U.S. patent application number 16/059476 was filed with the patent office on 2019-03-14 for assays for measuring binding kinetics and binding capacity of acceptors for lipophilic or amphiphilic molecules.
This patent application is currently assigned to Northwestern University. The applicant listed for this patent is Northwestern University. Invention is credited to RAJA KANNAN MUTHARASAN, C. Shad Thaxton.
Application Number | 20190079105 16/059476 |
Document ID | / |
Family ID | 56924957 |
Filed Date | 2019-03-14 |
United States Patent
Application |
20190079105 |
Kind Code |
A1 |
MUTHARASAN; RAJA KANNAN ; et
al. |
March 14, 2019 |
ASSAYS FOR MEASURING BINDING KINETICS AND BINDING CAPACITY OF
ACCEPTORS FOR LIPOPHILIC OR AMPHIPHILIC MOLECULES
Abstract
Aspects of the invention relate to methods for measuring the
binding constant of a lipophilic or amphiphilic molecule acceptor
for a lipophilic or amphiphilic molecule. Methods involve rapid,
cell-free competition assays including a labeled lipophilic or
amphiphilic molecule and nanoparticle.
Inventors: |
MUTHARASAN; RAJA KANNAN;
(Chicago, IL) ; Thaxton; C. Shad; (Chicago,
IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Northwestern University |
Evanston |
IL |
US |
|
|
Assignee: |
Northwestern University
Evanston
IL
|
Family ID: |
56924957 |
Appl. No.: |
16/059476 |
Filed: |
August 9, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15073941 |
Mar 18, 2016 |
10078092 |
|
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16059476 |
|
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62134788 |
Mar 18, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/92 20130101;
G01N 2800/324 20130101; G01N 33/54346 20130101; G01N 2800/50
20130101; G01N 33/553 20130101; G01N 2333/775 20130101; G01N 33/542
20130101; G01N 33/743 20130101 |
International
Class: |
G01N 33/92 20060101
G01N033/92; G01N 33/74 20060101 G01N033/74; G01N 33/553 20060101
G01N033/553; G01N 33/543 20060101 G01N033/543; G01N 33/542 20060101
G01N033/542 |
Claims
1.-9. (canceled)
10. A kit for measuring an equilibrium constant of an acceptor for
a lipophilic or amphiphilic molecule, comprising: (a) the
lipophilic or amphiphilic molecule having a detectable signal; and
(b) a structure, the structure comprising a nanostructure core and
a lipid layer surrounding and attached to the nanostructure core,
wherein the structure quenches the signal of the lipophilic or
amphiphilic molecule when the structure and the lipophilic or
amphiphilic molecule are proximate.
11. The kit of claim 10, further comprising instructions for use of
the kit for measuring the equilibrium constant of the acceptor for
the lipophilic or amphiphilic molecule.
12. A system for measuring an equilibrium constant of an acceptor
for a lipophilic or amphiphilic molecule, comprising: (a) a sample,
the sample comprising: (i) a lipophilic or amphiphilic molecule,
wherein the molecule can provide a detectable signal; (ii) a
structure, the structure comprising a nanostructure core and a
lipid layer surrounding and attached to the nanostructure core,
wherein the structure quenches the signal of the lipophilic or
amphiphilic molecule when the structure and the lipophilic or
amphiphilic molecule are proximate; (iii) an acceptor; and (b) a
detector for measuring the signal.
13. The system of claim 12, further comprising a device configured
to calculate the equilibrium constant of the acceptor for the
lipophilic or amphiphilic molecule based on the detected
signal.
14. The system of claim 12, further comprising a radiation source
configured to induce the signal.
15.-32. (canceled)
33. The kit of claim 10, wherein the equilibrium constant is
measured in a cell-free assay.
34. The kit of claim 10, wherein the lipophilic or amphiphilic
molecule comprises a steroid, a lipopolysaccharide, a cholestane,
cholane, pregnane, androstane, estrane or a derivative or analog
thereof.
35. The kit of claim 10, wherein the lipophilic or amphiphilic
molecule comprises or consists of BODIPY-cholesterol.
36. The kit of claim 10, wherein the signal is fluorescence.
37. The kit of claim 10, wherein the nanostructure core is an
inorganic material.
38. The kit of claim 10, wherein the nanostructure core is
gold.
39. The kit of claim 10, wherein the structure further comprises an
apolipoprotein bound to at least the outer surface of the lipid
layer.
40. The kit of claim 10, wherein the acceptor is a high-density
lipoprotein (HDL).
41. The system of claim 12, wherein the sample is a biological
sample and wherein the biological sample is not a cell.
42. The system of claim 12, wherein the lipophilic or amphiphilic
molecule comprises a steroid, a lipopolysaccharide, a cholestane,
cholane, pregnane, androstane, estrange, or BODIPY-cholesterol.
43. The system of claim 12, wherein the signal is detected by
positron emission tomography, SPECT medical imaging,
chemiluminescence, electron-spin resonance, ultraviolet/visible
absorbance spectroscopy, mass spectrometry, nuclear magnetic
resonance, magnetic resonance, flow cytometry, autoradiography,
scintillation counting, phosphoimaging, or an electrochemical
method.
44. The system of claim 12, wherein the signal is fluorescence.
45. The system of claim 12, wherein the nanostructure core is
gold.
46. The system of claim 12, wherein the structure further comprises
apolipoprotein bound to at least the outer surface of the lipid
layer.
47. The system of claim 12, wherein the acceptor is a high-density
lipoprotein (HDL).
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/073,941, entitled "ASSAYS FOR MEASURING
BINDING KINETICS AND BINDING CAPACITY OF ACCEPTORS FOR LIPOPHILIC
OR AMPHIPHILIC MOLECULES" filed Mar. 18, 2016 which claims priority
under 35 U.S.C. .sctn. 119(e) to U.S. Provisional Application Ser.
No. 62/134,788, entitled "ASSAYS FOR MEASURING BINDING KINETICS AND
BINDING CAPACITY OF ACCEPTORS FOR LIPOPHILIC OR AMPHIPHILIC
MOLECULES" filed on Mar. 18, 2015, which is herein incorporated by
reference in its entirety.
FIELD OF INVENTION
[0002] The present invention relates generally to the use of
nanoparticles in assays for monitoring the binding of lipophilic or
amphiphilic molecules.
BACKGROUND OF THE INVENTION
[0003] Coronary heart disease, which can manifest as heart attacks
or sudden death from lethal arrhythmias, is the number one killer
in the U.S. and worldwide. Accurate estimation of cardiovascular
disease risk is a critical first step in applying life-saving
preventative therapies to high risk populations. State-of-the-art
methods to ascertain risk, however, remain imperfect. Evidence
shows that measuring Cholesterol efflux capacity of a certain human
serum fraction significantly enhances prediction accuracy of
cardiovascular risk. Measurement of an aspect of Reverse
Cholesterol Transport (RCT) with an assay that involves cultured
cells and human serum samples improves the accuracy of clinical
risk assessment for heart disease (Khera et al. (2011) N. Engl. J.
Med. 364:127). However, because this assay entails tissue culture
techniques, radiolabeled cholesterol, and a 72-hour turnaround
time, its clinical utility is limited.
SUMMARY OF THE INVENTION
[0004] Quantifying cholesterol transport in complex biological
matrices is critically important because of the central role
cholesterol balance or overload plays in many human pathologies,
such as heart disease (Lusis (2000) Nature 407:233). Cholesterol is
a hydrophobic molecule minimally soluble in the aqueous milieu of
biological systems, yet it is found in millimolar quantities in
blood, owing to solubilization in lipid carriers known as
lipoproteins which bind and transport cholesterol. Despite the
important role cholesterol-lipoprotein interactions have in
understanding cholesterol transport, the dissociation constant
(K.sub.D) of cholesterol from lipoprotein-containing serum
fractions remains unknown. Of particular interest is the strength
of cholesterol interactions with high density lipoproteins (HDL),
as these structures mediate cholesterol clearance from peripheral
tissues, a process termed Reverse Cholesterol Transport (RCT)
(Rosenson et al. (2012) Circulation 125:1905). A simple,
automatable assay measuring serum affinity for lipophilic or
amphiphilic molecules, such as cholesterol is highly desirable.
[0005] Disclosed herein is a nanoparticle-enabled, rapid and
cell-free competition assay which directly measures the affinity of
lipophilic or amphiphilic molecule acceptors, including human
serum. This method was used to discover the dissociation constant
(K.sub.D) of serum and a serum fraction enriched in HDL for
cholesterol.
[0006] Aspects of the invention relate to a method for measuring
the equilibrium constant of an acceptor for a molecule, comprising:
providing a lipophilic or amphiphilic molecule, wherein the
molecule can provide a detectable signal; providing a structure,
the structure comprising a nanostructure core and a lipid layer
surrounding and attached to the nanostructure core, wherein the
structure quenches the signal of the molecule when the structure
and the molecule are proximate; providing an acceptor; allowing the
acceptor to compete with the structure for binding with the
molecule; and measuring the signal, wherein the level of the signal
correlates with the equilibrium constant of the acceptor for the
molecule.
[0007] In certain embodiments, the method further comprises
increasing the amount of acceptor provided. In certain embodiments,
the method further comprises increasing the amount of structure
provided.
[0008] In certain embodiments, increasing the amount of acceptor
provided leads to an increase in signal. In certain embodiments,
increasing the amount of structure provided leads to a decrease in
signal. In certain embodiments, increasing or decreasing the amount
of molecule improves the signal to noise ratio of the system.
[0009] In certain embodiments, the method is a component of an
assay. In certain embodiments, the method is a component of a
diagnostic assay. In certain embodiments, the method is a method
for assessing cardiovascular risk in a subject.
[0010] Other aspects of the invention relate to a kit for measuring
an equilibrium constant of an acceptor for a molecule, comprising:
a lipophilic or amphiphilic molecule, wherein the molecule can
provide a detectable signal; and a structure, the structure
comprising a nanostructure core and a lipid layer surrounding and
attached to the nanostructure core, wherein the structure quenches
the signal of the molecule when the structure and the molecule are
proximate.
[0011] In certain embodiments, the kit further comprises
instructions for use of the kit for measuring the equilibrium
constant of the acceptor for the molecule.
[0012] Other aspects of the invention relate to a system for
measuring an equilibrium constant of an acceptor for a molecule,
comprising: a sample, the sample comprising: a lipophilic or
amphiphilic molecule, wherein the molecule can provide a detectable
signal; a structure, the structure comprising a nanostructure core
and a lipid layer surrounding and attached to the nanostructure
core, wherein the structure quenches the signal of the molecule
when the structure and the molecule are proximate; an acceptor; and
a detector for measuring the signal.
[0013] In certain embodiments, the system described further
comprises a device configured to calculate the equilibrium constant
of the acceptor for the molecule based on the detected signal. In
certain embodiments, the system further comprises a radiation
source configured to induce the signal.
[0014] In certain embodiments of the method, kit, or system herein
described, the lipid layer is a bilayer. In certain embodiments of
the method, kit or system herein described, the molecule is a
steroid or a derivative or analog thereof.
[0015] In certain embodiments of the method, kit, or system herein
described, the molecule is a lipopolysaccharide or a derivative or
analog thereof. In certain embodiments of the method, kit, or
system herein described, the molecule is a steroid or a derivative
of analog thereof. Several non-limiting classes of steroids include
Cholestanes, Cholanes, Pregnanes, Androstanes and Estranes or
derivative or analog thereof.
[0016] In certain embodiments, of the method, kit, or system herein
described, the molecule is BODIPY-cholesterol. In certain
embodiments of the method, kit, or system herein described, the
signal is fluorescence. In certain embodiments of the method, kit,
or system herein described, the signal is fluorescence
polarization.
[0017] In certain embodiments of the method, kit, or system herein
described, the nanostructure core is an inorganic material. In
certain embodiments of the method, kit, or system herein described,
the nanostructure core is a metal. In certain embodiments of the
method, kit, or system herein described, the nanostructure core is
gold.
[0018] In certain embodiments of the method, kit, or system herein
described, the structure further comprises apolipoprotein bound to
at least the outer surface of the lipid layer. In certain
embodiments of the method, kit, or system herein described, the
apolipoprotein is apolipoprotein A-I, apolipoprotein A-II, or
apolipoprotein E.
[0019] In certain embodiments of the method, kit, or system herein
described, the acceptor is a lipoprotein. In certain embodiments of
the method, kit, or system herein described, the acceptor is a
high-density lipoprotein (HDL). In certain embodiments of the
method, kit, or system herein described, the acceptor is a
component of serum. In certain embodiments of the method, kit, or
system herein described, the serum is human serum. In certain
embodiments of the method, kit, or system herein described, the
serum is enriched for HDL. In certain embodiments of the method,
kit, or system herein described, the serum is depleted for
ApoB.
[0020] The subject matter of this application involves, in some
cases, interrelated products, alternative solutions to a particular
problem, and/or a plurality of different uses of structures and
compositions.
[0021] Other advantages and novel features of the present invention
will become apparent from the following detailed description of
various non-limiting embodiments of the invention when considered
in conjunction with the accompanying figures. In cases where the
present specification and a document incorporated by reference
include conflicting and/or inconsistent disclosure, the present
specification shall control. If two or more documents incorporated
by reference include conflicting and/or inconsistent disclosure
with respect to each other, then the document having the later
effective date shall control.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Non-limiting embodiments of the present invention will be
described by way of example with reference to the accompanying
figures, which are schematic and are not intended to be drawn to
scale. In the figures, each identical or nearly identical component
illustrated is typically represented by a single numeral. For
purposes of clarity, not every component is labeled in every
figure, nor is every component of each embodiment of the invention
shown where illustration is not necessary to allow those of
ordinary skill in the art to understand the invention. In the
figures:
[0023] FIG. 1 shows an exemplary binding experiment used to measure
binding and kinetics of a cholesterol acceptor, in this case
ApoB-depleted human serum. Baseline fluorescence intensity was
first recorded in a 6.25 uM solution of BODIPY cholesterol in 100
ul of 20% ethanol in water. Next 100 ul of 500 nM HDL AuNP was
added and fluorescence intensity was measured for 30 minutes at 2
minute read intervals. At this point the concentrations of BODIPY
cholesterol and HDL AuNP were 312.5 nM and 250 nM, respectively.
Finally, cholesterol acceptor was added and fluorescence was
measured over 60 minutes. FIG. 1 shows measurement of ApoB-depleted
serum at final assay concentrations of 0.00%, 0.16%, 0.32%, 0.49%,
and 0.65%, respectively (addition of 0, 5, 10, 15, and 20 ul of
ApoB-depleted human serum prepared as described above added to the
200 ul volume containing BODIPY cholesterol and HDL AuNP). The
amount of fluorescence recovery rises with the increasing amounts
of cholesterol acceptor added. Data points are the mean of wells
plated in triplicate.
[0024] FIG. 2 shows a lipidated nanoparticle templated with 5 nm
diameter gold which was titrated into 250 nM of fluorescent
BODIPY-Cholesterol. Fluorescence quenching was measured upon
binding. Nonlinear regression analysis modeling the
BODIPY-Cholesterol interaction with the lipidated nanoparticle as a
multiple binding site interaction with same K.sub.D and no
cooperativity yields a K.sub.D of 40.+-.9 nM and a binding site
number of 16.+-.1 binding sites. Error bars denote standard
deviations of technical quadruplicates.
[0025] FIG. 3 shows serum or ApoB-depleted serum that was titrated
into a solution of 20 nM NP and 125 nM BODIPY-Cholesterol. The
effective K.sub.D of serum in this case was calculated as
1.4.times.10.sup.-4, and of ApoB-depleted serum as
3.0.times.10.sup.-4. Error bars denote standard deviations of
technical quadruplicates.
[0026] FIG. 4 shows an illustration of lipidated nanoparticles
synthesis.
[0027] FIG. 5 shows the manipulate function in Mathematica that was
used to assess the performance and plausibility of the function
generated from solving a system of binding equations. This allowed
for assessment of function performance as well as manual estimation
of initial starting parameters for KD.
[0028] FIG. 6 shows an assay measuring patient serum affinity for
cholesterol binding. The data show a correlation between the
performance of the assay of the invention and a currently accepted
assay. The normalized efflux assay is plotted on the x-axis, and
can be seen to vary in magnitude among participants from
approximately 0.8 to approximately 1.4, approximately 1.0 to
approximately 2.0, or approximately 0.8 to approximately 2.4 with a
value of 1.0 being equivalent to the efflux value of the pooled
sample.
DETAILED DESCRIPTION
[0029] The invention is based, at least in part, on the development
of a rapid, cell-free competition assay for measuring binding
between lipophilic or amphiphilic molecules and a lipophilic or
amphiphilic molecule acceptor, such as serum or fractions thereof.
Methods described herein, using labeled lipophilic or amphiphilic
molecules, such as cholesterol analogs, and a lipidated
nanoparticle, are rapid and high throughput. These novel assays
have widespread applications including: assessing risk of
disorders, such as cardiovascular disorders, by measuring
lipophilic or amphiphilic molecule binding in patient samples;
conducting screens for agents that influence lipophilic or
amphiphilic molecule efflux; investigating lipophilic or
amphiphilic molecule binding kinetics and binding capacity; and
investigating small molecule binding and binding capacity to HDLs
(e.g., competition assays).
[0030] Aspects of the invention relate to a competition assay that
can detect the K.sub.D of lipophilic or amphiphilic molecule
acceptors, such as human serum, for lipophilic or amphiphilic
molecules, such as cholesterol. When the assay is applied to a
sample, such as a human serum sample, it can be used to assess the
strength of the interaction between lipophilic or amphiphilic
molecules, such as cholesterol, and high density lipoproteins (HDL)
within the serum sample. HDL plays a significant role in clearance
of cholesterol from peripheral tissues, in a process referred to as
Reverse Cholesterol Transport (RCT). Assessing the binding capacity
of HDL to cholesterol in a serum sample from a subject provides
valuable information on the subject's functional capacity for
RCT.
[0031] Novel assays described herein have multiple advantages over
previous methods for assessing RCT, including: i) Assays described
herein are automatable and involve simple and straightforward
pipetting steps that can be programmed into laboratory
hardware/robotic liquid handling systems. A high-throughput--96- or
384-well format can allow for dozens of samples to be read and
processed simultaneously; ii) No radiochemicals need to be used in
these assays. Radiolabelled cholesterol, used in previously
described assays had to be handled in a specific manner and had to
be disposed of carefully. Moreover, most clinical laboratories do
not have facilities to carry out radiometric tests; iii) Assays
described herein are very rapid--owing to the rapid binding
kinetics of both natural lipophilic or amphiphilic molecule
acceptors and materials described herein, this test can be
performed, including incubation times, within hours with minimal
human input. By contrast, previously described Cholesterol efflux
assays used in research take 4 days from start to finish; iv) very
low serum volumes are required--assays described herein require a
serum input on the order of 100 ul from ordinary blood draw tubes.
Furthermore, given the stability of lipophilic or amphiphilic
molecules, such as cholesterol acceptors in serum, this test has
the potential to be done as an "add-on" or "reflex" to routinely
collected specimens ordered for other purposes.
[0032] Methods described herein involve binding between a lipidated
nanoparticle and a labeled lipophilic or amphiphilic molecule, such
as a cholesterol analog. As used herein, a "lipidated nanoparticle"
refers to a nanoparticle and that is associated with one or more
lipids. Lipidated nanoparticles are described further in, and
incorporated by reference from PCT/US2009/002540, entitled
"Nanostructure Suitable for Sequestering Cholesterol and Other
Molecules."
[0033] It should be appreciated that any lipophilic or amphiphilic
molecules can be compatible with aspect of the invention. As used
herein, a lipophilic molecule refers to a molecule that can
dissolve in fats, oils, lipids, and non-polar solvents. Examples of
lipophilic groups include, but are not limited to, cholesterol, a
cholesteryl or modified cholesteryl residue, adamantine,
dihydrotesterone, long chain alkyl, long chain alkenyl, long chain
alkynyl, olely-lithocholic, cholenic, oleoyl-cholenic, palmityl,
heptadecyl, myrisityl, bile acids, cholic acid or taurocholic acid,
deoxycholate, oleyl litocholic acid, oleoyl cholenic acid,
glycolipids, phospholipids, sphingolipids, isoprenoids, such as
steroids, vitamins, such as vitamin E, fatty acids either saturated
or unsaturated, fatty acid esters, such as triglycerides, pyrenes,
porphyrines, Texaphyrine, adamantane, acridines, biotin, coumarin,
fluorescein, rhodamine, Texas-Red, digoxygenin, dimethoxytrityl,
t-butyldimethylsilyl, t-butyldiphenylsilyl, cyanine dyes (e.g. Cy3
or Cy5), Hoechst 33258 dye, psoralen, or ibuprofen. The cholesterol
moiety may be reduced (e.g. as in cholestan) or may be substituted
(e.g. by halogen). A combination of different lipophilic groups in
one molecule is also possible. The lipophilic molecule may be a
sterol, such as cholesterol.
[0034] As used herein, an amphiphilic molecule refers to a molecule
that possesses both hydrophilic and lipophilic properties. Several
non-limiting examples of amphiphilic compounds include
phospholipids, cholesterol, glycolipids, fatty acids, bile acids,
saponins and local anaesthetics.
[0035] In some embodiments, the molecule is a steroid or a
derivative or analog thereof. As used herein, a steroid refers to
an organic compound that contains four cycloalkane rings that are
joined to each other. Several non-limiting examples of classes of
steroids include Cholestane, Cholane, Pregnane, Androstane or
Estrane. In some embodiments, the molecule is a lipopolysaccharide
or a derivative or analog thereof. As used herein a
lipopolysaccharide refers to a molecule consisting of a lipid and a
polysaccharide joined by a covalent bond.
[0036] The lipophilic or amphiphilic molecules associated with
aspects of the invention produce a detectable signal. In some
embodiments, the lipophilic or amphiphilic molecule is labeled with
a fluorescent label. The terms "fluorescent label", "fluorescent
dye", and "fluorophore" are used interchangeably herein to refer to
moieties that absorb light energy at a defined excitation
wavelength and emit light energy at a different wavelength.
Examples of fluorescent labels include, but are not limited to:
Alexa Fluor dyes (Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor
532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor
633, Alexa Fluor 660 and Alexa Fluor 680), AMCA, AMCA-S, BODIPY
dyes (BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 493/503,
BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589,
BODIPY 581/591, BODIPY 630/650, BODIPY 650/665), Carboxyrhodamine
6G, carboxy-X-rhodamine (ROX), Cascade Blue, Cascade Yellow,
Coumarin 343, Cyanine dyes (Cy3, Cy5, Cy3.5, Cy5.5), Dansyl,
Dapoxyl, Dialkylaminocoumarin,
4',5'-Dichloro-2',7'-dimethoxy-fluorescein, DM-NERF, Eosin,
Erythrosin, Fluorescein, FAM, Hydroxycoumarin, IRDyes (IRD40, IRD
700, IRD 800), JOE, Lissamine rhodamine B, Marina Blue,
Methoxycoumarin, Naphthofluorescein, Oregon Green 488, Oregon Green
500, Oregon Green 514, Pacific Blue, PyMPO, Pyrene, Rhodamine B,
Rhodamine 6G, Rhodamine Green, Rhodamine Red, Rhodol Green,
2',4',5',7'-Tetra-bromosulfone-fluorescein, Tetramethyl-rhodamine
(TMR), Carboxytetramethylrhodamine (TAMRA), Texas Red, Texas Red-X,
5(6)-Carboxyfluorescein, 2,7-Dichlorofluorescein,
N,N-Bis(2,4,6-trimethylphenyl)-3,4:9,10-perylenebis(dicarboximide,
HPTS, Ethyl Eosin, DY-490XL MegaStokes, DY-485XL MegaStokes,
Adirondack Green 520, ATTO 465, ATTO 488, ATTO 495, YOYO-1,5-FAM,
BCECF, dichlorofluorescein, rhodamine 110, rhodamine 123, YO-PRO-1,
SYTOX Green, Sodium Green, SYBR Green I, Alexa Fluor 500, FITC,
Fluo-3, Fluo-4, fluoro-emerald, YoYo-1 ssDNA,
2',4',5',7'-Tetra-bromosulfone 1 dsDNA, YoYo-1, SYTO RNASelect,
Diversa Green-FP, Dragon Green, EvaGreen, Surf Green EX, Spectrum
Green, NeuroTrace 500525, NBD-X, MitoTracker Green FM, LysoTracker
Green DND-26, CBQCA, PA-GFP (post-activation), WEGFP
(post-activation), FlASH-CCXXCC, Azami Green monomeric, Azami
Green, green fluorescent protein (GFP), EGFP (Campbell Tsien 2003),
EGFP (Patterson 2001), Kaede Green,
7-Benzylamino-4-Nitrobenz-2-Oxa-1,3-Diazole, Bex1, Doxorubicin,
Lumio Green, and SuperGlo GFP. Those of ordinary skill in the art
will know of other suitable fluorescent labels for the assays
described herein, or will be able to ascertain such, using routine
experimentation.
[0037] In some embodiments, the fluorescent label is from the
difluoro-boraindacene (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)
family (BODIPY) family. In some embodiments, the labeled lipophilic
or amphiphilic molecule is BODIPY-Cholesterol, NBD-Cholesterol or
dansyl-Cholesterol or their associated cholesteryl esters. In other
embodiments, fluorescent cholesterol analogues that possess
intrinsic fluorescence (e.g., dehydroergosterol and
cholestatrienol) and those in which a fluorophore or photoreactive
group is attached (e.g., NBD-Cholesterol, BODIPY-Cholesterol, and
dansyl-cholestanol).
[0038] Nanoparticles described herein, such as lipidated gold
nanoparticles, have the ability to quench fluorescence emitted by a
fluorescently labeled lipophilic or amphiphilic molecules when the
fluorescently labeled lipophilic or amphiphilic molecule is in
close proximity to the nanoparticle. Accordingly, in assays
described herein, binding between the nanoparticle and the
fluorescently labeled lipophilic or amphiphilic molecule leads to a
reduction in the levels of lipophilic or amphiphilic molecule
measured in a sample.
[0039] Aspects of the invention involve lipophilic or amphiphilic
molecule acceptors. As used herein, a "lipophilic or amphiphilic
molecule acceptor" refers to a molecule that can bind to a
lipophilic or amphiphilic molecule. A lipophilic or amphiphilic
molecule acceptor may be involved in transporting a lipophilic or
amphiphilic molecule, such as cholesterol, to the liver from
peripheral tissues. A lipophilic or amphiphilic molecule acceptor
used in assays described herein can be serum, such as human serum.
The serum can be enriched for HDLs. In some embodiments, the serum
is depleted for Apolipoprotein B (ApoB). Addition of a lipophilic
or amphiphilic molecule acceptor, such as a cholesterol acceptor,
to assays described herein causes competition for cholesterol
binding with the nanoparticle and causes the fluorescent signal to
recover.
[0040] As used herein, an acceptor refers to a molecule that binds
cholesterol. Examples of an acceptor includes, but it is not
limited to, apolipoprotein A1 (ApoA1), high density lipoprotein
(HDL), albumin, serum, including human serum, or apolipoprotein B
(ApoB)-depleted human serum.
[0041] Measurement of fluorescence in assays described herein,
following addition of a cholesterol acceptor can be used to assay
levels of HDL in a sample and to determine binding and kinetics of
a cholesterol acceptor. Since a low HDL level in serum can
correlate with an increased risk of disorders associated with
cholesterol, such as cardiovascular disorders, assays described
herein can contribute to assessing a subject's risk of developing a
disorder associated with cholesterol, such as a cardiovascular
disorder.
[0042] It should be appreciated that in assays described herein a
detectable signal, such as fluorescence, can be measured according
to any method known in the art. In some aspects, samples are
processed in multi-well plates, such as 96-well or 384-well plates
and fluorescence is measured using a plate reader. Systems
associated with the invention can be configured such that
fluorescence is measured and then correlated with outputs such as
the binding constant of a lipophilic or amphiphilic molecule
acceptor for a lipophilic or amphiphilic molecule.
[0043] As used herein, "binding constant" or "association constant"
refers to a mathematical constant that describes the binding
affinity between two molecules at equilibrium. It should be
appreciated that methods described herein can also be used to
measure dissociation constants.
[0044] Any type of detectable label can be compatible with aspects
of the invention. As used herein, a detectable label refers to a
moiety, the presence of which can be ascertained directly or
indirectly. Generally, detection of a label involves an emission of
energy by the label. The label can be detected directly by its
ability to emit and/or absorb photons or other atomic particles of
a particular wavelength (e.g., radioactivity, luminescence, optical
or electron density, etc.). A label can be detected indirectly by
its ability to bind, recruit and, in some cases, cleave another
moiety which itself may emit or absorb light of a particular
wavelength (e.g., epitope tag such as the FLAG epitope, enzyme tag
such as horseradish peroxidase, etc.). An example of indirect
detection is the use of a first enzyme label which cleaves a
substrate into visible products. The label may be of a chemical,
peptide or nucleic acid molecule nature although it is not so
limited. Other detectable labels include radioactive isotopes such
as P32 or H3, luminescent markers such as fluorochromes, optical or
electron density markers, etc., or epitope tags such as the FLAG
epitope or the HA epitope, biotin, avidin, and enzyme tags such as
horseradish peroxidase, bb-galactosidase, etc. The label may be
bound to a peptide during or following its synthesis.
[0045] Non-limiting examples of the types of labels that can be
compatible with aspects of the claimed invention include enzymes,
radioisotopes, fluorescent compounds, colloidal metals,
chemiluminescent compounds, and bioluminescent compounds. Those of
ordinary skill in the art will know of other suitable labels for
the molecules described herein, or will be able to ascertain such,
using routine experimentation. Furthermore, the coupling or
conjugation of these labels to the molecules of the invention can
be performed using standard techniques common to those of ordinary
skill in the art.
[0046] Another labeling technique consists of coupling molecules
described herein to low molecular weight haptens. These haptens can
then be specifically altered by means of a second reaction. For
example, haptens such as biotin, can be used, which can react with
avidin, or dinitrophenol, pyridoxal, or fluorescein, which can
react with specific anti-hapten antibodies. Non-limiting examples
of haptens include digoxigenin, Alexa Fluor 488, Biotin-X SE,
Biotin-XX SE, Biotin-XX SSE, BODIPY FL-X SE, BODIPY FL STP ester,
Cascade Blue acetyl azide, Dansyl-X SE, DSB-X biotin SE, Lucifer
yellow iodoacetamide, 5(6)-TAMRA-X SE, Rhodamine Red-X SE, Texas
Red-X SE. Examples of corresponding antibodies for hapten
conjugation included nut are not limited to Anti-Alexa Fluor 488
dye, Anti-digoxigenin, Anti-biotin, Anti-BODIPY FL dye,
Anti-Cascade Blue dye, Anti-DNP, anti-DNP-KLH, anti-biotin,
Anti-fluorescein/Oregon Green dye, Anti-lucifer yellow,
Anti-fluorescein/Oregon Green dye and
Anti-tetramethylrhodamine.
[0047] A further category of detectable labels includes diagnostic
and imaging labels (generally referred to as in vivo detectable
labels) such as for example magnetic resonance imaging (MRI):
Gd(DOTA); for nuclear medicine: 201Tl, gamma-emitting radionuclide
99mTc; for positron-emission tomography (PET): positron-emitting
isotopes, (18)F-fluorodeoxyglucose ((18)FDG), (18)F-fluoride,
copper-64, gadodiamide, and radioisotopes of Pb(II) such as 203Pb;
111In.
[0048] The conjugations or modifications described herein employ
routine chemistry, which chemistry does not form a part of the
invention and which chemistry is well known to those skilled in the
art of chemistry. The use of protecting groups and known linkers
such as mono- and hetero-bifunctional linkers are well documented
in the literature and will not be repeated here. As used herein,
"conjugated" means two entities stably bound to one another by any
physiochemical means. In some aspects, it is important that the
nature of the attachment is such that it does not impair
substantially the effectiveness of either entity. Any covalent or
non-covalent linkage known to those of ordinary skill in the art
can be employed for conjugation. In some embodiments, covalent
linkage is preferred. Noncovalent conjugation includes hydrophobic
interactions, ionic interactions, high affinity interactions such
as biotin-avidin and biotin-streptavidin complexation and other
affinity interactions. Such means and methods of attachment are
well known to those of ordinary skill in the art.
[0049] A variety of methods may be used to detect the label,
depending on the nature of the label and other assay components.
For example, the label may be detected while bound to the solid
substrate or subsequent to separation from the solid substrate.
Labels may be directly detected through optical or electron
density, radioactive emissions, nonradiative energy transfers, etc.
or indirectly detected with antibody conjugates,
streptavidin-biotin conjugates, etc. Methods for detecting the
labels are well known in the art.
[0050] The term "secondary label" as used herein refers to moieties
such as biotin and various protein antigens that require the
presence of a second intermediate for production of a detectable
signal. For biotin, the secondary intermediate may include
streptavidin-enzyme conjugates. For antigen labels, secondary
intermediates may include antibody-enzyme conjugates. Some
fluorescent groups act as secondary labels because they transfer
energy to another group in the process of nonradiative fluorescent
resonance energy transfer (FRET), and the second group produces the
detected signal.
[0051] The term "mass-tag" as used herein refers to any moiety that
is capable of being uniquely detected by virtue of its mass using
mass spectrometry (MS) detection techniques. Examples of mass-tags
include electrophore release tags such as
N-[3-[4'-[(p-Methoxytetrafluorobenzy
oxyjphenylJ-S-methylglyceronylJisonipecotic Acid, 4'-[2,
3,5,6-Tetrafluoro-4-(pentafluorophenoxyl)]methyl acetophenone, and
their derivatives. The synthesis and utility of these mass-tags is
described in U.S. Pat. Nos. 4,650,750, 4,709,016, 5,360,8191,
5,516,931, 5,602,273, 5,604,104, 5,610,020, and 5,650,270. Other
examples of mass-tags include, but are not limited to, nucleotides,
dideoxynucleotides, oligonucleotides of varying length and base
composition, oligopeptides, oligosaccharides, and other synthetic
polymers of varying length and monomer composition. A large variety
of organic molecules, both neutral and charged (biomolecules or
synthetic compounds) of an appropriate mass range (100-2000
Daltons) may also be used as mass- tags.
[0052] Primary labels include but are not limited to radioisotopes
(e.g. tritium, 32P, 33P, 35S, 14C, 123I, 124I, 125I, or 131I),
mass-tags including, but not limited to, stable isotopes (e.g.,
13C, 2H, 17O, 18O, 15N, 19F, and 127I), positron emitting isotopes
(e.g., 11C, 18F, 13N, 124I, and 15O), and fluorescent labels are
signal generating reporter groups which can be detected without
further modifications. Detectable moities may be analyzed by
methods including, but not limited to fluorescence, positron
emission tomography, SPECT medical imaging, chemiluminescence,
electron-spin resonance, ultraviolet/visible absorbance
spectroscopy, mass spectrometry, nuclear magnetic resonance,
magnetic resonance, flow cytometry, autoradiography, scintillation
counting, phosphoimaging, and electrochemical methods.
[0053] The term "chemiluminescent group," as used herein, refers to
a group which emits light as a result of a chemical reaction
without the addition of heat. By way of example, luminol
(5-amino-2,3-dihydro-1,4-phthalazinedione) reacts with oxidants
like hydrogen peroxide (H2O2) in the presence of a base and a metal
catalyst to produce an excited state product (3-aminophthalate,
3-APA).
[0054] The term "chromophore," as used herein, refers to a molecule
which absorbs light of visible wavelengths, UV wavelengths or IR
wavelengths.
[0055] The term "dye," as used herein, refers to a soluble,
coloring substance which contains a chromophore.
[0056] The term "electron dense group," as used herein, refers to a
group which scatters electrons when irradiated with an electron
beam. Such groups include, but are not limited to, ammonium
molybdate, bismuth subnitrate, cadmium iodide, carbohydrazide,
ferric chloride hexahydrate, hexamethylene tetramine, indium
trichloride anhydrous, lanthanum nitrate, lead acetate trihydrate,
lead citrate trihydrate, lead nitrate, periodic acid,
phosphomolybdic acid, phosphotungstic acid, potassium ferricyanide,
potassium ferrocyanide, ruthenium red, silver nitrate, silver
proteinate (Ag Assay: 8.0-8.5%) "Strong", silver tetraphenylporphin
(S-TPPS), sodium chloroaurate, sodium tungstate, thallium nitrate,
thiosemicarbazide (TSC), uranyl acetate, uranyl nitrate, and
vanadyl sulfate.
[0057] The term "energy transfer agent," as used herein, refers to
a molecule which either donates or accepts energy from another
molecule. By way of example only, fluorescence resonance energy
transfer (FRET) is a dipole-dipole coupling process by which the
excited-state energy of a fluorescence donor molecule is
non-radiatively transferred to an unexcited acceptor molecule which
then fluorescently emits the donated energy at a longer wavelength.
The term "moiety incorporating a heavy atom," as used herein,
refers to a group which incorporates an ion of atom which is
usually heavier than carbon. In some embodiments, such ions or
atoms include, but are not limited to, silicon, tungsten, gold,
lead, and uranium.
[0058] The term "photoaffinity label," as used herein, refers to a
label with a group, which, upon exposure to light, forms a linkage
with a molecule for which the label has an affinity.
[0059] The term "photocaged moiety," as used herein, refers to a
group which, upon illumination at certain wavelengths, covalently
or non-covalently binds other ions or molecules.
[0060] The term "photoisomerizable moiety," as used herein, refers
to a group wherein upon illumination with light changes from one
isomeric form to another.
[0061] The term "radioactive moiety," as used herein, refers to a
group whose nuclei spontaneously give off nuclear radiation, such
as alpha, beta, or gamma particles; wherein, alpha particles are
helium nuclei, beta particles are electrons, and gamma particles
are high energy photons.
[0062] The term "spin label," as used herein, refers to molecules
which contain an atom or a group of atoms exhibiting an unpaired
electron spin (i.e. a stable paramagnetic group) that in some
embodiments are detected by electron spin resonance spectroscopy
and in other embodiments are attached to another molecule. Such
spin-label molecules include, but are not limited to, nitryl
radicals and nitroxides, and in some embodiments are single
spin-labels or double spin-labels.
[0063] The term "quantum dots," as used herein, refers to colloidal
semiconductor nanocrystals that in some embodiments are detected in
the near-infrared and have extremely high quantum yields (i.e.,
very bright upon modest illumination).
[0064] One of ordinary skill in the art will recognize that a
detectable moiety may be attached to a provided compound via a
suitable substituent. As used herein, the term "suitable
substituent" refers to a moiety that is capable of covalent
attachment to a detectable moiety. Such moieties are well known to
one of ordinary skill in the art and include groups containing,
e.g., a carboxylate moiety, an amino moiety, a thiol moiety, or a
hydroxyl moiety, to name but a few. It will be appreciated that
such moieties may be directly attached to a provided compound or
via a tethering moiety, such as a bivalent saturated or unsaturated
hydrocarbon chain. In some embodiments, detectable moieties are
attached to a provided compound via click chemistry. In some
embodiments, such moieties are attached via a 1,3-cycloaddition of
an azide with an alkyne, optionally in the presence of a copper
catalyst. Methods of using click chemistry are known in the art and
include those described by Rostovtsev et al., Angew. Chem. Int. Ed.
2002, 41, 2596-99 and Sun et al., Bioconjugate Chem., 2006, JJ,
52-57. In some embodiments, a click ready inhibitor moiety is
provided and reacted with a click ready -T-R1 moiety. As used
herein, "click ready" refers to a moiety containing an azide or
alkyne for use in a click chemistry reaction. In some embodiments,
the click ready inhibitor moiety comprises an azide. In certain
embodiments, the click ready -T-R1 moiety comprises a strained
cyclooctyne for use in a copper-free click chemistry reaction (for
example, using methods described in Baskin et al., Proc. Natl.
Acad. Sci. USA 2007, 104, 16793-16797).
[0065] Further aspects of the invention relate to using assays
described herein in screening methods for therapeutics. For
example, therapeutics or potential therapeutics can be added to
samples within the assays described herein and the effect of the
therapeutic or potential therapeutic on lipophilic or amphiphilic
molecules, such as cholesterol, binding, transport or efflux can be
measured.
Nanoparticles
[0066] Articles, compositions, kits, and methods relating to
nanostructures, including those that can sequester molecules such
as cholesterol, are provided. Certain embodiments described herein
include structures having a core-shell type arrangement; for
instance, a nanoparticle core may be surrounded by a shell
including a material, such as a lipid bilayer, that can interact
with cholesterol and/or other lipids. In some embodiments, the
structures, when introduced into a subject, can sequester
cholesterol and/or other lipids and remove them from circulation.
Accordingly, the structures described herein may be used to
diagnose, prevent, treat or manage certain diseases or bodily
conditions, especially those associated with abnormal lipid
levels.
[0067] Certain structures described herein can mimic circulating
lipoproteins such as high density lipoprotein (HDL) and low density
lipoprotein (LDL), commonly referred to as "good" and "bad"
cholesterol, respectively. One function of lipoproteins is to
transport cholesterol and other lipids in the body in the aqueous
blood, since these molecules do not normally dissolve in the blood.
Lipoproteins are also responsible for a number of important
pathologic functions such as atherosclerosis. These lipoproteins,
and other similar circulating particles (e.g., intermediate density
lipoproteins, very low density lipoproteins, etc.), include
nanostructures typically between 5 and 1000 nm. Each lipoprotein is
unique with regard to its surface chemistry, size and composition.
However, they also have in common an outer layer of phospholipids,
an inner core of hydrophobic moieties (e.g., cholesteryl esters and
triglycerides), and a surface protein that identifies individual
lipoprotein species and dictates physiology.
[0068] In some embodiments described herein, a core (e.g., a gold
nanoparticle) can be used as a scaffold to template and direct the
synthesis of structures of well defined size, shape, and surface
chemistry that are amenable to a wide variety of further surface
chemistry and tailorability. For example, a bottom-up,
size-specific, lipoprotein synthesis may be carried out by using a
nanostructure core to support a shell including a lipid bilayer
and/or other suitable components.
[0069] Articles and methods described herein involve the use of
nanostructure scaffolds for controllable synthesis of structures
with a high degree of reproducibility and with the potential for
massive scale-up. The resulting structures may be stable in a
variety of solvents, may have high in vivo circulation times, and
may be relatively inexpensive to fabricate. Additionally, as lipids
can be easily modified with commercially available linker
chemistries, the structures described herein are amenable to
further functionalization with potential pharmacological agents
and/or targeting/recognition agents such as antibodies, small
molecules and proteins. Further advantages are described in more
detail below.
[0070] Nanostructures compatible with aspects of the invention are
further described in, and incorporated by reference from
PCT/US2009/002540, the content of which is incorporated by
reference herein in its entirety.
[0071] In embodiments in which the core is a nanostructure, the
core includes a surface to which one or more components can be
optionally attached. For instance, in some cases, core is a
nanostructure surrounded by shell, which includes an inner surface
and an outer surface. The shell may be formed, at least in part, of
one or more components, such as a plurality of lipids, which may
optionally associate with one another and/or with surface of the
core. For example, components may be associated with the core by
being covalently attached to the core, physisorbed, chemisorbed, or
attached to the core through ionic interactions, hydrophobic and/or
hydrophilic interactions, electrostatic interactions, van der Waals
interactions, or combinations thereof. In one particular
embodiment, the core includes a gold nanostructure and the shell is
attached to the core through a gold-thiol bond.
[0072] Optionally, components can be crosslinked to one another.
Crosslinking of components of a shell can, for example, allow the
control of transport of species into the shell, or between an area
exterior to the shell and an area interior of the shell. For
example, relatively high amounts of crosslinking may allow certain
small, but not large, molecules to pass into or through the shell,
whereas relatively low or no crosslinking can allow larger
molecules to pass into or through the shell. Additionally, the
components forming the shell may be in the form of a monolayer or a
multilayer, which can also facilitate or impede the transport or
sequestering of molecules. In one exemplary embodiment, shell
includes a lipid bilayer that is arranged to sequester cholesterol,
as described in more detail below.
[0073] It should be understood that a shell which surrounds a core
need not completely surround the core, although such embodiments
may be possible. For example, the shell may surround at least 50%,
at least 60%, at least 70%, at least 80%, at least 90%, or at least
99% of the surface area of a core. In some cases, the shell
substantially surrounds a core. In other cases, the shell
completely surrounds a core. The components of the shell may be
distributed evenly across a surface of the core in some cases, and
unevenly in other cases. For example, the shell may include
portions (e.g., holes) that do not include any material in some
cases. If desired, the shell may be designed to allow penetration
and/or transport of certain molecules and components into or out of
the shell, but may prevent penetration and/or transport of other
molecules and components into or out of the shell. The ability of
certain molecules to penetrate and/or be transported into and/or
across a shell may depend on, for example, the packing density of
the components forming the shell and the chemical and physical
properties of the components forming the shell. As described
herein, the shell may include one layer of material, or multilayers
of materials in some embodiments.
[0074] Structure may also include one or more components such as
proteins, nucleic acids, and bioactive agents which may optionally
impart specificity to the structure. One or more components may be
associated with the core, the shell, or both; e.g., they may be
associated with surface of the core, inner surface of the shell,
outer surface of the shell, and/or embedded in the shell. For
example, one or more components may be associated with the core,
the shell, or both through covalent bonds, physisorption,
chemisorption, or attached through ionic interactions, hydrophobic
and/or hydrophilic interactions, electrostatic interactions, van
der Waals interactions, or combinations thereof. In one particular
embodiment, shell is in the form of a lipoprotein assembly or
structure which includes both proteins and lipids that are
covalently or non-covalently bound to one another. For example, the
shell may be in the form of an apolipoprotein assembly that serves
as an enzyme co-factor, receptor ligand, and/or lipid transfer
carrier that regulates the uptake of lipids. As described herein,
the components of structure may be chosen such that the surface of
the structure mimics the general surface composition of HDL, LDL,
or other structures.
[0075] It should be understood that components and configurations
other than those described herein may be suitable for certain
structures and compositions, and that not all of the components
described are necessarily present in some embodiments.
[0076] In some cases, core is hollow and therefore does not include
a nanostructure core. Thus, in some such and other embodiments,
structure includes a shell that can optionally allow components
(e.g., bioactive agents, cholesterol) to pass to and from core and
an environment outside of the shell. In contrast to certain
existing hollow structures (e.g., liposomes) which typically have a
largest cross-sectional dimension of greater than about 100 nm due
to the steric hindrance of the components forming the shell,
structures having a hollow core (e.g., a partially or wholly hollow
core) may be very small, e.g., having a largest cross-sectional
dimension of less than about 100 nm, or even less than about 50 nm.
For example, liposomes that include a lipid bilayer comprising
phospholipids are difficult to fabricate having a size of less than
100 nm since the phospholipids become limited sterically, thus
making it difficult or impossible to form bilayered hollow
structures with small radii of curvature. Using the methods
described herein, however, such and other structures having small
radii of curvature can be formed, as provided in more detail
below.
[0077] In one set of embodiments, a structure, whether including a
nanostructure core or a hollow core, is constructed and arranged to
sequester, transport, or exchange certain molecules to and/or from
a subject or a biological sample. For instance, a structure, when
introduced into a subject, may interact with one or more components
in the subject such as cells, tissues, organs, particles, fluids
(e.g., blood), and portions thereof. The interaction may take
place, at least in part, through the shell of a structure, and may
involve, for example, the exchange of materials (e.g., proteins,
peptides, polypeptides, nucleic acids, nutrients) from the one or
more components of the subject to a structure, and/or from a
structure to the one or more components of the subject. In some
such embodiments, the shell of a structure can be designed to
include components with properties that allow favorable interaction
(e.g., binding, adsorption, transport) with the one or more
materials from the subject. For example, the shell may include
components having a certain hydrophobicity, hydrophilicity, surface
charge, functional group, specificity for binding, and/or density
to facilitate particular interactions, as described in more detail
below. In certain embodiments, one or more materials from a subject
are sequestered by a structure, and a structure facilitates
excretion, breakdown, and/or transport of the material. The
excretion, breakdown, and/or transport of the material can lead to
certain beneficial and/or therapeutic effects. As such, the
structures described herein can be used for the diagnosis,
prevention, treatment or management of certain diseases or bodily
conditions.
[0078] In one particular set of embodiments, a structure, whether
including a nanostructure core or a hollow core, is constructed and
arranged to sequester cholesterol (and/or other lipids). Without
wishing to be bound by theory, it is hypothesized that a structure
sequesters cholesterol through hydrophobic interactions with a
hydrophobic layer (e.g., a lipid bilayer) of the structure. For
example, in some cases, cholesterol can bind to a surface of the
structure (e.g., to the outer surface of the shell) through
hydrophobic interactions. In other cases, the cholesterol can be
transported from an outer surface of the shell to an inner surface
of the shell and/or to the core of the structure. The cholesterol
can also be imbedded in the shell, e.g., between two layers of the
shell. Optionally, a structure may include one or more
apolipoproteins (e.g., apoliprotein-A1), proteins, or peptides,
which can facilitate the sequestering of cholesterol. A structure
may also sequester cholesterol by removing cholesterol and
phospholipids from a cell, or from other circulating lipoprotein
species. cholesterol sequestered by a structure may be esterified
enzymatically (e.g., by lecithin:acyl CoA transferase (LCAT)) to
form a cholesteryl ester that may migrate towards the center of the
structure. In the case of hollow core embodiments, the cholesteryl
ester may accumulate in the hollow core.
[0079] Additionally, without wishing to be bound by theory, it is
believed that the structures described herein can sequester
cholesterol from high concentrations of cholesterol (e.g., plaques)
and transfer it to the liver directly or indirectly. For example,
cholesterol may be sequestered from areas of high concentrations of
cholesterol (e.g., plaques) by direct efflux of cholesterol from
the plaque, or any components of the plaque, into or onto the
structures described herein. In some such embodiments, the
cholesterol that is sequestered by the structures is transported
directly to the liver by the structures. In other embodiments,
other circulating lipoprotein species (e.g., LDL) may participate
in cholesterol exchange. For example, in some cases, free
cholesterol or esterified cholesterol is transferred from other
lipoproteins to the structures described herein. In other cases,
once free cholesterol or esterified cholesterol is sequestered by
the structures described herein, the cholesterol can be transferred
from the structures to the other lipoprotein species, which may
ultimately end up in the liver. Thus, in such embodiments, the
structures described herein can augment reverse cholesterol
transport indirectly. Furthermore, in the case where free
cholesterol or esterified cholesterol is sequestered from the
structures described herein to other lipoprotein species, the
structures may further sequester cholesterol from, for example,
areas of high cholesterol content, plaques, circulating
lipoproteins, or other physiologic sites of high cholesterol
concentration. It should be understood, however, that the
structures described herein may remove cholesterol and/or other
molecules by other routes, such as through urine, and the invention
is not limited in this respect.
[0080] Accordingly, a structures may be used in the field of
cardiovascular disease for studying atherosclerosis and cholesterol
transport, and, generally, to diagnose, prevent, treat or manage
diseases or bodily conditions associated with abnormal lipid
levels, as described in more detail below.
[0081] The amount of a molecule (e.g., cholesterol or other lipids)
sequestered by a structure and/or a composition described herein
may depend on, for example, the size of the structure, the biology
and surface chemistry of the particle, as well as the method of
administration. As such, a single structure described herein, which
may be incorporated into a pharmaceutical composition or other
formulation, may be able to sequester any suitable number of a
particular type of molecule (e.g., lipids such as cholesterol;
steroids such as estrogen, progesterone, and testosterone; bile
salts, etc.) during use, e.g., at least 2, at least 5, at least 10,
at least 20, at least 30, at least 50, at least 100, at least 200,
at least 500, at least 1,000, at least 2,000, at least 5,000, or at
least 10,000 molecules, which may depend on the size (e.g., surface
area and/or volume) of the structure, the particular application,
and the method of administration. In some cases, such numbers of
molecules can be bound to the structure at one particular
instance.
[0082] In some cases, a single structure has a binding constant for
cholesterol, K.sub.d, of, for example, less than or equal to about
100 .mu.M, less than or equal to about 10 .mu.M, less than or equal
to about 1 .mu.M, less than or equal to about 0.1 .mu.M, less than
or equal to about 10 nM, less than or equal to about 7 nM, less
than or equal to about 5 nM, less than or equal to about 2 nM, less
than or equal to about 1 nM, less than or equal to about 0.1 nM,
less than or equal to about 10 pM, less than or equal to about 1
pM, less than or equal to about 0.1 pM, less than or equal to about
10 fM, or less than or equal to about 1 fM. Methods for determining
the amount of cholesterol sequestered and binding constants are
provided in more detail below.
[0083] In certain embodiments, the molecules that are sequestered
by the structures described herein cause the structure to grow in
size (e.g., cross-sectional area, surface area and/or volume),
e.g., depending on the number of molecules sequestered. The
molecules may associate with a surface of a structure, be imbedded
in a shell of a structure, be transported to a core of the
structure, or combinations thereof, as described herein. As such,
the size of a structure (e.g., cross-sectional area, surface area
and/or volume) can increase by at least 5%, at least 10%, at least
20%, at least 30%, at least 50%, at least 70%, or at least 100%,
from a time prior to sequestration compared to a time after/during
sequestration in some embodiments.
[0084] It should be understood, however, that while many of the
embodiments herein are described in the context of sequestering
cholesterol or other lipids, the invention is not limited as such
and the structures, compositions, kits, and methods described
herein may be used to sequester other molecules and/or to prevent,
treat, or manage other diseases or bodily conditions.
[0085] A core, whether being a nanostructure core or a hollow core,
may have any suitable shape and/or size. For instance, the core may
be substantially spherical, non-spherical, oval, rod-shaped,
pyramidal, cube-like, disk-shaped, wire-like, or irregularly
shaped. The core (e.g., a nanostructure core or a hollow core) may
have a largest cross-sectional dimension (or, sometimes, a smallest
cross-section dimension) of, for example, less than or equal to
about 500 nm, less than or equal to about 250 nm, less than or
equal to about 100 nm, less than or equal to about 75 nm, less than
or equal to about 50 nm, less than or equal to about 40 nm, less
than or equal to about 35 nm, less than or equal to about 30 nm,
less than or equal to about 25 nm, less than or equal to about 20
nm, less than or equal to about 15 nm, or less than or equal to
about 5 nm. In some cases, the core has an aspect ratio of greater
than about 1:1, greater than 3:1, or greater than 5:1. As used
herein, "aspect ratio" refers to the ratio of a length to a width,
where length and width measured perpendicular to one another, and
the length refers to the longest linearly measured dimension.
[0086] In embodiments in which a core includes a nanostructure
core, the nanostructure core may be formed from any suitable
material. For instance, in one embodiment, a nanostructure core
comprises an inorganic material. The inorganic material may
include, for example, a metal (e.g., Ag, Au, Pt, Fe, Cr, Co, Ni,
Cu, Zn, and other transition metals), a semiconductor (e.g.,
silicon, silicon compounds and alloys, cadmium selenide, cadmium
sulfide, indium arsenide, and indium phosphide), or an insulator
(e.g., ceramics such as silicon oxide). The inorganic material may
be present in the core in any suitable amount, e.g., at least 1 wt
%, 5 wt %, 10 wt %, 25 wt %, 50 wt %, 75 wt %, 90 wt %, or 99 wt %.
In one embodiment, the core is formed of 100 wt % inorganic
material. The nanostructure core may, in some cases, be in the form
of a quantum dot, a carbon nanotube, a carbon nanowire, or a carbon
nanorod. In some cases, the nanostructure core comprises, or is
formed of, a material that is not of biological origin. In some
embodiments, a nanostructure includes one or more organic materials
such as a synthetic polymer and/or a natural polymer. Examples of
synthetic polymers include non-degradable polymers such as
polymethacrylate and degradable polymers such as polylactic acid,
polyglycolic acid and copolymers thereof. Examples of natural
polymers include hyaluronic acid, chitosan, and collagen.
[0087] A structure, which may include a shell surrounding a core,
may also have any suitable shape and/or size. For instance, a
structure may have a shape that is substantially spherical, oval,
rod-shaped, pyramidal, cubed-like, disk-shaped, or irregularly
shaped. The largest cross-sectional dimension (or, sometimes, a
smallest cross-section dimension) of a structure may be, for
example, less than or equal to about 500 nm, less than or equal to
about 250 nm, less than or equal to about 100 nm, less than or
equal to about 75 nm, less than or equal to about 50 nm, less than
or equal to about 40 nm, less than or equal to about 35 nm, less
than or equal to about 30 nm, less than or equal to about 25 nm,
less than or equal to about 20 nm, less than or equal to about 15
nm, or less than or equal to about 5 nm. The structure may also
have an aspect ratio substantially similar to the aspect ratio of
the core.
[0088] Furthermore, a shell of a structure can have any suitable
thickness. For example, the thickness of a shell may be at least 10
Angstroms, at least 0.1 nm, at least 1 nm, at least 2 nm, at least
5 nm, at least 7 nm, at least 10 nm, at least 15 nm, at least 20
nm, at least 30 nm, at least 50 nm, at least 100 nm, or at least
200 nm (e.g., from the inner surface to the outer surface of the
shell). In some cases, the thickness of a shell is less than 200
nm, less than 100 nm, less than 50 nm, less than 30 nm, less than
20 nm, less than 15 nm, less than 10 nm, less than 7 nm, less than
5 nm, less than 3 nm, less than 2 nm, or less than 1 nm (e.g., from
the inner surface to the outer surface of the shell). Such
thicknesses may be determined prior to or after sequestration of
molecules as described herein.
[0089] Those of ordinary skill in the art are familiar with
techniques to determine sizes of structures and particles. Examples
of suitable techniques include dynamic light scattering (DLS)
(e.g., using a Malvern Zetasizer instrument), transmission electron
microscopy, scanning electron microscopy, electroresistance
counting and laser diffraction. Other suitable techniques are known
to those or ordinary skill in the art. Although many methods for
determining sizes of nanostructures are known, the sizes described
herein (e.g., largest or smallest cross-sectional dimensions,
thicknesses) refer to ones measured by dynamic light
scattering.
[0090] The shell of a structure described herein may comprise any
suitable material, such as a hydrophobic material, a hydrophilic
material, and/or an amphiphilic material. Although the shell may
include one or more inorganic materials such as those listed above
for the nanostructure core, in many embodiments the shell includes
an organic material such as a lipid or certain polymers. The
components of the shell may be chosen, in some embodiments, to
facilitate the sequestering of cholesterol or other molecules. For
instance, cholesterol (or other sequestered molecules) may bind or
otherwise associate with a surface of the shell, or the shell may
include components that allow the cholesterol to be internalized by
the structure. Cholesterol (or other sequestered molecules) may
also be embedded in a shell, within a layer or between two layers
forming the shell. The components of a shell may be charged, e.g.,
to impart a charge on the surface of the structure, or
uncharged.
[0091] In one set of embodiments, a structure described herein or a
portion thereof, such as a shell of a structure, includes one or
more natural or synthetic lipids or lipid analogs (i.e., lipophilic
molecules). One or more lipids and/or lipid analogues may form a
single layer or a multi-layer (e.g., a bilayer) of a structure. In
some instances where multi-layers are formed, the natural or
synthetic lipids or lipid analogs interdigitate (e.g., between
different layers). Non-limiting examples of natural or synthetic
lipids or lipid analogs include fatty acyls, glycerolipids,
glycerophospholipids, sphingolipids, saccharolipids and polyketides
(derived from condensation of ketoacyl subunits), and sterol lipids
and prenol lipids (derived from condensation of isoprene
subunits).
[0092] In one particular set of embodiments, a structure described
herein includes one or more phospholipids. The one or more
phospholipids may include, for example, phosphatidylcholine,
phosphatidylglycerol, lecithin, .beta.,
.gamma.-dipalmitoyl-.alpha.-lecithin, sphingomyelin,
phosphatidylserine, phosphatidic acid,
N-(2,3-di(9-(Z)-octadecenyloxy))-prop-1-yl-N,N,N-trimethylammonium
chloride, phosphatidylethanolamine, lysolecithin,
lysophosphatidylethanolamine, phosphatidylinositol, cephalin,
cardiolipin, cerebrosides, dicetylphosphate,
dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine,
dipalmitoylphosphatidylglycerol, dioleoylphosphatidylglycerol,
palmitoyl-oleoyl-phosphatidylcholine,
di-stearoyl-phosphatidylcholine,
stearoyl-palmitoyl-phosphatidylcholine,
di-palmitoyl-phosphatidylethanolamine,
di-stearoyl-phosphatidylethanolamine,
di-myrstoyl-phosphatidylserine, di-oleyl-phosphatidylcholine,
1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, and combinations
thereof. In some cases, a shell (e.g., a bilayer) of a structure
includes 50-200 natural or synthetic lipids or lipid analogs (e.g.,
phospholipids). For example, the shell may include less than about
500, less than about 400, less than about 300, less than about 200,
or less than about 100 natural or synthetic lipids or lipid analogs
(e.g., phospholipids), e.g., depending on the size of the
structure.
[0093] Non-phosphorus containing lipids may also be used such as
stearylamine, docecylamine, acetyl palmitate, and fatty acid
amides. In other embodiments, other lipids such as fats, oils,
waxes, cholesterol, sterols, fat-soluble vitamins (e.g., vitamins
A, D, E and K), glycerides (e.g., monoglycerides, diglycerides,
triglycerides) can be used to form portions of a structure
described herein.
[0094] A portion of a structure described herein such as a shell or
a surface of a nanostructure may optionally include one or more
alkyl groups, e.g., an alkane-, alkene-, or alkyne-containing
species, that optionally imparts hydrophobicity to the structure.
An "alkyl" group refers to a saturated aliphatic group, including a
straight-chain alkyl group, branched-chain alkyl group, cycloalkyl
(alicyclic) group, alkyl substituted cycloalkyl group, and
cycloalkyl substituted alkyl group. The alkyl group may have
various carbon numbers, e.g., between C.sub.2 and C.sub.40, and in
some embodiments may be greater than C.sub.5, C.sub.10, C.sub.15,
C.sub.20, C.sub.25, C.sub.30, or C.sub.35. In some embodiments, a
straight chain or branched chain alkyl may have 30 or fewer carbon
atoms in its backbone, and, in some cases, 20 or fewer. In some
embodiments, a straight chain or branched chain alkyl may have 12
or fewer carbon atoms in its backbone (e.g., C.sub.1-C.sub.12 for
straight chain, C.sub.3-C.sub.12 for branched chain), 6 or fewer,
or 4 or fewer. Likewise, cycloalkyls may have from 3-10 carbon
atoms in their ring structure, or 5, 6 or 7 carbons in the ring
structure. Examples of alkyl groups include, but are not limited
to, methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl,
tert-butyl, cyclobutyl, hexyl, cyclochexyl, and the like.
[0095] The alkyl group may include any suitable end group, e.g., a
thiol group, an amino group (e.g., an unsubstituted or substituted
amine), an amide group, an imine group, a carboxyl group, or a
sulfate group, which may, for example, allow attachment of a ligand
to a nanostructure core directly or via a linker. For example,
where inert metals are used to form a nanostructure core, the alkyl
species may include a thiol group to form a metal-thiol bond. In
some instances, the alkyl species includes at least a second end
group. For example, the species may be bound to a hydrophilic
moiety such as polyethylene glycol. In other embodiments, the
second end group may be a reactive group that can covalently attach
to another functional group. In some instances, the second end
group can participate in a ligand/receptor interaction (e.g.,
biotin/streptavidin).
[0096] In some embodiments, the shell includes a polymer. For
example, an amphiphilic polymer may be used. The polymer may be a
diblock copolymer, a triblock copolymer, etc., e.g., where one
block is a hydrophobic polymer and another block is a hydrophilic
polymer. For example, the polymer may be a copolymer of an
.alpha.-hydroxy acid (e.g., lactic acid) and polyethylene glycol.
In some cases, a shell includes a hydrophobic polymer, such as
polymers that may include certain acrylics, amides and imides,
carbonates, dienes, esters, ethers, fluorocarbons, olefins,
sytrenes, vinyl acetals, vinyl and vinylidene chlorides, vinyl
esters, vinyl ethers and ketones, and vinylpyridine and
vinylpyrrolidones polymers. In other cases, a shell includes a
hydrophilic polymer, such as polymers including certain acrylics,
amines, ethers, styrenes, vinyl acids, and vinyl alcohols. The
polymer may be charged or uncharged. As noted herein, the
particular components of the shell can be chosen so as to impart
certain functionality to the structures.
[0097] Where a shell includes an amphiphilic material, the material
can be arranged in any suitable manner with respect to the
nanostructure core and/or with each other. For instance, the
amphiphilic material may include a hydrophilic group that points
towards the core and a hydrophobic group that extends away from the
core, or, the amphiphilic material may include a hydrophobic group
that points towards the core and a hydrophilic group that extends
away from the core. Bilayers of each configuration can also be
formed.
[0098] The structures described herein may also include one or more
proteins, polypeptides and/or peptides (e.g., synthetic peptides,
amphiphilic peptides). In one set of embodiments, the structures
include proteins, polypeptides and/or peptides that can increase
the rate of cholesterol transfer or the cholesterol-carrying
capacity of the structures. The one or more proteins or peptides
may be associated with the core (e.g., a surface of the core or
embedded in the core), the shell (e.g., an inner and/or outer
surface of the shell, and/or embedded in the shell), or both.
Associations may include covalent or non-covalent interactions
(e.g., hydrophobic and/or hydrophilic interactions, electrostatic
interactions, van der Waals interactions).
[0099] An example of a suitable protein that may associate with a
structure described herein is an apolipoprotein, such as
apolipoprotein A (e.g., apo A-I, apo A-II, apo A-IV, and apo A-V),
apolipoprotein B (e.g., apo B48 and apo B100), apolipoprotein C
(e.g., apo C-I, apo C-II, apo C-III, and apo C-IV), and
apolipoproteins D, E, and H. Specifically, apo A.sub.1, apo
A.sub.2, and apo E promote transfer of cholesterol and cholesteryl
esters to the liver for metabolism and may be useful to include in
structures described herein. Additionally or alternatively, a
structure described herein may include one or more peptide
analogues of an apolipoprotein, such as one described above. A
structure may include any suitable number of, e.g., at least 1, 2,
3, 4, 5, 6, or 10, apolipoproteins or analogues thereof. In certain
embodiments, a structure includes 1-6 apolipoproteins, similar to a
naturally occurring HDL particle. Of course, other proteins (e.g.,
non-apolipoproteins) can also be included in structures described
herein.
[0100] Optionally, one or more enzymes may also be associated with
a structure described herein. For example, lecithin-cholesterol
acyltransferase is an enzyme which converts free cholesterol into
cholesteryl ester (a more hydrophobic form of cholesterol). In
naturally-occurring lipoproteins (e.g., HDL and LDL), cholesteryl
ester is sequestered into the core of the lipoprotein, and causes
the lipoprotein to change from a disk shape to a spherical shape.
Thus, structures described herein may include lecithin-cholesterol
acyltransferase to mimic HDL and LDL structures. Other enzymes such
as cholesteryl ester transfer protein (CETP) which transfers
esterified cholesterol from HDL to LDL species may also be
included.
[0101] In some cases, one or more bioactive agents are associated
with a structure or a composition described herein. The one or more
bioactive agents may optionally be released from the structure or
composition (e.g., long-term or short-term release). Bioactive
agents include molecules that affect a biological system and
include, for example proteins, nucleic acids, therapeutic agents,
vitamins and their derivatives, viral fractions,
lipopolysaccharides, bacterial fractions and hormones. Other agents
of interest may include chemotherapeutic agents.
[0102] In some embodiments, one or more nucleic acids is associated
with a structure described herein. A nucleic acids includes any
double strand or single strand deoxyribonucleic acid (DNA) or
ribonucleic acid (RNA) of variable length. Nucleic acids include
sense and anti-sense strands. Nucleic acid analogs such as
phosphorothioates, phosphoramidates, phosphonates analogs are also
considered nucleic acids and may be used. Nucleic acids also
include chromosomes and chromosomal fragments.
[0103] One or more sugar residues can optionally be associated with
structures described herein.
[0104] It should be understood that the components described
herein, such as the lipids, phospholipids, alkyl groups, polymers,
proteins, polypeptides, peptides, enzymes, bioactive agents,
nucleic acids, and species for targeting described above, may be
associated with a structure in any suitable manner and with any
suitable portion of the structure, e.g., the core, the shell, or
both. For example, one or more such components may be associated
with a surface of a core, an interior of a core, an inner surface
of a shell, an outer surface of a shell, and/or embedded in a
shell. Furthermore, such components can be used, in some
embodiments, to facilitate the sequestration, exchange and/or
transport of materials (e.g., proteins, peptides, polypeptides,
nucleic acids, nutrients) from one or more components of a subject
(e.g., cells, tissues, organs, particles, fluids (e.g., blood), and
portions thereof) to a structure described herein, and/or from the
structure to the one or more components of the subject. In some
cases, the components have chemical and/or physical properties that
allow favorable interaction (e.g., binding, adsorption, transport)
with the one or more materials from the subject.
[0105] Additionally, the components described herein, such as the
lipids, phospholipids, alkyl groups, polymers, proteins,
polypeptides, peptides, enzymes, bioactive agents, and nucleic
acids, may be associated with a structure described herein prior to
administration to a biological sample and/or after administration
to a biological sample. For example, in some cases a structure
described herein includes a core and a shell which is administered,
and the structure has a greater therapeutic effect after
sequestering one or more components (e.g., an apolipoprotein) from
a biological sample. That is, the structure may use natural
components from the biological sample to increase efficacy of the
structure after it has been administered.
[0106] In one aspect, methods of making structures described herein
are provided. In some embodiments, methods include providing a
fluid comprising a plurality of nanostructures (e.g., nanostructure
cores) and a first solvent, as well as a fluid comprising a
plurality of components and a second solvent. First solvent may be
chosen such that it stabilizes nanostructures, preventing the
nanostructures from precipitating out of solution. Second solvent
may be chosen so as to solubilize components. The first and second
solvents may be miscible in some embodiments, and immiscible in
other embodiments. In certain embodiments in which solvents and are
miscible with one another, the solvents may also be miscible with
water. Such and other solvents may be useful in a single-phase
synthesis. Solvents that are miscible or slightly miscible with
water are known to those or ordinary skill in the art and include,
for example, alcohols (e.g., ethanol, propanol), THF, DMF and DMSO.
Organic solvents that are immiscible with water can also be used
(e.g., in two-phase synthesis).
[0107] When components are combined with nanostructures, a shell
comprising components is formed on the surface. The shell can
include a monolayer of components, however, in other embodiments,
multi-layers can be formed (e.g., at least two or at least three
layers). If additional components are desired, the components can
be combined and the components may associate with at least a
portion of shell. For example, a second component present in a
third solvent may be combined with nanostructure to form a
structure including a shell in the form of a bilayer. The bilayer
may form due to favorable interaction between components, which may
be the same or different. In certain embodiments, components
interdigitate.
[0108] Optionally, all or a portion of nanostructure may be removed
from an assembled structure to form a partially or wholly hollow
core. Nanostructure can be removed from the structure by a variety
of methods, which may depend on the particular material used to
form nanostructure. For instance, where nanostructure is a metal
(e.g., gold) nanoparticle, solvents that are known to dissolve
certain metals, such as iodine, potassium cyanide, and strong acids
(e.g., nitric acid), can be used to remove the nanostructure core.
Accordingly, in some cases where the core is formed of a metal
(e.g., Au(0)), removal of the metal may include oxidizing the metal
to form a metal salt, e.g., Au(0) to Au.sup.+ and/or Au.sup.3+.
Electrochemical and redox methods can also be used to remove all or
portions of a core. In some cases, a portion, but not all of the
nanostructure core is removed, e.g., such that the nanostructure
core is now more porous than before the removal step. In other
cases, the core is released from the shell without removing a
portion of the core. For example, a shell that is bonded to a metal
core via sulfur-metal bonds can be released from the core by using
small molecules such as dithiothreitol (DTT), which can displace
the bonds. A suitable solvent or a chemical may be chosen such that
it can remove at least portions of a core material, and/or release
the shell from the core, without negatively affecting the shape
and/or configuration of the shell, and/or degrade (e.g., denature)
the components of the shell.
[0109] In certain embodiments, components are cross linked with one
another prior to removing all or a portion of the nanostructure
core. For example, components may be thiolated ligands which cross
link by forming disulfide bonds. Any suitable method for cross
linking can be used, such as photo cross linking, chemical cross
linking, and oxidation-reduction methods, as known to those of
ordinary skill in the art. The cross linking step may help to
stabilize shell in the same or a similar configuration as that
achieved when associated with nanostructure. In certain
embodiments, cross linking of components is performed at the same
time as the removal of nanostructure to form a partially or wholly
hollow structure.
[0110] A similar approach for removing all or a portion of
nanostructure can be used to form structure, which includes a shell
comprising a bilayer of components surrounding a hollow core.
[0111] In some cases, instead of forming multiple layers of
components on a nanostructure surface in separate steps,
multi-layers can be formed in a single step. For instance,
components may be combined in a single phase of a liquid, e.g., a
liquid that solubilizes and/or stabilizes the components and the
nanostructures. Such a liquid may, in some cases, comprise water,
or a solvent that is miscible with water. In some such embodiments,
at least a first layer including components and a second layer
including components are formed by self-assembly. The first and
second layers in such a process may, in some instances, be formed
substantially simultaneously. Additional layers can also be formed
by such a process. Each of the layers can include a single
component, or mixtures of components. To facilitate formation of
the layers, a portion of the liquid may be removed from the
mixture, e.g., by applying heat to evaporate a solvent having a low
boiling point.
[0112] The ratio of components and nanostructures can be tailored
depending on, for example, the type of components and
nanostructures, the solvent(s) used, and the method of fabrication
of the structures. For instance, to obtain solubility in aqueous
solution, a suitable ratio can be chosen such that there is an
ample amount of a component on the surface of the nanostructure so
as to maintain water solubility. Thus, in certain instances, if the
concentration of a component is too low, the structures will not be
stable. Furthermore, if the ratio is too high with certain
components, certain undesirable structures may be formed instead of
stable monodisperse structures. Those of ordinary skill in the art
can determine suitable ratios by simple experimentation in
combination with the description provided herein.
[0113] Furthermore, additional components such as proteins, nucleic
acids, polymers, bioactive agents (e.g., cholesterol lowering
agents) or other structures can be associated with the structures
at any step. For example, in some embodiments additional structures
are added at the same time as addition of components, prior to the
addition of components, or after the addition of components.
[0114] Advantageously, using the methods described herein,
liposome-like structures having a hollow core (or at least a
partially hollow core) can be formed in a size range that is unique
to certain existing liposomes. For example, for many existing
liposomes formed from a phospholipid bilayer and having a hollow
core, the liposomes are large enough (e.g., typically greater than
about 100 nm in diameter) such that the phospholipid bilayer is
capable of being formed. As one attempts to make liposomes of
smaller diameter, the packing of phospholipid moieties becomes
limited sterically thus making it difficult or impossible to form
bilayered liposomal structures with small radii of curvature (e.g.,
smaller than about 100 nm in diameter). Methods described herein,
however, can be used to form structures of smaller diameter (e.g.,
structures having a largest cross-sectional dimension of less than
about 100 nm, or less than or equal to about 50 nm), since the use
of a nanostructure core as a template allows the arrangement of
components in a shell that is dictated, at least in part, by the
size and shape of the nanostructure core. Such methods can be used
to make biologically relevant structures having a surface chemistry
that mimics certain molecules such as HDL and LDL.
[0115] Additionally, because structures described herein can be
formed by the use of nanostructures that serve as a template, and
because certain nanostructures can be provided (e.g., made or
purchased) having relatively high uniformity in size, shape, and
mass, the structures described herein may also have relatively high
uniformity in size, shape, and mass. That is, a mixture of
relatively uniform structures can be formed, where the plurality of
structures have a distribution of cross-sectional dimensions such
that no more than about 20%, 15%, 10%, or 5% of the structures have
a cross-sectional dimension greater than about 20%, 15%, 10%, or 5%
of the average cross-sectional dimension. Structures having
relatively high uniformity are useful in certain compositions and
methods described herein.
[0116] Furthermore, the structures that are formed using methods
described herein may disperse in a liquid, instead of forming
aggregates. Dispersions of structures described herein are useful
in certain compositions and methods described herein.
[0117] Those of ordinary skill in the art can choose appropriate
components, such as those cited in, and incorporated by reference,
from PCT/US2009/002540, nanostructure cores, and solvents useful
for the formation of structures described herein by, for example,
knowing the particular components and nanostructure cores that
would lead to favorable structures, the physical properties of the
components, nanostructures and solvents, and/or by a simple
screening test. One simple screening test may include adding
components (and/or nanostructures) to a solvent and determining
whether the component (or nanostructure) is soluble or stabilized
in the solvent and/or reacts with or is negatively affected by the
solvent. Other simple tests can be conducted by those of ordinary
skill in the art.
[0118] In one set of embodiments, the structures, compositions and
methods described herein are used to diagnose, prevent, treat or
manage diseases or bodily conditions associated with abnormal lipid
levels. For instance, high density lipoprotein is a dynamic serum
nanostructure protective against the development of atherosclerosis
and resultant illnesses such as heart disease and stroke.
Furthermore, in certain embodiments, diagnosis, prevention,
treatment or management of diseases or bodily conditions associated
with abnormal lipid levels may involve using the structures,
compositions and methods described herein.
[0119] Other diseases or bodily conditions associated with abnormal
lipid levels which could benefit from methods and/or compositions
described herein include, for example, atherosclerosis,
phlebosclerosis or any venous condition in which deposits of
plaques containing cholesterol or other material are formed within
the intima or inner media of veins, acute coronary syndromes,
angina including, stable angina, unstable angina, inflammation,
sepsis, vascular inflammation, dermal inflammation, congestive
heart failure, coronary heart disease (CHD), ventricular
arrythmias, peripheral vascular disease, myocardial infarction,
onset of fatal myocardial infarction, non-fatal myocardial
infarction, ischemia, cardiovascular ischemia, transient ischemic
attacks, ischemia unrelated to cardiovascular disease,
ischemia-reperfusion injury, decreased need for revascularization,
coagulation disorders, thrombocytopenia, deep vein thrombosis,
pancreatitis, non-alcoholic steatohepatitis, diabetic neuropathy,
retinopathy, painful diabetic neuropathy, claudication, psoriasis,
critical limb ischemia, impotence, dyslipidemia, hyperlipidemia,
hyperlipoproteinemia, hypoalphalipoproteinemia,
hypertriglyceridemia, any stenotic condition leading to ischemic
pathology, obesity, diabetes including both Type I and Type II,
ichtyosis, stroke, vulnerable plaques, lower-limb ulceration,
severe coronary ischemia, lymphomas, cataracts, endothelial
dysfunction, xanthomas, end organ dysfunction, vascular disease,
vascular disease that results from smoking and diabetes, carotid
and coronary artery disease, regress and shrink established
plaques, unstable plaques, vessel intima that is weak, unstable
vessel intima, endothelial injury, endothelial damage as a result
of surgical procedures, morbidity associated with vascular disease,
ulcerations in the arterial lumen, restenosis as a result of
balloon angioplasty, protein storage diseases (e.g., Alzheimer's
disease, prion disease), diseases of hemostasis (e.g., thrombosis,
thrombophilia, disseminated intravascular coagulation,
thrombocytopenia, heparin induced thrombocytopenia, thrombotic
thrombocytopenic purpura,), rheumatic diseases (e.g., multiple
sclerosis, systemic lupus erythematosis, sjogren's syndrome,
polymyositis/dermatomyositis, scleroderma), neuroligical diseases
(e.g., Parkinson's disease, Alzheimer's disease), and
subindications thereof.
[0120] Further non-limiting examples of conditions associated with
cholesterol levels include: Abetalipoproteinemia, Familial
Dysbetalipoproteinemia, Familial Lecithin cholesterol
Acyltransferase Deficiency, Familial lipoprotein Lipase Deficiency,
Hyperlipoproteinemias, Hypolipidemia, Tangier Disease, Alzheimers,
coronary sclerosis, high blood pressure, macular degeneration,
mixed dyslipidemia, primary hypercholesterolemia
[0121] In some embodiments, the disease or bodily condition is a
metabolic and/or degenerative disease. Several non-limiting
examples include: Alzheimer's disease, amyotrophic lateral
sclerosis, amyotrophic lateral sclerosis (ALS), angina pectoris,
arginase deficiency, atherosclerosis, cachexia, cancer,
carbamylphosphate synthase deficiency, carboxylase defects,
cataracts, chronic obstructive pulmonary disease, chronic traumatic
encephalopathy, citrullinaemia, congenital heart disease,
congenital lactic acidosis, congenital myopathy, coronary artery
disease, coronary heart disease, crginosuccinic aciduria,
cystinosis, diabetes, diabetic nephropathy, diabetic neuropathy,
diabetic retinopathy, dilated cardiomyopathy, erectile dysfunction,
essential tremor, facioscapulohumeral muscular dystrophy, familial
cardiomyopathy, Friedreich's ataxia, galactosemia, Gierke's
disease, glomerulosclerosis, glutaric acidemia type I & II,
glutaric aciduria type I, glycogenosis or dextrinosis, GSD type II
(acid maltase deficiency--Pompe's disease), GSD type III (glycogen
debrancher deficiency--Cori's disease or Forbe's disease), GSD type
IV (glycogen branching enzyme deficiency--Andersen disease), GSD
type V (muscle glycogen phosphorylase deficiency--McArdle disease),
GSD type VI (liver phosphorylase deficiency--Hers's disease), GSD
type VII (muscle phosphofructokinase deficiency--Tarui's disease),
GSD type XI (glucose transporter deficiency--Fanconi-Bickel
disease), heart disease, homocystinuria, Huntington's disease,
hyperinsulinemia, hyperlipidemia, hypertrophic cardiomyopathy,
impaired glucose metabolism, inflammatory bowel disease (IBD),
isovaleric acidemia, keratoconus, lactic acidosis (pyruvate
dehydrogenase complex defects), Lewy body disease, macular
degeneration, maple syrup urine disease, medium chain acyl CoA
dehydrogenase (MCAD) deficiency, methylmalonic acidemia,
mitochondrial myopathies, mitochondrial myopathy, mitochondrial
respiratory chain defects, multiple system atrophy, muscle wasting
syndrome, myotonic muscular dystrophy, n-acetyl glutamate
synthetase deficiency, Niemann-Pick disease, non-ketotic
hyperglycinaemia, ornithine carbamyl transferase deficiency,
osteoarthritis, osteoporosis, Parkinson's disease, phenylketonuria,
premenstrual syndrome, progressive supranuclear palsy, propionic
acidemia, prostatitis, restrictive cardiomyopathy, rheumatic heart
disease, rheumatoid arthritis, sarcopenia, spinal muscular atrophy,
Tay-Sachs disease, type 2 diabetes, tyrosinaemia (Type I),
ulcerative colitis, vascular restenosis, coronary heart
disease.
[0122] In one particular embodiment, structures, compositions and
methods described herein are used for assessing a subject's risk
for atherosclerosis. In some embodiments, assays described herein
can also be used to screen for agents to treat atherosclerosis.
Treating atherosclerosis may include performing a therapeutic
intervention that results in reducing the cholesterol content of at
least one atherosclerotic plaque, or prophylactically inhibiting or
preventing the formation or expansion of an atherosclerotic plaque.
Generally, the volume of the atherosclerotic plaque, and hence the
degree of obstruction of the vascular lumen, will also be reduced.
The present structures, compositions and methods are particularly
useful for treating atherosclerotic lesions associated with
familial hyperlipidemias.
[0123] Treatment of atherosclerosis may reduce the cholesterol
content of atherosclerotic plaques and/or the volume of
atherosclerotic plaques. The cholesterol content may be reduced by,
for example, at least 10%-30%, at least 30%-50%, and in some
instances at least 50%-85% or more. The volume of the
atherosclerotic plaques may also be reduced. The reduction in
plaque volume may be, for example, at least 5%-30%, often as much
as 50%, and in some instances 75% or more. Methods of determining
the reduction of cholesterol content of atherosclerotic plaques
and/or the volume of atherosclerotic plaques are known to those of
ordinary skill in the art, and include intravascular ultrasound and
magnetic resonance imaging.
[0124] In another embodiment, structures, compositions and methods
described herein are used for assessing a subject's risk of having
a vascular or a cardiovascular condition or assessing whether the
subject is at risk of developing a cardiovascular condition.
Vascular conditions are conditions that involve the blood vessels
(arteries and veins). Cardiovascular conditions are conditions that
involve the heart and the blood vessels associated with the heart.
Examples of vascular conditions include diabetic retinopathy,
diabetic nephropathy, renal fibrosis, hypertension,
atherosclerosis, arteriosclerosis, atherosclerotic plaque,
atherosclerotic plaque rupture, cerebrovascular accident (stroke),
transient ischemic attack (TIA), peripheral artery disease,
arterial occlusive disease, vascular aneurysm, ischemia, ischemic
ulcer, heart valve stenosis, heart valve regurgitation and
intermittent claudication. Examples of cardiovascular conditions
include coronary artery disease, ischemic cardiomyopathy,
myocardial ischemia, and ischemic or post-myocardial ischemia
revascularization.
[0125] Structures, compositions and methods described herein can
also be used for assessing a subject's risk of developing a
cardiovascular condition. The degree of risk of a cardiovascular
condition depends on the multitude and the severity or the
magnitude of the risk factors that the subject has. Risk charts and
prediction algorithms are available for assessing the risk of
cardiovascular conditions in a human subject based on the presence
and severity of risk factors. One commonly used algorithm for
assessing the risk of a cardiovascular condition in a human subject
based on the presence and severity of risk factors is the
Framingham Heart Study risk prediction score. A human subject is at
an elevated risk of having a cardiovascular condition if the
subject's 10-year calculated Framingham Heart Study risk score is
greater than 10%. Another method for assessing the risk of a
cardiovascular event in a human subject is a global risk score that
incorporates a measurement of a level of a marker of systemic
inflammation, such as CRP, into the Framingham Heart Study risk
prediction score. Other methods of assessing the risk of a
cardiovascular event in a human subject include coronary calcium
scanning, cardiac magnetic resonance imaging, and/or magnetic
resonance angiography.
[0126] The structures, compositions and methods described herein
may also be useful for prophylactic treatments.
[0127] Hyperlipidemias may also be assessed by the compositions and
methods described herein.
[0128] In certain embodiments, structures and compositions
described herein are used in a method involving the determination
of a disease or condition of a subject or biological sample. For
instance, a method may include introducing a composition comprising
a plurality of structures described herein to a biological sample
(e.g., in vitro), and exposing the plurality of nanostructures
and/or the subject or biological sample to testing conditions that
can determine a disease or condition of the subject or biological
sample.
[0129] It should be understood that any suitable structures
described herein can be used in such methods, including, for
example, structures having a nanostructure core comprising an
inorganic material and a shell substantially surrounding and
attached to the nanostructure core. In some cases, such structures
are adapted to sequester cholesterol. In other cases, the
structures are a marker for a disease or bodily condition.
[0130] In other embodiments, a composition is introduced to a
subject or a biological sample, and the structures of the
composition and/or the subject or biological sample are exposed to
assay conditions that can determine a disease or condition of the
subject or biological sample. At least a portion of the structures
may be retrieved from the subject or biological sample and an assay
may be performed with the structures retrieved. The structures may
be assayed for the amount and/or type of molecules bound to or
otherwise sequestered by the structures. For example, in one set of
embodiments, a competition assay is performed, e.g., where labeled
cholesterol is added and displacement of cholesterol is monitored.
The more measured uptake of labeled cholesterol, the less bound
un-labeled free cholesterol is present. This can be done, for
example, after a composition comprising the structures described
herein are administered to a subject or a biological sample, and
the structures are subsequently retrieved from the subject or
biological sample. This method can be used, for example, where the
structures are to be used as a diagnostic agent to see how much
cholesterol (unlabeled) it has sequestered in a subject or
biological sample.
[0131] Other methods can also be used to determine the amount of
cholesterol sequestered by structures described herein. In some
cases, labeled cholesterol (e.g., fluorescently-labeled cholesterol
such as NBD-cholesterol, BODIPY-cholesterol or radioactive
cholesterol) can be used. Labeled cholesterol can be added to the
structures either in vivo or in vitro. By adding structures without
labeled cholesterol and measuring the fluorescence increase upon
binding, one can calculate the binding constant of labeled
cholesterol to the structure. In addition, to remove the
cholesterol from the structure, one can dissolve the particle
(e.g., KCN) and then measure the resultant fluorescence in
solution. Comparing to standard curve can allow determination of
the number of cholesterol molecules per particle. Other methods
such as organic extraction and quantitative mass spectrometry can
also be used to calculate amount of cholesterol sequestered by one
or more structures described herein.
[0132] Further aspects of the invention relate to kits for
measuring binding between a cholesterol acceptor and cholesterol.
Kits can comprise one or more types of fluorescently labeled
cholesterol analogs and one or more types of lapidated gold
nanoparticles. Kits can further comprise instructions for use.
[0133] The kits described herein may also contain one or more
containers, which can contain components such as the structures,
signaling entities, and/or biomolecules as described. The kits also
may contain instructions for mixing, diluting, and/or
administrating the compounds. The kits also can include other
containers with one or more solvents, surfactants, preservatives,
and/or diluents (e.g., normal saline (0.9% NaCl), or 5% dextrose)
as well as containers for mixing, diluting or administering the
components to the sample or to the patient in need of such
treatment.
[0134] The compositions of the kit may be provided as any suitable
form, for example, as liquid solutions or as dried powders. When
the composition provided is a dry powder, the powder may be
reconstituted by the addition of a suitable solvent, which may also
be provided. In embodiments where liquid forms of the composition
are used, the liquid form may be concentrated or ready to use. The
solvent will depend on the particular inventive structure and the
mode of use or administration. Suitable solvents for compositions
are well known and are available in the literature.
[0135] The kit, in one set of embodiments, may comprise one or more
containers such as vials, tubes, and the like, each of the
containers comprising one of the separate elements to be used in
the method. For example, one of the containers may comprise a
positive control in the assay. Additionally, the kit may include
containers for other components, for example, buffers useful in the
assay.
[0136] As used herein, a "subject" or a "patient" refers to any
mammal (e.g., a human), for example, a mammal that may be
susceptible to a disease or bodily condition such as a disease or
bodily condition associated with abnormal lipid levels. Examples of
subjects or patients include a human, a non-human primate, a cow, a
horse, a pig, a sheep, a goat, a dog, a cat or a rodent such as a
mouse, a rat, a hamster, or a guinea pig. Generally, the invention
is directed toward use with humans. A subject may be a subject
diagnosed with a certain disease or bodily condition or otherwise
known to have a disease or bodily condition. In some embodiments, a
subject may be diagnosed as, or known to be, at risk of developing
a disease or bodily condition. In some embodiments, a subject may
be diagnosed with, or otherwise known to have, a disease or bodily
condition associated with abnormal lipid levels, as described
herein. In certain embodiments, a subject may be selected for
diagnosis and/or treatment on the basis of a known disease or
bodily condition in the subject. In some embodiments, a subject may
be selected for diagnosis and/or treatment on the basis of a
suspected disease or bodily condition in the subject. In some
embodiments, the presence of an existing disease or bodily
condition may be suspected, but not yet identified in a
subject.
[0137] A "biological sample," as used herein, is any cell, body
tissue, or body fluid sample obtained from a subject. Non-limiting
examples of body fluids include, for example, lymph, saliva, blood,
urine, and the like. Samples of tissue and/or cells for use in the
various methods described herein can be obtained through standard
methods including, but not limited to, tissue biopsy, including
punch biopsy and cell scraping, needle biopsy; or collection of
blood or other bodily fluids by aspiration or other suitable
methods.
[0138] The following examples are intended to illustrate certain
embodiments of the present invention, but are not to be construed
as limiting and do not exemplify the full scope of the
invention.
EXAMPLES
Example 1: Development of a Cell-Free Competition Assay to Measure
the Dissociation Constant of Cholesterol and Human Serum Using a
Biomimetic Nanostructure
Introduction
[0139] Reverse Cholesterol Transport (RCT) is a major function of
high density lipoproteins and may protect against atherosclerosis.
Measurement of cellular RCT improves assessment of cardiovascular
risk, but current methods of measurement are limited in clinical
applicability. Here, a rapid, cell-free competition assay to
measure the dissociation constant of human serum for cholesterol is
described using a fluorescently labeled cholesterol analog and a
biomimetic lipidated gold nanoparticle. Using this assay, the
K.sub.D of human serum as well as a high density
lipoprotein-enriched fraction of serum is reported. Because
cholesterol binding to serum cholesterol acceptors is a significant
step in cellular RCT, this assay has widespread applications for
assessing cardiovascular risk.
[0140] An intriguing property of colloidal gold is that it has the
ability to quench fluorescent signals when the fluorescent moeity
is in close physical proximity to the gold nanoparticle.
Boron-dipyrromethene (BODIPY)-labelled cholesterol is a highly
fluorescent molecule which we show (vide infra) to be strongly
quenched by AuNP-HDL in a dose-dependent and titratable manner.
Using assays described herein, it was found that BODIPY-Cholesterol
is reversibly bound to AuNP-HDL, and that the addition of a
cholesterol acceptor, (such as ApoB-depleted human serum, which
contains HDL) leads to recovery of the fluorescent signal in a
dose-dependent manner. Furthermore, because of the stability of the
fluorescent signal, using this method, the binding capacity and
kinetics of cholesterol binding to both HDL-AuNP and the
competitive natural cholesterol acceptor can be ascertained. An
effective K.sub.D of the cholesterol acceptor added, such as human
serum, can also be calculated. These parameters are fundamental
biochemical parameters that are highly related to the process of
reverse cholesterol transport. Measuring these functional
parameters in a clinical test is very useful in determining a
patient's functional capacity for reverse cholesterol
transport.
Materials and Methods
Synthesis of Lipidated Nanoparticles
[0141] Citrate-stabilized colloidal gold nanoparticles at 5 nm
diameter and 80 nM concentration were purchased from Ted Pella,
Redding, Calif. Phospholipids were purchased from Avanti Polar
Lipids, Alabaster, Ala., and 1 mM stock solutions of each were
made. 100% Ethanol (Sigma Aldrich, St. Louis, Mo.) as well as lipid
stocks were added to the gold nanoparticle such that the final
concentration of ethanol was 20%, the colloidal gold was 64 nM, and
250-fold excesses of each lipid were present. After a two hour room
temperature incubation, the solutions were purified by
diafiltration with a KrosFlo Research II tangential flow filtration
system (Spectrum Laboratories, Rancho Dominguez, Calif.). The
samples were first concentrated down to a minimum holdup volume of
approximately 5 ml, and were then exchanged with buffer (Ultrapure
18.2 M.OMEGA. water) continuously for at least 7 volumes (35 ml) to
remove excess lipid. Concentration of the samples was then assessed
by absorbance spectroscopy, using the extinction coefficient of 5
nm gold colloid of 9.696.times.10.sup.6. Prior to use in further
experiments the lipidated nanoparticle stock was diluted with
ultrapure water to a final concentration of 1.0 .mu.M.
[0142] In some embodiments, HDL AuNP constructs were synthesized as
follows. Briefly, an 80 nM solution of 5 nm colloidal gold was
incubated with a five-fold molar excess of purified ApoA1 protein.
Next 250-fold molar excess of
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(3-(2-pyridyldithio)pr-
oprionate (PDP PE) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
(DPPC) lipids were added and incubated with the ApoA1-Gold
Nanoparticle for 2 hours in 20% Ethanol. Finally, excess reagents
and ethanol were purified from the particles using a tangential
flow filtration system, resulting in stable HDL AuNPs in water. The
concentration of these particles was then determined using
absorbance at 525 nM, the absorption maximum for 5 nm colloidal
gold.
Fluorescence Measurements
[0143] All fluorescence measurements were made using black 96-well
plates (Costar, Tewksbury, Mass.) on a BioTek Synergy 2 plate
reading fluorometer (Winooski, Vt.) using read settings as follows:
Tungsten lamp; Excitation filter: 485/20 nm; Emission filter:
528/20 nm; Optics position: Top read 510 nm; Sensitivity: 100. All
reactions were carried out in a total volume of 200 .mu.l of water
at 37.degree. C. BODIPY Cholesterol stock solution used in these
experiments was 125 .mu.M in 100% Ethanol. Stock solution was made
stored at -20.degree. C.; working stocks were made fresh by
diluting a portion of the stock solution to 1.25 .mu.M or 2.5 .mu.M
in water. Serum samples used in this experiment were collected in
compliance with Institutional guidelines for human subjects
research.
Cellular RCT Assay
[0144] A cellular RCT assay was performed by plating J774 mouse
macrophages (ATCC, Bethesda, Md.) on day 1, labeling the cells with
.sup.3H-Cholesterol on day 2, upregulating membrane cholesterol
transport proteins with a cyclic AMP analog on day 3, and
incubating for 4 hours the radiolabeled cells with a cholesterol
acceptor solution, such as human serum, on day 4..sup.3,4 Percent
efflux was calculated as specific radioactivity in the
serum-containing media divided by total radioactivity in the cells
and the media.
[0145] Increased percent efflux to ApoB-depleted serum (vide
infra), a serum fraction relatively enriched in HDL,.sup.4
associates with coronary disease status even after adjusting for
total amount of HDL..sup.3 Thus, quantifying the function of HDL
independent of HDL concentration leads to improved diagnostics. The
cellular RCT assay may be conceptualized as being dependent upon
both cellular interactions with lipoproteins, and lipoprotein
interactions with cholesterol itself. Importantly, data demonstrate
the cell-independent component contributes most to overall
efflux..sup.5 The method herein demonstrated, allows measurement of
cholesterol binding with serum cholesterol acceptors, independent
of the cellular context, and provides a means to calculate K.sub.D
values.
[0146] In some embodiments, the RCT assay was performed as follows:
A 250 uM stock solution of BODIPY-Cholesterol in 100% Ethanol was
created. 5 ul of BODIPY stock solution was added to a well of a
96-well plate suitable for fluorescence measurements. 15 ul of
Ethanol and 80 ul of water were added. Baseline fluorescence was
then recorded using a plate-reading fluorometer using the
excitation and emission filters of 485/20 and 528/20, respectively.
100 ul of HDL AuNP at a concentration of 500 nM was then added and
fluorescence was recorded at 2 minute intervals for 30 minutes. An
approximately 70% decline in fluorescence was observed owing to
quenching of BODIPY-Cholesterol signal upon binding of the molecule
to the HDL-AuNP construct. A volume of approximately 20 ul of
cholesterol acceptor, such as purified ApoA1, Human HDL, or
ApoB-depleted human serum, was added and fluorescence was recorded
at 2 minute intervals for 60 minutes. Depending on the amount and
capacity of the cholesterol acceptor added, fluorescence recovery
was observed as the new (non-quenching) cholesterol acceptor
competes for BODIPY-Cholesterol bound to and quenched by the
HDL-AuNP construct. The amount of fluorescence recovery for a given
cholesterol acceptor monotonically increased as a function of the
concentration of that acceptor (FIG. 1).
[0147] Preparation of ApoB-depleted serum was completed as follows.
Polyethylene glycol with an average molecular weight of 8000 (PEG
8000) was prepared as a 20% weight/volume solution in 200 mM
glycine, pH 7.4. Serum and PEG solution were mixed in a 10:4 ratio
and incubated at room temperature for 20 minutes, then centrifuged
at 12,700.times.g for 30 minutes at 4 degrees Celsius to pellet the
ApoB fraction. The supernatant was essentially free of ApoB
containing lipoproteins, including low density lipoprotein (LDL),
and was therefore relatively enriched in HDL, free ApoA1, and
albumin, all of which are capable of binding cholesterol.
[0148] This assay technology may exquisitely calculate the ability
of human serum cholesterol acceptors (e.g. Apo A1, HDLs) to efflux
cholesterol in a non-radioactive and non-cell based assay.
Furthermore, the assay can be automated, scaled, is inexpensive,
rapid, and easy. There are no comparable tests on the market.
Results and Discussion
[0149] Methods described herein are based on a fluorescence-based
competition assay that harnesses the quenching capacity of the gold
core of a biomimetic lipid construct to measure the K.sub.D of
cholesterol from serum and fractions thereof. Data presented herein
demonstrate that this assay is capable of rapidly and
quantitatively measuring cholesterol binding to biomimetic
nanostructures. Using assays developed herein, interrogation of
cholesterol binding to complex biological matrices (e.g., human
serum and serum HDL fractions) is quantitative, rapid, and can be
automated for high-throughput screening. Finally, the assay can be
easily adapted for applications where cholesterol binding,
transport, or metabolism (or other similar fluorescently-tagged
analytes), is of interest.
[0150] In order to study cholesterol binding, lipidated
nanoparticles (NPs) were synthesized using a 5 nm diameter gold
nanoparticle template core (FIG. 4)..sup.7 Gold nanoparticles (Ted
Pella, 80 nM) were incubated with a 250-fold excess each of
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)pr-
opionate] (PDP PE) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
(DPPC) in 20% ethanol (v/v). Incubation took place for two hours at
room temperature. The NP conjugates were purified from unreacted
lipids by using tangential flow filtration (Spectrum Laboratories,
Rancho Dominguez, Calif.). Conjugates were characterized as
previously described..sup.7
[0151] To measure cholesterol binding, NPs were titrated into a
solution with 250 nM of fluorescent cholesterol analog,
23-(dipyrrometheneboron difluoride)-24-norCholesterol
(BODIPY-Cholesterol, Ex: 495 nm, Em: 507 nm). Fluorescence
quenching was measured as the BODIPY-Cholesterol bound to the
surface of the NPs. Binding was modeled as a receptor (NP) with
multiple binding sites (n.sub.NP,chol) with identical dissociation
constants (K.sub.D,NP). Binding isotherms were fit to the quadratic
form of the binding equilibrium.sup.11 (Equation 1) using
non-linear least-squares regression to estimate the parameters
K.sub.D,NP and n.sub.NP,chol:
[ Chol ] f = - ( n NP , Chol + K D , NP - [ Chol ] T ) + ( n NP ,
Chol [ NP ] + K D , NP - [ Chol ] T ) + 4 K D - [ Chol ] T 2
Equation 1 ##EQU00001##
[0152] Where [Chol].sub.f is the free concentration of
BODIPY-Cholesterol and [Chol].sub.T is the total concentration of
BODIPY-Cholesterol. The Klotz plot is shown in FIG. 2; regression
analysis demonstrated the K.sub.D,NP for NP-Cholesterol was 40.+-.9
nM, while n.sub.NP,chol was 16.+-.1. These values favorably
correspond to those reported for similar NPs..sup.7
[0153] To determine the effective K.sub.D of serum (K.sub.D,Serum)
for cholesterol, competition assays between NP and serum fractions
were performed. Two types of serum fraction were tested: (1) whole
serum, and (2) ApoB-depleted serum. ApoB-depleted serum was
prepared by incubation of serum in a 10:4 ratio with a solution of
20% w/v polyethylene glycol at a MW of 8000 and 200 mM glycine, pH
7.4 for 20 minutes at room temperature, followed by centrifugation
at 12,800.times.g for 30 minutes at 4.degree. C. This leaves a
serum supernatant depleted of LDL particles, and enriched in HDL
particles, lipid poor ApoA1, and albumin, a relevant fraction for
assessing reverse cholesterol transport..sup.3
[0154] Equation 2 gives a standard form of the competition
equilibrium relating K.sub.D,Serum to EC.sub.50:
K D , Serum = K D , NP n NP , Chol [ NP ] .times. EC 50 ( Equation
2 ) ##EQU00002##
[0155] EC.sub.50 is the fraction of serum in the reaction needed to
recover half-maximal fluorescence. Serum or ApoB-depleted serum
were titrated into solutions containing 20 nM lipidated NP and 125
nM BODIPY-Cholesterol, and incubated at 37.degree. C. for 4 hours,
by which point equilibrium was reached (FIG. 3). The EC.sub.50 for
whole serum was 1.1.times.10.sup.-3, and was 2.2.times.10.sup.-3
for ApoB-depleted serum, yielding an effective K.sub.D for whole
serum of 1.4.times.10.sup.-4 and an effective K.sub.D for
ApoB-depleted serum of 3.0.times.10.sup.-4 (dimensionless
units).
[0156] Because Equation 2 relies on several simplifying assumptions
regarding binding site occupancy and free ligand concentration,
further assessment of the validity of K.sub.D,Serum determinations
was performed by solving the exact cubic form of the competition
equilibrium algebraically, then using non-linear least-squares
regression to determine K.sub.D as described herein. The effective
K.sub.D for whole serum by this method was 1.4.times.10.sup.-4 and
for ApoB-depleted serum was 3.1.times.10.sup.-4. Thus K.sub.D,Serum
as assessed by Equation 2 was in excellent agreement with
determinations by fitting to the exact formula.
[0157] Using the method reported herein, it is possible to
calculate the K.sub.D of cholesterol on a molar basis to a given
cholesterol acceptor such as free ApoA1 or a pure fraction of HDL.
Using a concentration of .about.34 .mu.M HDL in serum,.sup.12 the
apparent K.sub.D for cholesterol per mol HDL is approximately 10
nM, which compares favorably to the K.sub.D of NP for
cholesterol.
[0158] Values for the K.sub.D of cholesterol interactions with
lipoproteins have not previously been reported. However, the
strength of the interaction reported here compares well to the
strength of porphyrin interactions with low density lipoprotein
(K.sub.D=20 nM), as measured by quenching of porphyrin ring
autofluoresence upon interaction with low density
lipoprotein..sup.13 Thus this technique allows for calculation of
previously unreported K.sub.D values, and may be extended to
further refine understanding of cholesterol binding equilibria with
lipoprotein species. However for clinical applications, simple
measurement of the K.sub.D in rapidly attainable bulk matrices such
as serum or ApoB-depleted serum may be sufficient or even
desirable. Reporting K.sub.D as an affinity constant (K.sub.A)
yields, in this example, a K.sub.A for whole serum of 7100 and for
ApoB-depleted serum of 3300, meaningful and readily comprehensible
integers with higher values meaning tighter binding, features that
make K.sub.A particularly well-suited for direct interpretation and
implementation in clinical practice. Finally, this method can be
extended to ascertain binding properties of complex matrices for
other lipophilic fluorescently-labeled ligands such as testosterone
or cortisol.
[0159] In summary, the synthesis of a lipid nanostructure has been
reported which allows for the measurement of K.sub.D of cholesterol
with serum and serum fractions. To our knowledge, K.sub.D of serum
for cholesterol has not been previously reported. Measurement of
this parameter in various fractions of serum will facilitate
construction of more accurate models of cholesterol flux.
Furthermore this method is rapid, straightforward, and automatable
and therefore will find clinical utility in improving prediction of
patients at risk for pathologies dependent upon cholesterol
overload, such as certain forms of heart disease.
Example 2: Calculations and Nonlinear Regression Analysis
[0160] [ Chol ] f = - ( n NP , Chol + K D , NP - [ Chol ] T ) + ( n
NP , Chol [ NP ] + K D , NP - [ Chol ] T ) + 4 K D - [ Chol ] T 2 (
Equation 1 ) ##EQU00003##
Equation 1 was derived from algebraic manipulation of the following
fundamental equations:
[ Chol ] f = [ Chol ] T + [ Chol ] Bound , NP ( Equation S1 ) K D ,
NP = [ Chol ] f ( n NP , Chol [ NP ] - [ Chol ] Bound , NP ) [ Chol
] Bound , NP ( Equation S2 ) ##EQU00004##
[0161] Where [Chol].sub.Bound,NP is the concentration of
cholesterol bound to the nanoparticle. Note that the term
(n.sub.Np,Chol[NP]-[Chol].sub.Bound,NP) denotes the concentration
of free cholesterol binding sites in the reaction.
[0162] Nonlinear regression analysis for Equation 1 was performed
using GraphPad Prism 6 (La Jolla, Calif.) for the parameters
n.sub.NP,chol and K.sub.D,NP by inputting a custom equation in the
non-linear regression fit package:
Y=((Ltotal-Kd-n*10 X+(4*Ltotal*Kd+(-Ltotal+Kd+n*10 X) 2) 0.5)/2)
(Equation S3)
Where Y is free cholesterol, X is log [NP], n is n.sub.NP,Chol,
Ltotal is [Chol].sub.T.
[0163] To test whether Equation 2 is valid under experimental
conditions used, where binding site occupancy may be a concern, the
exact cubic root of the following system of equations was found
using Mathematica 9.0 (Champaign, Ill.). These equations are given
below in a format suitable for direct programming into
Mathematica.
kdnpeq:=kdnp==(cf)(nnp*npt-cbnp)/cbnp;
kdseq:=kds==(cf)(ns*st-cbs)/cbs;
cteq:=ct==cf+cbs+cbnp;
This system of equations defines the following terms: kdnp--Kd of
the Nanoparticle cf--free cholesterol concentration nnp--number of
binding sites on the nanoparticle npt--total amount of nanoparticle
in the reaction cbnp--amount of cholesterol bound to the
nanoparticle==>(nnp*npt-cbnp) thus equals free binding sites on
the nanoparticle and accounts for binding site depletion.
Similarly:
[0164] kds--Kd of Serum ns--number of binding sites on serum. This
is for completeness we model\ this lumped sum parameter as 1
binding site st--total amount of serum cbs--cholesterol bound to
serum==>(ns*st-cbs) thus equals free binding sites in serum and
accounts for binding site depletion.
Finally:
[0165] ct=Total cholesterol cf=free cholesterol cbs=cholesterol
bound to serum cbnp=cholesterol bound to nanoparticle.
[0166] The system has 3 equations, and thus two variables can be
eliminated. cf and cbs were selected for elimination, as cbnp is
the direct read of quenching and the assay measurement. From prior
assay determinations we kdnp and nnp are known. ct and npt can be
controlled. st is manipulated as the titrate. ns in this instance
is set to 1. Thus, an equation can be solved for which only needs
kds to be fit as a parameter.
Eliminate[{kdnpeq, kdseq, cteq}, {cf, cbs}] Algebraically
eliminates cf and cbs from the system of equations, yielding the
cubic function:
ct (-cbnp kdnp+cbnp kds+kdnp nnp npt-2 kds nnp npt+(kds nnp 2 npt
2)/cbnp)/cbnp)==-cbnp 2 kdnp+cbnp kdnp 2+cbnp 2 kds-cbnp kdnp
kds+cbnp kdnp nnp npt-2 cbnp kds nnp npt+kdnp kds nnp npt+kds nnp 2
npt 2-cbnp kdnp ns st+kdnp nnp npt ns st
[0167] This is set to be an equation, cbnpeq, which is then solved
for cbnp, which is essentially a direct readout of the assay.
Solve[cbnpeq, cbnp] Yields one real root:
TABLE-US-00001 {cbnp -> -((-ct kdnp - kdnp{circumflex over ( )}2
+ ct kds + kdnp kds - kdnp nnp npt + 2 kds nnp npt + kdnp ns st)/(
3 (kdnp - kds))) - (2{circumflex over ( )}( 1/3) (-(-ct kdnp -
kdnp{circumflex over ( )}2 + ct kds + kdnp kds - kdnp nnp npt + 2
kds nnp npt + kdnp ns st){circumflex over ( )}2 + 3 (kdnp - kds)
(ct kdnp nnp npt - 2 ct kds nnp npt - kdnp kds nnp npt - kds
nnp{circumflex over ( )}2 npt{circumflex over ( )}2 - kdnp nnp npt
ns st)))/(3 (kdnp - kds) (2 ct{circumflex over ( )}3
kdnp{circumflex over ( )}3 + 6 ct{circumflex over ( )}2
kdnp{circumflex over ( )}4 + 6 ct kdnp{circumflex over ( )}5 + 2
kdnp{circumflex over ( )}6 - 6 ct{circumflex over ( )}3
kdnp{circumflex over ( )}2 kds - 18 ct{circumflex over ( )}2
kdnp{circumflex over ( )}3 kds - 18 ct kdnp{circumflex over ( )}4
kds - 6 kdnp{circumflex over ( )}5 kds + 6 ct{circumflex over ( )}3
kdnp kds{circumflex over ( )}2 + 18 ct{circumflex over ( )}2
kdnp{circumflex over ( )}2 kds{circumflex over ( )}2 + 18 ct
kdnp{circumflex over ( )}3 kds{circumflex over ( )}2 + 6
kdnp{circumflex over ( )}4 kds{circumflex over ( )}2 - 2
ct{circumflex over ( )}3 kds{circumflex over ( )}3 - 6
ct{circumflex over ( )}2 kdnp kds{circumflex over ( )}3 - 6 ct
kdnp{circumflex over ( )}2 kds{circumflex over ( )}3 - 2
kdnp{circumflex over ( )}3 kds{circumflex over ( )}3 - 3
ct{circumflex over ( )}2 kdnp{circumflex over ( )}3 nnp npt + 3 ct
kdnp{circumflex over ( )}4 nnp npt + 6 kdnp{circumflex over ( )}5
nnp npt + 12 ct{circumflex over ( )}2 kdnp{circumflex over ( )}2
kds nnp npt - 3 ct kdnp{circumflex over ( )}3 kds nnp npt - 15
kdnp{circumflex over ( )}4 kds nnp npt - 15 ct{circumflex over (
)}2 kdnp kds{circumflex over ( )}2 nnp npt - 3 ct kdnp{circumflex
over ( )}2 kds{circumflex over ( )}2 nnp npt + 12 kdnp{circumflex
over ( )}3 kds{circumflex over ( )}2 nnp npt + 6 ct{circumflex over
( )}2 kds{circumflex over ( )}3 nnp npt + 3 ct kdnp kds{circumflex
over ( )}3 nnp npt - 3 kdnp{circumflex over ( )}2 kds{circumflex
over ( )}3 nnp npt - 3 ct kdnp{circumflex over ( )}3 nnp{circumflex
over ( )}2 npt{circumflex over ( )}2 + 6 kdnp{circumflex over ( )}4
nnp{circumflex over ( )}2 npt{circumflex over ( )}2 - 3 ct
kdnp{circumflex over ( )}2 kds nnp{circumflex over ( )}2
npt{circumflex over ( )}2 - 12 kdnp{circumflex over ( )}3 kds
nnp{circumflex over ( )}2 npt{circumflex over ( )}2 + 12 ct kdnp
kds{circumflex over ( )}2 nnp{circumflex over ( )}2 npt{circumflex
over ( )}2 + 3 kdnp{circumflex over ( )}2 kds{circumflex over ( )}2
nnp{circumflex over ( )}2 npt{circumflex over ( )}2 - 6 ct
kds{circumflex over ( )}3 nnp{circumflex over ( )}2 npt{circumflex
over ( )}2 + 3 kdnp kds{circumflex over ( )}3 nnp{circumflex over (
)}2 npt{circumflex over ( )}2 + 2 kdnp{circumflex over ( )}3
nnp{circumflex over ( )}3 npt{circumflex over ( )}3 - 3
kdnp{circumflex over ( )}2 kds nnp{circumflex over ( )}3
npt{circumflex over ( )}3 - 3 kdnp kds{circumflex over ( )}2
nnp{circumflex over ( )}3 npt{circumflex over ( )}3 + 2
kds{circumflex over ( )}3 nnp{circumflex over ( )}3 npt{circumflex
over ( )}3 - 6 ct{circumflex over ( )}2 kdnp{circumflex over ( )}3
ns st - 12 ct kdnp{circumflex over ( )}4 ns st - 6 kdnp{circumflex
over ( )}5 ns st + 12 ct{circumflex over ( )}2 kdnp{circumflex over
( )}2 kds ns st + 24 ct kdnp{circumflex over ( )}3 kds ns st + 12
kdnp{circumflex over ( )}4 kds ns st - 6 ct{circumflex over ( )}2
kdnp kds{circumflex over ( )}2 ns st - 12 ct kdnp{circumflex over (
)}2 kds{circumflex over ( )}2 ns st - 6 kdnp{circumflex over ( )}3
kds{circumflex over ( )}2 ns st + 6 ct kdnp{circumflex over ( )}3
nnp npt ns st - 3 kdnp{circumflex over ( )}4 nnp npt ns st - 9 ct
kdnp{circumflex over ( )}2 kds nnp npt ns st + 9 kdnp{circumflex
over ( )}3 kds nnp npt ns st + 3 ct kdnp kds{circumflex over ( )}2
nnp npt ns st - 6 kdnp{circumflex over ( )}2 kds{circumflex over (
)}2 nnp npt ns st + 3 kdnp{circumflex over ( )}3 nnp{circumflex
over ( )}2 npt{circumflex over ( )}2 ns st - 12 kdnp{circumflex
over ( )}2 kds nnp{circumflex over ( )}2 npt{circumflex over ( )}2
ns st + 3 kdnp kds{circumflex over ( )}2 nnp{circumflex over ( )}2
npt{circumflex over ( )}2 ns st + 6 ct kdnp{circumflex over ( )}3
ns{circumflex over ( )}2 st{circumflex over ( )}2 + 6
kdnp{circumflex over ( )}4 ns{circumflex over ( )}2 st{circumflex
over ( )}2 - 6 ct kdnp{circumflex over ( )}2 kds ns{circumflex over
( )}2 st{circumflex over ( )}2 - 6 kdnp{circumflex over ( )}3 kds
ns{circumflex over ( )}2 st{circumflex over ( )}2 - 3
kdnp{circumflex over ( )}3 nnp npt ns{circumflex over ( )}2
st{circumflex over ( )}2 - 3 kdnp{circumflex over ( )}2 kds nnp npt
ns{circumflex over ( )}2 st{circumflex over ( )}2 - 2
kdnp{circumflex over ( )}3 ns{circumflex over ( )}3 st{circumflex
over ( )}3 + .quadrature.((2 ct{circumflex over ( )}3
kdnp{circumflex over ( )}3 + 6 ct{circumflex over ( )}2
kdnp{circumflex over ( )}4 + 6 ct kdnp{circumflex over ( )}5 + 2
kdnp{circumflex over ( )}6 - 6 ct{circumflex over ( )}3
kdnp{circumflex over ( )}2 kds - 18 ct{circumflex over ( )}2
kdnp{circumflex over ( )}3 kds - 18 ct kdnp{circumflex over ( )}4
kds - 6 kdnp{circumflex over ( )}5 kds + 6 ct{circumflex over ( )}3
kdnp kds{circumflex over ( )}2 + 18 ct{circumflex over ( )}2
kdnp{circumflex over ( )}2 kds{circumflex over ( )}2 + 18 ct
kdnp{circumflex over ( )}3 kds{circumflex over ( )}2 + 6
kdnp{circumflex over ( )}4 kds{circumflex over ( )}2 - 2
ct{circumflex over ( )}3 kds{circumflex over ( )}3 - 6
ct{circumflex over ( )}2 kdnp kds{circumflex over ( )}3 - 6 ct
kdnp{circumflex over ( )}2 kds{circumflex over ( )}3 - 2
kdnp{circumflex over ( )}3 kds{circumflex over ( )}3 - 3
ct{circumflex over ( )}2 kdnp{circumflex over ( )}3 nnp npt + 3 ct
kdnp{circumflex over ( )}4 nnp npt + 6 kdnp{circumflex over ( )}5
nnp npt + 12 ct{circumflex over ( )}2 kdnp{circumflex over ( )}2
kds nnp npt - 3 ct kdnp{circumflex over ( )}3 kds nnp npt - 15
kdnp{circumflex over ( )}4 kds nnp npt - 15 ct{circumflex over (
)}2 kdnp kds{circumflex over ( )}2 nnp npt - 3 ct kdnp{circumflex
over ( )}2 kds{circumflex over ( )}2 nnp npt + 12 kdnp{circumflex
over ( )}3 kds{circumflex over ( )}2 nnp npt + 6 ct{circumflex over
( )}2 kds{circumflex over ( )}3 nnp npt + 3 ct kdnp kds{circumflex
over ( )}3 nnp npt - 3 kdnp{circumflex over ( )}2 kds{circumflex
over ( )}3 nnp npt - 3 ct kdnp{circumflex over ( )}3 nnp{circumflex
over ( )}2 npt{circumflex over ( )}2 + 6 kdnp{circumflex over ( )}4
nnp{circumflex over ( )}2 npt{circumflex over ( )}2 - 3 ct
kdnp{circumflex over ( )}2 kds nnp{circumflex over ( )}2
npt{circumflex over ( )}2 - 12 kdnp{circumflex over ( )}3 kds
nnp{circumflex over ( )}2 npt{circumflex over ( )}2 + 12 ct kdnp
kds{circumflex over ( )}2 nnp{circumflex over ( )}2 npt{circumflex
over ( )}2 + 3 kdnp{circumflex over ( )}2 kds{circumflex over ( )}2
nnp{circumflex over ( )}2 npt{circumflex over ( )}2 - 6 ct
kds{circumflex over ( )}3 nnp{circumflex over ( )}2 npt{circumflex
over ( )}2 + 3 kdnp kds{circumflex over ( )}3 nnp{circumflex over (
)}2 npt{circumflex over ( )}2 + 2 kdnp{circumflex over ( )}3
nnp{circumflex over ( )}3 npt{circumflex over ( )}3 - 3
kdnp{circumflex over ( )}2 kds nnp{circumflex over ( )}3
npt{circumflex over ( )}3 - 3 kdnp kds{circumflex over ( )}2
nnp{circumflex over ( )}3 npt{circumflex over ( )}3 + 2
kds{circumflex over ( )}3 nnp{circumflex over ( )}3 npt{circumflex
over ( )}3 - 6 ct{circumflex over ( )}2 kdnp{circumflex over ( )}3
ns st - 12 ct kdnp{circumflex over ( )}4 ns st - 6 kdnp{circumflex
over ( )}5 ns st + 12 ct{circumflex over ( )}2 kdnp{circumflex over
( )}2 kds ns st + 24 ct kdnp{circumflex over ( )}3 kds ns st + 12
kdnp{circumflex over ( )}4 kds ns st - 6 ct{circumflex over ( )}2
kdnp kds{circumflex over ( )}2 ns st - 12 ct kdnp{circumflex over (
)}2 kds{circumflex over ( )}2 ns st - 6 kdnp{circumflex over ( )}3
kds{circumflex over ( )}2 ns st + 6 ct kdnp{circumflex over ( )}3
nnp npt ns st - 3 kdnp{circumflex over ( )}4 nnp npt ns st - 9 ct
kdnp{circumflex over ( )}2 kds nnp npt ns st + 9 kdnp{circumflex
over ( )}3 kds nnp npt ns st + 3 ct kdnp kds{circumflex over ( )}2
nnp npt ns st - 6 kdnp{circumflex over ( )}2 kds{circumflex over (
)}2 nnp npt ns st + 3 kdnp{circumflex over ( )}3 nnp{circumflex
over ( )}2 npt{circumflex over ( )}2 ns st - 12 kdnp{circumflex
over ( )}2 kds nnp{circumflex over ( )}2 npt{circumflex over ( )}2
ns st + 3 kdnp kds{circumflex over ( )}2 nnp{circumflex over ( )}2
npt{circumflex over ( )}2 ns st + 6 ct kdnp{circumflex over ( )}3
ns{circumflex over ( )}2 st{circumflex over ( )}2 + 6
kdnp{circumflex over ( )}4 ns{circumflex over ( )}2 st{circumflex
over ( )}2 - 6 ct kdnp{circumflex over ( )}2 kds ns{circumflex over
( )}2 st{circumflex over ( )}2 - 6 kdnp{circumflex over ( )}3 kds
ns{circumflex over ( )}2 st{circumflex over ( )}2 - 3
kdnp{circumflex over ( )}3 nnp npt ns{circumflex over ( )}2
st{circumflex over ( )}2 - 3 kdnp{circumflex over ( )}2 kds nnp npt
ns{circumflex over ( )}2 st{circumflex over ( )}2 - 2
kdnp{circumflex over ( )}3 ns{circumflex over ( )}3 st{circumflex
over ( )}3){circumflex over ( )}2 + 4 (-(-ct kdnp - kdnp{circumflex
over ( )}2 + ct kds + kdnp kds - kdnp nnp npt + 2 kds nnp npt +
kdnp ns st){circumflex over ( )}2 + 3 (kdnp - kds) (ct kdnp nnp npt
- 2 ct kds nnp npt - kdnp kds nnp npt - kds nnp{circumflex over (
)}2 npt{circumflex over ( )}2 - kdnp nnp npt ns st)){circumflex
over ( )}3)){circumflex over ( )}(1/3)) + 1/(3 2{circumflex over (
)}( 1/3) (kdnp - kds)) (2 ct{circumflex over ( )}3 kdnp{circumflex
over ( )}3 + 6 ct{circumflex over ( )}2 kdnp{circumflex over ( )}4
+ 6 ct kdnp{circumflex over ( )}5 + 2 kdnp{circumflex over ( )}6 -
6 ct{circumflex over ( )}3 kdnp{circumflex over ( )}2 kds - 18
ct{circumflex over ( )}2 kdnp{circumflex over ( )}3 kds - 18 ct
kdnp{circumflex over ( )}4 kds - 6 kdnp{circumflex over ( )}5 kds +
6 ct{circumflex over ( )}3 kdnp kds{circumflex over ( )}2 + 18
ct{circumflex over ( )}2 kdnp{circumflex over ( )}2 kds{circumflex
over ( )}2 + 18 ct kdnp{circumflex over ( )}3 kds{circumflex over (
)}2 + 6 kdnp{circumflex over ( )}4 kds{circumflex over ( )}2 - 2
ct{circumflex over ( )}3 kds{circumflex over ( )}3 - 6
ct{circumflex over ( )}2 kdnp kds{circumflex over ( )}3 - 6 ct
kdnp{circumflex over ( )}2 kds{circumflex over ( )}3 - 2
kdnp{circumflex over ( )}3 kds{circumflex over ( )}3 - 3
ct{circumflex over ( )}2 kdnp{circumflex over ( )}3 nnp npt + 3 ct
kdnp{circumflex over ( )}4 nnp npt + 6 kdnp{circumflex over ( )}5
nnp npt + 12 ct{circumflex over ( )}2 kdnp{circumflex over ( )}2
kds nnp npt - 3 ct kdnp{circumflex over ( )}3 kds nnp npt - 15
kdnp{circumflex over ( )}4 kds nnp npt - 15 ct{circumflex over (
)}2 kdnp kds{circumflex over ( )}2 nnp npt - 3 ct kdnp{circumflex
over ( )}2 kds{circumflex over ( )}2 nnp npt + 12 kdnp{circumflex
over ( )}3 kds{circumflex over ( )}2 nnp npt + 6 ct{circumflex over
( )}2 kds{circumflex over ( )}3 nnp npt + 3 ct kdnp kds{circumflex
over ( )}3 nnp npt - 3 kdnp{circumflex over ( )}2 kds{circumflex
over ( )}3 nnp npt - 3 ct kdnp{circumflex over ( )}3 nnp{circumflex
over ( )}2 npt{circumflex over ( )}2 + 6 kdnp{circumflex over ( )}4
nnp{circumflex over ( )}2 npt{circumflex over ( )}2 - 3 ct
kdnp{circumflex over ( )}2 kds nnp{circumflex over ( )}2
npt{circumflex over ( )}2 - 12 kdnp{circumflex over ( )}3 kds
nnp{circumflex over ( )}2 npt{circumflex over ( )}2 + 12 ct kdnp
kds{circumflex over ( )}2 nnp{circumflex over ( )}2 npt{circumflex
over ( )}2 + 3 kdnp{circumflex over ( )}2 kds{circumflex over ( )}2
nnp{circumflex over ( )}2 npt{circumflex over ( )}2 - 6 ct
kds{circumflex over ( )}3 nnp{circumflex over ( )}2 npt{circumflex
over ( )}2 + 3 kdnp kds{circumflex over ( )}3 nnp{circumflex over (
)}2 npt{circumflex over ( )}2 + 2 kdnp{circumflex over ( )}3
nnp{circumflex over ( )}3 npt{circumflex over ( )}3 - 3
kdnp{circumflex over ( )}2 kds nnp{circumflex over ( )}3
npt{circumflex over ( )}3 - 3 kdnp kds{circumflex over ( )}2
nnp{circumflex over ( )}3 npt{circumflex over ( )}3 + 2
kds{circumflex over ( )}3 nnp{circumflex over ( )}3 npt{circumflex
over ( )}3 - 6 ct{circumflex over ( )}2 kdnp{circumflex over ( )}3
ns st - 12 ct kdnp{circumflex over ( )}4 ns st - 6 kdnp{circumflex
over ( )}5 ns st + 12 ct{circumflex over ( )}2 kdnp{circumflex over
( )}2 kds ns st + 24 ct kdnp{circumflex over ( )}3 kds ns st + 12
kdnp{circumflex over ( )}4 kds ns st - 6 ct{circumflex over ( )}2
kdnp kds{circumflex over ( )}2 ns st - 12 ct kdnp{circumflex over (
)}2 kds{circumflex over ( )}2 ns st - 6 kdnp{circumflex over ( )}3
kds{circumflex over ( )}2 ns st + 6 ct kdnp{circumflex over ( )}3
nnp npt ns st - 3 kdnp{circumflex over ( )}4 nnp npt ns st - 9 ct
kdnp{circumflex over ( )}2 kds nnp npt ns st + 9 kdnp{circumflex
over ( )}3 kds nnp npt ns st + 3 ct kdnp kds{circumflex over ( )}2
nnp npt ns st - 6 kdnp{circumflex over ( )}2 kds{circumflex over (
)}2 nnp npt ns st + 3 kdnp{circumflex over ( )}3 nnp{circumflex
over ( )}2 npt{circumflex over ( )}2 ns st - 12 kdnp{circumflex
over ( )}2 kds nnp{circumflex over ( )}2 npt{circumflex over ( )}2
ns st + 3 kdnp kds{circumflex over ( )}2 nnp{circumflex over ( )}2
npt{circumflex over ( )}2 ns st + 6 ct kdnp{circumflex over ( )}3
ns{circumflex over ( )}2 st{circumflex over ( )}2 + 6
kdnp{circumflex over ( )}4 ns{circumflex over ( )}2 st{circumflex
over ( )}2 - 6 ct kdnp{circumflex over ( )}2 kds ns{circumflex over
( )}2 st{circumflex over ( )}2 - 6 kdnp{circumflex over ( )}3 kds
ns{circumflex over ( )}2 st{circumflex over ( )}2 - 3
kdnp{circumflex over ( )}3 nnp npt ns{circumflex over ( )}2
st{circumflex over ( )}2 - 3 kdnp{circumflex over ( )}2 kds nnp npt
ns{circumflex over ( )}2 st{circumflex over ( )}2 - 2
kdnp{circumflex over ( )}3 ns{circumflex over ( )}3 st{circumflex
over ( )}3 + .quadrature.((2 ct{circumflex over ( )}3
kdnp{circumflex over ( )}3 + 6 ct{circumflex over ( )}2
kdnp{circumflex over ( )}4 + 6 ct kdnp{circumflex over ( )}5 + 2
kdnp{circumflex over ( )}6 - 6 ct{circumflex over ( )}3
kdnp{circumflex over ( )}2 kds - 18 ct{circumflex over ( )}2
kdnp{circumflex over ( )}3 kds - 18 ct
kdnp{circumflex over ( )}4 kds - 6 kdnp{circumflex over ( )}5 kds +
6 ct{circumflex over ( )}3 kdnp kds{circumflex over ( )}2 + 18
ct{circumflex over ( )}2 kdnp{circumflex over ( )}2 kds{circumflex
over ( )}2 + 18 ct kdnp{circumflex over ( )}3 kds{circumflex over (
)}2 + 6 kdnp{circumflex over ( )}4 kds{circumflex over ( )}2 - 2
ct{circumflex over ( )}3 kds{circumflex over ( )}3 - 6
ct{circumflex over ( )}2 kdnp kds{circumflex over ( )}3 - 6 ct
kdnp{circumflex over ( )}2 kds{circumflex over ( )}3 - 2
kdnp{circumflex over ( )}3 kds{circumflex over ( )}3 - 3
ct{circumflex over ( )}2 kdnp{circumflex over ( )}3 nnp npt + 3 ct
kdnp{circumflex over ( )}4 nnp npt + 6 kdnp{circumflex over ( )}5
nnp npt + 12 ct{circumflex over ( )}2 kdnp{circumflex over ( )}2
kds nnp npt - 3 ct kdnp{circumflex over ( )}3 kds nnp npt - 15
kdnp{circumflex over ( )}4 kds nnp npt - 15 ct{circumflex over (
)}2 kdnp kds{circumflex over ( )}2 nnp npt - 3 ct kdnp{circumflex
over ( )}2 kds{circumflex over ( )}2 nnp npt + 12 kdnp{circumflex
over ( )}3 kds{circumflex over ( )}2 nnp npt + 6 ct{circumflex over
( )}2 kds{circumflex over ( )}3 nnp npt + 3 ct kdnp kds{circumflex
over ( )}3 nnp npt - 3 kdnp{circumflex over ( )}2 kds{circumflex
over ( )}3 nnp npt - 3 ct kdnp{circumflex over ( )}3 nnp{circumflex
over ( )}2 npt{circumflex over ( )}2 + 6 kdnp{circumflex over ( )}4
nnp{circumflex over ( )}2 npt{circumflex over ( )}2 - 3 ct
kdnp{circumflex over ( )}2 kds nnp{circumflex over ( )}2
npt{circumflex over ( )}2 - 12 kdnp{circumflex over ( )}3 kds
nnp{circumflex over ( )}2 npt{circumflex over ( )}2 + 12 ct kdnp
kds{circumflex over ( )}2 nnp{circumflex over ( )}2 npt{circumflex
over ( )}2 + 3 kdnp{circumflex over ( )}2 kds{circumflex over ( )}2
nnp{circumflex over ( )}2 npt{circumflex over ( )}2 - 6 ct
kds{circumflex over ( )}3 nnp{circumflex over ( )}2 npt{circumflex
over ( )}2 + 3 kdnp kds{circumflex over ( )}3 nnp{circumflex over (
)}2 npt{circumflex over ( )}2 + 2 kdnp{circumflex over ( )}3
nnp{circumflex over ( )}3 npt{circumflex over ( )}3 - 3
kdnp{circumflex over ( )}2 kds nnp{circumflex over ( )}3
npt{circumflex over ( )}3 - 3 kdnp kds{circumflex over ( )}2
nnp{circumflex over ( )}3 npt{circumflex over ( )}3 + 2
kds{circumflex over ( )}3 nnp{circumflex over ( )}3 npt{circumflex
over ( )}3 - 6 ct{circumflex over ( )}2 kdnp{circumflex over ( )}3
ns st - 12 ct kdnp{circumflex over ( )}4 ns st - 6 kdnp{circumflex
over ( )}5 ns st + 12 ct{circumflex over ( )}2 kdnp{circumflex over
( )}2 kds ns st + 24 ct kdnp{circumflex over ( )}3 kds ns st + 12
kdnp{circumflex over ( )}4 kds ns st - 6 ct{circumflex over ( )}2
kdnp kds{circumflex over ( )}2 ns st - 12 ct kdnp{circumflex over (
)}2 kds{circumflex over ( )}2 ns st - 6 kdnp{circumflex over ( )}3
kds{circumflex over ( )}2 ns st + 6 ct kdnp{circumflex over ( )}3
nnp npt ns st - 3 kdnp{circumflex over ( )}4 nnp npt ns st - 9 ct
kdnp{circumflex over ( )}2 kds nnp npt ns st + 9 kdnp{circumflex
over ( )}3 kds nnp npt ns st + 3 ct kdnp kds{circumflex over ( )}2
nnp npt ns st - 6 kdnp{circumflex over ( )}2 kds{circumflex over (
)}2 nnp npt ns st + 3 kdnp{circumflex over ( )}3 nnp{circumflex
over ( )}2 npt{circumflex over ( )}2 ns st - 12 kdnp{circumflex
over ( )}2 kds nnp{circumflex over ( )}2 npt{circumflex over ( )}2
ns st + 3 kdnp kds{circumflex over ( )}2 nnp{circumflex over ( )}2
npt{circumflex over ( )}2 ns st + 6 ct kdnp{circumflex over ( )}3
ns{circumflex over ( )}2 st{circumflex over ( )}2 + 6
kdnp{circumflex over ( )}4 ns{circumflex over ( )}2 st{circumflex
over ( )}2 - 6 ct kdnp{circumflex over ( )}2 kds ns{circumflex over
( )}2 st{circumflex over ( )}2 - 6 kdnp{circumflex over ( )}3 kds
ns{circumflex over ( )}2 st{circumflex over ( )}2 - 3
kdnp{circumflex over ( )}3 nnp npt ns{circumflex over ( )}2
st{circumflex over ( )}2 - 3 kdnp{circumflex over ( )}2 kds nnp npt
ns{circumflex over ( )}2 st{circumflex over ( )}2 - 2
kdnp{circumflex over ( )}3 ns{circumflex over ( )}3 st{circumflex
over ( )}3){circumflex over ( )}2 + 4 (-(-ct kdnp - kdnp{circumflex
over ( )}2 + ct kds + kdnp kds - kdnp nnp npt + 2 kds nnp npt +
kdnp ns st){circumflex over ( )}2 + 3 (kdnp - kds) (ct kdnp nnp npt
- 2 ct kds nnp npt - kdnp kds nnp npt - kds nnp{circumflex over (
)}2 npt{circumflex over ( )}2 - kdnp nnp npt ns st)){circumflex
over ( )}3)){circumflex over ( )}( 1/3)}
[0168] To assess the performance of the function and how it
performs with real parameters, the manipulate function is used
(FIG. 5). For example:
Manipulate[LogLinearPlot[ct-(Cubic Root Argument noted above), {st,
1* -5, 1* 0}, PlotRange->{0, 250*10 -9}], {ct, 1* -9, 500* -9,
Appearance->"Labeled" }, {nnp, 1, 100, Appearance->"Labeled"
}, {kdnp, 0.1* -9, 7* -8, Appearance->"Labeled"}, {npt, 1* -9,
100* -9, Appearance->"Labeled" }, {kds, 0.0000001, 0.001,
Appearance->"Labeled" }, {ns, 1, 10, Appearance->"Labeled"
}]
[0169] This allows assessment of function performance as well as
allows manual estimation of initial starting parameters for
K.sub.D.
Next non-linear regression is performed. Although the root of the
function is real, it contains imaginary parts which makes the
built-in NonlinearModelFit function return an error because
rounding errors lead to the return of a complex number with an
extremely small imaginary part, which disallows correct execution
of the function.
[0170] Therefore, optimization was done semi-manually as follows
using the following standard method for least-squares error
functions:
Error = i n ( observed i - predicted i ) 2 ( Equation S4 )
##EQU00005##
[0171] To find the minimum value of this function, K.sub.D was
stepped over a range of values until the minimum was found to a
precision of 4 digits for K.sub.D. Initial choice of the range of
K.sub.D to test is guided by K.sub.D values generated by standard
EC.sub.50 analysis, for example as implemented in GraphPad
Prism.
[0172] An example of the stepping argument which generates a list
of Errors for incrementally different values of K.sub.D:
TABLE-US-00002 Do[Print[j] Print[Sum[(data[[i, 2]] -
f3[250.*{circumflex over ( )}-9, 15., 40.*{circumflex over ( )}-9,
20.*{circumflex over ( )}-9, j, 1, data[[i, 1]]]){circumflex over (
)}2, {i, 1, 48}]], {j, 0.0001, 0.0005, 0.000005}]
[0173] Where data is an array containing the experimental data with
serum concentration in column 1 and free cholesterol readings in
column 2. The argument {i, 1, 48} steps the function f3 (which
contains the binding equation solution) through the 48 data points
in this data set. The argument .omicron.j, 0.0001, 0.0005,
0.0000051 steps the value of K.sub.D from 0.0001 to 0.0005 in
0.000005 increments. A list of errors is printed and the minimum
error is found. Using this exact method, K.sub.D for serum and
ApoB-depleted serum were found as reported herein. The values found
by Equation 2 compared very favorably to these.
Example 3: Assay Measuring Patient Serum Affinity for Cholesterol
Binding
[0174] An experiment was performed to compare the methods of the
invention with currently used methods. The data suggest a
correlation between the two assays. The assay of the invention
performed consistently and in a similar manner to the currently
accepted assay.
[0175] In this experiment, 14 healthy donors donated 10 ml serum
for further study. This serum was collected in the conventional
manner using needles and a vacutainer tube. Using this serum, a
conventional cholesterol efflux assay using J774 macrophages and
radiolabled cholesterol (.sup.3H-labelled cholesterol) was
performed. (A. V. Khera, M. Cuchel, M. de la Llera-Moya, A.
Rodrigues, M. F. Burke, K. Jafri, B. C. French, J. A. Phillips, M.
L. Mucksavage, R. L. Wilensky, E. R. Mohler, G. H. Rothblat and D.
J. Rader, The New England journal of medicine, 2011, 364,
127-135.)
[0176] The radiolabelled cholesterol assay was performed as
follows:
[0177] Sample Preparation for Cholesterol Efflux Assay.
[0178] Frozen serum aliquots from participants were gently thawed
by room temperature incubation. 60 .mu.l of serum was pipetted into
a microcentrifuge tube. To precipitate ApoB-containing lipoproteins
from the sample, 24 .mu.l of PEG solution (20% w/v of Polyethylene
Glycol 8000 (Sigma-Aldrich, Saint Louis, Mo.), 200 mM glycine, pH
7.4) was added and mixed by gently flicking the tube. After a 20
minute room temperature incubation, the mixture was centrifuged at
12,700.times.g for 30 minutes at 4.degree. C. To create the final
efflux media, 50.4 .mu.l of the supernatant was then added to 1750
.mu.l MEM media with 25 mM HEPES buffer, 30 .mu.M of cAMP analog
(8-(4-Cholorphenylthio)adenosine 3',5'-cyclic monophosphate sodium
salt, Sigma-Aldrich, Saint Louis, Mo.), and 2 .mu.g/ml Acyl-CoA
Cholesterol Acyl Transferase (ACAT) inhibitor (Sandoz 58-035,
Sigma-Aldrich, Saint Louis, Mo.). This results in an efflux medium
with 2.0% final concentration of Apo-B depleted serum, in
sufficient volume for technical triplicates at 500 .mu.l each.
[0179] Efflux Assay.
[0180] Cholesterol efflux from cells to patient serum was measured
using the assay developed by Rothblat, et al. J774 mouse
macrophages were plated on 24-well plates at 150,000 cells per well
on day 1. On day 2, the cells were radiolabeled with
.sup.3H-Cholesterol at 2 .mu.Ci/ml by incubating each well in 500
ul of RPMI media with 5% FBS and .sup.3H-Cholesterol (Perkin-Elmer,
Waltham, Mass.). After overnight incubation, to upregulate
cholesterol efflux, cells were treated with 2 ug/ml ACAT inhibitor,
300 .mu.M cAMP analog in RPMI medium with 5% bovine serum albumin
for 18 hours. On day 4, the efflux assay was performed. Efflux
medium prepared from participant serum was placed on cells in
technical triplicate at a volume of 500 .mu.l per well. After four
hours, 300 ul of the medium was aspirated and filtered to remove
cellular debris. 250 ul of the filtered medium was assayed for
radioactivity by scintillation counting. Total radioactivity was
assessed by counting radioactivity in cells after isopropanol
extraction. Percent efflux for each sample was calculated as
follows: (radioactivity in the medium after efflux--radioactivity
in blank medium lacking serum)/(total radioactivity in labeled
cells). The percent efflux for the technical triplicates was
averaged together. To normalize these efflux results across sample
runs, a serum sample pool drawn from healthy volunteers was run
with each sample. Normalized efflux was calculated as percent
efflux for the sample divided by percent efflux for the sample
pool. This normalized efflux value for each participant was then
used for further association and correlation analysis.
[0181] In this example, the normalized efflux assay was plotted on
the x-axis, and could be seen to vary in magnitude among
participants from approximately 0.8 to approximately 1.4, with a
value of 1.0 being equivalent to the efflux value of the pooled
sample.
[0182] Nanoparticle Assay:
[0183] In parallel experiments, the nanoparticle-based sensor assay
discussed here was performed. In this particular experiment,
nanoparticles at a concentration of 30 uM, BODIPY-cholesterol at a
concentration of 250 nM, and Apo-B depleted serum (prepared as
described above) at a final concentration of 2% were mixed together
in a total volume of 200 ul and allowed to incubate overnight in a
96-well plate. Mixtures were prepared in duplicate for each of the
14 participants. Background control wells lacking any serum (to
measure maximum quenched signal in the presence of nanoparticle)
were also prepared. After overnight incubation, fluorescence was
read using a 96-well plate reading fluorimeter, at an excitation
wavelength of 485 nm and an emission wavelength of 520 nm.
[0184] For each sample, from these fluorescence values was
subtracted the background control fluorescence value to arrive at
signal for each serum, with higher signal reflecting enhanced
ability of the participant's serum to compete with the nanoparticle
for BODIPY-cholesterol. This value was normalized to the
background-subtracted fluorescence value of the plasma pool, and
plotted on the y-axis. It can be seen that the normalized
nanoparticle-based sensor assay value ranges from approximately 1.0
to 2.0. It can further be seen from the graph that there is a
correlation between the two assays, with an r value of 0.39, and a
slope of 0.51, and a p=0.17, which is close to statistical
significance.
[0185] The analysis method in this example is different than the
one described above. It can be readily appreciated that serum with
a greater ability to compete with nanoparticle for binding
cholesterol will at any given serum concentration be equal to that
of serum with a lesser ability to compete with nanoparticle for
binding cholesterol, lead to greater dequenching of serum
BODIPY-cholesterol binding, and lead to a higher fluorescence value
after correcting for background.
[0186] While several embodiments of the present invention have been
described and illustrated herein, those of ordinary skill in the
art will readily envision a variety of other means and/or
structures for performing the functions and/or obtaining the
results and/or one or more of the advantages described herein, and
each of such variations and/or modifications is deemed to be within
the scope of the present invention. More generally, those skilled
in the art will readily appreciate that all parameters, dimensions,
materials, and configurations described herein are meant to be
exemplary and that the actual parameters, dimensions, materials,
and/or configurations will depend upon the specific application or
applications for which the teachings of the present invention
is/are used. Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. It is, therefore, to be understood that the foregoing
embodiments are presented by way of example only and that, within
the scope of the appended claims and equivalents thereto, the
invention may be practiced otherwise than as specifically described
and claimed. The present invention is directed to each individual
feature, system, article, material, kit, and/or method described
herein. In addition, any combination of two or more such features,
systems, articles, materials, kits, and/or methods, if such
features, systems, articles, materials, kits, and/or methods are
not mutually inconsistent, is included within the scope of the
present invention.
[0187] All definitions, as defined and used herein, should be
understood to control over dictionary definitions, definitions in
documents incorporated by reference, and/or ordinary meanings of
the defined terms.
[0188] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one."
[0189] It should also be understood that, unless clearly indicated
to the contrary, in any methods claimed herein that include more
than one step or act, the order of the steps or acts of the method
is not necessarily limited to the order in which the steps or acts
of the method are recited.
[0190] In the claims, as well as in the specification above, all
transitional phrases such as "comprising," "including," "carrying,"
"having," "containing," "involving," "holding," "composed of," and
the like are to be understood to be open-ended, i.e., to mean
including but not limited to. Only the transitional phrases
"consisting of" and "consisting essentially of" shall be closed or
semi-closed transitional phrases, respectively, as set forth in the
United States Patent Office Manual of Patent Examining Procedures,
Section 2111.03.
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