U.S. patent application number 12/845177 was filed with the patent office on 2011-02-10 for biofunctionalized quantum dots for biological imaging.
This patent application is currently assigned to The Government of the United State of America, as Represented by the Secretary, Department of Health. Invention is credited to Joseph J. Barchi, JR., Serge A. Svarovsky.
Application Number | 20110033954 12/845177 |
Document ID | / |
Family ID | 34651896 |
Filed Date | 2011-02-10 |
United States Patent
Application |
20110033954 |
Kind Code |
A1 |
Barchi, JR.; Joseph J. ; et
al. |
February 10, 2011 |
BIOFUNCTIONALIZED QUANTUM DOTS FOR BIOLOGICAL IMAGING
Abstract
Novel biofunctionalized quantum dots include a mercaptoalkanoic
acid linked to the surface of a nanocrystalline core and a
biofunctional group linked to the surface. Biofunctionalized
quantum dots are made by a novel synthesis method.
Biofunctionalized quantum dots can be used in imaging or therapy
applications.
Inventors: |
Barchi, JR.; Joseph J.;
(Frederick, MD) ; Svarovsky; Serge A.; (North
Potomac, MD) |
Correspondence
Address: |
Woodcock Washburn LLP;Ott-NIH
Cira Centre, 12th Floor, 2929 Arch Street
Philadelphia
PA
19104
US
|
Assignee: |
The Government of the United State
of America, as Represented by the Secretary, Department of
Health
Rockville
MD
|
Family ID: |
34651896 |
Appl. No.: |
12/845177 |
Filed: |
July 28, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10578405 |
May 5, 2006 |
7790473 |
|
|
PCT/US03/34897 |
Nov 5, 2003 |
|
|
|
12845177 |
|
|
|
|
Current U.S.
Class: |
436/529 ;
977/774; 977/906 |
Current CPC
Class: |
A61K 2039/55555
20130101; A61K 39/0011 20130101; A61K 49/0058 20130101; A61K
49/0052 20130101; A61K 49/0067 20130101; B82Y 5/00 20130101 |
Class at
Publication: |
436/529 ;
977/906; 977/774 |
International
Class: |
G01N 33/544 20060101
G01N033/544 |
Claims
1. A biofunctionalized quantum dot, comprising: a nanocrystalline
core exhibiting quantum confinement and having a band gap and a
surface; a mercaptoalkanoic acid linked to the surface; and a
biofunctional group linked to the surface, wherein the
biofunctional group comprises a saccharide or the mercaptoalkanoic
acid is linked to the surface of the nanocrystalline core without a
shell layer.
2. The biofunctionalized quantum dot of claim 1, the
mercaptoalkanoic acid having exactly one carboxyl group and
comprising less than seven carbon atoms.
3. The biofunctionalized quantum dot of claim 1, the
mercaptoalkanoic acid comprising mercaptoacetic acid.
4. The biofunctionalized quantum dot of claim 1, further
comprising: a shell layer overcoating the nanocrystalline core.
5. The biofunctionalized quantum dot of claim 4, the shell layer
comprising cadmium sulfide or mercury sulfide; and the
nanocrystalline core comprising cadmium telluride or cadmium
selenide or mercury telluride or mercury selenide.
6. The biofunctionalized quantum dot of claim 1, the saccharide not
comprising mannose or dextran.
7. The biofunctionalized quantum dot of claim 1, the saccharide
being selected from the group consisting of a tumor-associated
antigen and Thomsen-Friedenreich disaccharide.
8. The biofunctionalized quantum dot of claim 1, the saccharide
linked to a sulfur atom; and the sulfur atom linked to the surface
of the nanocrystalline core.
9. The biofunctionalized quantum dot of claim 1, the saccharide
linked to a linking group; the linking group linked to a sulfur
atom; and the sulfur atom linked to the surface of the
nanocrystalline core.
10. The biofunctionalized quantum dot of claim 9, the linking group
comprising a carbon atom.
11. The biofunctionalized quantum dot of claim 1, wherein the
biofunctionalized quantum dot is stable in aqueous solution under
storage in the dark at 4.degree. C. for at least 4 months with
respect to luminescence, precipitation, flocculation, and leaching
of the biofunctional group.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 10/578,405, "Biofunctionalized Quantum Dots
for Biological Imaging", filed May 5, 2006, now allowed, which
claims the benefit of PCT/US2003/034897, "Biofunctionalized Quantum
Dots for Biological Imaging", filed Nov. 5, 2003, the entirety of
each application is incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to biofunctionalized quantum
dots, which can be used, for example, in biological research,
medical research, medical imaging, and medical therapy.
[0003] Quantum dots are small semiconductor particles that exhibit
quantum confinement. See "Overview," Quantum Dot Corp., (2003)
http://www.qdots.com/new/technology/overview.html. A semiconductor
has a characteristic band gap, which is the difference in energy
between an electron in the valence band and an electron in the
conduction band of the semiconductor material. When energy is
applied to the material, for example in the form of a photon having
a quantum of energy greater than or equal to the band gap, an
electron can be stimulated to jump from the valence band to the
conduction band. The missing electron in the valence band is
referred to as a "hole". See H. B. Gray, "Chemical Bonds," (W. A.
Benjamin, Inc., 1973), pp. 208-218. When an electron falls back
into a "hole" in the valence band, a photon having a quantum of
energy equal to the band gap, and thus a particular wavelength, can
be emitted. Thus, materials in which high energy photons can cause
electrons to jump into the conduction band, after which electrons
can fall back into the valence band, emitting a photon, can exhibit
the phenomenon of fluorescence. See A. E. Siegman, "Lasers,"
University Science Books, 1986), pp. 6-15.
[0004] Quantum confinement refers to a phenomenon observed when the
physical size of the semiconductor is smaller than the typical
radius of the electron-hole pair (Bohr radius). In this case, the
wavelength of light emitted through electron-hole recombination is
shorter than the wavelength of light emitted by the semiconductor
in bulk. The wavelength of light emitted by a semiconductor
exhibiting quantum confinement can be termed the characteristic
wavelength. Quantum dots can be made to fluoresce at their
characteristic wavelength by exposing them to light having a
wavelength shorter than the characteristic wavelength. The
wavelength of light emitted is dependent on the size of the quantum
dot: a smaller size results in a shorter wavelength. Therefore, the
characteristic wavelength of a quantum dot can be "tuned" by
adjusting the size of the quantum dot. Furthermore, techniques
exist for producing quantum dots with narrow monodispersity in
size, so that the light emitted from a number of quantum dots has a
narrow bandwidth. See "Overview," Quantum Dot Corp., (2003)
http://www.qdots.com/new/technology/overview.html.
[0005] The essential part of a quantum dot is a nanocrystalline
core, a semiconductor in a crystalline state which has a
characteristic size of from about 1 to about 100 nm. Quantum dots
used for their fluorescing properties can have a size range of from
about 1 to about 10 nm. See "Anatomy", Quantum Dot Corp., (2003)
http://www.qdots.com/new/technology/dottech.html.
[0006] The quantum efficiency refers to the ratio of the number of
photons emitted to the number of photons to which the quantum dot
is exposed and which stimulate light emission.
[0007] To increase the quantum efficiency of a nanocrystalline
core, and thereby enhance the intensity of fluorescence, the
nanocrystalline core can be overcoated with a shell layer of a
semiconductor material which has a band gap greater than the band
gap of the nanocrystalline core. Bawendi et al, U.S. Pat. No.
6,306,610. A shell layer can also serve to protect the
nanocrystalline core from the surrounding environment. If
protection of the nanocrystalline core from the environment is
important, but enhancement of quantum efficiency is not, a
non-semiconductor material can be used for the shell layer. A
quantum dot having both a nanocrystalline core and a shell layer
can be referred to as a core/shell quantum dot.
[0008] Chemical groups, including chemical groups which have an
effect on a biological system, can be bound to the surface of a
quantum dot. This capacity to be functionalized, together with
chemical stability and tunable fluorescing properties, makes
quantum dots of great interest in the development of new materials
and techniques for biological research and medical diagnosis.
Furthermore, quantum dots are much less prone to photobleaching
than many conventional dyes.
[0009] For most biological or medical applications, in order to be
useful, a quantum dot must be rendered hydrophilic and have a
biofunctional group attached to its surface. Chan and Nie linked
mercaptoacetic acid to cadmium selenide core/zinc sulfide shell
quantum dots. They bonded the protein transferrin to the linked
mercaptoacetic acid groups by using
ethyl-3-(dimethylaminopropyl)carbodiimide. Chan and Nie found that
the transferrin linked to the quantum dot was recognized by
receptors on a cell surface. See Chan and Nie, "Quantum Dot
Bioconjugates for Ultrasensitive Nonisotopic Detection", Science,
v. 281 (1998) p. 2016.
[0010] Akerman et al. used cadmium selenide core/zinc sulfide shell
quantum dots coated with trioctylphosphine (TOPO), rendered them
water soluble, and coated them with mercaptoacetic acid. Thiolated
peptides were then linked to the surface of the quantum dots.
Akerman et al. also made quantum dots in which thiolated
polyethylene glycol and thiolated peptides were linked to
mercaptoacetic acid coated quantum dots. They found that the
peptide-functionalized quantum dots coupled with corresponding
peptide receptors expressed by cells. See Akerman et al.,
"Nanocrystal targeting in vivo", Proc. National Academy of
Sciences, v. 99(2) (2002) p. 12617.
[0011] Larson et al. encapsulated a cadmium selenide core/zinc
sulfide shell quantum dot within a amphiphilic polymer to render
the quantum dot hydrophilic. They were able to image fluorescing
quantum dots through the skin. Larson et al. suggested that the
cadmium selenide core/zinc sulfide shell quantum dots leave the
body before breakdown because there were no noticed toxic effects
from the cadmium on mice into which they were injected. See Larson
et al., "Water-Soluble Quantum Dots for Multiphoton Fluorescence
Imaging in Vivo", Science, v. 300 (2003) p. 1434.
[0012] Semiconductor nanocrystals can attach trioctylphosphine
oxide (TOPO) as a ligand, rendering the semiconductor nanocrystals
soluble in organic solvents such as chloroform and toluene, but not
soluble in polar solvents such as water and ethanol. In an
approach, a cadmium selenide core/zinc sulfide shell quantum dot
was first coordinated with TOPO. Molecules in which mannose groups
were covalently bonded to a phosphine oxide were then used to
replace the TOPO groups on the cadmium selenide core/zinc sulfide
shell, rendering the quantum dot hydrophilic. See Tamura et al.,
"Synthesis of Hydrophilic Ultrafine Nanoparticles Coordinated with
Carbohydrate Cluster", J. Carbohydrate Chemistry, v. 21(5) (2002)
p. 445. However, it is doubtful whether the functionalized quantum
dots produced were stable. In another approach, cadmium selenide
core/zinc sulfide shell structures coordinated with TOPO were
treated with a silathiane and mercaptosuccinic acid. The quantum
dots were treated with a solutions of carboxymethyl dextran and of
polylysine and treated with 1-ethyl-3-(3)-dimethylaminopropyl
carbodiimide, which acts as a crosslinking agent. See Chen et al.,
"Synthesis of Glyconanospheres Containing Luminescent CdSe--ZnS
Quantum Dots", Nano Letters, v. 3(5) (2003) p 581.
[0013] The applicants attempted to displace a TOPO layer on a
cadmium selenide core/zinc sulfide shell quantum dot commercially
available from Evident Technologies with a hydrophilic thiol
compound using the modified phase-transfer procedure developed by
Wang et al. See Wang et al., J. Am. Chem. Soc., v. 106 (2002) p.
2293. However, either the displacement was incomplete or the
resultant functionalized quantum dots were fragile and did not
survive mild ultrafiltration or dialysis and precipitated or
flocculated shortly after the hydrophilic thiol compound was
removed from the solution.
[0014] Bawendi et al. functionalized quantum dots with proteins and
with oligonucleotides. The procedure used started with TOPO-capped
cadmium selenide core/zinc sulfide shell quantum dots with which
the proteins or oligonucleotides were linked. Bawendi et al., U.S.
Pat. No. 6,306,610.
[0015] Gaponik et al. synthesized hydrophilic cadmium telluride
core/cadmium sulfide shell quantum dots using an aqueous synthesis
approach. In the approach, a cadmium salt and a mercapto-compound
were mixed in an aqueous solution through which hydrogen telluride
was bubbled. Cadmium telluride nanocrystals were formed which were
capped at the surface with the mercapto compound. The
mercapto-compound was linked to the cadmium telluride core through
the sulfur atom. Thus, the cadmium telluride core was understood to
be surrounded by a layer of sulfur atoms, which also were present
deeper in the core, and which bonded to the cadmium atoms to form a
cadmium sulfide shell layer. The hydrophilic cadmium telluride
core/cadmium sulfide shell quantum dots exhibited good
photostability; i.e., fluoresced over a long duration of
illumination. Gaponik et al., "Thiol-Capping of CdTe Nanocrystals:
An alternative to Organometallic Synthetic Routes", J. Phys. Chem.
B, v. 106 (2002) p. 7177.
[0016] For a preparation of quantum dots with biofunctional groups
linked to their surfaces to be useful in biological research,
medical diagnostic, and medical therapeutic applications, the
quantum dots must fluoresce brightly, be hydrophilic, and be stable
in water not containing excess biofunctional groups for prolonged
periods of time.
[0017] Coupling of receptors to cell-surface saccharides mediates
many relevant biological processes, including differentiation,
motility, adhesion, tumor progression, and metastasis. Therefore,
quantum dots functionalized with saccharides are of interest for
biological research, medical diagnostic, and medical therapeutic
applications. However, quantum dots suitable for such applications
have up until now not been developed.
[0018] There thus remains a need for quantum dots which fluoresce
brightly, have biofunctional groups linked to their surfaces, are
hydrophilic, and are stable in aqueous solution. There is also a
continuing need for quantum dots which have saccharides linked to
their surfaces.
SUMMARY OF THE INVENTION
[0019] It is therefore an object of the present invention to
provide novel biofunctionalized quantum dots which fluoresce
brightly, are hydrophilic, and are stable in aqueous solution. It
is further an object of the present invention to provide quantum
dots which have saccharides linked to their surfaces.
[0020] An embodiment of a biofunctionalized quantum dot according
to the invention includes a nanocrystalline core exhibiting quantum
confinement and having a band gap and a surface, a mercaptoalkanoic
acid linked to the surface, and a biofunctional group linked to the
surface. The ratio of mercaptoalkanoic acid molecules to
biofunctional group molecules linked to the surface can be in the
range of from about 1:1 to about 5:1. The mercaptoalkanoic acid can
be chosen from a set of mercaptoalkanoic acids not including
mercaptosuccinic acid. The mercaptoalkanoic acid can be chosen to
have only one carboxyl group and comprising less than seven carbon
atoms. The mercaptoalkanoic acid can be mercaptoacetic acid.
[0021] In an embodiment, the biofunctional group is chosen to have
a molecular weight greater than a molecular weight of the
mercaptoalkanoic acid. The biofunctional group can be chosen to
have a molecular volume greater than a molecular volume of the
mercaptoalkanoic acid.
[0022] In another embodiment of a biofunctionalized quantum dot
according to the invention, a shell layer overcoats a
nanocrystalline core. The shell layer can include cadmium sulfide
and the nanocrystalline core can include cadmium telluride, cadmium
selenide, mercury telluride, and mercury selenide.
[0023] The biofunctional group on a quantum dot according to the
invention can be a saccharide. For example, the saccharide can be a
tumor-associated carbohydrate antigen. The saccharide can be
Thomsen-Friedenreich disaccharide. The biofunctional group on a
quantum dot according to the invention can be chosen from a set of
saccharides not comprising mannose or dextran. The saccharide can
be directly linked to a sulfur atom, the sulfur atom being linked
to the surface of the nanocrystalline core. The saccharide can be
linked to a linking group, the linking group linked to a sulfur
atom, and the sulfur atom linked to the surface of the
nanocrystalline core. The linking group can include a carbon
atom.
[0024] In another embodiment, the biofunctionalized quantum dot is
stable in aqueous solution under storage in the dark at 4.degree.
C. for at least 4 months with respect to luminescence,
precipitation, flocculation, and leaching of the biofunctional
group.
[0025] In an embodiment, a formulation includes a liquid, a
biofunctionalized quantum dot, a mercaptoalkanoic acid linked to
the surface of the nanocrystalline core of the quantum dot, and a
biofunctional group linked to the surface and the biofunctionalized
quantum dot is dissolved or suspended in the liquid and does not
precipitate or flocculate.
[0026] In an embodiment, a biofunctionalized quantum dot is made by
refluxing a biofunctional group-thiol of Formula III with a cadmium
salt, hydrogen-alkali-telluride or hydrogen-alkali-selenide, and a
suitable solvent to produce a quantum dot in a solution. The
R.sub.1 group includes at least one carbon atom. Suitable solvents
include water and N,N-dimethylformamide (DMF). The refluxing can be
conducted in a range of from about 24 to about 48 hours. The
refluxed mixture can further include a mercaptoalkanoic acid, for
example, mercaptoacetic acid. The biofunctional group can be a
saccharide, for example, Thomsen-Friedenreich disaccharide. The
refluxing can be carried out with Thomsen-Friedenreich disaccharide
and mercaptoacetic acid in a molar ratio of from about 1:1 to about
5:1. After refluxing, the solution can be purified and dried to
obtain a biofunctionalized quantum dot preparation. The purifying
can include separating the biofunctionalized quantum dot from the
remainder of the solution by filtration through an ultrafiltration
membrane with a cutoff of about 50 kilodaltons. The purified and
dried biofunctionalized quantum dot preparation can be dissolved or
suspended in an aqueous solvent.
##STR00001##
[0027] A biofunctional group-thiol of Formula III can be made by
reacting a glycoside of Formula I with an alkylthio acid in the
presence of a catalyst to produce a thioester of Formula II,
debenzylidenating the thioester of Formula II, and hydrolyzing the
thioester of Formula II to produce the biofunctional group-thiol of
Formula III; the group R.sub.2 includes at least one carbon
atom.
##STR00002##
[0028] In an embodiment, a biofunctionalized quantum dot is made as
follows. A glycoside of Formula IV is reacted with an alkylthio
acid in the presence of 2,2'-azobisisobutyronitrile in 1,4-dioxane
at about 75.degree. C. to produce a thioester of Formula V,
debenzylidinating the thioester of Formula V. The thioester of
Formula V is debenzylidinated and the debenzylidinated thioester of
Formula V is hydrolyzed to produce a Thomsen-Friedenreich-thiol of
Formula VI. The Thomsen-Friedenreich-thiol of Formula VI is
refluxed with cadmium perchlorate, mercaptoacetic acid, hydrogen
sodium telluride, and a suitable solvent, either water or
N,N-dimethylformamide, to produce a
Thomsen-Friedenreich-functionalized quantum dot in a solution.
##STR00003##
[0029] The debenzylidination step can include treating the
thioester of Formula V with aqueous acetic acid at about 60.degree.
C. and evaporating to obtain the debenzylidinated thioester.
Alternatively, the debenzylidination step can include treating the
thioester of Formula V with acetyl chloride in methanol, adding
pyridine to the thioester of Formula V with acetyl chloride in
methanol for quenching the reaction, and evaporating to obtain
debenzylidinated thioester. The hydrolyzing step can include
treating the debenzylidinated thioester with sodium methoxide in
methanol to produce the Thomsen-Friedenreich-thiol of Formula VI.
Alternatively, the hydrolyzing step can include treating the
debenzylidinated thioester with sodium methoxide in methanol while
bubbling air through the debenzylidinated thioester, sodium
methoxide, and methanol to produce a Thomsen-Friedenreich-disulfide
of Formula VII and treating the Thomsen-Friedenreich-disulfide of
Formula VII with dithiothreitol in water to produce the
Thomsen-Friedenreich-thiol of Formula VI.
##STR00004##
[0030] In an embodiment, a biofunctionalized quantum dot is used
for imaging. The biofunctionalized quantum dot, of which the
biofunctional group includes a saccharide, or which includes a
mercaptoalkanoic acid linked to the nanocrystalline surface, is
contacted with a biological material. The biological material is
exposed to light having a wavelength effective to cause the quantum
dot to fluoresce and the fluorescing quantum dots are imaged. The
biofunctional group can be Thomsen-Friedenreich disaccharide. The
biological material can include a cell culture or can include a
tissue. The biofunctionalized quantum dot can be dissolved or
suspended in a biocompatible aqueous solvent. Contacting the
biofunctionalized quantum dot with biological material can included
injecting the biofunctionalized quantum dot into tissues of a
living animal.
[0031] The fluorescing quantum dot adhered to secretions of the
biological material can be imaged. Tissue which imaging identifies
as tissue to which the biofunctional group exhibits high affinity
can be identified as tissue in a diseased or abnormal state, for
example, a cancerous state.
[0032] In an embodiment, several types of biofunctionalized quantum
dots are used for imaging. The biofunctional groups of the
biofunctionalized quantum dots include a saccharide, or the
biofunctionalized quantum dots include a mercaptoalkanoic acid
linked to the nanocrystalline surface. Each type of
biofunctionalized quantum dot has a characteristic wavelength
distinct from the other types. Each type of quantum dot is
functionalized with a different antigen or a different set of
antigens. The several types of biofunctionalized quantum dots are
contacted with a biological material, the biological material is
exposed to light having a wavelength effective to cause the quantum
dots to fluoresce, and the fluorescing quantum dots are imaged.
[0033] In an embodiment, a biofunctionalized quantum dot is used
for therapy. The biofunctional group of the biofunctionalized
quantum dot includes a saccharide, or the biofunctionalized quantum
dot includes a mercaptoalkanoic acid linked to the nanocrystalline
surface. The biofunctionalized quantum dot is contacted with a
biological material and thereby treats a disease. The biofunctional
group can be an immune-response-stimulating group. The
biofunctional group can be a tumor-associated antigen. The
biofunctional group can be Thomsen-Friedenreich disaccharide. The
contacting with a biological material can include injecting the
biofunctionalized quantum dot into tissues of a living animal in
order to treat cancer.
[0034] A biofunctionalized quantum dot used for therapy can have a
therapeutic agent linked to the surface. The shell layer or the
nanocrystalline shell of a biofunctionalized quantum dot used for
therapy can include a therapeutic agent.
[0035] In an embodiment, a biofunctionalized quantum dot is used to
coat a device which, when not coated, is in contact with a
biological material. The biofunctional group of the
biofunctionalized quantum dot includes a saccharide, or the
biofunctionalized quantum dot includes a mercaptoalkanoic acid
linked to the nanocrystalline surface.
[0036] In an embodiment, a cell-quantum dot complex includes a
biofunctionalized quantum dot linked to a cell. The biofunctional
group of the biofunctionalized quantum dot includes a saccharide,
or the biofunctionalized quantum dot includes a mercaptoalkanoic
acid linked to the nanocrystalline surface. The biofunctional group
can be Thomsen-Friedenreich disaccharide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1 is a schematic of cadmium telluride nanocrystal
functionalized with mercaptoacetic acid and with a biofunctional
group-thiol.
[0038] FIG. 2 is a graph of the absorption spectra of growing
Thomsen-Friedenreich-functionalized cadmium telluride quantum dots
at different times.
[0039] FIG. 3 shows the NMR spectra of a Thomsen-Friedenreich-thiol
and of Thomsen-Friedenreich-functionalized cadmium telluride
quantum dots.
[0040] FIG. 4 shows the NMR spectra of mercaptoacetic acid, of a
Thomsen-Friedenreich-thiol, and of
Thomsen-Friedenreich-mercaptoacetic-acid-functionalized cadmium
telluride quantum dots.
[0041] FIG. 5 shows the absorption spectrum of
Thomsen-Friedenreich-mercaptoacetic-acid-functionalized cadmium
telluride quantum dots.
DETAILED DESCRIPTION
[0042] Embodiments of the invention are discussed in detail below.
In describing embodiments, specific terminology is employed for the
sake of clarity. However, the invention is not intended to be
limited to the specific terminology so selected. A person skilled
in the relevant art will recognize that other equivalent components
can be employed and other methods developed without parting from
the spirit and scope of the invention. All references cited herein
are incorporated by reference as if each had been individually
incorporated.
[0043] In an embodiment of a biofunctionalized quantum dot, a
biofunctional group is linked to the surface of a nanocrystalline
core exhibiting quantum confinement. Examples of core materials
included in the nanocrystalline core include zinc sulfide, zinc
selenide, zinc telluride, cadmium sulfide, cadmium selenide,
cadmium telluride, mercury sulfide, mercury selenide, mercury
telluride, magnesium telluride, aluminum phosphide, aluminum
arsenide, aluminum antimonide, gallium nitride, gallium phosphide,
gallium arsenide, gallium antimonide, indium nitride, indium
phosphide, indium arsenide, indium antimonide, aluminum sulfide,
lead sulfide, lead selenide, germanium, or silicon. Core materials
also include other group II-group VI compounds, group III-group V
compounds, and group IV compounds. Core materials also include
other semiconductor materials. The core material may also be formed
of an alloy, compound, or mixture of these compounds and elements
which are suitable core materials. For example, the core material
can be a mercury-cadmium sulfide compound. The core material can
also be doped with one or more suitable dopants.
[0044] In an embodiment, a biofunctionalized quantum dot includes a
shell layer overcoating and surrounding a nanocrystalline core. The
shell layer can include a single layer of a shell material
different from the core material which forms the nanocrystalline
core. The shell layer can include a semiconductor material with a
band gap greater than the band gap of the nanocrystalline core.
Examples of shell materials included in the shell layer include
zinc oxide, zinc sulfide, zinc selenide, zinc telluride, cadmium
oxide, cadmium sulfide, cadmium selenide, cadmium telluride,
mercury oxide, mercury sulfide, mercury selenide, mercury
telluride, magnesium telluride, aluminum nitride, aluminum
phosphide, aluminum arsenide, aluminum antimonide, gallium nitride,
gallium phosphide, gallium arsenide, gallium antimonide, indium
nitride, indium phosphide, indium arsenide, indium antimonide,
aluminum sulfide, lead sulfide, lead selenide, germanium, or
silicon. Shell materials also include other group II-group VI
compounds, group III-group V compounds, and group IV compounds.
Shell materials also include other semiconductor materials. The
shell material may also be formed of an alloy, compound, or mixture
of these compounds and elements which are suitable shell materials.
The term quantum dot may refer to a nanocrystalline core without a
shell layer, or a to the composite structure of a nanocrystalline
core with a shell layer. The core material can also be doped with
one or more suitable dopants.
[0045] A shell layer can include a single layer of the atoms which
form the shell material. For example, a cadmium selenide or cadmium
telluride nanocrystalline core can be overcoated with a cadmium
sulfide shell. The cadmium sulfide shell can be formed of sulfur
atoms bonded to cadmium atoms on the surface of or within the
cadmium selenide or cadmium telluride nanocrystalline core. As
another example, a mercury selenide or mercury telluride
nanocrystalline core can be overcoated with a mercury sulfide
shell. The mercury sulfide shell can be formed of sulfur atoms
bonded to mercury atoms on the surface of or within the mercury
selenide or mercury telluride nanocrystalline core.
[0046] A quantum dot is biofunctionalized when the quantum dot has
molecules, referred to as biofunctional groups, linked to its
surface which act to change the response of a biological system
from that resulting from contact with a non-functionalized
nanocrystalline core or shell. The term "link" refers to an
attractive association of an atom or molecule with another atom or
molecule, for example, a covalent bond, an ionic bond, a hydrogen
bond, or a bond or interaction of another type. As an example,
biofunctional groups may be attached to the surface of a
nanocrystalline core or a shell which stimulate an immunological
response, allow the quantum dot as a whole to adhere to biological
material or secretions of the biological material, e.g.,
antibodies, and render the quantum dot as a whole biologically
inert so that the biological system does not "see" the quantum dot
and does not respond. A biofunctional group which stimulates an
immunological response can be referred to as an
immune-response-stimulating group.
[0047] In another embodiment, a biofunctional group is linked to
the surface of a nanocrystalline core and a mercaptoalkanoic acid
is linked to the surface of the nanocrystalline core. In an
embodiment, the mercaptoalkanoic acid has one mercapto group, one
carboxyl group and from one to six carbon atoms. For example, the
mercaptoalkanoic acid can be mercaptoacetic acid.
[0048] The biofunctional group can be directly linked to the
nanocrystalline core, or it can be linked to a shell layer which
overcoats the nanocrystalline core. Certain saccharides are
biofunctional groups. In this application, the term "saccharide"
refers to mono-, di-, tri-, and oligosaccharides. The saccharide
can be a saccharide found in nature, or can be a saccharide which
is not found in nature. A saccharide may be, for example, an
antigen found on the membrane of a tumor cell or a bacterium. For
example, Thomsen-Friedenreich disaccharide is found on the surface
of many human cancer cells but not on the surface of normal human
cells. A saccharide found on the surface of cancer cells, but not
on the surface of normal human cells can be referred to as a
tumor-associated carbohydrate antigen.
[0049] A biofunctional group can be directly linked to a
nanocrystalline core or a shell layer. A biofunctional group can
also be linked to an atom which has high affinity for or integrates
with a nanocrystalline core or a shell layer so that the
biofunctional group is linked through the atom to the
nanocrystalline core or shell layer. A biofunctional group can also
be linked to a "linking group", which is in turn linked to the
nanocrystalline core or the shell layer. A linking group may play a
number of roles. For example, a linking group may act as a "spacer"
between the nanocrystalline core or shell layer and the
biofunctional group so that the biofunctional group can assume a
conformation required to stimulate or suppress the response of a
biological system as desired. A linking group can also act to
separate charge in or on the nanocrystalline core or shell layer
from the biofunctional group. A linking group can facilitate a
method of linking a biofunctional group to a nanocrystalline core
or shell layer. A biofunctional group can be linked to a linking
group, the linking group in turn linked to an atom which has a high
affinity for and thus links to the nanocrystalline core or shell
layer or which integrates with the nanocrystalline core or shell
layer. For example, a biofunctional group can be linked to a sulfur
atom and the sulfur atom in turn linked to the surface of a
nanocrystalline core. As another example, a saccharide which is a
biofunctional group can be linked to a linking group comprising a
chain of at least one carbon atom. The linking group can in turn be
linked to a sulfur atom. The sulfur atom can then be linked to a
nanocrystalline core, for example, a cadmium selenide or cadmium
telluride nanocrystalline core. In an embodiment, a
Thomsen-Friedenreich disaccharide is covalently bonded to a chain
of five carbon atoms, which is in turn bonded to a sulfur atom,
which is in turn bonded to a nanocrystalline core of cadmium
telluride or cadmium selenide.
[0050] In an embodiment, a quantum dot is functionalized with a
biofunctional group and with a mercaptoalkanoic acid. The
biofunctional group and the mercaptoalkanoic acid are selected so
that the biofunctional group has a molecular weight greater than
the molecular weight of the mercaptoalkanoic acid. Alternatively,
the biofunctional group and the mercaptoalkanoic acid are selected
so that the biofunctional group has a molecular volume greater than
the molecular volume of the mercaptoalkanoic acid. Such a selection
of the biofunctional group and the mercaptoalkanoic acid can be
made to ensure that the mercaptoalkanoic acid groups on the surface
of the quantum dot do not shield or screen the biofunctional groups
from the environment, for example, from molecules or structures in
a biological material, such as in a living animal.
[0051] An embodiment of a method for making a biofunctionalized
quantum dot is now described. A biofunctional group-thiol of
Formula III, in which R.sub.1 represents a group containing one or
more carbon atoms, can be refluxed with a cadmium salt, e.g.,
cadmium perchlorate, a hydrogen alkali telluride, e.g., hydrogen
sodium telluride, and a suitable solvent, e.g., water or
N,N-dimethylformamide, to produce a quantum dot in which the
biofunctional group-thiol of Formula III is linked to the surface
of a nanocrystal of cadmium telluride. A hydrogen alkali selenide,
e.g., hydrogen alkali selenide, can be used instead of a hydrogen
alkali telluride to produce a quantum dot in which the
biofunctional group-thiol is linked to the surface of a nanocrystal
of cadmium selenide. In an embodiment, the biofunctional
group-thiol of Formula III can be a Thomsen-Friedenreich-thiol. In
general, the longer refluxing is conducted, the larger the
biofunctionalized quantum dots produced will be. In an embodiment,
refluxing is conducted for a duration of from about 24 hours to
about 48 hours. For example, refluxing can be conducted for 39
hours.
##STR00005##
[0052] In another embodiment, the mixture which is refluxed also
contains a mercaptoalkanoic acid, e.g., mercaptoacetic acid. A
biofunctionalized quantum dot is thereby formed in which the
biofunctional group-thiol and a mercaptoalkanoic acid group are
linked to the surface of a nanocrystal of cadmium telluride when a
hydrogen alkali telluride is used, as shown in FIG. 1. The
biofunctional group-thiol and a mercaptoalkanoic acid group can
also be linked to the surface of a nanocrystal of cadmium selenide
when a hydrogen alkali selenide is used. In an embodiment, the
biofunctional group is Thomsen-Friedenreich disaccharide, the
mercaptoalkanoic acid is mercaptoacetic acid, and the
Thomsen-Friedenreich-thiol and the mercaptoacetic acid are present
in a molar ratio of from about 1:1 to about 5:1 in the mixture. For
example, they can be in a molar ratio of about 3.4:1.
[0053] In an embodiment, the biofunctional group-thiol of Formula
III can be formed by reacting a glycoside of Formula I with a
alkylthio acid in the presence of a catalyst to produce a thioester
of Formula II, in which R.sub.2 represents a group containing one
or more carbon atoms. The thioester of Formula II can then be
debenzylidinated and hydrolyzed to produce the biofunctional
group-thiol of Formula III in solution. In an embodiment, the
glycoside can be selected to produce a Thomsen-Friedenreich-thiol
for the compound of Formula III.
##STR00006##
[0054] In an embodiment, the solution containing biofunctionalized
quantum dots illustrated in FIG. 1 can be purified, and the
purified solution can be dried to isolate a preparation of
biofunctional group-functionalized quantum dots. For example, the
solution can be filtered through a membrane with a cutoff in the
range of 10 to 100 kilodaltons. The cutoff can be selected so that
only the desired quantum dots less than a certain size pass through
and larger quantum dots and particles are retained; in this case
the permeate passing through the filter is dried to obtain isolated
biofunctionalized quantum dots. Alternatively, the cutoff can be
selected so that the desired quantum dots of greater than a certain
size are retained and smaller quantum dots and particles pass
through; in this case the retentate retained by the filter is dried
to isolate biofunctionalized quantum dots. The solution containing
the quantum dots can also be forced through a filter with a larger
cutoff, the permeate then passed through a filter with a smaller
cutoff, and the retentate of the filter with the smaller cutoff
then dried to isolate biofunctionalized quantum dots. Membranes of
various types can be used, for example, an ultrafiltration membrane
can be used or a dialysis membrane can be used. As an example, the
solution containing the quantum dots can be passed through an
ultrafiltration membrane with a cutoff of about 50 kilodaltons and
the retentate dried to isolate biofunctionalized quantum dots. The
isolated biofunctionalized quantum dots can be redissolved or
resuspended in an aqueous solvent, for example, a biocompatible
aqueous solvent, for further use. A biocompatible aqueous solvent
could be a solvent containing components in addition to water and
the quantum dots which improve the performance of the
water-dissolved or water-suspended quantum dots when they are
applied to a biomaterial. For example, a biocompatible aqueous
solvent may be adjusted to have similar salinity and pH as a tissue
into which it is to be injected.
[0055] In an embodiment, a biofunctionalized quantum dot is linked
to a cell to form a cell-quantum dot complex. For example, the
biofunctional group on the quantum dot may act as a ligand which
couples with a receptor on the surface of a cell. The biofunctional
group on the quantum dot can be, for example, a saccharide, such as
Thomsen-Friedenreich disaccharide. For example, the
Thomsen-Friedenreich disaccharide may act as a ligand which couples
with a receptor protein, galectin-3, on an endothelial cell. In
addition to a biofunctional group, the quantum dot may have other
groups on its surface, such as a mercaptoalkanoic acid, e.g.,
mercaptoacetic acid.
[0056] In an embodiment, the biofunctionalized quantum dots are in
the form of a formulation. Such a formulation includes a liquid and
biofunctionalized quantum dots dissolved or suspended in the liquid
so that the solution or suspension does not precipitate or
flocculate. The biofunctionalized quantum dots according to the
invention, when mixed with water, form a solution which is clear,
although it may be colored. Thus it appears that the quantum dots
dissolve in water. However, the literature on hydrophilic quantum
dots often refers to a suspension of quantum dots, it may be that
although when mixed with water, the resultant composition is clear,
the term "suspension" is used because of the greater size of
quantum dots with respect to low molecular weight molecules.
[0057] In an embodiment, the biofunctionalized quantum dots in a
formulation have a mercaptoalkanoic acid, e.g., mercaptoacetic
acid, linked to their surfaces. The biofunctional group can be a
saccharide, for example, Thomsen-Friedenreich disaccharide.
[0058] Biofunctionalized quantum dots can be used in systems for
assessing characteristics of a biological material. For example,
biofunctionalized quantum dots can be used to diagnose disease
states of tissue. Such tissue could be evaluated in vivo, i.e.,
while still in an organism, or in vitro, e.g., a biopsy sample
could be evaluated. A biological material may either be living,
i.e., exhibiting metabolism, or nonliving. A non-exhaustive list of
examples of biological materials include isolated cells, a number
of cells which do not act cooperatively, cells in a cell culture,
cells in or removed from a multicellular organism, e.g., an animal,
tissue in or removed from a multicellular organism, e.g., portions
of organs such as liver, structures in or removed from an organism,
e.g., hair, contents of cells, and material secreted by cells or by
an organism, e.g., serum, mucus, proteins, or antibodies.
[0059] Biofunctionalized quantum dots can be used in biological or
medical imaging applications. In an embodiment, a biofunctionalized
quantum dot is contacted with a biological material. The
biofunctionalized quantum dots and biological material are then
exposed to light having a wavelength effective to cause the quantum
dot to fluoresce, i.e., light with a wavelength shorter than the
characteristic wavelength of the quantum dot. The biofunctionalized
quantum dots and biological material can then be imaged, e.g.,
through chemical photography or a video camera. The fluorescing
regions of the biological material are regions to which the
biofunctional groups on the quantum dots adhere. By noting
differences in fluorescence intensity resulting from different
number density of quantum dots in different regions of the
biological material, differences in characteristics of these
regions may be detected. Such differences in characteristics can be
used to identify tissue in a diseased or abnormal state, for
example, cancerous tissue or tissue infected by bacteria,
parasites, or viruses.
[0060] Scientists from the University of Missouri have shown that
cancer-associated carbohydrate T antigen, e.g.,
Thomsen-Friedenreich disaccharide, plays a leading role in docking
breast and prostate cancer cells onto endothelium by specifically
interacting with an endothelium-expressed protein, galectin-3. The
presence of cancer cells in the body may stimulate expression of
galectin-3 in endothelial cells.
[0061] Biofunctionalized quantum dots according to the invention
can be injected into an organism, for example, into the tissues,
including the circulatory system, of a living animal. For example,
the biofunctionalized quantum dots can be dissolved or suspended in
a biocompatible aqueous solvent, and the solution or suspension
then injected into the body. The
Thomsen-Friedenreich-functionalized quantum dots of the invention
would adhere to cells which express galectin-3, in particular,
endothelial cells which have been stimulated to express large
amounts of galectin-3. The body or a biopsy of tissue from the body
can then be exposed to light which causes the quantum dots to
fluoresce, the body or biopsy sample can then be imaged. By noting
which regions of tissue fluoresce most intensely, the state of
advancement of a tumor, for example, a metastasizing tumor, can be
determined See Glinsky et al., "The role of Thomsen-Friedenreich
antigen in adhesion of human breast and prostate cancer cells to
the endothelium", Cancer Res., 61 (12): 4851-4857, Jun. 15, 2001.
The fact that the biofunctionalized quantum dots of the present
application are water-soluble and biocompatible makes them
particularly advantageous for use in evaluating tissue in vivo or
in vitro.
[0062] Quantum dots can be functionalized with biological receptors
which couple with antigens on cancer cells, these antigens either
not being present in normal cells or being present on cancer cells
in much greater concentration than in normal cells. Similarly,
quantum dots can be functionalized with antigens which couple with
receptors on cancer cells, these receptors either not being present
in normal cells or being present on cancer cells in much greater
concentration than in normal cells. By contacting the quantum dots
with tissue in the body or in an in vitro sample and imaging,
regions of tissue in which cancer cells have proliferated can be
detected.
[0063] In an embodiment, biofunctionalized quantum dots of the
invention are used in a biological or medical analysis system. For
example, a quantum dot can be functionalized with an antigen to
which a pathogen sought to be detected has affinity, e.g., through
a receptor on the pathogen. A biological material or substance
secreted from a biological material can be brought into contact
with the biofunctionalized quantum dot. Coupling of a pathogen to
the quantum dot can be detected, for example, by passing a fluid
containing the quantum dots and pathogens over an assay plate on
which the antigen is fixed. A pathogen to which a quantum dot is
coupled and having affinity to an antigen will then couple to the
antigen fixed to the plate. By shining light of a shorter
wavelength than the characteristic wavelength of the quantum dot,
any quantum dots in a pathogen-quantum dot complex affixed to the
plate is made to fluoresce. Such fluorescence is then indicative of
the presence of the pathogen.
[0064] Similarly, different types of quantum dots can be produced,
each functionalized with a different antigen corresponding to an
antigen fixed to a specific region of an assay plate. The quantum
dots can then be combined with the sample suspected of containing
pathogens. A fluid containing the sample and the quantum dots is
then passed over the assay plate. A pathogen bearing a receptor
will couple to a quantum dot having the corresponding antigen and
to the region of the assay plate having the corresponding antigen.
When the quantum dots are made to fluoresce, the fluorescing
regions on the plate can be noted. Because the antigen
corresponding to a region of the plate is known, the presence of a
number of pathogens bearing receptors specific to antigens can be
identified.
[0065] As another example, the quantum dots can be functionalized
with several antigens. In an embodiment, a number of types of
quantum dots are made, each type having a specific size and being
made of a specific material so that each type fluoresces at a
different wavelength. Each type can be functionalized with a
different antigen or with a different set of antigens. The antigens
present on the quantum dots can then be distributed over and fixed
to an assay plate. Pathogens binding to antigens on the quantum
dots would then couple to antigens on the plate surface. By shining
light of a shorter wavelength than the characteristic wavelengths
of the quantum dots, the quantum dots are made to fluoresce. By
determining the wavelengths of the light emitted from the quantum
dot--pathogen complexes coupled to the plate surface, the presence
of pathogens bearing receptors specific to antigens can be
identified. Such assay plates can be in a microchip format to form
a "lab on a chip" used in small analytical devices or even
implanted in the body.
[0066] Biofunctionalized quantum dots of the invention can also be
used together with an assay plate as follows. An antibody is fixed
to an assay plate. A sample which may contain antigens or pathogens
bearing antigens is brought into contact with the assay plate.
Quantum dots are functionalized with the same antibody and brought
into contact with the assay plate. Light of a shorter wavelength
than the characteristic wavelength of the quantum dots is then
shown on the assay plate. Fluorescence from the quantum dots
indicates the presence of the antigen or the pathogen-bearing
antigen. This method can be extended to assay plates on which more
than one type of antibody is fixed, each antibody being fixed to a
specific region of the assay plate. The method can also be extended
to a method in which several types of quantum dots fluorescing at
different frequencies are functionalized, each type with a
different antibody or set of antibodies, the different antibodies
are distributed over and fixed to an assay plate, a sample which
may contain antigens or pathogen-bearing antigens is brought into
contact with the assay plate, and the antibody-functionalized
quantum dots are brought into contact with the assay plate.
[0067] Biofunctionalized quantum dots can be used in therapeutic
applications. For example, cancer cells may express antigens which
couple with receptors on normal cells. Such coupling can play a
role in metastasis of cancer cells or other interactions of cancer
cells with the body. In an embodiment, quantum dots are
functionalized with the same antigens which the cancer cells
express, the quantum dots may bind to receptors on normal cells and
thereby block adhesion of cancer cells to the normal cells. For
example, as discussed above, cancer-associated carbohydrate T
antigen, e.g., Thomsen-Friedenreich disaccharide, plays a leading
role in docking breast and prostate cancer cells onto endothelium
by specifically interacting with an endothelium-expressed protein,
galectin-3. Thomsen-Friedenreich-functionalized quantum dots could
be injected into the body to adhere to endothelial cells which
express galectin-3, in particular, endothelial cells which have
been stimulated to express large amounts of galectin-3, and thereby
block adhesion of the cancer cells to the endothelium. Such therapy
could delay or prevent the metastasis of cancer cells. See Glinsky
et al., "The role of Thomsen-Friedenreich antigen in adhesion of
human breast and prostate cancer cells to the endothelium", Cancer
Res., 61 (12): 4851-4857, Jun. 15, 2001.
[0068] It is thought that multiple presentations of antigenic
saccharides to receptor proteins, i.e., a high concentration of
antigenic saccharides, may dramatically increase the strength of
coupling between the particle or cell with the antigenic
saccharides and the particle or cell with the receptor proteins;
this is known as the cluster glycoside effect. Thus, quantum dots
can advantageously be used as vehicles to provide antigenic
saccharides to receptors proteins, because the antigenic
saccharides are present in high concentrations on the surface of
the quantum dots.
[0069] The biofunctionalized quantum dots presented in this
application can be especially useful in that they can be used
simultaneously for therapy and diagnosis. For example,
biofunctionalized quantum dots can be injected into the body for
therapy, and then induced to fluoresce and imaged to monitor the
response of the body, especially of diseased tissue, to the
therapy.
[0070] As discussed above, quantum dots functionalized with an
antigen can bind with diseased cells, e.g., cancer cells, which
express a receptor for the antigen, and quantum dots functionalized
with a receptor can bind with diseased cells, e.g., cancer cells,
which express an antigen which couples with the receptor. In an
embodiment, the quantum dot, in addition to the biofunctional
antigen or receptor, has a therapeutic agent linked to it. By
injecting such a quantum dot, site-specific drug delivery can be
achieved. Such site-specific therapeutic agent delivery is of great
interest in cancer therapy, as the therapeutic agents used can be
toxic to normal as well as cancerous cells. The therapeutic agent
delivered can be a drug, e.g., a drug to stimulate an immune
response, a chemotherapeutic agent, e.g., for killing or weakening
a cancer cell, or a radiotherapeutic agent for killing or weakening
a cancer cell. Alternatively, the nanocrystalline core or the shell
layer of the quantum dot may itself serve as the therapeutic agent.
For example, radioisotopes may be used as elements in the formation
of the semiconductor nanocrystalline core or of the shell layer.
Non-radioactive elements or compounds may be selected for their
toxicity to cancer cells and selected so that the semiconductor
nanocrystalline core or the shell layer which they form degrades
over time, exposing the cancer cells to which the quantum dot is
bound to these toxic elements or compounds. Drug-functionalized,
radioactive, or chemotoxic quantum dots functionalized with an
antigen can also be used to selectively weaken or destroy cells in
the body which cancer cells co-opt for their growth or
proliferation.
[0071] In an embodiment, biofunctionalized quantum dots are used as
a component of an immunogenic composition. Tumor-associated
antigens expressed by cancerous cells, for example, antigenic
saccharides such as Thomsen-Friedenreich disaccharide, can be used
to functionalize quantum dots. Introduction of
tumor-associated-antigens alone usually fails to stimulate an
immune response because of immune self-tolerance. However, multiple
and dense presentation of tumor-associated-antigens on the surface
of a quantum dot may be recognized by the immune system as
distinctly unnatural so that an immune response is stimulated. When
injected into the body, these tumor-associated
antigen-functionalized quantum dots may stimulate an immune
response and thus spur the immune system in attacking the cancerous
cells.
[0072] In an embodiment, biofunctionalized quantum dots are used to
coat surfaces of devices which come into contact with biological
material. Examples of such devices are implants or extracorporeal
devices, e.g., dialysis machines. For example, the biofunctional
groups on the quantum dots can be selected so that the biological
material, e.g., blood or tissue, recognizes the biofunctionalized
quantum dots on the device surface as "self" so that an immune or
inflammatory response is not stimulated. The coating of foreign
surfaces with biofunctionalized quantum dots could be used in a
therapeutic, e.g., for coating implants, and in a research
context.
Example 1
[0073] A solution of a glycoside of Formula IV (120 mg) in
anhydrous 1,4-dioxane (4 ml) was purged with argon for 20 min. To
this solution was added triply distilled thiolacetic acid (1.4 ml)
followed by 2,2'-azobisisobutyronitrile (30 mg). The reaction was
left to stir under an argon atmosphere at 75.degree. C. for 12
hours and quenched with cyclohexene (0.1 ml). The solution was
co-evaporated with xylenes under reduced pressure. Flash column
chromatography of the residue on silica gel with a solution of
ethyl acetate and hexanes in a volume ratio of 3:1 provided a
thioester of Formula V (125 mg).
##STR00007##
[0074] The thioester of Formula V was then debenzylidinated. A
first approach for debenzylidination was carried out as follows.
The thioester of Formula V (110 mg) was dissolved in a solution of
80% acetic acid in water (3 ml) was stirred at 60.degree. C. for 16
hours. The reaction solution was concentrated at reduced pressure
and co-evaporated twice with xylenes. The residue was purified by
flash column chromatography on silica gel using a solution of 7%
methanol in methylene chloride to provide a debenzylidinated
thioester (69 mg).
[0075] In a second, alternative approach for debenzylidination, the
thioester of Formula V (600 mg) was dissolved in methanol (14 ml)
and treated with 3 drops of acetyl chloride. After 30 minutes, the
reaction was quenched with pyridine (1 ml) and evaporated. The
residue was purified by flash column chromatography using a solvent
of 5% to 10% methanol on methylene chloride to yield a
debenzylidinated thioester (475 mg).
[0076] The debenzylidinated thioester was then hydrolyzed. A first
approach for hydrolysis was carried out as follows. A solution of
debenzylidinated thioester (30 mg) in methanol (5 ml) was treated
with a solution of sodium methoxide in methanol (25% w/v, 25 ml)
and allowed to react for 30 minutes. The solution was then
neutralized with strongly acidic Amberlite.RTM.-120 ion-exchange
resin, filtered, and concentrated. Purification was performed on a
Strata.RTM. SI-1 silica gel cartridge with an eluting solvent of
20% methanol in methylene chloride to yield the
Thomsen-Friedenreich-thiol of Formula VI (20 mg) as a white
solid.
##STR00008##
[0077] In a second, alternative approach for hydrolysis, the
debenzylidinated thioester (300 mg) was dissolved in methanol (5
ml). The solution was treated with a solution of sodium methoxide
in methanol (25% (w/v), 30 .mu.l). Air was bubbled through the
solution and the solution was stirred at room temperature and
allowed to react for 24 hours. The solution was then neutralized
with strongly acidic Amberlite.RTM.-120, and evaporated under
reduced pressure at 50.degree. C. to yield the
Thomsen-Friedenreich-disulfide of Formula VII (200 mg). The
Thomsen-Friedenreich-disulfide of Formula VII was purified by
reverse phase flash chromatography with aqueous methanol to yield
purified Thomsen-Friedenreich-disulfide of Formula VII (187 mg) as
a white powder which was soluble in water and in methanol. The
Thomsen-Friedenreich-disulfide of Formula VII (130 mg) was then
dissolved in distilled water (1 ml) and degassed with argon for 20
minutes. Dithiothreitol (130 mg) was added and the solution allowed
to react for 20 minutes. The excess dithiothreitol was then removed
by several extractions with ethyl acetate. The residue was then
purified by reverse phase flash chromatography on a C-18 column
with an aqueous solution of methanol (10%-40% (v/v)) to yield the
Thomsen-Friedenreich-thiol of Formula VI. The
Thomsen-Friedenreich-thiol of Formula VI could be stored under
argon at -20.degree. C. without significant dimerization for weeks
but normally was used immediately since it oxidizes to the
Thomsen-Friedenreich-disulfide of Formula VII upon standing at room
temperature.
##STR00009##
Example 2
[0078] Hydrogen telluride gas was generated by reacting aluminum
telluride (Al.sub.2Te.sub.3, 123 mg) with aqueous sulfuric acid
(0.5M, 10 ml). The hydrogen telluride was then passed with a slow
flow of argon through a deaerated solution of sodium hydroxide in
water (50 mM, 10 ml) to yield a solution of hydrogen sodium
telluride (NaHTe, 50 mM).
[0079] The Thomsen-Friedenreich-thiol of Formula VI (28 mg) was
then dissolved in an aqueous solution of cadmium perchlorate (16
mM, 700 .mu.l) and was purged with argon for 20 minutes. The
freshly prepared hydrogen sodium telluride solution (115 .mu.l) was
then quickly added to this mixture. The mixture was then refluxed
in the open air. During the refluxing, 50 .mu.l aliquots were
collected and analyzed for UV absorption. The absorption spectra
during the first 2 hours of the synthesis are shown in FIG. 2, in
which curve A represents an aliquot taken at 30 minutes, curve B
represents an aliquot taken at 60 minutes, curve C represents an
aliquot taken at 90 minutes, and curve D represents an aliquot
taken at 120 minutes. Rapid growth of the nanocrystals during these
first 2 hours is evident from the shift of the absorption maxima to
longer wavelengths (see Gaponik et al., J. Phys. Chem. B, (2002) v.
106, p. 7177). After 48 hours of refluxing, faint green
luminescence was observed. The solution was cooled to ambient
temperature, diluted with water, and purified from the low
molecular weight impurities on Centriplus.RTM. YD-30 (MWCO 30 KDa)
cartridges. Drying of the purified solution yielded
Thomsen-Friedenreich-functionalized cadmium telluride quantum dots
as a pale yellow fluffy substance that was freely soluble in water.
Comparison of the .sup.1H NMR spectra of the
Thomsen-Friedenreich-thiol of Formula VI (label A) and the
Thomsen-Friedenreich-functionalized cadmium telluride quantum dots
(label B) in deuterium oxide solution is shown in FIG. 3. The
absence of sharp peaks in the spectrum of the
Thomsen-Friedenreich-functionalized cadmium telluride quantum dots
in deuterium oxide indicates that no free ligands are present in
solution.
[0080] In a modified procedure, the Thomsen-Friedenreich-thiol of
Formula VI, the cadmium perchlorate, and the hydrogen sodium
telluride solution can be dissolved in N,N-dimethylformamide and
the solution can be refluxed.
Example 3
[0081] Hydrogen telluride gas was generated by reacting aluminum
telluride (Al.sub.2Te.sub.3, 123 mg) with aqueous sulfuric acid
(0.5M, 10 ml). The hydrogen telluride was then passed with a slow
flow of argon through a deaerated solution of sodium hydroxide in
water (50 mM, 10 ml) to yield a solution of hydrogen sodium
telluride (NaHTe, 50 mM).
[0082] The Thomsen-Friedenreich-thiol of Formula VI (12.3 mg) and
mercaptoacetic acid (3 ml) were dissolved in an aqueous solution of
cadmium perchlorate (16 mM, 1400 .mu.l) and purged with argon for
20 minutes. The freshly prepared hydrogen sodium telluride solution
(230 .mu.l) was then quickly added to this mixture under argon. The
mixture was then refluxed in the open air. Aliquots were taken
after 15, 21, 27, and 39 hours; the intensity of fluorescence was
observed to increase with time. After 39 hours of refluxing, bright
yellow luminescence was observed. The solution was cooled to
ambient temperature, diluted with water, and purified from low
molecular weight impurities on Centriplus.RTM. YD-50 (MWCO 50 KDa)
cartridges. Drying of the purified solution yielded
Thomsen-Friedenreich-mercaptoacetic-acid-functionalized cadmium
telluride quantum dots (4 mg) as a yellow fluffy substance that was
freely soluble in water and dimethylsulfoxide.
[0083] The .sup.1H NMR spectra of mercaptoacetic acid (label A),
the Thomsen-Friedenreich-thiol of Formula VI (label B), and the
Thomsen-Friedenreich-mercaptoacetic-acid-functionalized cadmium
telluride quantum dots (label C) in deuterium oxide solution are
shown in FIG. 4. The broad peaks in the spectrum of the quantum
dots (label C) are a result of increased relaxation rates due to
effective molecular weight (>50 KDa) of the nanoparticles and
the close packing of the mercaptoacetic acid and the
Thomsen-Friedenreich-thiol groups on their surface. The downfield
shift of the --CH.sub.2S-- methylene triplet (.delta. 2.6, spectrum
B) may be attributed to the close proximity of C--S to the
semiconductor surface which results in strong electronic
interaction. Interestingly, this methylene signal completely
disappeared when the Thomsen-Friedenreich-thiol of Formula VI was
attached to gold nanoparticles. The chemical shifts of the
remaining protons confirmed that the bonded Thomsen-Friedenreich
groups have the same structure as in the free Thomsen-Friedenreich
thiol. Also noteworthy is the fact that, although a three-fold
excess of the Thomsen-Friedenreich-thiol over mercaptoacetic acid
was used in the synthesis, the NMR shows that approximately 1.5
molecules of mercaptoacetic acid were incorporated into a quantum
dot per molecule of the Thomsen-Friedenreich-thiol incorporated, as
calculated by integration of the methylene signal of mercaptoacetic
acid (.delta. 3.1) and the methyl group on the acetamide group
(.delta. 2.1) of the Thomsen-Friedenreich-thiol. This effect of
preferential binding affinity of one ligand over another was
reported before in the synthesis of hybrid sugar-bearing gold
nanoparticles. See Barrientos et al., Chem. Eur. J., v. 9 (2003) p.
1909. The absence of sharp peaks in the spectrum of the
Thomsen-Friedenreich-mercaptoacetic-acid-functionalized cadmium
telluride quantum dots in deuterium oxide solution indicates that
no free mercaptoacetic acid or free Thomsen-Friedenreich-thiol of
Formula VI is present in solution. The absorption spectrum of
Thomsen-Friedenreich-mercaptoacetic-acid-functionalized cadmium
telluride quantum dots is shown in FIG. 5 in which the first
excitonic maximum at 460 nm is apparent.
[0084] Coupling between
Thomsen-Friedenreich-mercaptoacetic-acid-functionalized cadmium
telluride quantum dots and a monoclonal anti-Thomsen-Friedenreich
antibody was observed. Imaging with a laser scanning confocal
microscope clearly revealed time-dependent aggregation of the
quantum dots over time after addition of the antibody. This result
confirms that the functional integrity of the Thomsen-Friedenreich
antigen is conserved while the antigen is linked to the quantum
dot.
[0085] The Thomsen-Friedenreich-mercaptoacetic-acid-functionalized
cadmium telluride quantum dot samples showed prolonged stability of
their luminescence against oxidation. Thus, solutions of pure
Thomsen-Friedenreich-mercaptoacetic-acid-functionalized cadmium
telluride quantum dots in water stored in the dark at 4.degree. C.
for at least 4 months showed no signs of decreased luminescence or
precipitation or flocculation. NMR analysis of samples indicated
that there was no leaching of the mercaptoacetic acid or
Thomsen-Friedenreich groups from the quantum dot into the water.
This stability is remarkable. Similarly prepared mercatoacetic acid
or mercaptoproprionic acid capped quantum dots, which were not
capped with a saccharide group completely flocculated in a few days
when stored in aqueous solution in the absence of free ligand.
[0086] In a modified procedure, the Thomsen-Friedenreich-thiol of
Formula VI, the mercaptoacetic acid, the cadmium perchlorate, and
the hydrogen sodium telluride solution can be dissolved in
N,N-dimethylformamide and the solution refluxed.
[0087] In summary, a simple aqueous synthesis of robust,
luminescent tumor-associated-carbohydrate-antigen-encapsulated
cadmium telluride quantum dots is reported for the first time.
[0088] The embodiments illustrated and discussed in this
specification are intended only to teach those skilled in the art
the best way known to the inventors to make and use the invention.
Nothing in this specification should be considered as limiting the
scope of the present invention. All examples presented are
representative and non-limiting. The above-described embodiments of
the invention may be modified or varied, without departing from the
invention, as appreciated by those skilled in the art in light of
the above teachings. It is therefore to be understood that, within
the scope of the claims and their equivalents, the invention may be
practiced otherwise than as specifically described.
* * * * *
References