U.S. patent application number 11/844864 was filed with the patent office on 2008-03-13 for method of measuring propulsion in lymphatic structures.
This patent application is currently assigned to BAYLOR COLLEGE OF MEDICINE. Invention is credited to Kristen Adams, John Rasmussen, Eva Sevick-Muraca, Ruchi Sharma.
Application Number | 20080064954 11/844864 |
Document ID | / |
Family ID | 39107737 |
Filed Date | 2008-03-13 |
United States Patent
Application |
20080064954 |
Kind Code |
A1 |
Adams; Kristen ; et
al. |
March 13, 2008 |
METHOD OF MEASURING PROPULSION IN LYMPHATIC STRUCTURES
Abstract
Novel methods and imaging agents for functional imaging of lymph
structures are disclosed herein. Embodiments of the methods utilize
highly sensitive optical imaging and fluorescent spectroscopy
techniques to track or monitor packets of organic dye flowing in
one or more lymphatic structures. The packets of organic dye may be
tracked to provide quantitative information regarding lymph
propulsion and function. In particular, lymph flow velocity and
pulse frequency may be determined using the disclosed methods.
Inventors: |
Adams; Kristen; (Houston,
TX) ; Sharma; Ruchi; (Houston, TX) ;
Rasmussen; John; (Spring, TX) ; Sevick-Muraca;
Eva; (Montgomery, TX) |
Correspondence
Address: |
CONLEY ROSE, P.C.;David A. Rose
P. O. BOX 3267
HOUSTON
TX
77253-3267
US
|
Assignee: |
BAYLOR COLLEGE OF MEDICINE
One Baylor Plaza
Houston
TX
77030
|
Family ID: |
39107737 |
Appl. No.: |
11/844864 |
Filed: |
August 24, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60840256 |
Aug 25, 2006 |
|
|
|
60823481 |
Aug 24, 2006 |
|
|
|
Current U.S.
Class: |
600/431 |
Current CPC
Class: |
A61B 5/0059 20130101;
A61M 5/007 20130101; A61K 49/0054 20130101; A61K 49/0032 20130101;
A61B 5/41 20130101; A61B 5/415 20130101; A61K 49/0034 20130101;
A61B 5/0064 20130101; A61K 47/61 20170801; A61B 5/0071 20130101;
A61K 49/0041 20130101; A61B 5/418 20130101 |
Class at
Publication: |
600/431 |
International
Class: |
A61B 6/00 20060101
A61B006/00 |
Claims
1. A method of measuring lymph propulsion comprising: a)
administering an organic dye to one or more lymph structures, the
organic dye having an excitation wavelength; b) illuminating the
tissue surface with excitation light to excite the organic dye; c)
detecting an emission from the organic dye and capturing a
plurality of images for a time period ranging from about 1 minute
to about 30 minutes; and e) tracking one or more packets of organic
dye flowing through the lymph structure from the plurality of
images of the one or more lymph structures to quantitatively
measure lymph propulsion.
2. The method of claim 1 wherein (a) comprises injecting the
organic fluorescent dye subcutaneously to the one or more lymph
structures.
3. The method of claim 1 wherein (a) comprises using a catheter to
inject the organic dye intradermally into the one or more lymph
structures.
4. The method of claim 1 wherein the organic dye comprises
tricarbocyanine dyes, bis(carbocyanine)dyes, dicarbocyanine dyes,
indol-containing dyes, polymethine dyes, acridines, anthraquinones,
benzimidazols, indolenines, napthalimides, oxazines, oxonols,
polyenes, porphins, squaraines, styryls, thiazols, xanthins, or
combinations thereof.
5. The method of claim 1 wherein the organic dye is indocyanine
green (ICG).
6. The method of claim 1 wherein the organic dye has an excitation
wavelength ranging from about 750 nm to about 900 nm.
7. The method of claim 1 wherein the excitation light has a
wavelength ranging from about 700 nm to about 800 nm.
8. The method of claim 1 wherein (c) comprises capturing each image
at an integration time ranging from about 10 ms to about 1 s.
9. The method of claim 1 wherein (c) comprises capturing a
plurality of images for at least 30 min.
10. The method of claim 1 wherein (c) and (d) comprise using an
intensified charge-coupled camera.
11. The method of claim 1 wherein the one or more lymphatic
structures is at least about 3 cm beneath the tissue surface.
12. The method of claim 1 wherein (b) comprises illuminating the
tissue surface with an excitation light source selected from group
consisting of laser diodes, semiconductor laser diodes, gas lasers,
light emitting diodes, and combinations thereof.
13. The method of claim 1 wherein the one or more packets of
organic dye causes a peak in fluorescent intensity.
14. The method of claim 1 wherein (d) comprises measuring a peak in
fluorescent intensity at a target region to determine pulse
frequency of the one or more lymph structures.
15. The method of claim 1 wherein (d) comprises defining a
plurality of target regions and measuring a change in fluorescent
intensity at each target region to determine lymph flow
velocity.
16. The method of claim 1 wherein (c) comprises using tomographic
imaging techniques to capture three-dimensional images of lymph
propulsion.
17. A method of determining pulse frequency in one or more lymph
structures comprising: a) administering an organic dye to the one
or more lymph structures under a tissue surface; b) illuminating
the tissue surface with excitation light to excite the organic dye;
c) determining a target region of the one or more lymph structures;
d) continuously measuring fluorescent intensity from the organic
dye at the target region for a time period of at least 1 min; and
e) determining a number of fluorescent intensity peaks over the
time period, the intensity peaks representing one or more packets
of organic dye being propelled through the one or more lymphatic
structures, and dividing the number of intensity peaks by the time
period to determine the pulse frequency of the one or more lymph
structures.
18. A method of determining lymph flow velocity in one or more
lymph structures comprising: a) administering an organic dye to the
one or more lymph structures under a tissue surface; b)
illuminating the tissue surface with excitation light to excite the
organic dye; c) determining at least a first and a second target
region along the one or more lymph structures having a distance
between the first and second target regions; d) measuring a time
period for a packet of organic dye to pass through the target
regions; e) dividing the distance by the time period to determine
the lymph flow velocity in the one or more lymph structures.
19. The method of claim 18 wherein (d) comprises measuring the time
period elapsed between detecting a fluorescent intensity peak at
each target region.
20. The method of claim 18 wherein the distance comprises the
entire length of the one or more lymph structures.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of U.S.
Provisional Application Ser. No. 60/840,256, filed Aug. 25, 2006,
and U.S. Provisional Application Ser. No. 60/823,481, filed Aug.
24, 2006, each of which is herein incorporated by reference in
their entireties for all purposes. U.S. application Ser. No.
11/844,807 entitled Imaging Agents for Functional Imaging of
Lymphatic Structures, filed Aug. 24, 2007, Attorney Docket No.
1373-03901, is herein incorporated by reference in its entirety for
all purposes.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This work was supported by the National Institutes of Health
(R01 CA112679).
BACKGROUND
[0003] 1. Field of the Invention
[0004] This invention relates generally to the field of biomedical
imaging. More specifically, the invention relates to a method of
functionally imaging and measuring lymphatic function by assessing
lymph propulsion in lymphatic structures.
[0005] 2. Background of the Invention
[0006] The lymphatic system is made of vessels or ducts that begin
in tissues and are designed to carry lymph fluid to local lymph
nodes where the fluid is filtered and processed and sent to the
next lymph node down the line until the fluid reaches the thoracic
duct where it enters the blood stream. Lymph vessels infiltrate all
tissues and organs of the body. Lymph fluid is generated from
capillaries which, because of tissue motion and hydrostatic
pressure, enters the lymph vessels carrying with it local and
foreign substances and materials from the tissues. These local and
foreign molecular, micromolecular, and macromolecular substances
include antigens, infectious agents, particles and cells. Lymph
nodes, the lymph "filters," consist of essentially two major
compartments: the fluid spaces (or sinuses) and the cellular
elements. There is one major sinus at the outer margin of the node
that feeds a maze of sinuses that serve to percolate the fluid
slowly towards the hilum of the node from where it is carried
downstream. The sinuses are lined by macrophages that phagocytose
materials carried by the fluid, particularly if the materials have
certain surface charges or specific shapes. The remainder of the
cellular elements in the lymph node performs the immunologic
function of the node. In this regard, the lymph nodes process fluid
by sieving and phagocytosis to remove particulate and cell
materials delivered by the lymphatic vessels, thereby cleaning it
before it is returned to the blood stream.
[0007] The impairment of lymphatic transport capacity occurs due to
either 1) lymph vessel damage and subsequent insufficient repair
processes, or 2) congenital defects leading to abnormal lymph
vessel development. Regardless of the cause, the impairment causes
fluid and protein accumulation, which in turn leads to lymphedema.
Lymphedema is a lifelong condition progressing from swelling and
scarring to immune dysregulation and malnutrition. No curative
treatment exists for lymphedema, which afflicts 300 million people
worldwide. Congenital or primary lymphedema afflicts 1 in every
6,000 newborns and can also appear at the onset of puberty.
Acquired or secondary lymphedema is caused by the filaria parasite
(in a condition referred to as elephantiasis) or by trauma due to
radiation therapy, infiltrating cancer, surgery, or infection. In
developing-world countries, 100 million people are afflicted
worldwide by filariasis. In Western countries, acquired lymphedema
afflicts 3 to 5 million people. The etiology for trauma-associated,
acquired lymphedema is thought to arise from the interruption of
lymph channels coupled with postsurgical infection or
radiation-induced skin reaction. The onset of symptoms, however,
can occur from days, weeks, to years following the initial trauma,
striking at a rate cited between 6 and 62.5% of breast cancer
survivors who have undergone axillary lymph node dissection, up to
64% of all patients who undergo groin dissections, and 25% of all
radical hysterectomy patients. Little is known about the molecular
or functional basis of acquired lymphedema or which persons could
be at risk for the condition. There is a paucity of strategies for
predicting or managing lymphedema due in part to the lack of
diagnostic imaging approaches to noninvasively and routinely
measure lymphatic function. Since lymph function is also implicated
in diseases of significant prevalence (e.g. diabetes, obesity,
cancer, and asthma), the ability to quantitatively image lymph
function could have substantial impact on the health of the world's
population.
[0008] The ability to functionally image the lymphatic system
non-invasively may be clinically relevant for the prevention,
diagnosis, treatment, and research of lymphatic diseases. However,
there are presently very few technologies with the ability to
non-invasively image the lymphatic system in vivo and in real
time.
[0009] Consequently, there is a need for a non-invasive imaging
methods and imaging agents for dynamically assessing lymph function
in vivo.
BRIEF SUMMARY
[0010] Novel methods and imaging agents for functional imaging of
lymph structures are disclosed herein. Embodiments of the methods
utilize highly sensitive optical imaging and fluorescent
spectroscopy techniques to track or monitor packets of organic,
soluble dyes being propelled through one or more lymphatic
structures. The packets of organic dye may be tracked to provide
quantitative information regarding lymph propulsion and function.
Thus, the disclosed methods provide non-invasive ways of assessing
lymph function in deep lymph structures. The organic dyes may be
excited at the near-infrared wavelength regime of 750-800 nm with
fluorescence >800 nm allowing for deep tissue imaging of
lymphatic function.
[0011] In addition, novel imaging agents targeted to lymph
endothelial cell receptors are disclosed. The disclosed imaging
agents incorporate biological molecules such as hyaluronic acid
which bind to lymph endothelial cell receptors. Lymph endothelial
cell receptor expression may be related to the beginnings of tumor
formation. As such, embodiments of the imaging agents may be used
to stain lymph structures for detailed imaging of lymph
architecture as well as serving as potential markers for tumor
angiogenesis, tumor metastases, etc. Further advantages and
features of the methods and imaging agents are described in more
detail below.
[0012] In an embodiment, a method of measuring lymph propulsion
comprises administering an organic, soluble dye to one or more
lymph structures. The organic dye generally has an excitation
wavelength. The method further comprises illuminating the tissue
surface with excitation light to excite the organic dye. In
addition, the method comprises detecting the emission from the
organic dye. The method comprises capturing a plurality of images
of the one or more lymph structures for a period ranging from about
1 minute to about 30 minutes. The method also comprises tracking
one or more packets of organic dye flowing through the lymph
structure from the plurality of images of the one or more lymph
structures to quantitatively measure lymph propulsion. The method
may be performed non-invasively in intact living subjects.
[0013] In another embodiment, a method of determining pulse
frequency in one or more lymph structures comprises administering
an organic dye to the one or more lymph structures under a tissue
surface. The method additionally comprises illuminating the tissue
surface with excitation light to excite the organic dye. Moreover,
the method comprises determining a target region of the one or more
lymph structures. Furthermore, the method comprises continuously
measuring fluorescent intensity from the organic dye at the target
region for a time period of at least about 1 minute. The method
also comprises determining a number of fluorescent intensity peaks
over the time period, the intensity peaks representing one or more
packets of organic dye being propelled through the one or more
lymphatic structures, and dividing the number of intensity peaks by
the time period to determine the pulse frequency of the one or more
lymph structures.
[0014] In an embodiment, a method of determining lymph flow
velocity in one or more lymph structures comprises administering an
organic dye to the one or more lymph structures under a tissue
surface. Additionally, the method comprises illuminating the tissue
surface with excitation light to excite the organic dye. The method
further comprises determining at least a first and a second target
region along the one or more lymph structures having a distance
between the first and second target regions. Moreover, the method
comprises measuring a time period for a packet of organic dye to
pass through the target regions. The method also comprises dividing
the distance by the time period to determine the lymph flow
velocity in the one or more lymph structures.
[0015] The foregoing has outlined rather broadly the features and
technical advantages of the invention in order that the detailed
description of the invention that follows may be better understood.
Additional features and advantages of the invention will be
described hereinafter that form the subject of the claims of the
invention. It should be appreciated by those skilled in the art
that the conception and the specific embodiments disclosed may be
readily utilized as a basis for modifying or designing other
structures for carrying out the same purposes of the invention. It
should also be realized by those skilled in the art that such
equivalent constructions do not depart from the spirit and scope of
the invention as set forth in the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] For a detailed description of the preferred embodiments of
the invention, reference will now be made to the accompanying
drawings in which:
[0017] FIG. 1 is a flow diagram of an embodiment of a method of
measuring lymph propulsion;
[0018] FIG. 2 illustrates a schematic of a system that may be used
in conjunction with embodiments of the method;
[0019] FIG. 3 is a plot of intensity versus time of the target
region of interest (ROI) illustrating the pulsatile motion of
functioning lymph vessels after organic dye was intradermally
administered;
[0020] FIG. 4 is a plot of average intensity in ROI's at increasing
distance along a lymph vessel away from the administration site
away at 28, 30, and 35 seconds from time of injection;
[0021] FIG. 5A shows the structure of an exemplary organic dye,
indocyanine green;
[0022] FIG. 5B shows an embodiment of a modified imaging agent
comprising a fluorescent dye conjugated to a polysaccharide such as
hyaluronic acid;
[0023] FIG. 6 shows a schematic of an embodiment of modifying an
imaging agent;
[0024] FIGS. 7A-C show organic dye trafficking in abdominal lymph
vessel of swine: A) organic dye packet (circle) collected in a
vessel segment; B) Organic dye packet pushed forward to next vessel
segment; C) Still from a movie at 60 seconds after image taken in
(A);
[0025] FIG. 8 is a snapshot of organic dye being propelled from the
hindlimb to the middle iliac lymph node in swine;
[0026] FIG. 9 shows the position of ROIs selected along the length
of a channel to track averaged intensity change at each region
selected along the length of the channel for the complete duration
of imaging;
[0027] FIGS. 9B-9C are three dimensional plots of intensity profile
of organic dye as a function of vessel length and time: (B) Front
view of a three-dimensional plot of intensity profile at selected
ROIs as a function of length of vessel and time of imaging; (C) Top
view of the same 3D plot that depicts trails of trafficking dye
along the channel length in real time;
[0028] FIGS. 10A-10C show a mean fluorescent intensity plot of leg
lymph vessel stained with HA-NIR and indocyanine green (ICG) at a
fixed ROI for dynamic imaging time. FIG. 10A shows ROI selected on
leg lymph vessel stained with HA-NIR dye. FIG. 10B shows ROI
selected on leg lymph vessel with trafficking ICG. FIG. 10C shows a
comparison of intensity profile as a function of time for selected
ROI;
[0029] FIGS. 11A-B are fluorescent image of swine's leg lymph
vessel stained with an embodiment of a modified imaging agent
(HA-NIR);
[0030] FIG. 11C is a three dimensional plot of intensity (arbitrary
units (a.u.)) vs time(s) vs length (cm) of the vessel that shows
change in intensity along the length of a channel during dynamic
imaging of modified imaging agent staining the vessel wall;
[0031] FIG. 11D is a three dimensional plot for unmodified organic
dye flowing in the leg lymph vessel;
[0032] FIG. 12 shows images of lymph propulsion in a human subject;
and
[0033] FIG. 13 shows different concentrations of modified imaging
agent (HA-modified IR-783) incubated with different concentration
of hyaluronidase.
NOTATION AND NOMENCLATURE
[0034] Certain terms are used throughout the following description
and claims to refer to particular system components. This document
does not intend to distinguish between components that differ in
name but not function.
[0035] In the following discussion and in the claims, the terms
"including" and "comprising" are used in an open-ended fashion, and
thus should be interpreted to mean "including, but not limited to .
. . ". Also, the term "couple" or "couples" is intended to mean
either an indirect or direct electrical connection. Thus, if a
first device couples to a second device, that connection may be
through a direct electrical connection, or through an indirect
electrical connection via other devices and connections.
[0036] As used herein, the term "lymphatic structure(s)" refers to
all or a portion of structures that make up a mammalian lymphatic
system including without limitation, lymph nodes, collecting
vessels, lymph trunks, lymph ducts, capillaries, or combinations
thereof.
[0037] As used herein, the term "near-infrared" refers to
electromagnetic radiation at wavelengths ranging from about 750 nm
to about 900 nm.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0038] FIG. 1 illustrates a flow diagram of a method 100 of
measuring propulsion in one or more lymphatic structures. As used
herein, the term "propulsion" and other tenses and forms thereof
refers to the act of pumping or driving forward a fluid. In 102, a
dose or bolus of fluorescent organic dye may be administered to one
or more lymph structures under the tissue surface of a patient. The
organic dye generally has an excitation wavelength at which it will
fluoresce or emit light at an emission wavelength. An excitation
light encompassing the excitation wavelength may then be
illuminated on the target tissue surface to excite the organic dye
in 104. Upon illumination, emissions from the organic dye may be
continuously sensed or detected at the excitation wavelength in 106
to capture fluorescent images of the one or more lymphatic
structures. Packets or masses of organic dye may be tracked,
observed or imaged being propelled through the lymph structures in
108. The images that have been continuously acquired or captured
are used to create a sequence of images (i.e. a movie) to track the
packets of organic dye for quantitative assessment and measurement
of lymph propulsion.
[0039] FIG. 2 illustrates an example of a system 200 that may be
used to implement embodiments of the disclosed method including an
excitation light source 201. Briefly, an excitation light source
201 may be mounted on a stepper motor 203 to enable scanning across
the tissue surface 213 (i.e. patient) at the desired target tissue
region 215. The excitation light may be shaped using a lens 205.
Images may be acquired by an intensified CCD camera 207. An image
intensifier 209 and filter 211 may be placed in front of the lens
of CCD camera 207. Filter 211 may comprise any suitable filter to
pass only the emitted light at the excitation wavelength from the
organic dye. The captured images may be processed and stored in
computer 260. Further examples of and variations on such a system
may be found in U.S. Pat. Nos. 5,865,754 and 7,054,002,
incorporated herein by reference in their entireties for all
purposes.
[0040] In embodiments, the imaging agent used in conjunction with
the disclosed methods is preferably an organic dye. As used herein,
the term "organic dye" refers to all non-particulate, compounds
that do not contain mineral or inorganic components and are capable
of fluorescence. Preferably, the organic dye is soluble in liquid
solvents such as water. Examples of suitable organic dyes include
without limitation, tricarbocyanine dyes, bis(carbocyanine) dyes,
dicarbocyanine dyes, indol-containing dyes, polymethine dyes,
acridines, anthraquinones, benzimidazols, indolenines,
napthalimides, oxazines, oxonols, polyenes, porphins, squaraines,
styryls, thiazols, xanthins, or combinations thereof. In a specific
embodiment, the organic dye is indocyanine green. The organic dyes
may have excitation wavelengths ranging from about 700 nm to about
1000 nm, preferably from about 750 nm to about 900 nm, more
preferably from about 780 nm to about 800 nm. In particular, the
organic dyes may have excitation wavelengths in the near-infrared
range (NIR).
[0041] The organic dye is typically diluted in a liquid solution
such as saline solution. The concentration of organic dye in
solution may range from about 1 .mu.M to about 400 .mu.M,
preferably from about 10 .mu.M to about 200 .mu.M, more preferably
from about 25 .mu.M to about 100 .mu.M. In addition, any
appropriate amount of the organic dye may be administered to the
lymph structure(s). In some embodiments, the amount of organic dye
administered may range from <1 .mu.g to 10 mg, preferably from
about <1 .mu.g to about 1 mg, more preferably from about <1
.mu.g to about 100 .mu.g.
[0042] Referring back to FIG. 1, the organic dye may be
administered to the lymphatic system through any suitable means
102. For example, the organic dye may be delivered using a syringe.
In particular, the organic dye may be administered intradermally or
into the skin. Without being limited by theory, the lymphatic
plexus may pick up packets or masses of the organic dye and transit
the packets of organic dye through lymph vessels to the lymph
nodes. Alternatively, the organic dye is administered using a
catheter system inserted directly into the lymphatic system.
Because of the high sensitivity of the disclosed methods, lymphatic
structures deep beneath the tissue surface may be imaged. In
particular, the lymph structures may be located at a depth of at
least about 1 cm below the tissue surface, preferably at least
about 2 cm, more preferably at least about 3 cm below the tissue
surface.
[0043] To excite the organic dye in the lymphatic system, an
excitation light may be illuminated on the tissue surface over the
targeted lymph nodes and/or channels in 104 by an excitation light
source 201 as shown in FIG. 2. Excitation light source 201 may be
any light source known to those of skill in the art. Examples of
suitable light sources include without limitation laser diodes,
semiconductor laser diodes, gas lasers, light emitting diodes
(LEDs), or combinations thereof. In an embodiment, excitation light
source may comprise a Gaussian light source. As defined herein, a
Gaussian light source is a light source in which the spatial
distribution of the emitted light is a Gaussian distribution.
[0044] Preferably, the excitation light source 201 is a continuous
wave light source which emits a continuous wave light. The light
source may emit light having wavelengths ranging from about 700 nm
to about 800 nm, preferably from about 725 nm to about 775 nm, more
preferably from about 745 nm to about 755 nm. Alternatively, the
excitation light source 201 may be a time varying light source.
Thus, the intensity of the excitation light source 201 may vary
with time. In other words, the excitation light source may emit an
intensity-modulated light beam. The intensity modulation of
excitation light source may comprise without limitation,
sinusoidal, square wave, or ramp wave modulation. In addition, the
excitation light source 201 may also be pulsed at certain
frequencies and repetition rates. The frequency and repetition
rates may also be varied with time. The time variation of the
excitation light source may be about 1 to about 3 orders of
magnitude of the lifetime of the organic dyes used in conjunction
with embodiments of the method.
[0045] Upon illumination of the tissue surface by the excitation
light, the organic dye administered to the lymphatic system emits
fluorescent light. A sensor may be used to detect or sense the
emissions from the fluorescent organic dye. The sensor is
preferably capable of detecting fluorescent light emitted from the
fluorescent targets and detecting excitation light reflected from
the medium. In an embodiment, the sensors may comprise an
intensified charge-coupled camera. Other examples of suitable
sensors include without limitation, gated or non-gated electron
multiplying (EM)-CCD or intensified (ICCD) cameras. The sensor may
further comprise any suitable filters or polarizers necessary to
measure the appropriate wavelengths of light required for
fluorescent optical tomography and imaging.
[0046] In an embodiment, fluorescent emissions from the organic dye
may be continuously detected in 106. The emissions may be
continuously detected by continuously capturing or acquiring images
of the emitted light from the organic dye to create a sequence of
real-time images (i.e. a movie or video) of lymph propulsion
through the lymph structures. The image may be captured for a time
period ranging from about 100 milliseconds to about 30 minutes,
preferably from about 1 minute to about 20 minutes, more preferably
from about 5 minutes to about 15 minutes. Moreover, the images may
be captured or recorded at any suitable integration time ranging
from about 1 millisecond to about 5 seconds, preferably from about
10 milliseconds to about 1 second, and more preferably from about
100 milliseconds to about 800 milliseconds. Accordingly, depending
on the time period and the frame rate, the images collected may
range anywhere from 100 images to over 1,000 images.
[0047] An aspect of embodiments of the disclosed method is the
ability to track or monitor, in real-time, packets of organic dye
being propelled or trafficked through the one or more target lymph
structures. It is believed that the packets of organic dye
represent real-time physiological propulsion of fluids through the
lymph structures which has previously never been seen before using
any imaging technique. By tracking packets of organic dye as they
are pumped through the lymph structures, lymph propulsion and
function may be quantitatively and accurately measured. In
addition, the sequence of recorded images provides a permanent
optical recording of one or more packets or masses of organic dye
being propelled or trafficked through the lymph structure upon
which further analysis may be performed to assess lymph
functionality.
[0048] To quantify pulsatile lymph flow, a stationary target area
or region may be identified on a fluorescent lymph vessel as shown
in FIG. 3A. The target area or region is a point along the lymph
structure at which measurements may specifically be taken. The
fluorescent intensity at the specified target area may then be
measured continuously over a given period of time. As a packet of
organic dye passes through the lymph structure, a corresponding
spike or peak in fluorescent intensity may be measured. A plot of
intensity over time as shown in FIG. 3B provides a good
illustration of the measurement. The pulse frequency of the lymph
structure may then be quantified by dividing the number of pulse
measured by the measurement time period. For example, if eight
intensity peaks were measured over a time period of 5 minutes, the
pulse frequency would equal about 1.6 pulses/min.
[0049] In another embodiment, the functional status of lymph
channels may be characterized by measuring the velocity of the
transiting organic dye "packets." A plurality of target regions may
be assigned along the length of the target lymph structure.
Furthermore, the plurality of target regions may comprise any
distribution along the lymph structure. For example, a target
region may be assigned every 2 mm along a lymph structure.
[0050] As a further illustration, FIG. 4 shows a plot of
"snapshots" of different target regions of fluorescent intensity as
a function of vessel length from the site of administration at
approximately 28, 30, and 35 seconds. Superimposed on each plot are
the time-averaged target region fluorescent intensities with
standard deviations plotted as a function of vessel length. As
depicted in the image frames, a packet of organic dye, as indicated
by the dashed curve, may be clearly differentiated from the time
averaged baseline fluorescent intensity, illustrated by the
asterisks with error bars.
[0051] To determine the velocity of propelled packet, a first and
second target region on the lymph structure may be defined. The
target regions may be any distance apart on the lymph structure. In
some embodiment, the target region may be the entry of the lymph
structure and the second target region may be the exit of the lymph
structure such that the distance is the entire length of the lymph
structure. Once the target regions have been defined, the peak of
the fluorescent intensity representing a packet of organic dye may
be tracked along the known vessel distance. A time period is
determined as a packet is propelled through a first target region
at time "t" and then propelled through the second target region at
time "t+x," where x is a known elapsed time. The velocity may then
be calculated as the distance between the target regions divided by
the elapsed time between the packet passing through the first and
second target regions. The resulting lymph velocity may be
calculated using any suitable units (e.g. m/s, in/s, etc). Thus,
the above disclosed methods provide a quantitative, simple, and
non-invasive way to assess lymph functionality using pulse
frequency and/or lymph flow velocity.
[0052] In further embodiments, it is envisioned that the disclosed
methods may be used in conjunction with tomographic imaging to
produce three dimensional images of lymph propulsion. Tomographic
techniques with patterned illumination as disclosed in U.S. patent
application Ser. No. 11/688,732, incorporated herein by reference
in its entirety for all purposes, may be used to acquire deep
tissue images of lymph propulsion.
[0053] FIG. 5 illustrates an embodiment of an imaging agent that
may be used to stain one or more lymph structures for the
quantitative assessment of lymph function. In an embodiment, the
imaging agent comprises an organic dye coupled to one or more
binding moieties. The one or more binding moieties may provide the
ability to bind the organic dye to lymph structures and provide
detail of the lymph architecture, detect lymph nodes, and assess
tumor lymphangiogenesis.
[0054] The organic dye may be any of the organic dyes described
previously above. In particular, the organic dye is a compound that
exhibits fluorescence at near-infrared wavelengths when exposed to
excitation light. Examples of the organic dye which may be used in
conjunction with the one or more binding moieties include without
limitation, indol-containing dyes, carbocyanine-containing dyes,
polymethine dyes, acridines, anthraquinones, benzimidazols,
indolenines, napthalimides, oxazines, oxonols, polyenes, porphins,
squaraines, styryls, thiazols, xanthins, other NIR dyes known to
those of skill in the art, or combinations thereof. In an
embodiment, the organic dye is IR-783 as shown in FIG. 5.
Furthermore, the organic dye preferably has an excitation
wavelength in the near-infrared range. In particular, the organic
dye may have excitation wavelengths ranging from about 700 nm to
about 1000 nm, preferably from about 720 nm to about 900 nm, more
preferably from about 750 nm to about 850 nm.
[0055] The binding moiety may comprise any molecule or compound
which preferentially binds to lymph endothelial cells. Examples of
suitable targeting moieties include without limitation, amino
acids, receptors, enzymes, signaling molecules, peptides, proteins,
oligopeptides, or combinations thereof. Furthermore, binding moiety
may comprise biological molecules such as without limitation,
proteoclycans, glycosaminoglycans (GAGs), carbohydrates,
polysaccharides, or combinations thereof. In an embodiment,
targeting moiety comprises hyaluronic acid or hyaluranon (HA) as
shown in FIG. 5. HA is a high molecular weight polysaccharide found
in extracellular matrix of all types of tissues. It is a linear
polymer of (1-.beta.-4) D-glucoronic acid (1-.beta.-3)
N-acetyl-D-glucosamine. The HA coupled to the organic dye may have
a molecular weight ranging from about 1,000 Da to about 25,000 Da.
The subscripts "m" and "n" in FIG. 5 denote integers representing
the number of repeating units. The ratio of "m" and "n" preferably
may range from 1:1 to 2:100. In addition, "m" and "n" may comprise
integers ranging from 1,000 to 25,000. HA facilitates mitosis, cell
migration during wound healing and inflammation and embryonic
morphogenesis. Turnover rate of the HA varies from 0.5 to a few
days depending on the tissue type. HA is naturally transported
through the lymph system to lymph nodes where 90% of the
glucosaminoglycan is degraded and the remaining 10% is broken down
in the liver. Consequently, the conjugated HA-dye is a particular
imaging agent directed to lymph.
[0056] Generally, the binding moiety is capable of binding to a
hyaluronic acid binding receptor or receptors which bind to HA. In
a particular embodiment, the binding moiety is specific to a lymph
vascular cell receptor such as without limitation, LYVE-1 receptor
(Lymphatic Vessel Endothelial Receptor 1). LYVE-1 is a receptor
that is a specific marker of lymph endothelium. Without being bound
by theory, the LYVE-1 receptor on the lymphatic endothelium may
sequester HA on the lymph vessel endothelium. Hence, HA conjugated
to a near infra-red fluorescent organic dye may be used to stain
one or more lymphatic structures. It is envisioned that an organic
dye may be coupled to one or more binding moieties which
preferentially bind to other lymph endothelial cell receptors
besides LYVE-1. The binding moiety may also bind to hyaluranon cell
receptors such as without limitation, hyaluranon receptor for
endocytosis (HARE), which may be responsible for polysaccharide
uptake in the lymph nodes.
[0057] Since the imaging agent is bound to the lymph structure, the
structure is "stained" and the architecture of the lymph structure
may be quantitatively imaged. Thus, an advantage of the disclosed
imaging agents is their ability to remain in the lymph structure
for long periods of time as opposed to other imaging agents which
are quickly cleared from the lymphatic system after injection.
Embodiments of the imaging agent are particularly useful in
targeting the lymph node as it is known to degrade polysaccharides
such as hyaluronic acid. The ability to image the architecture of
lymphatics and specifically, the lymph nodes, is clinically
relevant for the prevention, diagnosis, treatment, and research of
lymphatic diseases and cancer, as well as for surgical
planning.
[0058] Therefore, in another embodiment, the imaging agent modified
with the one or more binding moieties may be used in a method of
imaging one or more lymph structures. In particular, the imaging
agent may be used to stain and image the lymph nodes. System 200 as
shown in FIG. 2 may be used to image the stained lymph
structures.
[0059] The modified imaging agent (e.g. IR-783 functionalized with
HA) may be administered to one or more lymph structures under the
tissue surface. The modified imaging agent may be administered
either (i) intradermally into the interstitial space for transport
into the lymphatics, (ii) directly into the lymphatics via
cannulation of the lymphatic vessels, or (iii) intravenously, for
transport across the vasculature into the lymph nodes.
[0060] The imaging agent may be diluted in a solution such as
without limitation, saline solution. The concentration of the
modified imaging agent in solution may range from about 1 .mu.M to
about 400 .mu.M, preferably from about 10 .mu.M to about 200 .mu.M,
more preferably from about 25 .mu.M to about 100 .mu.M.
Furthermore, different amounts of the imaging agent may be
administered or delivered to the one or more lymph structures. For
example, the amounts administered may range from <1 .mu.g to 10
mg, preferably from about <1 .mu.g to about 1 mg, more
preferably from about <1 .mu.g to about 100 .mu.g.
[0061] The modified imaging agent may bind to the endothelial
lining of the one or more lymph structures and stain the
structures. The tissue surface may then be illuminated with
excitation light to excite the imaging agent at its excitation
wavelength. Emissions from the imaging agent may be detected and
captured as fluorescent images.
[0062] Moreover, in addition to staining the lymph structures, due
to the preferential binding of the imaging agent to lymph
endothelial cell receptors, embodiments of the imaging agent may be
used to detect and/or measure the expression of such receptors.
[0063] Embodiments of the disclosed imaging agents may be used to
sense hyaluronidase, an enzyme which degrades HA. Studies have
shown that hyaluronidase may be a marker for cancer. Accordingly,
the ability to optically detect an increase in hyaluronidase
activity may be useful in early detection of metastases and/or
tumor formation. Thus, a method of sensing hyaluronidase may
comprise administering a concentration of a modified imaging agent
(e.g. HA-modified organic dye). The modified imaging agent binds
and stains the lymph structures. The initial fluorescent intensity
may be measured and recorded. Over time, the fluorescent intensity
may be monitored. An increased hyaluronidase may be sensed by a
corresponding increase in the fluorescent intensity. Without being
limited by theory, it is believed that the close spacing of the dye
molecules may cause fluorescent quenching. The cleavage of the HA
polysaccharide may result in a reduction in the quenching and an
increase in fluorescent yield. Thus, an increase in fluorescence
over time may signal the presence of hyaluronidase.
[0064] By altering the concentration of modified or conjugated
imaging agent, the fluorescent intensity of the imaging agent may
be tuned to sense hyaluronidase. Since hyaluronan degradation
occurs primarily in the lymph nodes, this may be used to provide a
selective method for imaging lymph nodes with this specificity and
sensitivity.
[0065] Now referring to FIG. 6, in an embodiment, a method of
making a lymph binding imaging agent comprises modifying or
functionalizing an organic fluorescent dye such as IR-783 dye. In
particular, an organic dye is functionalized to contain an amine
group. Any method known to one of skill in the art may be used to
aminate (i.e. attach an amine group) the organic dye. In a specific
embodiment, the organic dye may be reacted with a mercapto or thiol
compound such as mercaptobenzoic acid to form an intermediate
linking group. Other mercapto compounds such as
mercaptohexadecanoic acid may be used form an intermediate linking
group. The intermediate linking group may be modified with a trityl
amine-containing compound (e.g. mono-trityl 1,6-diaminohexan acetic
acid salt) to form an alkyl diamine linkage group. The diamine
linkage group may contain an alkyl chain having from 2 to 10 carbon
atoms. In a specific embodiment, the linkage group is a hexane
diamine linkage group. The binding moiety (e.g. hyaluronic acid)
may then be coupled to the organic dye via the alkyl diamine
group.
[0066] To further illustrate various illustrative embodiments of
the invention, the following examples are provided.
Example 1
Material and Methods
Animal Models
[0067] Four, two-month old, 60 lb white Yorkshire swine (K Bar
Livestock, HC 69 Box 270 Sabinal, Tex.) were imaged using protocols
which were approved by the Baylor College of Medicine Institutional
Animal Care and Use Committee. The animals were anesthetized,
intubated, and maintained with isoflurane. Animal body temperature
was maintained at 100.degree. F. using a warming blanket. At the
end of the procedure, the animals were euthanized and lymph nodes
resected with fluorescence guidance. Swine were chosen for the
lymph mapping study because swine dermis and lymphatic plexus of
swine is considered most comparable to humans.
NIR Fluorescence Enhanced Imaging
[0068] Continuous-wave optical imaging of the fluorescent NIR dyes
was performed with a custom built intensified charged-coupled
device (CCD). Reynolds J S, Troy T L, and Sevick-Muraca E M.
Multipixel techniques for frequency-domain photon migration
imaging. Biotechnol Prog 13: 669-680, 1997., incorporated herein by
reference in its entirety for all purposes. Briefly, the device had
three principal components: 1) a NIR-sensitive image intensifier
(model FS9910C ITT Night Vision, Roanoke, Va.); 2) a 16-bit dynamic
range, frame transfer CCD camera (Roper Scientific, Tucson, Ariz.);
and 3) a 785-nm laser diode (Thorlabs) used to provide the
excitation light for activating ICG and hyaluronan-NIR. The 785-nm
laser diode beam was expanded by using a planoconvex lens and a
holographic optical diffuser such that approximately 0.08 m.sup.2
of the swine's body was illuminated. A 785-nm holographic notch
band rejection filter (model HNPF-785.0-2.0, Kaiser Optical
Systems, Ann Arbor, Mich.) and an 830-nm image quality bandpass
filter (model 830.0-2.0, Andover, Salem, N.H.) were placed before
the 28-mm Nikkor lens (Nikon) to selectively reject the excitation
light and pass the emitted 830-nm wavelength. A total of 400 to
1,000 images with 512.times.512 resolution and 200 ms exposure time
were acquired enabling near-real time visualization of ICG
trafficking. For image registration, white light images were
acquired by replacing the holographic and bandpass filters with a
neutral density filter and illuminating the surface of the swine
with a low-power lamp.
Image Analysis
[0069] Images were processed using MATLAB (The Mathworks, Natick,
Mass.), ImageJ (National Institutes of Health, Bethesda, Md.), and
V++ (Digital Optics, Auckland, New Zealand). ImageJ is an image
analysis and processing program and supports optical imaging file
formats. V++ is the Roper Scientific CCD camera software interface
with programmable modules for ICCD operation. MATLAB was used to
compute average velocity and frequency of pulsatile lymph flow. To
reveal the frequency of the pulsatile flow, which is a
characteristic of pumping lymphatics, the mean fluorescence
intensity from a fixed region of interest on a lymph channel was
selected and plotted as a function of imaging time. Average
velocity was computed by tracking the position of a "bolus" of ICG
pulse moving along the length of a lymph channel.
Results
Real Time Imaging of Lymph Function
[0070] FIG. 7 represents a typical set of image frames from a movie
depicting nonspecific ICG trafficking in the lymph vessels of the
swine abdomen after four 200-.mu.L injections of 32 .mu.M ICG using
the "research catheter set" at the level of the third teats. Each
imaged lymph vessel was associated with a single injection site.
Lymph propulsion was visualized immediately upon administration of
ICG. The vessels 1 and 4 in FIG. 2 drain to the superficial mammary
nodes (also known as the inguinal nodes), and the vessels 2 and 3
drain to the subiliac lymph nodes that are located 2.5- to 3-cm
deep. The H&E and NIR fluorescence micrographs of resected
fluorescent tissues confirmed dye deposition within the lymph
nodes. The still frames were taken at intervals of 36 and 60
seconds, and the circle in FIG. 7 depicts a typical "packet" of ICG
transiting the 12-cm long lymph vessel. Whereas ICG propulsion is
not directly evident in all the lymph vessels in the still frames,
quantitative analysis of the movie demonstrates that similar
trafficking was seen in the three other lymph vessels.
[0071] FIG. 8 presents an overlay of white light image of the
swine's anterior hindlimb and a typical fluorescent image frame
from a movie depicting ICG trafficking in leg lymph vessel. Upon
intradermal delivery of 200 .mu.l of 32 .mu.M ICG using a
"microcone" device, lymph flow immediately progressed from the site
of injection to the middle iliac node and continued for 4 hours,
the duration for which the animal was anesthetized. The middle
iliac node was already fluorescent before the intradermal injection
in the hindlimb due to ICG drainage from the subiliac nodes from
previous abdominal imaging as shown in FIG. 2. The movie also
demonstrated propulsive lymph flow in the hindlimb as seen in the
abdominal area.
Analysis of Lymph Function
[0072] To quantify pulsatile lymph flow, a stationary, circular
region of interest (ROI) was identified on a fluorescent lymph
vessel as shown in FIG. 3A. FIG. 3B depicts the fluorescent
intensity counts within the ROI as a function of time over 7
minutes of imaging time. The fluorescence intensity profile
illustrates the typical, spontaneous propulsive lymph flow as
indicated by the fluorescence intensity peaks repeating on an
average of every 46 seconds when an ICG "packet" was propelled from
the injection site toward the lymph node in the second (from left)
abdominal afferent lymph vessel. As depicted in FIG. 9A, ROIs were
identified across the length of the vessel. FIGS. 9B and 9C
illustrate different views of a three-dimensional plot of
fluorescence intensity as a function of vessel length and elapsed
time after intradermal injection of 200 .mu.l of 32 .mu.M ICG
within the second abdominal lymph vessel as shown in Figure. The
plot depicts eight trails of ICG packets (enumerated in FIGS. 9B
and 9C) that were propelled from the injection site (at length of
about 0 cm) to the lymph node (at length of about 12.5 cm). For
example, at time 225 seconds, an ICG packet was present at 5 cm
from the injection site. Lymphangion contractions propelled the ICG
forward, and after 4 additional seconds, the bolus was at 9.3 cm
from the injection site. There was only one ICG packet present at
any one time in one segment of the lymph vessel. As can be seen in
FIG. 9B, each packet consistently accumulated within a certain
segment of the lymph vessel before being emptied into a downstream
section of the vessel. FIG. 9C depicts the top view of FIG. 9B.
FIG. 9C (shown only for added clarity) illustrates eight ICG
packets transiting along the lymph channels as a function of
time.
[0073] The functional status of lymph channels was also
characterized by measuring velocity of transiting ICG "packets."
FIG. 4 is a plot of ROI fluorescent intensity as a function of
vessel length from the site of administration at 28 seconds, 30
seconds, and 35 seconds. Superimposed on each plot are the
time-averaged ROI fluorescent intensities with standard deviations
plotted as a function of vessel length.
[0074] The values of lymph flow velocities and pulse frequencies
were computed from imaging on six abdomens and three hindlimb
regional lymph vessels. Upon intradermal injection of ICG in the
swine's leg, the leg lymph vessel was observed to immediately take
up the dye and propel the ICG "packets" toward the middle iliac
lymph node at a frequency ranging from 3.3 to 6.2 pulses of ICG per
minute and at a velocity ranging from 0.33 to 0.46 cm/s. Four hours
after an injection on the leg, the lymph vessel still depicted
active lymphangion contraction by propelling ICG "packets" at a
rate of 1.3 pulses/min and at a velocity of 0.62.+-.0.23 cm/s. We
also observed a range of velocities from 0.23 to 0.75 cm/s for ICG
"packets" transiting in the abdominal lymph vessels, and each bolus
of ICG dye or pulse of ICG passed through a fixed location on the
vessel at a frequency of 0.5-1.3 pulses/min following 200 .mu.l
injection of 32 .mu.M ICG. The rate of propulsion did not correlate
to respiration or heart rate.
Dynamic Imaging of Lymph Propulsion in Humans
[0075] The above disclosed method of imaging lymphatic function was
successfully performed on normal human subjects. The
instrumentation required for normal human subjects consisted of a
frame transfer CCD camera, a near-infrared sensitive image
intensifier, optics for efficiently collecting and filtering the
emitted fluorescence, and incident excitation light from a laser
diode illuminating the tissues with less than 1.9 mW/cm2 of tissue.
Imaging of lymph function was performed on the arms and legs of
human subjects.
[0076] FIG. 12 contains a series of white light photographs of the
ventral (inner) arm with the elbow in the center of the image and
fluorescent overlays of lymph flow at times of 0, 1.8, 4.21, 5.46
seconds (from top to bottom). Four interdigit, intradermal
injections of 25 micrograms of ICG resulted in lymph propulsion
through several lymphatic vessels (denoted by the green overlay).
Lymph flow occurred from the interdigit injection sites (located at
the top but out of the field of view), to the cubital lymph nodes
within the elbow, towards the upper arm and axillary lymph nodes.
The white circles denote packets of ICG flowing through the
lymphatic system. Measurement of velocity of these packets may
provide a quantitative measure of lymph sufficiency in humans.
Example 2
Modification of IR-783 Dye
[0077] IR-783 was combined with 4 mercaptobenzoic acid in presence
of ethyldiisopropylamine (DIPEA). The mixture was stirred for eight
hours under nitrogen atmosphere and dimethyl formic acid solvent
was removed. The compound 1 (IR-783-S-Ph-COOH) was purified by
flash chromatography.
[0078] Compound 1 was treated with hydroxysuccinamide (HOSu) in
dimethylformamide (DMF) and purified to obtain compound 2
(IR-783-S-Ph-COOSu). Compound 2 was combined with mono-Trityl
1,6-diaminohexan acetic acid salt in DMF and DIPEA. The mixture was
stirred overnight to remove solvent and the residue (compound 3)
was dissolved in methanol and purified by flash chromatography.
Finally, compound 3 was mixed with 40% TFA (trifluoro acetic acid)
in DCM (dichloromethane). The mixture was stirred for 30 minutes to
remove the solvent. The residue was washed with ether several times
and purified by HPLC (high pressure liquid chromatography.
Conjugation of Modified IR-783 to HA
[0079] Modified IR-783, hyaluronan, N-Hydroxybenzotriazole (HOBt)
and benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate (PyBop) were combined in dimethylformamide
(DMF). DIPEA was added to this mixture. The mixture was stirred for
four hours. After solvent removal residue was washed with ether and
ethyl acetate several times. The final compound was subjected to
TLC, thin layer chromatography to identify the purified
compound.
Staining of Lymph Vessels With HA-NIR Molecule
[0080] FIGS. 10A and 10B, represent fluorescent images arising from
intradermal injection of 150 .mu.l of 64 .mu.M hyaluronan conjugate
(HA-NIR) and 32 .mu.M ICG in the swine hindlimb vessel. Unlike the
free, nonspecific ICG dye, HA-NIR "stained" the lymph vessel walls
as it filled the vessel. Propulsion of packets of HA-NIR was not
observed as was observed for ICG.
[0081] Instead, HA-NIR uniformly demarcated the lymphatic vessels
and lymph nodes for as long as the imaging study was conducted (4
h). With the use of the ROIs indicated on FIGS. 10A and 10B, the
normalized fluorescent intensity values were plotted for the lymph
vessels imaged with HA-NIR and ICG. Whereas fluctuation and net
reduction of intensity as a function of time was seen for
fluorescence owing to ICG lymph trafficking, FIG. 10C shows that
the fluorescence intensity due to HA-NIR was almost constant and
remained unchanged for the duration of image acquisition 6 min. The
observation was consistent with the binding of HA-NIR to LYVE-1
present on the lymph endothelium.
[0082] To better display the differences between HA-NIR and ICG,
FIGS. 11C and 11D show the three-dimensional plots of fluorescent
intensity as a function of vessel length and time for HA-NIR dye
and the nonspecific ICG in swine lymph vessels. For HA-NIR, the
intensity remained constant with time but diminished along the
length of the vessel at increasing distances away from the
injection site. This observation may be explained by either the
variation in the depth of the vessel as it drained into the lymph
node or due to the binding of HA-NIR to LYVE-1 and subsequent
depletion of unbound HA-NIR as it transited along the lymph vessel
away from the site of injection. On the other hand, the
fluorescence intensity due to ICG varies with time and length along
the vessel, showed "spikes" associated perhaps with the spontaneous
lymphangion contractions and a reduction of intensity owing to its
exit from the lymph channel.
Use of HA-Modified Imaging Agent as a Report of Hyaluronidase
Activity
[0083] FIG. 13 shows different concentrations of conjugated imaging
agents incubated with different concentrations of hyaluronidase.
The vertical lanes consist of unmodified imaging agent alone (lane
1) and increasing concentration of conjugated imaging agents (lanes
1-12). Without hyaluronidase present (top row), increased
fluorescence quenching occurred with increased dye loading. In the
presence of 12.5 units of hyaluronidase, conjugates in lanes 5
through 11 had reduced quenching and increased fluorescent yield.
The table below shows the different imaging agent concentration in
each lane. TABLE-US-00001 TABLE 1 Concentrations of modified
imaging agent in lanes 1-12 for FIG. 13 1: dye 2: HA-dye 1% 3:
HA-dye 2% 4: HA-dye 4% 5: HA-dye 5% 6: HA-dye 6% 7: HA-dye 7% 8:
HA-dye 8% 9: HA-dye 9% 10: HA-dye 12% 11: HA-dye 17% 12: HA-dye
100%
[0084] When the molar percentage of modified IRDye783 was decreased
(in lanes shown in FIG. 13), the fluorescent yield was reduced,
suggesting the close spacing of the dye molecules may have caused
quenching (as seen in the top horizontal row in FIG. 13). However,
when hyaluronidase, the natural enzyme of HA was added (rows 2-5),
the cleavage of the HA polysaccharide may have resulted in a
reduction in the quenching and an increase in fluorescent
yield.
[0085] While embodiments of the invention have been shown and
described, modifications thereof can be made by one skilled in the
art without departing from the spirit and teachings of the
invention. The embodiments described and the examples provided
herein are exemplary only, and are not intended to be limiting.
Many variations and modifications of the invention disclosed herein
are possible and are within the scope of the invention.
Accordingly, the scope of protection is not limited by the
description set out above, but is only limited by the claims which
follow, that scope including all equivalents of the subject matter
of the claims.
[0086] The discussion of a reference in the Description of the
Related Art is not an admission that it is prior art to the present
invention, especially any reference that may have a publication
date after the priority date of this application. The disclosures
of all patents, patent applications, and publications cited herein
are hereby incorporated herein by reference in their entirety, to
the extent that they provide exemplary, procedural, or other
details supplementary to those set forth herein.
* * * * *