such as chemotherapeutics, e.g., cisplatin, polypeptides, including
growth
factors and antibodies, and nucleic acids, such as oligonucleotides, DNA, RNA,
and plasmids, local anesthetics, such as lidocaine and benzocaine. The
compounds may optionally be heated prior to generation of the stress wave to
facilitate their transport into the biofilms.
In general, differential drug localization can be achieved using
guidelines for administration determined using standard techniques known in
the
field of pharmacology. Preferably, the compound dosage and time course are
such that a 2:1 or greater concentration ratio is achieved in the treated
tissues,
cells, or other treated sites, compared to the surrounding, untreated tissues
or sites.
Determining the appropriate dosage for a specific compound, and for a
particular subject or patient (human or animal) is a routine matter for one
skilled
in the art of pharmaceutical administration. Two approaches are commonly used
to assay directly the quantity of drug in the diseased (treated) and
surrounding
sites. First, tissue samples are obtained from animals (e.g., pigs) or
patients who
have been treated with different dosage and timing protocols. The quantity of
drug in each sample is then measured either chemically, or if there is a
unique
optical signal such as fluorescence, then by quantitative microscopy or laser-
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induced fluorescence. The results are tabulated to determine a scale of
optimum
drug dosages and types of stress waves for a given biofilm, the structure
attached
to the biofilm, and the compound.
Compounds which have a toxic effect at higher dosages can be
administered to a patient using guidelines for administration that will
produce
greater concentrations of the drugs in the treated tissues or cells compared
to the
surrounding tissues, while maintaining adequate levels of the drug in these
treated
tissues or cells.
Topical application and delivery of compounds by the new methods
allow the compounds to be localized to a site of interest. Thus, the compound,
e.g., a drug, is more concentrated at the site of action and has a minimal, if
any,
systemic concentration. This enhances the therapeutic effect of the drug and
simultaneously minimizes systemic side effects. Another advantage compared to
systemic administration is that compounds transported through biofilms bypass
systemic deactivation or degradation (e.g., hepatic "first-pass" effects).
Gastrointestinal incompatibility and potential toxicological risks are also
minimized relative to systemic administration. In addition, drugs developed
for
topical application can be designed so that they are deactivated
systematically
(i.e., the "soft drug" concept), using standard techniques. Topical
administration
may also be desired when the compound is rare or expensive.
EXAMPLES
The invention will be further described in the following examples,
which do not limit the scope of the invention described in the claims.
Example 1 - Stress Waves Enhance the Penetration of Methylene Blue in A.
Viscosus Biofilms
The ability of stress waves to enhance penetration of a compound into
a biofihn was demonstrated by comparing the penetration of methylene blue into
a
biofilm in the presence and absence of an applied laser stress wave.
The biofilm was generated by culturing A. viscous on an enamel
surface. Enamel surfaces measuring 5 x 5 x 2 mm were sterilized and suspended
in trypticase soy agar with 5% sheep blood in wells of 24-well plates with
cultures
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of A. viscosus. Plates containing the suspended enamel surfaces v~-ere
incubated in
an anaerobic chamber at 35°C. Fresh medium containing A. viscosus
cultures was
added twice per week until a 1.0-1.5 mm thick biofilm was formed.
Specimens were incubated with methylene blue (Sigma, St. Louis,
MO), followed by exposure to a laser pulse, or incubated with methylene blue
only for 5 minutes in the absence of light.
Specimens to be exposed to a laser pulse were placed in a reservoir as
shown in Fig. 1. A reservoir 10 contained the enamel surface 12, to which the
A.
viscosus biofilm 14 was adhered. The biofilm 14 was bathed in a solution 16
containing 50 ug/ml methylene blue for 5 minutes, after which a sterile black
plastic (unexpanded polystyrene) target 18 ( 1 cm in diameter and 1 mm thick)
was
placed over the top of the reservoir 10 in contact with the solution 14.
Biofilms
were exposed to a single laser pulse 20, which, after ablating the target 16
generated a stress wave 22 through the solution 16. The laser pulse was
generated
with a Q-switched ruby laser (not shown). The operating parameters of the
laser
were 694 nm, 2.1 J, and 23 ns pulse duration.
The temporal waveform of a stress wave generated by ablation of the
polystyrene target by 694 nm radiation from the Q-switched laser is shown Fig.
2.
The stress wave was measured by a calibrated piezoelectric transducer and had
a
rise time of 50 nsec, 110 nsec duration, and peak pressure of 600 bar. The
beam
size was about 6 mm in diameter and provided a fluence of about 7.0 J/cm2.
The specimens were then placed in petri dishes, covered with PBS,
after which biofilms were viewed with a Leica TCS NTTM fluorescence scanning
confocal microscope. The microscope was equipped with a lOx water immersion
objective lens. An argon laser (476 nm) was used as the excitation source for
methylene blue. Sections were collected at 100 um intervals and analyzed by
image-processing to assess the distribution of methylene blue within the
biofilm
matrices.
The images demonstrated that the fluorescence from the biofilm where
laser-generated stress waves were applied was much stronger than the control,
indicating that the methylene blue had penetrated into the biofilm. In
addition, the
fluorescent intensity of a control slice was increased to a depth of no more
than
400 um, whereas florescence was observed to a depth of 600-700 um in the
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specimens subjected to stress waves. A significant increase of fluorescence
intensity obtained from the specimens exposed to stress waves was observed as
compared to controls.
These results demonstrate that application of stress waves to a biofilm
facilitates penetration of the biofilm with a bioactive agent.
Example 2 -Stress Waves Enhance Bactericidal Effects of a Photosensitizer
on Bacterial Biofilms
To demonstrate that stress waves can enhance the bacteriocidal effects
of photosensitizing agents on biofilms, A. viscosus biofilms were exposed to
methylene blue, which exerts phototoxic effects upon exposure to light.
The biofilms were grown on enamel surfaces as described in Example
1. Biofilms were incubated with methylene blue for 1 or 5 minutes and exposed
to stress waves (one pulse), after which they were exposed to 666 mn red light
with a fluence of 15 J/cm2 at an irradiance of 50 mW/cm2. In control samples,
biofilms were treated only with methylene blue, or were treated with methylene
blue and exposed to red light, but without stress waves. An argon ion laser
with
an emission of 514.5. nm was used to pump a dye laser. The laser light was
coupled into a 1.0 mm quartz fiber and appropriate spot sizes were created
with an
objective lens.
After illumination, adherent bacteria from the samples as treated above
were scraped from the enamel surfaces with a sterile blade and dispersed in
trypticase soy broth. Serial dilutions were prepared, and 100 u1 aliquots were
spread over the surfaces of blood agar plates. Survival fractions from each
biofilm were calculated by counting the colonies on the plates and dividing by
the
number of colonies from dark controls incubated with the drug and kept at room
temperature for periods equal to irradiation time. Other controls were: 1 )
biofilms untreated with methylene blue, light, or stress waves; 2) biofilms
exposed
only to a stress wave; 3) biofilms exposed to light without methylene blue;
and 4)
biofilms exposed to light after incubation with methylene blue.
As shown in Fig. 3, 99% of the bacteria associated with biofilms were
killed when subjected to a stress wave and red light after a five-minute
incubation
with methylene blue, whereas only 57% of control bacteria were killed after
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exposure to red light in the absence of an applied stress wave. In addition, a
one-
minute incubation in methylene blue followed by a stress wave killed 96.5% of
bacteria whereas the same conditions without a stress wave killed only 34% of
bacteria. These results demonstrate that application of a photosensitizer
along
with stress waves to a biofilin enhances the phototoxic effect of the
photosensitizer.
Example 3 -- Delivery of Bioactive Agents into Biofilms in Periodontal
Pockets
A solution of metronidazole is applied to a periodontal pocket of a
patient with chronic destructive periodontitis. Stress waves generated by
ablation
of a polystyrene target material with a Q-switched ruby laser are used to
permeabilize biofilms (dental plaque) attached to the tooth root.
A stainless steel needle (0.8 mm inner diameter by 1.3 mm outer
diameter) is used as a hollow waveguide. To deliver the laser pulse to the
target,
the blunt end of the needle is sealed with the target that absorbs the laser
pulse.
The target is chosen so that it absorbs the laser pulse completely.
Upon delivery of the laser pulse, the target emits a stress wave that
temporarily increases the permeability of the biofilm. As a result, the
metronidazole passes into the biofilm, thereby facilitating metronidazole-
mediated
destruction of cells in the biofilm.
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in
conjunction with the detailed description thereof, the foregoing description
is
intended to illustrate and not limit the scope of the invention, which is
defined by
the scope of the appended claims. Other aspects, advantages, and modifications
are within the scope of the following claims.
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