U.S. patent application number 16/548491 was filed with the patent office on 2019-12-19 for composition and method for monitoring lipid.
The applicant listed for this patent is Memorial Sloan-Kettering Cancer Center. Invention is credited to Daniel A. HELLER, Prakrit JENA, Daniel ROXBURY.
Application Number | 20190383744 16/548491 |
Document ID | / |
Family ID | 54699781 |
Filed Date | 2019-12-19 |
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United States Patent
Application |
20190383744 |
Kind Code |
A1 |
JENA; Prakrit ; et
al. |
December 19, 2019 |
COMPOSITION AND METHOD FOR MONITORING LIPID
Abstract
A method of detecting a condition in a subject comprises the
steps of contacting cells of the subject with single-walled carbon
nanotubes (SWCNTs), monitoring photoluminescence emitted by SWCNTs
internalized into the cells and generating an SWCNT emission
profile, comparing the SWCNT emission profile to a control emission
profile for the SWCNTs to produce a result, and determining a
likelihood of having the condition in the subject based on the
result from the comparing step. Also disclosed is a method for
screening agents capable of changing endocytic environment using
SWCNTs.
Inventors: |
JENA; Prakrit; (New York,
NY) ; HELLER; Daniel A.; (New York, NY) ;
ROXBURY; Daniel; (New York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Memorial Sloan-Kettering Cancer Center |
New York |
NY |
US |
|
|
Family ID: |
54699781 |
Appl. No.: |
16/548491 |
Filed: |
August 22, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15313580 |
Nov 23, 2016 |
10401295 |
|
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PCT/US2015/032891 |
May 28, 2015 |
|
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16548491 |
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62004122 |
May 28, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B82Y 5/00 20130101; A61B
5/1455 20130101; A61B 5/14546 20130101; G01N 21/6428 20130101; G01N
21/6486 20130101; G01N 21/6489 20130101; B82Y 15/00 20130101; B82Y
30/00 20130101 |
International
Class: |
G01N 21/64 20060101
G01N021/64; A61B 5/1455 20060101 A61B005/1455; A61B 5/145 20060101
A61B005/145; B82Y 15/00 20060101 B82Y015/00; B82Y 30/00 20060101
B82Y030/00; B82Y 5/00 20060101 B82Y005/00 |
Goverment Interests
[0002] This invention was made with Government support under
National Institutes of Health Grants DP2-HD075698 and R37-DK27083.
The Government has certain rights in the invention.
Claims
1. A method of detecting a condition in a subject using
single-walled carbon nanotubes (SWCNTs), comprising: contacting
cells of said subject with SWCNTs; monitoring photoluminescence
emitted by SWCNTs internalized into said cells and generating an
SWCNT emission profile; comparing the SWCNT emission profile to a
control emission profile for the SWCNTs to produce a result; and
determining a likelihood of having said condition in said subject
based on the result from said comparing step.
2. (canceled)
3. (canceled)
4. The method of claim 1, wherein the SWCNTs are semi-conductive
SWCNTs.
5. The method of claim 1, wherein the SWCNTs are
oligonucleotide-coated non-covalently encapsulated SWCNTs.
6. The method of claim 1, wherein the SWCNTs are encapsulated by
DNA oligonucleotides, optionally the DNA oligonucleotides are
ss(GT).sub.6 oligonucleotides.
7. The method of claim 1, wherein the SWCNTs have chiral indices
(n,m) of (6,5) or (8,6).
8. The method of claim 1, wherein the SWCNTs are purified (8,6)
SWCNTs encapsulated with ss(GT).sub.6 oligonucleotides.
9. The method of claim 1, wherein the condition is a disease linked
to elevated lipid and cholesterol content and wherein a blue-shift
in the SWCNT emission profile from the control emission profile is
an indication of the present of the condition in the subject.
10. The method of claim 9, wherein the condition is
atherosclerosis, fatty liver disorder or cancer.
11. The method of claim 9, wherein the condition is
hypercholesterolemia.
12. The method of claim 1, wherein the cells contacted with SWCNTs
are cultured in vitro.
13. The method of claim 1, wherein the cells contacted with SWCNTs
are fibroblasts, microphages or hepatocytes.
14. The method of claim 1, wherein the cells contacted with SWCNTs
are located in a subject in vivo.
15. The method of claim 1, further comprising the step of:
extracting cells from the subject; and culturing the extracted
cells in vitro to produce culture cells, wherein the cells
contacted with SWCNTs are the cultured cells from the culturing
step.
16. The method of claim 3, wherein the sample is a biological
sample.
17. The method of claim 16, wherein the sample is a plasma
sample.
18.-22. (canceled)
23. A kit for detecting changes in lipid accumulation, comprising:
SWCNTs; and oligonucleotides suitable for encapsulating SWCNTs.
24. The kit of claim 23, wherein the SWCNTs are (8,6) carbon
nanotubes and the oligonucleotides are ss(GT).sub.6
oligonucleotides.
Description
CROSS-REFERENCE TO PRIOR APPLICATIONS
[0001] This application claims priority of U.S. Provisional
Application No. 62/004,122, filed on May 28, 2014. The entirety of
the aforementioned application is incorporated herein by
reference.
FIELD
[0003] The present disclosure relates generally to devices and
methods for monitoring lipid and, in particular, lipid in a
biological sample or intracellular lipid using carbon
nanotube-based optical reporters.
BACKGROUND
[0004] Lysosomes are vacuolar organelles responsible for the
breakdown of lipids, proteins, sugars, and other cellular material
into their constituent components. To degrade intracellular
organelles, lysosomes fuse with autophagosomes to form
autolysosomes, while extracellular cargo marked for further
processing and degradation is directed to the lysosome from the
endolysosomal pathway itself. In addition, lysosomes are involved
in nutrient sensing of engulfed amino acids. During starvation,
mTORC1 is inhibited and autophagy is induced, thus indicating
lysosomes as a link between nutrient availability and signaling
pathways related to cell growth.
[0005] A growing body of work implicates lysosomal dysfunction in a
range of pathologies. Dysfunction in the ability of lysosomes to
catabolize or export their contents results in lysosomal storage
disorders, a family of diseases characterized by pathologies caused
by the accumulation of undigested substrates. Lysosomal cholesterol
accumulation is causal during inflammation in both atherosclerosis
and non-alcoholic steatohepatitis. Defects in the fusion of
lysosomes with autophagosomes leads to an accumulation of
autolysosomes in the neurons of patients with amyotrophic lateral
sclerosis (ALS), while impaired autolysosomal proteolysis is
implicated in both Alzheimer's and Parkinson's diseases. The
ability to observe changes in the lipid content of lysosomes is
crucial to understanding the distinct roles played by the lysosomes
in such a variety of diseases. However, no one has developed and/or
applied imaging or any other method to observe endogenous
cholesterol and/or other lipids in live cells and animals. It would
be advantageous to do so for a number of reasons including, for
example, to measure and detect cholesterol and other lipids in
early stage disease detection.
[0006] Spectral imaging is a powerful tool for detection,
validation, separation, and quantification in applications ranging
from mineral assessment of geological satellite images to
semiconductor material characterization. In contrast to
multi-spectral imaging in discrete wavelength bands, hyper-spectral
imaging produces a full, quasi-continuous emission spectrum at
every spatial pixel. Several methods exist for hyperspectral data
acquisition, including pixel-by-pixel, line-by-line acquisition of
spectra, or globally by acquiring separate images at each
wavelength. Recent applications of global hyperspectral imaging
have used volume Bragg gratings (VBG) to acquire spectrally-defined
images from the scanned wavelength space, for the mapping of solar
cell saturation currents and in astronomical imaging.
SUMMARY
[0007] One aspect of the application relates to a method of
detecting a condition in a subject using single-walled carbon
nanotubes (SWCNTs). The method comprises the steps of contacting
cells of a subject with SWCNTs, monitoring photoluminescence
emitted by SWCNTs internalized into the cells and generating an
SWCNT emission profile, comparing the SWCNT emission profile to a
control emission profile for the SWCNTs, and determining a
likelihood of having the condition in the subject based on a result
from the comparing step.
[0008] Another aspect of the application relates to a method for
screening agents capable of changing endocytic dielectric
environment. The method comprises the steps of contacting testing
cells with a candidate agent, wherein the testing cells contain
internalized SWCNTs, monitoring photoluminescence emitted by
internalized SWCNTs and generating a SWCNT emission profile, and
comparing the SWCNT emission profile to a control emission profile
for the SWCNTs to produce a result, wherein the candidate agent is
an agent that is capable of changing endocytic dielectric
environment if the result shows a significant difference between
the SWCNT emission profile to a control emission profile.
[0009] Another aspect of the application relates to a method for
measuring lipid content in a sample. The method comprises the steps
of mixing the sample with SWCNTs, monitoring photoluminescence
emitted by the SWCNTs and generating a sample SWCNT emission
profile, comparing the sample SWCNT emission profile to a control
SWCNT emission profile or emission value, and determining a lipid
content in the sample based on the result of said comparing
step.
[0010] In some embodiments, the SWCNTs are semi-conductive SWCNTs.
In other embodiments, the SWCNTs are polymer-coated non-covalently
encapsulated SWCNTs. In other embodiments, the SWCNTs are
encapsulated by DNA oligonucleotides, wherein the DNA
oligonucleotides are ss(GT).sub.6 oligonucleotides. In other
embodiments, the SWCNTs have chiral indices (n,m) of (6,5) or
(8,6). In other embodiments, the SWCNTs are ss(GT).sub.6 (8,6)
SWCNTs.
[0011] In some embodiments, the SWCNT emission profile detected is
a solvatochromatic response which indicates that photoliuminescence
emitted by SWCNTs is blue-shifted, wherein the blue-shifted
emission indicates substantial lipid (such as cholesterol)
accumulation in the local dielectric environment.
[0012] In some embodiments, the condition is a disease linked to
elevated lipid or cholesterol content. In other embodiments, the
condition is a lysosomal storage disorder selected from the group
consisting of Niemann-Pick, Tay-Sachs and Gaucher's disease. In
another embodiment, the condition is hypercholesterolemia. In
another embodiment, the condition is a disease selected from the
group consisting of atherosclerosis, coronary heart diseases,
stroke, diabetes, fatty liver disease and cancer. In another
embodiment, the cells contacted with SWCNTs are cultured in vitro.
In another embodiment, the cells contacted with SWCNTs are located
in a subject in vivo.
[0013] In another embodiment, the cells contacted with SWCNTs are
cells extracted from the subject. The method further comprises the
steps of: extracting cells from the subject; incubating the
extracted cells with SWCNTs; and monitoring a wavelength-shifted
solvatochromatic response of the SWCNTs. In some embodiments, the
wavelength-shifted solvatochromatic response is a blue-shifted
emission by the SWCNTs. In some embodiments, the cells are
fibroblast cells. In other embodiments, the cells are microphages.
In other embodiments, the cells are hepatocytes. In other
embodiments, the cells are cancer cells. In other embodiments, the
cells are lymphocytes or blood cells.
[0014] The SWCNTs function as a carbon nanotube optical reporter
(CNOR) via solvatochromic shifting of its emission in response to a
change in the immediate dielectric environment, allowing
solvatochromic measurements which are free of artifacts induced by
photobleaching, concentration, and movement of the emitter through
the optical field. When applied to live cells, the CNOR wavelength
transiently and reversibly reflected the changing dielectric
environment of the maturing endosome. In some embodiments, the CNOR
measures a drug-induced increase in endosomal cholesterol or lipid
via an enhanced blue-shift and is capable of identifying the high
free cholesterol or lipid content in human patient fibroblasts with
the NPC1 mutation and benchmarked disease-state reversal on
treatment with cyclodextrin. Applications of the CNOR using
hyperspectral imaging will allow new functional measurements of
analytes in living systems for the study of disease, drug screening
and clinical applications.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The accompanying drawings illustrate one or more embodiments
of the present disclosure and, together with the written
description, serve to explain the principles of the exemplary
embodiments of the present disclosure.
[0016] FIG. 1 illustrates various aspects of reporter response to
lipidic molecules. Panel A illustrates normalized absorption and
emission spectra of the ss(GT).sub.6-encapsulated (8,6) SWCNTs;
Panel B illustrates response of the emission wavelength of
surface-adsorbed ssGT.sub.6-(8,6) SWCNTs to solvent dielectric
(error bar is from triplicate technical replicates); Panel C
illustrates emission wavelength of ssGT.sub.6-(8,6) SWCNTs in
solution as a function of added LDL concentration; Panel D
illustrates overly of transmitted light image and hyperspectral
image of the reporter in RAW 264.7 cells incubated in LPDS, and in
LPDS with Ac-LDL and U18666A added (color legend maps to nanotube
emission peak); Panel E illustrates histogram from the combined
emission from ss(GT).sub.6-(8,6) SWCNTs under the two conditions
previously listed; Panel F illustrates MD simulation 5 image of
ss(GT).sub.6-(8,6) SWCNTs in water, and in the presence of
additional cholesterol molecules; Panel G illustrates water density
on the nanotube surface for ss(GT).sub.6-(8,6) SWCNTs, and in the
presence of added cholesterol.
[0017] FIG. 2 illustrates various aspects of an exemplary SWCNT
solvatochromic response to endosomal dielectric environment. Panel
A is an illustration of exemplary time-course histograms of
ss(GT).sub.6-encapsulated (8,6) SWCNTs emission from live HeLa
cells showing that at 30 minutes, a narrow distribution of emission
wavelengths was observed, centered at 1200.7.+-.0.4 nm and after 6
hours of incubation, SWCNTs in the majority of endosomes were
blue-shifted, and 24 hours after incubation, a single blue-shifted
population remained; Panel B is an illustration of exemplary center
wavelengths of SWCNTs emission ROIs overlaid on transmitted light
images of the HeLa cells wherein a progressive blue-shifting in
individual endosomes was observed; Panel C is an illustration of
exemplary microtubule polymerization inhibitor nocodazole prevented
blue-shifting, while the steroid U18666A exacerbated blue-shifting
of SWCNTs emission in HeLa cells relative to control conditions
(mean.+-.SD, n=3 trials); Panel D is an illustration of exemplary
overlays of SWCNTs emission over brightfield images (top) and
filipin staining (bottom) for the same experimental conditions;
[0018] FIG. 3 illustrates various aspects of an exemplary live-cell
reporting of Niemann-Pick C disease and its therapeutic reversal in
lysosomal storage organelles. Panel A is an illustration of an
exemplary (8,6) nanotube in wild-type (WT) fibroblasts exhibited a
narrow, red-shifted distribution at the 24 hour time point showing
nanotubes incubated in NPC fibroblasts reported both a red
population and a broad, blue-shifted population; Panel B is an
illustration of an exemplary nanotube emission overlaid on
transmitted light images show the spatial distribution of this
spectral heterogeneity; Panel C shows (a) the SWCNTs in WT
fibroblasts maintained their red-shifted emission over 48 hours,
(b) that after 48 hours in NPC fibroblasts, the SWCNTs emission
became broad and blue-shifted, and (c) that after 24 hours of
treatment with 100 .mu.M HP.beta.CD (48 hours after the experiment
began), the SWCNTs population in NPC fibroblasts had red-shifted to
near-WT levels; Panel D is an illustration of an exemplary
nanotube/transmitted light overlays showing the SWCNTs response and
reversal quantitatively and spatially and showing that filipin
staining of fixed cells under similar conditions confirms the
accumulation of cholesterol in NPC cells without HP.beta.CD; Panel
E is an illustration of an exemplary nanotube spectral differences
in WT, NPC, and cyclodextrin-treated NPC cells plotted with
standard error (mean.+-.SD, n=3);
[0019] FIG. 4 illustrates various aspects of detection of lysosomal
storage disorders. Panel A illustrates mean nanotube emission from
wild-type fibroblasts at 24 hours and 48 hours, from
patient-derived NPC1 fibroblasts at 24 hours and 48 hours, and from
NPC1 fibroblasts treated with cyclodextrin for 24 hours, 24 hours
after nanotube addition; Panel B illustrates histograms of the
nanotube emission from single lysosomes at 48 hours, from wild-type
fibroblasts, NPC1 and NPC1 cells treated with cyclodextrin for 24
hours; Panel C illustrates mean filipin intensity from WT
fibroblasts, NPC1 and NPC1 cells treated with cyclodextrin for 24
hours, at 48 hours after nanotube addition; Panel D illustrates
mean emission from nanotubes in WT fibroblasts, NPC1, and NPC1
pretreated with cyclodextrin for 24 hours prior to nanotube
addition; Panel E illustrates mean emission from nanotubes in RAW
264.7 macrophages in DMEM+10% FBS media, in media with 3 ug/L
U18666A, and in media with 10 uM Lalistat; Panel F illustrates the
histograms corresponding to Panel E; Panel G illustrates model for
nanotube emission shift in normal lysosomes, lysosomes with NPC1
phenotype induced with U18666A, and in lysosomes with Wolman's
disease induced with Lalistat (all errors bars are S.E.M. from 3
triplicate experiments).
[0020] FIG. 5 illustrates various aspects of single cell kinetics
of lipid accumulation. Panel A illustrates RAW macrophages in
lipoprotein depleted serum (LPDS), incubated with nanotubes for 3
hours (Ac-LDL (100 ug/mL) and U18666A (3 ug/mL) added at t=0
minutes); Panel B illustrates mean emission from 4 independent time
courses and single cell trajectories of lysosomal lipid
accumulation (error bars=standard deviation, from 4 independent
experiments); Panel C illustrates distribution of time constants
from single cells undergoing lysosomal lipid accumulation, fit with
a log-normal distribution; Panel D illustrates a scatter plot of
the starting nanotube emission wavelength against the time constant
for lipid accumulation for 60 single cells.
[0021] FIG. 6 illustrates various aspects of spatially and
temporally correlating lysosomal lipid content to macrophage
differentiation. Panel A illustrates changes in macrophage
differentiation markers CD11b and F4/80, and the monocyte marker
GR1+ on BMDM differentiating in the presence of CSF1; Panel B
illustrates transmitted light and fluorescent image of nanotubes
localized within the lysosomes of BMDM; Panel C illustrates
hyperspectral image of nanotubes in BMDM at days 3 and 5; Panel D
illustrates histogram of the mean nanotube emission from BMDM cells
at days 3 and 5; Panel E illustrates hyperspectral image of two
BMDM cells at day 3, and their corresponding normalized Simpson's
Index; Panel F illustrates scatter plot of normalized Simpson's
Index with the mean emission from individual cells.
DETAILED DESCRIPTION
[0022] The following detailed description is presented to enable
any person skilled in the art to use the present methods and kits.
For purposes of explanation, specific nomenclature is set forth to
provide a thorough understanding of the present methods and kits.
However, it will be apparent to one skilled in the art that these
specific details are not required to practice the use of the
methods and kits. Descriptions of specific applications are
provided only as representative examples. The present methods and
kits are not intended to be limited to the embodiments shown, but
are to be accorded the widest possible scope consistent with the
principles and features disclosed herein.
[0023] Headings used herein are for organizational purposes only
and are not meant to be used to limit the scope of the description
or the claims. As used throughout this application, the word "may"
is used in a permissive sense (i.e., meaning having the potential
to), rather than the mandatory sense (i.e., meaning must). The
terms "a" and "an" herein do not denote a limitation of quantity,
but rather denote the presence of at least one of the referenced
items.
[0024] One aspect of the application relates to a method of
detecting a condition in a subject using single-walled carbon
nanotubes (SWCNTs). The method comprises the steps of contacting
cells of a subject with SWCNTs, monitoring photoluminescence
emitted by SWCNTs internalized into said cells and generating an
SWCNT emission profile; comparing the SWCNT emission profile to a
control emission profile for the SWCNTs; and determining a
likelihood of having said condition in said subject based on a
result from said comparing step. In some embodiments,
photoluminescence emitted by SWCNTs is monitored by hyperspectral
imaging.
Nanotubes
[0025] As used herein, the term "single-walled carbon nanotubes
(SWCNTs)" refers to allotropes of carbon with a cylindrical
nanostructure. Most SWCNTs have a diameter of close to 1 nanometer,
with a tube length that can be many millions of times longer. The
structure of a SWCNT can be conceptualized by wrapping a
one-atom-thick layer of graphite called graphene into a seamless
cylinder. The way the graphene sheet is wrapped is represented by a
pair of indices (n,m). The integers n and m denote the number of
unit vectors along two directions in the honeycomb crystal lattice
of graphene. If m=0, the nanotubes are called zigzag nanotubes, and
if n=m, the nanotubes are called armchair nanotubes. Otherwise,
they are called chiral nanotubes. SWCNTs are an important variety
of carbon nanotube because most of their properties change
significantly with the (n,m) values, and this dependence is
non-monotonic. In particular, their band gap can vary from zero to
about 2 eV and their electrical conductivity can show metallic or
semiconducting behavior. For a given (n,m) nanotube, if n=m, the
nanotube is typically metallic; if n-m is a multiple of 3, then the
nanotube is typically semiconducting with a very small band gap,
otherwise the nanotube is typically a moderate semiconductor.
Carbon nanotubes are available from a variety of manufacturers, and
may be obtained as sets of unpurified nanotubes within which there
are a variety of distinct species present.
[0026] In some embodiments, the SWCNTs are semiconducting SWCNTs.
Semiconducting SWCNTs exhibit intrinsic photoluminescence which is
narrow-band and uniquely photostable. The intrinsic
photoluminescence of semiconducting SWCNTs are sensitive to changes
in the local dielectric environment, which may result in intensity
modulation as well as wavelength shifting (i.e. solvatochromism) of
the photoluminescence profile of the semiconducting SWCNTs. The
instant application utilizes spatial measurement of the spectral
diversity and response of semiconducting SWCNTs in complex
biological environments to determine the presence or absence of
certain diseased conditions. In some embodiments, the SWCNTs have
chiral indices of (6,5) or (8,6).
[0027] The SWCNTs may be a mixture of SWCNTs with different chiral
indices. In some embodiments, the SWCNTs are a mixture of 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more SWCNT species of
different chiral indices. In other embodiments, purified or
partially purified SWCNT species is used. In some embodiments, the
SWCNTs have chiral indices of (8,6) or (6,5).
[0028] In some embodiments, the SWCNTs of the present application
have an average diameter of 0.1-2 nm, 0.3-1.6 nm or 0.6-1.3 nm. In
some embodiments, the SWCNTs of the present application produce
fluorescence with a wavelength between about 400-1800 nm, 600-1600
nm, 900-1500 nm, or 1000-1200 nm in an appropriate endocytic
dielectric environment. In some embodiments, fluorescence within
the claimed ranges is produced using excitation light in the
wavelength range of 700-900 nm. In some embodiments, the excitation
light has a wavelength of about 730 nm. is detected with wavelength
between about 730-880 nm. In some embodiments, a specialized
detector is used for fluorescent light with wavelength above 1050
nm.
[0029] Nanotubes are not water-soluble, and need to be solubilized
or suspended to facilitate intake by cells. In some embodiments,
SWCNTs are encapsulated with oligonucleotides (such as single
stranded DNA) or synthetic polymers (such as polycarbodiimide or
other amphiphilic polymers). Lipids and surfactants may all be used
to create a stable suspension of nanotubes.
Solubilization/suspension of nanotubes occurs by mixing the chosen
encapsulating agent, such as an oligonucleotide or a synthetic
polymer, with the nanotube in a weight ratio ranging from 1:1 to
4:1, followed with sonication. Sonication may be performed in a
variety of ways, including probe tip ultrasonication and the milder
bath sonication.
[0030] A large range of DNA oligonucleotides, both in terms of
length and sequence, can be used to stably suspend nanotubes. In
some embodiments, the oligo nucleotides are single stranded (ss)
oligo nucleotides with a length of 2-90 nucleotides. In some
embodiments, the oligonucleotides have sequences of ss(TAT).sub.n,
ss(AC).sub.n, ss(ACT).sub.n and ss(GT).sub.n, with n=2-40. In some
embodiments, the oligos are ss(GT).sub.6, ss(GT).sub.15,
ss(ACT).sub.6 or ss(TAT).sub.4, ss(TAT).sub.15. In some
embodiments, the oligo nucleotides are double stranded oligo
nucleotides. In other embodiments, SWCNTs are suspended in
detergents, such as sodium deoxycholate (SDC), sodium cholate (SC),
sodium dodecylbenzene sulfonate (SDBS) or sodium dodecyl sulfate
(SDS). In some embodiments, SWCNTs are encapsulated with
oligonucleotides by sonication with oligonucleotides in a buffer
solution, such as phosphate buffered saline (PBS). In some
embodiments, the SWCNTs are ss(GT).sub.6 encapsulated (8,6) SWCNTs
(also referred to as "ss(GT).sub.6-(8,6) nanotubes") or
ss(GT).sub.6 encapsulated (6,5) SWCNTs (also referred to as
"ss(GT).sub.6-(6,5) nanotubes").
[0031] Certain nanotube species are better sensors for lipids than
others, One of ordinary skill in the art will understand that the
present invention is not limited to any one specific chirality.
Nanotubes that are encapsulated with short DNA sequences are
preferred for detection of lipids within a particular biological
environment. One of ordinary skill will understand that the (8,6)
nanotube has been chosen simply as an non-limiting example. There
is no limitation upon the present invention of the nature or
complexity of the purification technique used to obtain a
particular nanotube species, and any technique which may enable
independent control of DNA sequence (such as synthetic biology
approaches) and or control of nanotube chirality may be used. There
is also no limitation upon the present invention to only pure
samples of a particular nanotube species, the present invention may
use either mixed or pure samples of polymer-encapsulated carbon
nanotubes at the discretion of researchers employing the disclosed
technique.
Disease Conditions
[0032] The compositions, devices and methods of the present
application may be used for the detection of disease conditions
such as atherosclerosis, coronary heart diseases, stroke, diabetes,
cancer, lysosomal storage disorders, hypercholesterolemia and other
diseases linked to elevated lipid and cholesterol content.
[0033] The term "lysosomal storage disorder" may refer to any of a
group of diseases resulting from abnormal metabolism resulting in
accumulation of a substrate, such as glycolipids, in the lysosome.
Lysosomal storage disorders are caused by lysosomal dysfunction
usually as a consequence of deficiency of a single enzyme required
for the metabolism of lipids, glycoproteins or mucopolysaccharides.
These diseases include, but are not limited to the following: Pompe
Disease, Hurler syndrome (Mucopolysaccharidosis type I; MPS-1),
Hunter syndrome (MPS-II), Sanfilippo syndrome A (MPS-IIIA),
Sanfilippo syndrome B (MPS-IIIB), Sanfilippo syndrome C (MPS-IIIC),
Sanfilippo syndrome D (MPS-IIID), classic Morquio syndrome
(MPS-IVA), Morquio B Disease (MPS-IVB), Maroteaux-Lamy syndrome
(MPS-VI), Sly syndrome (MPS-VII), Sialidosis (Mucolipidosis type I;
ML-I), I-cell Disease (ML-II), Pseudo-Hurler Polydystrophy
(ML-III), Schindler Disease/Kanzaki Disease, .alpha.-Mannosidosis,
.beta.-Mannosidosis, .alpha.-Fucosidosis, Aspartylglucosaminuria,
Niemann-Pick Disease Type C, Niemann-Pick Disease Type D, Neuronal
ceroid lipofuscinoses, Wolman Disease, Acid lipase disease, Fabry's
Disease, Niemann-Pick-Disease Type A, Niemann-Pick Disease Type B,
Gaucher's Disease, Krabbe's Disease, GM1 Gangliosidosis, Tay-Sachs
Disease, Sandhoff Disease, Metachromatic Leukodystrophy, Farber
Disease, Multiple sulfatase deficiency, and Galactosialidosis.
[0034] As used herein the term "cancer" refers to any of the
various malignant neoplasms characterized by the proliferation of
cells that have the capability to invade surrounding tissue and/or
metastasize to new colonization sites, including but not limited to
carcinomas, sarcomas, melanoma and germ cell tumors. Exemplary
cancers include bladder cancer, brain cancer, breast cancer,
ovarian cancer, cervix cancer, colon cancer, head and neck cancer,
kidney cancer, lung cancer, mesothelioma, prostate cancer, stomach
cancer and uterus cancer.
[0035] As used herein the term "hypercholesterolemia" refers to the
presence of high levels of cholesterol in the blood. Cholesterol is
a sterol which is precursor of the steroid hormones, bile acids and
vitamin D. Elevated levels of LDL-cholesterol is associated with an
increased risk of atherosclerosis and coronary heart disease.
[0036] As used herein the term "hyperspectral imaging" is a method
of imaging spectroscopy that generates a map of a region of
interest based on local chemical composition. In hyperspectral
imaging, a two-dimensional image is created having a spectral data
inherent in each pixel. These stacks of images comprise a
"hypercube." It is possible to correlate the spectrum of each pixel
with the presence and concentration of various chemical species.
This data can then be construed as a "gradient map" of these
species in a surface.
[0037] A "subject" refers to either a human or non-human animal.
Examples of non-human animals include vertebrates, e.g., mammals,
such as non-human primates (particularly higher primates), dogs,
rodents (e.g., mice, rats, or guinea pigs), pigs and cats, etc. In
a preferred embodiment, the subject is a human. A "biological
sample" may refer to a tissue, cell, blood or plasma sample from a
subject.
[0038] Another aspect of the application relates to a method of
monitoring the local dielectic environment in a tissue culture
using SWCNTs. The method comprises the steps of contacting cells of
a tissue culture of a subject with SWCNTs, monitoring
photoluminescence emitted by SWCNTs internalized into said cells
and generating an SWCNT emission profile; comparing the SWCNT
emission profile to a control emission profile for the SWCNTs; and
determining that a change in the local dielectric environment has
occurred within the cells of the tissue culture because a
blue-shift or red-shift is observed in the wavelength of light
reflected by the SWCNTs.
[0039] As used herein, the term "blue-shift" refers to a decrease
in wavelength, with a corresponding increase in frequency, of
electromagnetic waves. In visible light, this shifts the color from
the red end of the spectrum to the blue end. The term also applies
when photons outside the visible spectrum (e.g., X rays and radio
waves) are shifted toward shorter wavelengths, as well as to shifts
in the de Broglie wavelength of particles. In some embodiments, a
"blue-shift" is defined as a decrease of average wavelength or
median wavelength of at least 0.01, 0.03, 0.05, 0.07, 0.09, 0.1,
0.3, 0.5, 0.7, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15 or 20, 30, 40, 50 nm.
[0040] As used herein, the term "red-shift" refers to an increase
in wavelength, with a corresponding decrease in frequency, of
electromagnetic waves. In visible light, this shifts the color from
the blue end of the spectrum to the red end. The term also applies
when photons outside the visible spectrum (e.g., X rays and radio
waves) are shifted toward longer wavelengths, as well as to shifts
in the de Broglie wavelength of particles. In some embodiments, a
"red-shift" is defined as an increase of average wavelength or
median wavelength of at least 0.01, 0.03, 0.05, 0.07, 0.09, 0.1,
0.3, 0.5, 0.7, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15 or 20, 30, 40, 50 nm
Control Emission Profile
[0041] The control emission profile is the emission profile of a
given SWCNT or an oligo-encapsulated SWCNT at a predetermined
condition. In some embodiments, the control emission profile is the
emission profile of an internalized SWCNT or a plurality of
internalized SWCNTs in a normal cell or in a cell that is free from
a given condition. In other embodiments, the control emission
profile is a emission profile obtained at an earlier time point
(e.g., prior to exposure to an test agent, or prior to the
development of a condition, or prior to the initiation of a
treatment regimen) in the same cell or cells.
Method of Detection of a Lysosomal Storage Disorder
[0042] In some embodiments, the present disclosure pertains to a
method for detecting lysosomal storage disorder by measuring the
endocytic dielectric environment in fibroblasts obtained from a
subject using SWCNTs encapsulated in a specific short
oligonucleotide. A decrease in the dielectric constant of the
endosomal environmental was detected via a distinct solvatochromic
blue-shift in emission. A spectrometer, fluorometer, plate reader
apparatus, or combinations thereof may be employed for the
detection of solvatochromic shift. In one specific embodiment, a
spectral imaging/hyperspectral microscope is employed.
[0043] In one embodiment, the invention is a method of detection of
a lysosomal storage disorder comprising: contacting a tissue, cell,
blood or plasma sample from a subject with SWCNTs, determining the
fluorescence profile of the SWCNTs and comparing the profile to a
control profile of the SWCNTs. If exposure to the tissue, cell,
blood or plasma results in a solvatochromic blue-shift of the
fluorescence profile of the SWCNTs from the control profile, the
subject is at risk of the lysosomal storage disorder.
[0044] SWCNTs when used as CNORs undergo a shift in their emission
wavelength. In particular, when an SWCNT undergoes a shift in
emission wavelength when it interacts with lipids in close
proximity, including cholesterol and lipoproteins. When in
solution, the SWCNTs of the present invention can detect LDL and
total lipid content in solution, whole blood, and patient serum
with high sensitivity. When on a surface (e.g. immobilized on a
surface as part of a device), the SWCNTs of the present invention
can detect LDL and total lipid content at low concentrations. When
in cells, the SWCNTs can detect lipid accumulation in the endosomes
and lysosomes of live cells. In particular, SWCNTs can detect the
direct increase in lipid accumulation in lysosomal storage
disorders, such as Niemann Pick Type C or Wolman's Disease. The
SWCNTs of the present invention can be used as a screening tool in
live cells obtained from patients who have cells with lipid
accumulation. This provides an alternative to techniques such as
filipin staining used for NPC1.
[0045] Lysosomal lipid dysfunction, is correlated with a variety of
diseases. For example, non-alcoholic fatty liver disorder (NAFLD,
NASH, steatosis) has been shown to cause lysosomal lipid
dysfunction in hepatocytes, kupffer cells, and stellate cells from
human patients. In certain neurological disorders lipid
accumulation in the lysosomes is implicated due to autophagy
increasing lipid content in the lysosomes, and has been directly
implicated in a variety of diseases. In certain instances, onset of
atherosclerosis is associated with lipid droplet formation, which
occurs via the lysosomal pathway. Accordingly, all these diseases
or any other disease that is caused by or correlated with lysosomal
lipid accumulation may be detectable by the methods disclosed
herein.
Method of Screening for Drugs to Treat Lysosomal Storage
Disorders
[0046] In another aspect, the instant application utilizes spatial
measurement of the spectral diversity and response of
semiconducting SWCNTs in complex biological environments to
determine the effect of an agent on the local dielectric
environment of a cell.
[0047] In one embodiment, a method of screening for drugs to treat
lysosomal storage disorders comprises the steps of introducing
semiconducting SWCNTs into cells obtained from a subject with a
lysosomal storage disorder, determining the fluorescence profile of
the SWCNTs, exposing the cells to a candidate agent, determining
the fluorescence profile of the SWCNTs after exposure to the agent,
and identifying the agent as a candidate for treatment of lysosomal
storage disorders when the exposure to the agent results in a
red-shift of the fluorescence profile of the SWCNTs.
[0048] In another embodiment, a method of analyzing the efficacy of
treatment of lysosomal storage disorders comprises the steps of
introducing semiconducting SWCNTs into cells obtained from a
subject with a lysosomal storage disorder at different stages of
treatment, determining the fluorescence profile of internalized
SWCNTs at different stages of treatment, wherein a red-shift of the
fluorescence profile of the SWCNTs during the course of treatment
indicates efficacy of the treatment. In some embodiments, the
semiconducting SWCNTs are implanted in vivo in the subject and the
fluorescence profile of internalized SWCNTs at different stages of
treatment is determined in vivo.
[0049] The nanotube emissions from solutions or cells in a 96/384
well plate format may be obtained by an appropriately designed
instrument. Such an instrument can be used for high throughput
screening for drugs that increase or decrease lipid accumulation in
the lysosomes. For example, the present invention may be used for
detection and screening for phospholipidosis, a condition
characterized by cholesterol accumulation in the lysosomes, which
is a side effect in human cells and induced by a variety of drugs
currently in human use.
[0050] Nanotube emissions are also detectable in small animal in
vivo models via appropriately designed pre-clinical instruments. In
vivo models for disease progression may be assayed via lysosomal
lipid accumulation tracked by the techniques disclosed herein.
Disease onset for a variety of disorders as discussed herein, and
methods of intervention to treat such diseases can be assayed by
acquiring the nanotube emissions from animal models over a period
of time using the techniques disclosed herein.
[0051] The particular design of the instruments for detection of
nanotube emissions as disclosed herein is not limiting on the
present invention. For example, a hand-held probe, such as a
wand-like device, has been used to detect nanotube emissions (and
related shifts in emission wavelengths) in animal models and may be
used in human patients. Nanotube emission in the near-infra red is
preferred for detection through human tissue than emission at other
wavelengths.
Kits
[0052] Another aspects of the instant application relates to a kit
for detecting endocytic dielectric environment. In some
embodiments, the kit comprises SWCNTs and oligo nucleotides in an
amount sufficient to encapsulate the single-walled carbon
nanotubes. In some embodiments, the kit contains SWCNTs
encapsulated in oligo nucleotides. In some embodiments, the kit
contains ss(GT).sub.6-(8,6) nanotubes.
[0053] Another aspect of the instant application relates to
oligonucleotide encapsulated SWCNTs. In some embodiments, the
oligonucleotide encapsulated SWCNTs are selected from the groups
consisting of ss(GT).sub.6-(8,6) SWCNTs, ss(GT).sub.6-(6,5) SWCNTs,
ss(GT).sub.15-(8,6) SWCNTs, ss(GT).sub.15-(6,5) SWCNTs,
ss(ACT).sub.6-(8,6) SWCNTs, ss(ACT).sub.6-(6,5) SWCNTs,
ss(TAT).sub.4-(8,6) SWCNTs, ss(TAT).sub.4-(6,5) SWCNTs,
ss(TAT).sub.15-(8,6) SWCNTs and ss(TAT).sub.15-(6,5) SWCNTs. In
other embodiments, the oligonucleotide encapsulated SWCNTs are
ss(TAT).sub.n--(8,6) SWCNTs, ss(AC).sub.n--(8,6) SWCNTs,
ss(ACT).sub.n-(8,6) SWCNTs, ss(GT).sub.n--(8,6) SWCNTs,
ss(TAT).sub.n--(6,5) SWCNTs, ss(AC).sub.n--(6,5) SWCNTs,
ss(ACT).sub.n-(6,5) SWCNTs, ss(GT).sub.n--(6,5) SWCNTs, wherein n
is an integer between 2-40.
[0054] The description herein is for the purpose of teaching the
person of ordinary skill in the art how to practice the present
disclosure, and it is not intended to detail all those obvious
modifications and variations of it which will become apparent to
the skilled worker upon reading the description. The specific
embodiments of the present application have been presented for
purposes of illustration and description. They are not intended to
be exhaustive or to limit the application and method of use to the
precise forms disclosed. Obviously many modifications and
variations are possible in light of the above teaching. It is
understood that various omissions or substitutions of equivalents
are contemplated as circumstance may suggest or render expedient,
but is intended to cover the application or implementation without
departing from the spirit or scope of the claims of the present
application.
EXAMPLES
Example 1: Materials and Methods
[0055] The below materials and methods were used in the examples
herein.
[0056] Hyperspectral Microscope System Excitation: A continuous
wave (CW) 730 nm diode laser with an output power of 1 W was
injected into a multimode fiber to produce the excitation source
for photoluminescence experiments. To ensure a homogenous
illumination over the entire microscope field of view, the
excitation beam passed through a custom beam shaping module to
produce a top hat intensity profile within 20% variation on the
surface of the sample under test. A long pass dichroic mirror with
a cut-on wavelength of 880 nm was aligned to reflect the laser into
an Olympus IX-71 inverted microscope equipped with a 100.times.
(UAPON100XOTIRF, NA=1.49) oil objective (Olympus, USA).
[0057] Hyperspectral Filter Construction: The hyperspectral filter
was situated in the system between the long-pass dichroic mirror
and the camera. To decrease the amount of photons produced by
elastic laser scattering at the surface of the sample, a long-pass
filter with cut-on wavelength of 815 nm was placed at the filter
optical input. To ensure that the beam entirely passed through the
volume Bragg grating (VBG) for filtering, the pupil of the optical
system was imaged between two VBG passes by the first tube lens.
Non-resonant light passes through a VBG un-diffracted. Only the
wavelength that complies with the Bragg condition will resonate
with the grafting and be diffracted in the direction of the corner
cube. The corner cube was situated to reflect the filtered beam
onto the VBG for a second pass in order to cancel chromatic
dispersion induced by the first pass and to narrow filtered
bandwidth to 3.7 nm. The second tube lens formed a
spectrally-filtered image on the IR camera sensor.
[0058] To acquire the spectrally filtered image at different
wavelengths, the corner cube and the VBG were positioned on
rotation stages to continuously tune the diffracted wavelength. An
automatic wavelength calibration process was designed to guarantee
the relation between the angle of the rotation stage and the
absolute wavelength of the grating. Using a xenon spectral lamp
(Newport 6033), absolute wavelength accuracy of 0.5 nm was
obtained.
[0059] Hyperspectral Microscope System Camera: A near-infrared
hyperspectral fluorescence microscope was constructed by
incorporating a volume Bragg grating to spectrally resolve the
entire emitted image. A deep-cooled short-wave infrared (SWIR)
camera was designed for the hyperspectral system. By scanning the
turret-mounted grating with respect to the collimated emission, a
continuous stack of 3.7 nm bandwidth images was constructed by a 2D
InGaAs camera. A 2D InGaAs sensor array (nIR camera), operational
between 900 nm and 1700 nm and with a quantum efficiency superior
to 70%, was used. The array, consisting of 320.times.256 pixels
with 30 .mu.m pitch, was coupled with a four-stage TE cooler to
keep the sensor operating temperature below 190 K. The full-well
capacity of the camera was 168000 electrons in high gain and 3.5
million electrons in low gain with 65 dB S/N ratio and 346 frames
per second capability (full frame). The dynamic range was 14 bits
and the readout noise was 57 electrons at 346 fps in high gain.
Images in the visible (400-700 nm) range were acquired using a
QIClick digital CCD camera (Qimaging, Surrey, BC, Canada) attached
to a separate port of the microscope. Hyperspectral cubes were
acquired from 900=1400 nm for each of the 256.times.320 pixels,
thus providing 81,920 spectra per acquisition.
[0060] Hyperspectral Cube Rectification: Each point source produced
a collimated beam having a different incident angle on the VBG, as
an image is a sum of point sources issued from different positions
of the object seen by the microscope objective. Therefore, the
angular selectivity of the grating resulted in a gradient in
wavelength across the field of view in the dimension parallel to
the dispersion axis. The filtered image produced on the InGaAs
camera was composed of a series of vertical lines each with a
specific wavelength. To obtain a monochromatic image, several
frames at contiguous wavelengths must be scanned through in order
to retrieve the wavelength of interest for each image. The
reconstruction was performed using cubic interpolation on every
pixel for each monochromatic image according to the wavelength
calibration parameters.
[0061] Nanotube Sample Preparation: Standard chemical reagents were
purchased from Sigma-Aldrich (St. Louis, Mo., US) and Fisher
Scientific (Pittsburgh, Pa., US). Single-walled carbon nanotubes
used throughout the study were commercially purchased and produced
by the HiPco process (Unidym, Sunnyvale, Calif., US). Aqueous
dispersions were created by the probe-tip ultrasonication of
(Sonics & Materials, Inc.) 2 mg of the specified
oligonucleotide (IDT DNA, Coralville, Iowa) with 1 mg of raw SWCNT
in 1 mL of PBS (Gibco) for 30 minutes or 10 seconds at 40% of the
maximum amplitude. Following ultrasonication, the dispersions were
ultracentrifuged (Sorvall Discovery 90SE) for 30 minutes at
280,000.times.g. The top 3/4 of the resultant supernatant was
collected and its concentration was determined with a UV/Vis/nIR
spectrophotometer (Jasco, Tokyo, Japan) using the extinction
coefficient A.sub.910=0.02554 Lmg.sup.-1cm.sup.-1. To remove free
DNA, 100 kDa Amicon centrifuge filters (Millipore) were used to
concentrate and re-suspend the DNA-nanotube complexes.
[0062] To obtain near-pure (6,5) or (8,6)-nanotubes, HiPco sample
was dispersed with either ss(TAT).sub.4 or ss(GT).sub.6
oligonucleotides, respectively, and purified according to a
previously documented procedure (see X. M. Tu, S. Manohar, A.
Jagota, M. Zheng, DNA sequence motifs for structure-specific
recognition and separation of carbon nanotubes. Nature 460, 250
(Jul. 9, 2009). Briefly, 1 mL of DNA-nanotube solution, after
sonication and ultracentrifugation, was fed into a Hydrocell
CNT-NS1500 anion-exchange column (Biochrom) and eluted with a
linearly increasing salt gradient of NaSCN using an HPLC (Agilent
Technologies, CA, US). Sample purity was confirmed with absorption
and estimated at 90% (6,5) and 70% for (8,6).
[0063] Cell Lines and Cell Culture Procedures: HeLa CCL-2 cells
(ATCC, Manassas, Va., US) were grown under standard incubation
conditions at 37.degree. C. and 5% CO.sub.2 in sterile-filtered
DMEM with 10% heat-inactivated FBS, 2.5% HEPES, 1% Glutamine, and
1% Penicillin/Streptomycin (all Gibco). For studies performed with
homozygous mutant NPC, compound mutant heterozygote NPC, or
wild-type fibroblasts, the cell lines GM18453, GM03123 or GM05659
(Coriell, Camden, N.J., US), respectively, were cultured in MEM
with 10% FBS, 2.5% HEPES, and 1% Glutamine. Cells were plated on
glass-bottom petri dishes, or lysine-covered glass dishes (MatTek)
for fibroblasts. Unpurified SWCNT were incubated at 1 mg/L and
separated SWCNT were incubated at 0.25 mg/L in media with cells for
30 minutes at 37.degree. C. Depending on the experiment, cells were
imaged immediately, or trypsinized (Gibco) and re-plated on a fresh
glass-bottom petri dish followed by hyperspectral imaging.
[0064] Experimental Data Acquisition: For glass surface-based
measurements, DNA-nanotubes at 1 mg/L, were incubated on a
glass-bottom 35 mm petri dish (Mattek) for 10 seconds and
immediately withdrawn by pipette. The surface was washed with PBS
and fluorescence data were taken with 9 mL of fresh PBS covering
the surface. While focusing on the surface, the individual SWCNTs
were excited with a 730 nm laser, .about.350 mW power at the
sample, and wavelength data were taken from 900 to 1400 nm with 4
nm step sizes and 4 s exposure time. Hyperspectral data from blank
surfaces were also taken to be used post-processing in background
subtraction.
[0065] Data from hyperspectral cubes were analyzed by manually
selecting fluorescent nanotube spots with 3.times.3 pixel ROIs. The
mean intensity of a region devoid of ROIs was defined as the
background signal. Subtracting the background value from each ROI
resulted in an approximately zero baseline for the spectrum from
each nanotube.
[0066] Pharmacological treatments on HeLa cells were performed with
Nocodazole (Sigma, 10 .mu.g/mL added after SWCNT incubation) or
U18666A (Sigma, 3 .mu.g/mL added 6 hours prior to SWCNT
incubation). Fibroblast cells were incubated with
hydroxypropyl-.beta.-cyclodextrin (Sigma, 100 .mu.M) 6 hours prior
to SWCNT incubation.
[0067] Filipin staining was conducted by fixing cells with 1.5%
paraformaldehyde in PBS for 20 minutes. Cells were then labeled
with 50 .mu.g/mL filipin for 45 minutes. After washing, filipin
images were acquired using 350/50 nm excitation and 460/50 mm
emission filters with a 400 nm dichroic long-pass filter
(Olympus).
[0068] AFM imaging was conducted using freshly cleaved mica (Pelco
Mica Disc, V1, Ted Pella) treated with an aqueous MgCl.sub.2 (0.5
M) solution for 30 seconds. Excess solution was removed and 10
.mu.L aqueous suspension of purified ss(GT).sub.6-SWCNT (.about.5
mg/L) was deposited onto the mica and allowed to stand for 20
seconds. The mica surface was then rinsed with water two times to
remove unbound carbon nanotubes. The mica was then dried under
ultrapure nitrogen stream prior to AFM imaging. AFM images were
collected using an Asylum MFD-3D-BIO in AC mode using AC240TS tips
(Asylum Research). The typical scan size was 2-5 .mu.m; scan lines
and points were 512, and the scan rate was 0.5 Hz-1.95 Hz.
[0069] Analysis Software: Hyperspectral data and visible data
acquisition, as well as cube rectification, were performed using
the PhySpec software (Photon etc, Montreal, Canada) that also
controls the hyperspectral microscope. Image processing and (region
of interest) ROI selection were conducted using ImageJ and FIJI
using the Time Series Analyzer plugin and custom macros. Data
analysis, curve fitting, and simulation programs were written in
Matlab 2011 (The MathWorks, MA, US). Statistical analysis and
graphs were generated using OriginPro 8.6 (OriginLab, Northampton,
Mass.). AFM images were acquired and processed in IgorPro
(Wavemetrics, OR, US).
[0070] Sodium deoxycholate (SDC)-suspended HiPCO SWCNTs adsorbed
onto a glass surface, were imaged in 0.1% SDC. After fitting each
nanotube spectrum with a Voigt function, fits with an R.sup.2 value
of less than 0.8 were rejected from all following analyses. The
wavelength emission peak value was divided by the standard
deviation of the baseline (not including the emission peak waveform
itself) to obtain the SNR for the particular nanotube. A
distribution of the SNR for the sample was created to extract
statistical parameters.
[0071] Sorting the nanotube emission peaks in ascending wavelength
order separated individual nanotube species, either by obvious gaps
in the wavelength axis or via a k-means clustering algorithm. The
exercise resolved 14 (n,m) nanotube species and provided an
absolute count of the nanotube population. Separated populations
were assigned (n,m) values based on an empirical Kataura plot as
follows:
TABLE-US-00001 # (n, m) .lamda.start .lamda.end 1 (8, 3) 960 970 2
(6, 5) 984 993 3 (7, 5) 1028 1038 4 (11, 0) 1042 1054 5 (10, 2)
1059 1076 6 (9, 4) 1107 1116 7 (8, 4) 1117 1125 8 (7, 6) 1129 1145
9 (12, 1) 1179 1184 10 (8, 6) 1188 1195 11 (11, 3) 1206 1216 12
(10, 3) 1270 1275 13 (10, 5) 1276 1282 14 (8, 7) 1284 1300
[0072] The SWCNTs, adsorbed to a glass substrate, was imaged in the
presence of DMEM+10% FBS (D10) before 0.1% (sodium deoxycholate)
SDC was introduced to the buffer. The mean wavelength of the SWCNTs
was 1201.5 nm. Hyperspectral cubes obtained after 10 minutes showed
the SWCNTs emission to reach 1190.5 nm. The shifting was reversed
to 1199.1 nm on rinsing the surface with water to remove SDC.
[0073] As these nanotubes responded to lipoproteins in solution and
on a surface, they could be used to detect lipid accumulation in
live cells. Nanotubes encapsulated with either ss(GT).sub.6 or
ss(GT).sub.15 sequences were incubated in media with HeLa cells at
1 mg/L for 30 minutes at 37.degree. C. before being washed and
imaged in fresh media. The spectra were analyzed to obtain the peak
emission wavelengths of five nanotube species at different time
points. Data was reported as a change from the bulk wavelength
value (nm) versus time after initial incubation.
[0074] Cells were incubated for 30 minutes with 0.25 mg/L of the
SWCNTs before rinsing and replacing with fresh media. Hyperspectral
imaging was conducted immediately afterwards and 24 hours
later.
[0075] Nanotubes with fluorophore-labeled Cy3-ss(GT).sub.6 DNA were
encapsulated and co-localized with the Cy3 emission with
lysotracker to determine that nanotubes remained localized within
the endolysosomal pathway. It was confirmed that visible Cy3
emission also remains co-localized with the nIR emission from the
nanotubes themselves. At a 2 pM incubation concentration, no effect
of the nanotubes on the proliferation or viability of the cells was
observed.
[0076] Brightfield images of HeLa cells, and fluorescence images of
LysoTracker dye (Deep Red LysoTracker (ex/em 647/668 nm)), were
obtained using a QIMAGING CCD camera attached to the hyperspectral
microscope. Images of nanotube emission in the same cells were
obtained using the InGaAs camera on the same microscope. The two
cameras had different aspect ratios and pixel sizes, which were
scaled to the same final image size and cropped to represent the
same sample area. Emission from the SWCNT, (which shows as blue),
was localized within the same regions as the emission from
Lysotracker (which shows as red).
[0077] HeLa cells were incubated with purified (8,6) nanotubes for
30 minutes. Cells were then trypsinized and re-plated onto
glass-bottom Petri dishes. A second incubation was performed with
(6,5) nanotubes for 30 minutes (24 hours after the initial
incubation). The live cells were then imaged over the 900-1400 nm
range with the hyperspectral microscope.
[0078] In a similar procedure to the experiment with homozygous NPC
cells, fibroblasts with the compound heterozygous mutation of the
NPC1 gene were plated on a poly-d-lysine-coated glass-bottomed
petri-dish and imaged at different time points after an initial 30
minute incubation with the SWCNTs. Histograms of the wavelength
distribution profile of the SWCNTs were obtained.
Example 2: Analysis of Nanotube Emission
[0079] SWCNTs were non-covalently encapsulated in a single stranded
oligonucleotide sequence to generate water soluble DNA-nanotube
complexes. To test the hypothesis that an optimally short DNA
sequence could partially encapsulate the nanotube while leaving
enough exposed nanotube surface for hydrophobic molecules to bind
to a 12 base long oligonucleotide, GT.sub.6, was selected. GT.sub.6
also functions as the recognition sequence for the (8,6) chirality.
On encapsulating nanotubes with GT.sub.6, a mixture of
GT.sub.6-nanotubes composed of over 15 fluorescent nanotube
chiralities were formed. Ion exchange chromatography was performed
on this mixture to extract only the (8,6) chirality, so obtaining a
pure GT.sub.6-(8,6) sample. This GT.sub.6-(8,6) sample was free of
metallic and carbonaceous impurities, and consisted of a single
absorption peak at 730 nm and an emission peak at 1200 nm (FIG. 1,
panel A).
[0080] Surface adsorbed ss(GT).sub.6-(8,6) nanotubes were dried
with ultrapure N.sub.2 to remove any associated solution from the
nanotube surface. Hyperspectral images from single nanotubes in
different solvent environments were acquired to obtain the mean
nanotube emission value for each solvent (FIG. 1, panel B). The
emission wavelength of ss(GT).sub.6-(8,6) correlated well with the
solvent dielectric (Spearman correlation of 0.89,
p<0.01)--indicating that as the dielectric value of the
environment around the nanotube decreases, the nanotube emission
blue shifts.
[0081] The ss(GT).sub.6-encapsulated (8,6) nanotubes were
introduced to a solution of low density lipoprotein (LDL) to
determine if their emission wavelengths respond to whole
lipoprotein. The emission shifted monotonically with LDL
concentration in solution (FIG. 1, panel C), indicating that the
nanotubes were sensitive to this lipid-rich bio-assembly. The
response of single nanotubes on a surface to lipids was
characterized and the detection of lipidic molecules by the
nanotube was found to be immediate and reversible.
[0082] Single-walled carbon nanotubes (HiPco), suspended with
surfactant and adsorbed onto a glass substrate, were imaged under
730 nm excitation. A hyperspectral cube of nanotube
photoluminescence was acquired, spectrally resolving 14 distinct
species indicated by their unique chiral indices (n,m). Spectra of
individual nanotubes displayed a high signal to noise ratio
(average 41.71) and were fit as Voigt profiles to obtain each
nanotube's center wavelength, full width at half maximum (FWHM) and
intensity. A population analysis of 238 individual nanotubes, using
a k-means clustering algorithm, demarcated nanotube species such
that their approximate quantities resembled those in the bulk HiPco
sample. Analysis of the nanotube emission showed that the FWHM of
all species in the 950-1350 nm range fell between 18-23 nm and
exhibited a known positive correlation with nanotube wavelength
(Pearson's correlation=0.84 with p<0.005). Spectra from
DNA-encapsulated (6,5) nanotubes (N=273), separated by ion exchange
chromatography, were obtained to determine the intrinsic
variability in peak emission wavelength and distribution of FWHMs
for a single nanotube (n,m) species. The wavelength distribution
centered at 990.22.+-.0.35 nm with an average FWHM of 21.54.+-.0.28
nm.
Example 3: Analysis of Nanotube Localization
[0083] Hyperspectral imaging of live HeLa cells incubated with
nanotubes resolved 14 distinct internalized (n,m) species. HeLa
cells were incubated with 1 mg/L DNA-encapsulated HiPco for 10
minutes and unbound nanotubes were washed away prior to imaging. In
agreement with previous studies, the DNA-dispersed nanotubes
entered cells via endocytosis and remained in late endosomes and
lysosomes. After 30 minutes of incubation, the cells were found to
average one photoluminescent nanotube per endosome, measured by
counting the number of spatially-resolved emission bands per
endosome. Two separated (n,m) nanotube species, were used to label
sub-cellular structures. DNA-encapsulated (6,5) nanotubes were
imaged 24 hours after internalization within HeLa cells; the
nanotubes localized within lysosomes in the perinuclear region.
Subsequently, (8,6) nanotubes were imaged immediately after a
30-minute incubation with the same cells, resulting in the
localization of these nanotubes within endosomes only. The labeling
thus spatially distinguished endosomes and lysosomes by two, narrow
near-infrared spectral bands (FWHM .about.26 nm), separated by 210
nm.
[0084] A DNA-encapsulated nanotube complex, undergoing a continuous
solvatochromic shifting response, spatially and temporally tracked
endosomal maturation in live HeLa cells. Separated (8,6) nanotubes
encapsulated by an ss(GT).sub.6 oligonucleotide, with a median
length of 236 nm, were incubated with HeLa cells at 0.25 mg/L for
30 minutes before rinsing and replacing with fresh media. The
wavelength distribution of the nanotube complex in endosomes,
measured immediately after uptake, was centered at 1200.7.+-.0.4
nm, near the bulk value observed in water. In lysosomes, after 6
hours post-incubation, the wavelength distribution broadened before
blue-shifting to 1194.3.+-.0.7 nm at the 24-hour timepoint (FIG. 2,
panel A). A small red-shifted nanotube population, with a mean
wavelength of 1205 nm, appeared at the 6-hour time point but
disappeared by 24 hours. The relative mean wavelength of each
emissive region of interest (ROI) was laid over a transmitted light
image of the cells, resulting in spatial visualization of the
spectral distribution (FIG. 2, panel B). Both the inter- and
intra-cellular heterogeneity of emission wavelengths progressively
increased with endosomal maturation. The nanotube
wavelength-shifting phenomenon was found to differ among cell
types; MCF-10A epithelial cells caused blue-shifting similar to the
observation in HeLa cells, nanotubes in RAW 264.7 macrophages
exhibited little change relative to bulk nanotubes suspended in
water, and GM05659 human fibroblasts produced a red-shifted
nanotube population. The interaction of GT.sub.6-(8,6) nanotubes
with RAW 264.7 macrophages was characterized, by incubating 2 pM of
nanotubes in cell media for 30 minutes at 37.degree. C. Under these
conditions, the majority of cells efficiently took up nanotubes via
energy-dependent mechanisms.
Example 4: Identifying a Carbon Nanotube Optical Reporter of Local
Dielectric Environment
[0085] As nanotube photoluminescence exhibits blue-shifting
behavior in lower dielectric environments, the sensitivity of the
ss(GT).sub.6 oligonucleotide-encapsulated (8,6) nanotube complex to
solvents with known dielectric constants was directly tested. The
complex was incubated with butanol (.epsilon.=18), resulting in a
14.6.+-.0.9 nm blue shift from the corresponding emission in water
(.epsilon.=80). In contrast, ss(TAT).sub.4-encapsulated, separated
(6,5) nanotube complexes were relatively less sensitive to the
local dielectric environment (2.2.+-.0.2 nm blue shift in butanol)
and reported negligible wavelength-shifting in live HeLa cells,
even at 24 hours. Therefore, the ss(GT).sub.6-(8,6) complex
functions as a carbon nanotube optical reporter (CNOR) of local
dielectric environment.
[0086] Pharmacological inhibition of endosomal maturation by the
microtubule polymerization inhibitor nocodazole prevented the
blue-shifting of CNOR emission in HeLa cells (FIG. 2, panels C-D).
The maturation process of endosomes containing the CNOR by adding
nocodazole to the cell media at 10 .mu.g/mL 30 minutes prior to the
incubation with the CNOR was halted; these CNORs did not blue-shift
for up to 24 hours after internalization (1200.07.+-.1.4 nm mean
emission compared to 1194.28.+-.0.73 for control). This result
indicates that blue-shifting of the CNOR requires the endosome to
lysosome transition and the corresponding change in dielectric
environment.
[0087] As the CNOR transitions from early endosomes to lysosomes,
the environmental pH decreases from pH 6.6 to pH 4.5. To probe the
effect of pH on CNOR emission wavelength shifting,
NH.sub.4Cl--known to increase endosomal pH--was used to alkalinize
endocytic organelles in HeLa cells. On addition of 50 mM NH.sub.4Cl
to cells 6 hours after incubation with the CNOR, the blue-shifted
population remained unaffected while the red-shifted population was
depleted. This result, combined with the observed red-shift of the
CNOR with lowering pH suggests that the lowering of pH directly
causes red-shifting of DNA-nanotube emission but is nut responsible
for the blue-shifting phenomenon.
[0088] The blue-shifting of CNOR emission was enhanced by the drug
U18666A, an amphiphilic aminosteroid known to increase the
cholesterol content of late endosomes (FIG. 2, panels C-D). HeLa
cells treated with 3 .mu.g/mL of the drug for six hours before
incubation with the CNOR resulted in a further 3.8.+-.0.7 nm
increase in blue shift at 24 hours than in untreated control
cells.
[0089] A similar effect was observed in RAW 264.7 macrophages; the
CNOR exhibited a blue-shift of 11.1.+-.1.1 nm in drug-treated cells
relative to a drug-free control. RAW macrophages were incubated in
lipoprotein depleted serum (LPDS) for 24 hours. Incubation in these
conditions reduces the lipid content of the lysosomes and increases
the number of surface receptors for acetylated LDL (Ac-LDL) on the
cell surface. To induce lipid accumulation in the lysosomes, Ac-LDL
and U18666A (a cholesterol transport inhibitor) were introduced to
cells incubating in the lipoprotein depleted serum. After 3 hours
of incubation, the combination of Ac-LDL and U18666A results in a
significant accumulation of unesterified cholesterol in the
lysosomes of treated cells. Nanotubes were added in normal cell
media for 30 minutes, before free nanotubes were washed away and
the endocytosed nanotubes were given 3 hours to localize to the
lysosomes. Hyperspectral images of the nanotubes within cells were
acquired, and the images color coded to the peak emission
wavelength from each pixel. The hyperspectral images from nanotubes
in cells incubated in LPDS were mostly orange and red, while the
cells treated with Ac-LDL and U18666A appeared blue and green (FIG.
1, panel D). The histogram of the total nanotube emission from
cells in each condition indicates that the nanotube emission from
lipid-deficient lysosomes is .about.1200 nm while the emission from
lipid-rich lysosomes is .about.1192 nm (FIG. 1, panel E).
[0090] To understand why the accumulation of cholesterol within the
constrained volume of a lysosome could induce a blue shift in the
emission of the GT.sub.6-(8,6) nanotube, all-atom replica exchange
molecular dynamics simulations were performed of the nanotube, and
the nanotube with cholesterol in its vicinity. The equilibrium
configurations obtained after 100 ns indicate that cholesterol
partially self-assembles and binds to exposed regions on the
nanotube surface. In contrast to the starting configuration,
cholesterol also induces rearrangement of DNA on the nanotube
surface (FIG. 1, panel F). The net effect of cholesterol binding
and DNA rearrangement is a significant decrease in the density of
water molecules near nanotube surface (FIG. 1, panel G). Consistent
with the shift in nanotube emission due to environmental dielectric
(FIG. 1, panel B), displacement of water from the nanotube surface
results in a relative blue shift.
[0091] Labeling unesterified cholesterol using filipin, which
required fixing the cells, displayed a marked qualitative
difference between control and drug-exposed cells. In control HeLa
cells, filipin strongly illuminated the cholesterol-rich plasma
membrane with only a small amount of cholesterol-bound filipin
emission appearing from within the cell, while the intensity of
filipin in the cell interior increased greatly upon U18666A
exposure (FIG. 2, panel D). These results indicate that the CNOR
emission undergoes blue-shifting in the presence of endosomal
lipids including free cholesterol, the major species up-regulated
by the drug.
[0092] To confirm the ability of a water-soluble cholesterol
derivative to directly affect the emission of the nanotube
reporter, the CNOR with sodium deoxycholate (SDC) was probed. The
reporter, pre-incubated with cell culture media and serum, was
adsorbed to a glass coverslip. A 0.1% solution of SDC was
introduced, resulting in an average blue-shift of 11.0.+-.0.7 nm
from 1201.5 nm to 1190.5 nm. The blue-shifted CNOR largely reverted
back to its initial emission wavelength upon removing SDC. By
successively acquiring hyperspectral cubes, the solvatochromic
response was found to reach equilibrium in less than 300 seconds.
This experiment demonstrates the intrinsic reversibility and rapid
response of the reporter to changes in dielectric environment. It
suggests that in a cellular environment, the DNA-encapsulated
nanotube leaves enough exposed surface such that sterols or other
amphipathic molecules can reversibly bind, resulting in an
accumulative solvatochromic response to the change in the
dielectric environment of the CNOR.
[0093] The emission from other (n,m) species in an unsorted
ss(GT).sub.6 oligonucotide-encapsulated nanotube sample exhibited
similar results to the (8,6) nanotube, with varying degrees of
blue-shift observed as a function of time after uptake by HeLa
cells. Changing the encapsulating oligonucleotide sequence from
ss(GT).sub.6 prevented the blue-shifting phenomenon, indicating
sequence-specificity of the optical response. To test whether the
CNOR remains intact within the lysosome, ss(GT).sub.6-encapsulated
nanotubes was labeled with Cy3 and the organic fluorophore was
found to remain quenched by the nanotube surface. This finding
confirms the stability of the sensor, as the encapsulating DNA
remains associated with the nanotube.
Example 5: Identifying Lysosomal Storage Disorders
[0094] Niemann Pick Type-C(NPC) is a lysosomal storage disorder
characterized by massive accumulation of unesterified cholesterol
in the lysosomes of patient fibroblasts. The solvatochromic
shifting of nanotubes clearly distinguished wild-type (WT)
fibroblasts from fibroblasts acquired from a patient with NPC. The
NPC cells accumulate cholesterol and other lipids within their late
endosomes and lysosomes due to a homozygous mutation of the NPC1
gene. At 24 hours after the initial 30 minute incubation with the
CNOR, its emission blue-shifted by approximately 10 nm in NPC cells
compared to WT fibroblasts (1203.1.+-.0.48 nm for NPC vs.
1209.9.+-.0.32 nm for WT, FIG. 3, panel A). The solvatochromic
response was monitored spatially across the cells, showing
variations among endosomal compartments (FIG. 3, panel B), but
exhibiting clear, quantitative differences between diseased and
non-diseased cells (p-value <0.005 for spectral identification
of NPC and WT cells).
[0095] Treatment of homozygous NPC cells with cyclodextrin
(HP.beta.CD), a molecule which removes free cholesterol from the
lumen of the late endosome/lysosome, caused a reversal of the CNOR
blue-shift, thus benchmarking the therapeutic action in live cells.
Subsequent to imaging at 24 hours after incubation with nanotubes,
NPC fibroblasts were incubated with HP.beta.CD for an additional 24
hours (histograms and overlays in FIG. 3, panels C-D). The effect
of HP.beta.CD treatment on CNOR emission from NPC cells was
dramatic; the blue-shifted nanotube population transitioned to a
red-shifted population and appeared nearly identical to nanotubes
in WT cells (FIG. 3, panel E). This result correlated well with
staining by filipin, indicating that cyclodextrin removed free
cholesterol from the lumen of the late endosome/lysosome, likely
driving cholesterol off the nanotube surface and thereby reversing
its blue-shifted emission state. Pre-treatment of NPC cells with
HP.beta.CD prevented CNOR fluorescence from shifting in diseased
cells, resulting in emission that was spectrally identical
(p<0.005) to that in WT fibroblasts. Thus, the excess free
cholesterol in the late endosome/lysosome of NPC cells is
responsible for the blue shift observed when compared to healthy
fibroblasts, and removal of the excess free cholesterol inhibits
the blue-shift of the CNOR.
[0096] The blue-shifting phenomenon was also demonstrated on
fibroblasts from a different patient, with the compound
heterozygous mutations of the NPC1 gene, which also exhibit
substantial cholesterol accumulation. The lysosomes in NPC1 cells
were heterogeneous in their lipid content, in contrast to WT
fibroblasts. The reporter was incubated for 30 minutes with cells
acquired from a 10-year old patient, before free nanotubes were
washed away. Measurements at 24 hours indicated nanotubes in the
lysosomes of NPC1 cells to be blue shifted by 6 nm, compared with
WT fibroblasts (FIG. 4, panel A). To test the ability of the
reporter to detect disease reversal, the NPC1 cells were treated
with 100 uM cyclodextrin for 24 hours. This significantly
red-shifted the nanotube emission by approximately 4 nm--and
indicated that within the lysosomes of live cells, the reporter is
reversible and accurately detected the decrease in cholesterol due
to the therapeutic effect of cyclodextrin. By looking at histograms
obtained from single lysosomes from cells at 48 hours, WT
fibroblast lysosomes were found to be uniformly lipid-poor, with a
homogeneous distribution centered at .about.1210 nm (FIG. 4, panel
B). In contrast, the lysosomes from NPC1 cells showed a broad
distribution, and included a lipid-poor fraction, indicating that
not all lysosomes demonstrate lipid accumulation. The cyclodextrin
treated NPC cells showed a narrow homogeneous distribution of
lysosomal lipid content, similar to the distribution observed in WT
fibroblasts.
[0097] The nanotube results were confirmed by staining the cells
with filipin, a fluorescent fixed cell marker with high specificity
for cholesterol. Though filipin is not organelle specific,
cholesterol accumulation in NPC1 is limited to the lysosomes and
thus increased filipin intensity indicates an increase in the
cholesterol content of affected lysosomes. Filipin staining
confirmed the nanotube emission data as being accurate, with higher
filipin intensity observed in blue-shifted cells (FIG. 4, panel C).
To confirm that the reporter was specifically detecting
cholesterol, NPC1 cells were pre-treated with cyclodextrin. After
treatment, the lipid content of these lysosomes should approximate
WT cells. Consistent with this finding, nanotubes added to
cyclodextrin pretreated cells, and to WT fibroblasts displayed
identical emission values, which were significantly red-shifted
from untreated NPC1 fibroblasts (FIG. 4, panel D).
[0098] The reporter detected Wolman's disease, a lysosomal storage
disorder characterized by the accumulation of esterified
cholesterol in the lysosomes due to a non-functioning lysosomal
acid lipase (LAL). Lalistat, a specific inhibitor of LAL, was used
to prevent the hydrolysis of esterified cholesterol. As a positive
control in RAW macrophages, the NPC1 phenotype was induced by
preventing cholesterol efflux from lysosomes with U18666A. Compared
with the control macrophages, both U18666A and Lalistat induced a 8
nm blue shift (FIG. 4, panel E). Therefore, the reporter
blue-shifts due to an accumulation of lipidic molecules in the
lysosomes of cells. The lysosomal accumulation of unesterfied
cholesterol in NPC1 or esterified cholesterol in Wolman's diseases
both result in the binding of lipid molecules to the nanotube
surface and induce a blue shift in the nanotube emission (FIG. 4,
panel G).
[0099] Cells can accumulate lipids within their lysosomes at
different rates, and the reporter was used to understand the
parameters of lipid accumulation in individual cells. The reporter
was localized in the lysosomes of RAW macrophages incubated in
lipoprotein depleted serum. The emission from nanotubes in these
lipid-poor lysosomes was at .about.1200 nm. Upon adding both AcLDL
and U18666A to the media, hyperspectral images of the same cells
were acquired every 10 minutes for a two hour period. Cells
progressively blue shifted due to unesterified cholesterol
accumulation within their lysosomes (FIG. 5, panel A). Single cells
across hyperspectral images were tracked and the mean nanotube
emission from each cell for every time point was extracted. The
rate of blue shifting of individual cells can be described as a lag
period of no shifting, followed by a single exponential decay. Each
single exponential decay can be fit to obtain the time constant for
each cell (FIG. 5, panel B)--the average time constant of lipid
accumulation is 40 minutes. The distribution of time constants from
individual cells follows a log normal distribution, which is
consistent with the rate of lipid accumulation within the lysosomes
of a single cell being a convolution of multiple independent
processes (FIG. 5, panel C). Cells show remarkable heterogeneity in
their rate of lipid accumulation, with the slowest and fastest time
constants differing by an order of magnitude.
[0100] The slowest cells were found to be the ones that started
with the most lipid-deficient lysosomes. The time constant of lipid
accumulation correlated with the starting wavelength (Spearman
correlation of 0.33, p<0.01), but interestingly, all slow cells
(defined as cells with a time constant greater than 90 minutes)
started off with an emission greater than 1202 nm. The time lag
before the single exponential blue shifting commenced did not
correlate to the starting emission value, the final emission value,
the total emission shift or the time constant of decay for single
cells. Therefore, cells accumulate lipids in the lysosomes at a
rate independent of a lag period preceding lipid accumulation. The
rate of lipid accumulation is heterogeneous, and cells slow to
accumulate lipids within their lysosomes also start with the most
lipid-deficient lysosomes. Lipid accumulation due to U18666A action
in the presence of AcLDL uptake is a complex process. Using the
reporter directly measures the phenomenon of interest, lysosomal
lipid accumulation.
Example 6: Identifying Differentiation State of Bone Marrow
[0101] The reporter can correlate lysosomal lipid content with the
differentiation state of bone marrow derived macrophages (BMDM).
Monocytes in the presence of colony-stimulating factor (CSF1)
differentiate into macrophages, as observed by an increase in the
fraction of cells expressing the macrophage markers CD11b and F4/80
and a decrease in the fraction of cells expressing the monocyte
marker GR1 (FIG. 6, panel A). Nanotubes taken up by BMDM under
standard experimental conditions localize in the lysosomes and
appear as punctate spots under 100.times. magnification (FIG. 6,
panel B). Each emissive pixel is fitted to obtain the nanotube
emission peak, and thus generates a lysosomal lipid map of
individual cells (FIG. 6, panel C). The average nanotube emission
from each pixel within a cell gives a mean lysosomal lipid value
for that particular cell. A collection of these mean nanotube
emission values thus benchmarks a population of cells by the degree
of lipid accumulation within their lysosomes. As macrophages
differentiate from day 3 to day 5, the nanotube emission
significantly blue shifts by .about.5 nm (FIG. 6, panel D),
indicating that day 5 macrophages have significantly greater
lysosome lipid content than day 3 macrophages.
[0102] The process of lysosomal lipid accumulation was observed
within single cells, by quantifying the intra cellular
heterogeneity in lysosomal lipid content. The nanotube emission
from lysosomes within a cell was analyzed with two parameters--an
average emission value, representing the overall state of lysosomal
lipid accumulation within that cell, and a normalized Simpson's
Index, to quantify the degree of heterogeneity between lysosomes
within that cell. Two typical cells from day 3, showing a
relatively homogeneous red cell with a normalized Simpson's Index
(nSI) value of OAR, and a more heterogeneous blue cell with a nSI
of 0.74 are shown (FIG. 6, panel E). Plotting the mean emission
value of a cell against its nSI demonstrates a clear partitioning,
as all blue cells are highly heterogeneous (FIG. 6, panel F). Blue
cells being the most heterogeneous indicates that as cells mature
and transition from red (lipid deficient lysosomes) to blue (lipid
rich lysosomes), individual lysosomes within a single cell also
transition from red to blue, which results in an increase in the
heterogeneity (both red and blue lysosomes) observed within single
cells. The application of the reporter to study differentiating
macrophages informs us that lysosomal lipid content increases as
monocytes differentiate into macrophages, and this process is
observable at the single cell level.
[0103] In summary, the above-described non-photobleaching
fluorescent probe, localizes to the lysosome, and reports
variations in the lipid content of individual lysosomes within live
cells. Spatially localizing the nanotube emission generates a
endolysosomal lipid map of a cell, and changes in the lysosomal
lipid content can be measured over prolonged periods of time with
single cell and single lysosome resolution. This carbon nanotube
optical reporter indicates that changes in the lysosomal lipid
content can occur both due to dysfunctions such as lysosomal
storage disorders and normal cellular processes such as macrophage
differentiation.
* * * * *