U.S. patent application number 14/213290 was filed with the patent office on 2014-10-09 for polymer dot compositions and related methods.
This patent application is currently assigned to University of Washington through its Center for Commercialization. The applicant listed for this patent is University of Washington through its Center for Commercialization. Invention is credited to Daniel T. Chiu, Wei Sun, Changfeng Wu, Fangmao Ye, Jiangbo Yu.
Application Number | 20140302516 14/213290 |
Document ID | / |
Family ID | 51581401 |
Filed Date | 2014-10-09 |
United States Patent
Application |
20140302516 |
Kind Code |
A1 |
Chiu; Daniel T. ; et
al. |
October 9, 2014 |
POLYMER DOT COMPOSITIONS AND RELATED METHODS
Abstract
Lyophilized polymer dot compositions are provided. Also
disclosed are methods of making and using the lyophilized
compositions and kits supplying the compositions.
Inventors: |
Chiu; Daniel T.; (Seattle,
WA) ; Sun; Wei; (Seattle, WA) ; Yu;
Jiangbo; (Seattle, WA) ; Wu; Changfeng;
(Changchun, CN) ; Ye; Fangmao; (Seattle,
WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Washington through its Center for
Commercialization |
Seattle |
WA |
US |
|
|
Assignee: |
University of Washington through
its Center for Commercialization
Seattle
WA
|
Family ID: |
51581401 |
Appl. No.: |
14/213290 |
Filed: |
March 14, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61785293 |
Mar 14, 2013 |
|
|
|
Current U.S.
Class: |
435/7.1 ;
428/402; 525/54.1 |
Current CPC
Class: |
C08G 61/02 20130101;
C09K 11/025 20130101; C09K 11/06 20130101; C09B 69/109 20130101;
Y10T 428/2982 20150115; G01N 33/587 20130101; C08G 75/32 20130101;
G01N 33/533 20130101; G01N 2650/00 20130101; C09B 69/101 20130101;
G01N 33/54393 20130101; G01N 33/582 20130101; C08G 2261/11
20130101; G01N 33/588 20130101; C08G 2261/18 20130101; C08G 73/00
20130101; C09B 69/105 20130101 |
Class at
Publication: |
435/7.1 ;
525/54.1; 428/402 |
International
Class: |
G01N 33/569 20060101
G01N033/569 |
Claims
1. A lyophilized composition comprising fluorescent nanoparticles,
the fluorescent nanoparticles comprising at least one condensed
conjugated polymer.
2. The lyophilized composition of claim 1, further comprising a
carbohydrate.
3. The lyophilized composition of claim 2, wherein the carbohydrate
comprises a monosaccharide, a disaccharide, an oligosaccharide, a
polysaccharide, or a combination thereof.
4. The lyophilized composition of claim 1, further comprising an
alditol, hydroxypropyl-cyclodextrin, BSA, or a combination
thereof.
5. The lyophilized composition of claim 1, further comprising a
disaccharide.
6. The lyophilized composition of claim 5, wherein the disaccharide
is present between about 1% w/v and 50% w/v.
7. The lyophilized composition of claim 5, wherein the disaccharide
is present between about 10% w/v and 20% w/v.
8. The lyophilized composition of claim 5, wherein the disaccharide
is selected from the group consisting of sucrose, trehalose
dihydrate, maltose monohydrate, and lactose monohydrate.
9. The lyophilized composition of claim 5, wherein the disaccharide
is sucrose.
10. The lyophilized composition of claim 9, wherein sucrose is
present between about 10% w/v and 20% w/v.
11. The lyophilized composition of claim 1, wherein at least some
of the fluorescent nanoparticles are conjugated to a
biomolecule.
12. The lyophilized composition of claim 11, wherein the
biomolecule comprises a protein, an antibody, a nucleic acid
molecule, a lipid, a peptide, an aptamer, or a drug.
13. The lyophilized composition of claim 11, wherein the
biomolecule comprises streptavidin.
14. The lyophilized composition of claim 1, wherein the fluorescent
nanoparticles comprise the same or increased quantum yield when
dispersed in an aqueous solution as compared to the fluorescent
nanoparticles prior to lyophilization.
15. The lyophilized composition of claim 1, wherein the fluorescent
nanoparticles comprise the same particle diameter when dispersed in
an aqueous solution as compared to the particle diameter of
fluorescent nanoparticles prior to lyophilization.
16. The lyophilized composition of claim 1, wherein the at least
one condensed conjugated polymer comprises a semiconducting
polymer.
17. The lyophilized composition of claim 1, wherein the at least
one condensed conjugated polymer is selected from the group
consisting of a fluorene polymer, a flourene-based polymer or
copolymer, a phenylene vinylene polymer or copolymer, a phenylene
ethynylene polymer or copolymer, a Bodipy-based polymer or
copolymer.
18. The lyophilized composition of claim 1, wherein the at least
one condensed conjugated polymer is selected from the group
consisting of poly(9,9-dihexylfluorenyl-2,7-diyl) (PDHF),
Poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO),
poly[{9,9-dioctyl-2,7-divinylene-fluorenylene}-alt-co-{2-methoxy-5-(2-eth-
ylhexyloxy)-1,4-phenylene}] (PFPV),
poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1,3}-thiadiazole)]
(PFBT),
poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(4,7-Di-2-thienyl-2,1,3-b-
enzothiadiazole)] (PFTBT),
poly[(9,9-dioctylfluorenyl-2,7-diyl)-9-co-(4,7-Di-2-thienyl-2,1,3-benzoth-
iadiazole)] (PF-0.1TBT)), and
poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene]
(MEH-PPV),
poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylene-1,4-phenylene)]
(CN-PPV), BODIPY 570, BODIPY 590, and BODIPY 690.
19. The lyophilized composition of claim 1, wherein the fluorescent
nanoparticles have an average diameter of less than about 30 nm as
measured by dynamic light scattering.
20. The lyophilized composition of claim 1, wherein the fluorescent
nanoparticles includes a plurality of polymers.
21. The lyophilized composition of claim 20, wherein at least 50%
of the plurality of polymers includes conjugated polymers.
22. The lyophilized composition of claim 1, wherein the quantum
yield of the fluorescent particles after dispersion in a solution
is higher than the unlyophilized fluorescent particles.
23. The lyophilized composition of claim 1, wherein the full width
half maximum of the emission bandwidth of the fluorescent particles
after dispersion in a solution is narrower than the full width half
maximum emission bandwidth of the unlyophilized fluorescent
particles.
24. A method of producing a lyophilized composition, the method
comprising: lyophilizing a suspension comprising fluorescent
particles, thereby forming the lyophilized composition of
fluorescent nanoparticles, wherein the fluorescent nanoparticles
are polymer dots each including at least one condensed conjugated
polymer.
25. The method of claim 24, wherein lyophilizing comprises freezing
the suspension at a temperature below about -10.degree. C., below
about -20.degree. C., below about -30.degree. C., or below about
-40.degree. C.
26. The method of claim 24, wherein lyophilizing comprises freezing
the suspension at a temperature at or around -80.degree. C.
27. The method of claim 24, wherein before lyophilizing, the method
includes combining (i) a liquid comprising fluorescent
nanoparticles with (ii) a first aqueous solution, thereby forming
the suspension comprising fluorescent nanoparticles.
28. The method of claim 27, wherein the first aqueous solution
comprises a lyophilization agent.
29. The method of claim 24, wherein the suspension comprises a
carbohydrate.
30. The method of claim 29, wherein the carbohydrate comprises a
monosaccharide, a disaccharide, an oligosaccharide, a
polysaccharide, or a combination thereof.
31. The method of claim 24, wherein the suspension includes an
alditol, hydroxypropyl-cyclodextrin, bovine serum albumin or a
combination thereof.
32. The method of claim 24, wherein the suspension comprises a
disaccharide.
33. The method of claim 32, wherein the disaccharide is present
between about 1% w/v and 50% w/v.
34. The method of claim 32, wherein the disaccharide is present
between about 10% w/v and 20% w/v.
35. The method of claim 32, wherein the disaccharide is selected
from the group consisting of sucrose, trehalose dihydrate, maltose
monohydrate, and lactose monohydrate.
36. The method of claim 32, wherein the disaccharide is
sucrose.
37. The method of claim 36, wherein sucrose is present between
about 10% w/v and 20% w/v.
38. The method of claim 24, further comprising mixing the
lyophilized composition with a second aqueous solution, thereby
forming a second suspension comprising the fluorescent
nanoparticles dispersed in the second aqueous solution.
39. The method of claim 38, wherein the dispersed fluorescent
nanoparticles in the second aqueous solution have the same or
increased quantum yield as compared to unlyophilized fluorescent
nanoparticles.
40. The method of claim 38, wherein the dispersed fluorescent
nanoparticles in the second aqueous solution have the same particle
diameter as compared to the particle diameter unlyophilized
fluorescent nanoparticles.
41. The method of claim 24, wherein the dispersed fluorescent
nanoparticles have an average diameter of less than about 30 nm as
measured by dynamic light scattering.
42. The method of claim 24, wherein the at least one condensed
conjugated polymer comprises a semiconducting polymer.
43. The method of claim 24, wherein the at least one condensed
conjugated polymer is selected from the group consisting of a
fluorene polymer, a flourene-based polymer, a phenylene vinylene
polymer, and a phenylene ethynylene polymer.
44. The method of claim 24, wherein the at least one condensed
conjugated polymer is selected from the group consisting of
poly(9,9-dihexylfluorenyl-2,7-diyl) (PDHF),
Poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO),
poly[{9,9-dioctyl-2,7-divinylene-fluorenylene}-alt-co-{2-methoxy-5-(2-eth-
ylhexyloxy)-1,4-phenylene}] (PFPV),
poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1,3}-thiadiazole)]
(PFBT),
poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(4,7-Di-2-thienyl-2,1,3-b-
enzothiadiazole)] (PFTBT),
poly[(9,9-dioctylfluorenyl-2,7-diyl)-9-co-(4,7-Di-2-thienyl-2,1,3-benzoth-
iadiazole)] (PF-0.1TBT)), and
poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene]
(MEH-PPV),
poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylene-1,4-phenylene)]
(CN-PPV), BODIPY 570, BODIPY 590, and BODIPY 690.
45. The method of claim 24, wherein the quantum yield of the
fluorescent particles after dispersion in a solution is higher than
the unlyophilized fluorescent particles.
46. The method of claim 24, wherein the full width half maximum of
the emission bandwidth of the fluorescent particles after
dispersion in a solution is narrower than the full width half
maximum emission bandwidth of the unlyophilized fluorescent
particles.
47. A kit comprising a lyophilized composition of claim 1.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/785,293, filed Mar. 14, 2013, which application
is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Fluorescence-based techniques are playing an increasingly
important role in the study of biological systems. New fluorescent
probes ranging from small organic fluorophores to nanoparticles,
such as quantum dots (Qdots), and various forms of genetically
encoded green fluorescent proteins (GFPs) have been developed.
These fluorescent probes have made new measurements and advances
possible but they have their limitations, such as low brightness,
insufficient photostability, or toxicity concerns. As a result,
there continues to be a need for probes that improve upon the
existing fluorescent labels or at least complement them.
[0003] Polymer dots (Pdots) have been developed as a new class of
fluorescent nanoparticles. Compared to organic dyes and fluorescent
proteins, Pdots can possess orders of magnitude greater brightness
and are more resistant to photobleaching. When comparing to Qdots,
for example, Pdots can be an order of magnitude brighter. Moreover,
the dimensions of Pdots can be tuned from several to tens of
nanometers without affecting their spectral properties. Pdots with
small sizes are desirable in situations where labeling with large
nanoparticles may perturb the native behavior of the tagged
biomolecules. The small Pdots may also be useful in crowded
cellular or intercellular spaces where they can better penetrate
and distribute themselves. Various schemes have been developed to
control the surface properties and bioconjugation of Pdots, which
have provided use of Pdots for cell-surface and subcellular
labeling. In addition, Pdot-based ratiometric sensors have been
developed, including ones for pH, temperature, and ions, such as
iron and copper.
[0004] Although Pdots represent a promising new class of
fluorescent probes, there is a continued need to for developing
methods and compositions involving the use of polymer dots, e.g.,
methods and compositions for storing polymer dots.
SUMMARY OF THE INVENTION
[0005] The present invention provides lyophilized polymer dot
compositions and related methods.
[0006] For example, the present invention includes lyophilized
polymer dot compositions including fluorescent nanoparticles, the
fluorescent nanoparticles comprising at least one condensed
conjugated polymer. The present invention also includes methods of
producing polymer dot lyophilized compositions. For example, the
methods can include lyophilizing a suspension comprising
fluorescent particles, thereby forming the lyophilized composition
of fluorescent nanoparticles, wherein the fluorescent nanoparticles
are polymer dots each including at least one condensed conjugated
polymer.
INCORPORATION BY REFERENCE
[0007] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0009] FIG. 1 shows a schematic depicting an experimental
procedure, in accordance with an embodiment of the present
invention.
[0010] FIGS. 2A-2D provides example dynamic light scattering
measurements showing size distributions of streptavidin-conjugated
PFBT Pdots. FIG. 2A depicts the size distribution of Pdots before
lyophilization. FIGS. 2B-2D provides size distributions for
rehydrated Pdots after lyophilization with (B) 0%, (C) 1%, and (D)
10% sucrose.
[0011] FIGS. 3A-3E show normalized absorption and fluorescence
emission spectra of lyophilized and unlyophilized Pdots stored for
up to 6 months. Pdots were made of PFBT (A), CNPPV (B), PFBT-COOH
(C), PFO (D) and PF.sub.10BT (E). Pdots A, B, D and E were stored
at -80.degree. C. for 6 months. Pdots C were stored at -80.degree.
C. for 1 month. Solid curves are absorption. Dotted curves are
fluorescence emission. The x-axis is wavelength with unit nm.
[0012] FIG. 4 shows flow cytometry measurements of cells labeled
with lyophilized and unlyophilized Pdots stored for up to 6 months.
Column A: Strep-CNPPV; column B: Strep-PFBT; column C:
Strep-PFBT-COOH. The x-axis is the relative fluorescence intensity.
Pdots used in top figures were stored for 1 day. Pdots used in
bottom figures were stored for a longer term: Strep-CNPPV 6 months;
Strep-PFBT 6 months; Strep-PFBT-COOH 1 month. (1 D: 1 day; 1 M: 1
month; 6 M: 6 months.)
[0013] FIG. 5 depicts example main chain structures of tested Pdots
composed of five different conjugated polymers.
[0014] FIGS. 6A and B show results from flow cytometry studies of
Pdot-tagged MCF-7 cells in the presence and absence of sucrose
during cell labeling. FIG. 6A is a population of MCF-7 cells
belonging to the active gate as shown by the side scatter versus
forward scatter plot. FIG. 6B shows a fluorescence intensity
distribution of MCF-7 cells labeled with Pdot-streptavidin in
presence of 10% and 0% sucrose in the Pdot solutions used for cell
labeling. The primary antibody used was biotinylated anti-EpCAM;
the negative control was carried out under identical conditions as
in the cell labeling experiments, but in the absence of the
biotinylated primary antibody.
[0015] FIG. 7 shows data associated related to lyophilized polymer
dot compositions having trehalose dihydrate. The Pdots were made of
BODIPY-based conjugated polymer with emission peak at 590 nm
(BODIPY-590). As shown after 1 week of storage, the emission
bandwidth of Pdots without going through the lyophilization process
became wider than when the Pdots were freshly prepared. The
lyophilized Pdots, however, showed a narrower emission
bandwidth.
[0016] FIG. 8 shows the hydrodynamic diameter of Pdots with
lyophilization under different sucrose concentrations (0%, 1%, 10%,
20% and 50%) (upper panel) and the quantum yield of Pdots with
lyophilization under different sucrose concentrations (1%, 10%, 20%
and 50%) (lower panel). The result of unlyophilized Pdots was
included. All the Pdots were stored in -80.degree. C. freezer for 1
day before dispensing into aqueous solution. (Lyoph.: lyophilized;
Unlyoph.: unlyophilized.)
[0017] FIG. 9 shows the chemical structures of lyophilization
agents used for the Pdots (upper panel) and the Pdot diameter (from
DLS) and quantum yield for unlyophilized and lyopholized (with
different lyophilization agents and at different concentrations)
Pdots (lower panel). The storage time was 1 day in a -80.degree. C.
freezer. Two types of Pdots were used, one was PFBT-COOH 2%, the
other was BODIPY-690.
[0018] FIG. 10 (upper panel) shows example main chain structures of
tested Pdots composed of three different BODIPY based conjugated
polymers. The middle panel of FIG. 10 shows the Pdot diameter (from
DLS) and quantum yield for three types (with centered emission at
570 nm, 590 nm and 690 nm, respectively) of unconjugated/conjugated
(to biomolecules) and unlyophilized/lyophilized BODIPY based Pdots.
The sucrose concentration was 10% and the storage time was up to 6
months in -80.degree. C. freezer. The lower panel of FIG. 10 shows
the optical properties of unconjugated/conjugated and
unlyophilized/lyophilized BODIPY-690 Pdots.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The present invention provides lyophilized polymer dot
compositions and related methods. The present invention in-part is
based on the surprising discovery that polymer dots can be
lyophilized and stored while still retaining optical properties,
colloidal stability, and, for polymer dot bioconjugates,
cell-targeting capability during storage. Polymer dots are
fluorescent polymer-based particles and can contain a hydrophobic
core and thus potentially may aggregate if water is driven from the
core of the polymer dots. While not being limited to any particular
theory, during lyophilization, lyoprotectant molecules can form a
surface layer, and diffuse into the Pdot and or Pdot shell as water
is driven out of the particle. As a result, the colloidal stability
and photophysical properties of the particles were retained, and in
some instances, improved after lyophilization and being
reconstituted into a solution after storage. Lyoprotectant
molecules in Pdot and or Pdot shells can, e.g., reduce chain-chain
interactions. Therefore, in some instances fluorescence quantum
yield was increased and emission bandwidth was narrowed.
[0020] The lyophilized compositions and methods can provide several
useful results for polymer dots. For example, by using flow
cytometry, lyophilized Pdot bioconjugates retained their biological
targeting properties and were able to effectively label cells. In
one example, cells labeled with lyophilized Pdot bioconjugates
composed of PFBT, which were stored for 6 months at -80.degree. C.,
were .about.22% brighter than those labeled with identical but
unlyophilized Pdot bioconjugates. These results among others
indicate lyophilization can be a useful approach for storing and
shipping Pdot bioconjugates, which is an important practical
consideration for ensuring Pdots are widely adopted in biomedical
research.
[0021] As described further herein, the present invention relates
to lyophilized compositions of a new class of fluorescent
nanoparticles or polymer dots that have unique properties. By way
of background, a brief description of semiconducting polymers is
provided to describe some general properties of polymer dots. The
majority of organic polymers are insulators. However, when they
have .pi.-conjugated structures, electrons can move along the
polymer backbone through overlaps in .pi.-electron clouds by
hopping, tunneling, and related mechanisms. In general, these
.pi.-conjugated polymers can include wide-bandgap semiconductors,
the so-called semiconducting polymers.
[0022] Organic conjugated polymers and oligomers can be metallic
upon heavy doping, a term derived from inorganic semiconductor
chemistry. The doping in a conjugated polymer can include an
oxidation or a reduction of the .pi.-electronic system and is
called p-doping and n-doping, respectively. Semiconducting polymers
can exhibit a direct band gap, which leads to an efficient
(allowed) absorption or emission at the band edge. Depending on the
polymer species, a semiconducting polymer can exhibit strong
fluorescence, which can be described in terms of semiconductor band
theory. Upon photoexcitation, an electron is excited from the
highest occupied energy band (the .pi. band) to the lowest
unoccupied energy band (the .pi.* band), thus forming a bound state
(exciton) of the excited electron and hole in the .pi. band. The
recombination of the excited electron with the hole results in a
fluorescent photon. The wavelength of the absorbed light is
determined by the .pi.-.pi.* energy gap and can be tuned by
altering the molecular structure of the polymer.
[0023] Semiconducting polymers have been developed with emission
colors that span the full range of the visible spectrum. Important
examples of fluorescent semiconducting polymers include
polyfluorene (such as PDHF and PFO), poly(phenylene ethynylene)
(such as PPE), poly(phenylene vinylene) (such as MEH-PPV and
CN-PPV), fluorene-based copolymers (such as PFPV, PFBT, and
PF-DBT5), and BODIPY based copolymers, and related derivatives. In
many cases, photogenerated electron-hole pairs can dissociate to
form free carriers which migrate through the system. The free
carriers can either combine to form triplets or deactivate by other
nonradiative processes (unwanted processes for fluorescence). They
can also be collected to generate electric current (desirable
processes for photovoltaics).
[0024] As used herein, the term "polymer dot" or "Pdot" refers to a
structure including one or more conjugated polymers (e.g.,
semiconducting polymers) that have been collapsed into a stable
sub-micron sized particle. Polymer dots include fluorescent
nanoparticles having at least one condensed conjugated polymer. The
term "conjugated polymer" is recognized in the art. Electrons,
holes, or electronic energy, can be conducted along the conjugated
structure. In some embodiments, a large portion of the polymer
backbone can be conjugated. In some embodiments, the entire polymer
backbone can be conjugated. In some embodiments, the polymer can
include conjugated structures in their side chains or termini. In
some embodiments, the conjugated polymer can have conducting
properties, e.g. the polymer can conduct electricity. In some
embodiments, the conjugated polymer can have semiconducting
properties, e.g. the polymers can exhibit a direct band gap,
leading to an efficient absorption or emission at the band edge. In
some aspects, the polymer dots can be described as nanoparticles
including at least one condensed (or collapsed) conjugated polymer
(e.g., semiconducting polymer) to form the nanoparticle structure.
The polymer dots provided herein may be formed by any method known
in the art for collapsing polymers, including without limitation,
methods relying on precipitation, methods relying on the formation
of emulsions (e.g. mini or micro emulsion), and methods relying on
condensation. In some embodiments, the polymer dots described
herein can be formed by nanoprecipitation.
[0025] The polymer dots can be formed using a variety of polymers.
Non-limiting examples of semiconducting polymers include fluorene
polymers (e.g., Poly(9,9-dihexylfluorenyl-2,7-diyl) (PDHF),
Poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO)), fluorene based
copolymers (e.g.,
Poly[{9,9-dioctyl-2,7-divinylene-fluorenylene}-alt-co-{2-methoxy-5-
-(2-ethylhexyloxy)-1,4-phenylene}] (PFPV),
Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1,3}-thiadiazole)]
(PFBT),
Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(4,7-Di-2-thienyl-2,1,3-b-
enzothiadiazole)] (PFTBT),
Poly[(9,9-dioctylfluorenyl-2,7-diyl)-9-co-(4,7-Di-2-thienyl-2,1,3-benzoth-
iadiazole)] (PF-0.1TBT)), phenylene vinylene polymers (e.g.,
Poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene]
(MEH-PPV)),
poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylene-1,4-phenylene)]
(CN-PPV), BODIPY 570, BODIPY 590, BODIPY 690, and other polymers
that are used to make narrow band polymer dots (e.g., BODIPY based
polymer dots) such as those described in PCT/US12/71767, which is
herein incorporated by reference in its entirety). Other suitable
polymers and polymer dots are provided, e.g., in WO2011/057295,
which is herein incorporated by reference in its entirety. As
provided, e.g., in WO2011/057295, the polymers in the polymer dots
can be physically blended or chemically bonded (or chemically
crosslinked). For example, the physically blended polymer dots can
include polymers that are blended in the polymer dot and held
together by non-covalent interactions. Chemically bonded polymer
dots can include polymers that are covalently attached to each
other in the polymer dot. The chemically bonded polymers can be
covalently attached to each other prior to formation of the polymer
dots. In some embodiments, the polymers and polymer dots can
include those disclosed and claimed, e.g. in PCT/US11/56768. For
example, the polymer dots can include those that are directly
functionalized and/or have low density functionalization.
[0026] In some embodiments, the polymer dots can include a
semiconducting copolymer having at least two different chromophoric
units. For example, a conjugated copolymer may contain both
fluorene and benzothiazole chromophoric units present at a given
ratio. Typical chromophoric units used to synthesize semiconducting
copolymers include, but are not limited to fluorene unit, phenylene
vinylene unit, phenylene unit, phenylene ethynylene unit,
benzothiazole unit, thiophene unit, carbazole fluorene unit,
boron-dipyrromethene unit, and derivatives thereof. The different
chromophoric units may be segregated, as in a block copolymer, or
intermingled. As used herein, a chromophoric copolymer is
represented by writing the identity of the major chromophoric
species. For example, PFBT is a chromophoric polymer containing
fluorene and benzothiazole units at a certain ratio. In some cases,
a dash is used to indicate the percentage of the minor chromophoric
species and then the identity of the minor chromophoric species.
For example, PF-0.1 BT is a chromophoric copolymer containing 90%
PF and 10% BT.
[0027] In certain embodiments, the polymer dots can include a blend
of semiconducting polymers. The blends may include any combination
of homopolymers, copolymers, and oligomers. Polymer blends used to
form polymer dots may be selected in order to tune the properties
of the resulting polymer dots, for example, to achieve a desired
excitation or emission spectra for the polymer dot.
[0028] In some embodiments, the polymer dots can include at least
one functional group to facilitate conjugation to other moieties,
such as, e.g., biomolecules. As used herein, the term "functional
group" refers to any chemical unit that can be attached, such as by
any stable physical or chemical association, to the chromophoric
polymer, thereby rendering the surface of the chromophoric polymer
dot available for conjugation (e.g., bioconjugation). Non-limiting
examples of functional groups include, carboxylic acid, amino,
mercapto, azido, alkyne, aldehyde, hydroxyl, carbonyl, sulfate,
sulfonate, phosphate, cyanate, succinimidyl ester, alkyne, strained
alkyne, azide, diene, alkene, cyclooctyne, and phosphine groups,
substituted derivatives thereof, and combinations thereof. In some
embodiments, the polymers and/or biomolecules can include a
functional group to facilitate conjugations of the polymer dots to
the biomolecules.
[0029] The lyophilized compositions can include a variety of
lyophilization agents (e.g., cryoprotectants and/or
lyoprotectants). The constituents can include molecules that are
soluble in water, e.g., at a concentration sufficient to provide
lyophilization of the polymer dots. As used herein, the term
"carbohydrate" refers to, e.g., monosaccharides, oligosaccharides
(e.g., disaccharides), and polysaccharides, as well as compounds
derived from monosaccharides, oligosaccharides, and
polysaccharides.
[0030] As used herein, the term "monosaccharide" refers to, e.g.,
molecules having the general formula: C.sub.x(H.sub.2O).sub.y,
x.gtoreq.3. Examples of monosaccharides can include, but are not
limited to, glucose, fructose, galactose, xylose, ribose, and the
like.
[0031] As used herein, the term "oligosaccharide" refers to, e.g.,
a short monosaccharide polymer that contains, e.g., between 2 to 30
monosaccharide units. An oligosaccharide can include, e.g., a
"disaccharide" that refers to, e.g., molecules that are formed when
two monosaccharides are joined together and, e.g., a molecule of
water is removed. Examples of disaccharides can include, but are
not limited to, sucrose, lactulose, lactose, maltose, trehalose,
cellobiose, and the like. Other oligosaccharides can include, but
are not limited to, trisaccharides (e.g., raffinose),
tetrasaccharides (e.g., stachyose), and pentasaccharides (e.g.,
verbacose).
[0032] As used herein, the term "polysaccharide" refers to, e.g., a
monosaccharide polymer beyond the length of the oligosaccharide,
e.g., a polymer including more than 30 monosaccharide units.
[0033] As used herein, the term "sugar alcohol" refers to, e.g., a
hydrogenated form of carbohydrate, whose carbonyl group (aldehyde
or ketone, reducing sugar) has been reduced to a primary or
secondary hydroxyl group. Sugar alcohols have the general formula
H(HCHO).sub.n+1H. Example sugar alcohols can include, but are not
limited to, alditols (e.g., xylitol, mannitol or sorbitol).
[0034] The present invention further includes other cryoprotectants
and/or lyoprotectants that can be used in the lyophilized
compositions provided herein. Example lyoprotectants can include,
e.g., glycine, hydroxypropyl-.beta.-cyclodextrin, gelatin and
aerosil.
[0035] Other constituents can also be included in the solutions and
lyophilized compositions described herein. For example,
polyethylene glycol or other water soluble polymers can be used.
Buffers (e.g., Tris, HEPES, and other known buffers) and salts
(e.g., NaCl) can also be used.
[0036] As described further herein, the present invention provides
lyophilized polymer dot compositions. For example, the present
invention includes a lyophilized composition including fluorescent
nanoparticles, the fluorescent nanoparticles comprising at least
one condensed conjugated polymer. The various conjugated polymers
(e.g., semiconducting polymers) are described further herein. In
some embodiments, the lyophilized polymer dot compositions can
include functionalized polymer dots. The functionalized polymer
dots can have at least one functional group available for
conjugation (e.g., bioconjugation). In some embodiment, the
lyophilized polymer dot compositions can include a polymer
dot/biomolecule conjugate, wherein the biomolecule comprises a
protein, an antibody, a nucleic acid molecule, a lipid, a peptide,
an aptamer, and/or a drug. The biomolecule can be attached to the
polymer dot by any stable physical or chemical association. In some
embodiments, the lyophilized polymer dot compositions can include a
variety of constituents, such as, but not limited to, a
monosaccharide, an oligosaccharide (e.g., a disaccharide), and/or a
polysaccharide. Sugar alcohols and/or other suitable
cryoprotectants and/or lyoprotectants can be used. The constituents
added to facilitate lyophilization (e.g., cryoprotectants and/or
lyoprotectants), e.g., can be present in a polymer dot solution
prior to lyophilization at concentrations ranging between about 1%
to about 50%, between about 5% to about 40%, between about 10% to
about 30%, between about 1% and about 20%, and between about 10%
and about 20%. In some embodiments, several different types (e.g.,
two or more) of cryoprotectants and/or lyoprotectants can be
present at the same or different concentrations in the polymer dots
solutions prior to lyophilization.
[0037] In one aspect, the present invention includes lyophilized
polymer dot compositions including a disaccharide, such as, but not
limited to, sucrose, trehalose, maltose, lactose, and any
acceptable salt or hydrated forms. In some embodiments, one type of
disaccharide is used (e.g., sucrose). In certain embodiments, at
least two types of disaccharide can be used (e.g., trehalose and
sucrose). The disaccharide(s) can be added to a solution of polymer
dots prior to lyophilization. In some embodiments, the
concentration of the disaccharide(s) in the solution can vary over
a wide range that can be tailored through known techniques to
produce useful lyophilized polymer dot compositions that when
reconstituted provide polymer dots having about the same particle
diameter. In certain embodiments, when the polymer dots in the
lyophilized compositions are reconstituted optical properties are
the same or improved in comparison to the polymer dots in the
solution prior to lyophilization. For example, after lyophilization
in combination with a disaccharide, the polymer dots can
unexpectedly exhibit the same or increased quantum yield. A variety
of concentration ranges can be used for the disaccharides. The
disaccharides, e.g., can be present in a polymer dot solution prior
to lyophilization at concentrations ranging between about 1% to
about 50%, between about 5% to about 40%, between about 10% to
about 30%, between about 1% and about 20%, and between about 10%
and about 20%. In some embodiments, e.g., sucrose can be present in
a polymer dot solution prior to lyophilization at between about 10%
w/v to about 20% w/v. Concentrations of the various disaccharides
can be optimized using the techniques described herein. For
example, a polymer dot solution can be prepared, lyophilized,
resuspended, and then the properties (e.g., size) of the polymer
dots can be analyzed to confirm that no aggregation occurred due to
lyophilization.
[0038] The process of lyophilization can be performed in a variety
of ways that are generally well known in the art. Lyophilization
can, e.g., include a dehydration process used to preserve the
polymer dots described herein and to, e.g., make them more
convenient for transport. Lyophilization generally works by
freezing the polymer dots and then, e.g., reducing the surrounding
pressure to allow the frozen water in the material to sublimate
directly from the solid phase to the gas phase. The present
invention includes methods for lyophilizing the polymer dots to
form lyophilized polymer dot compositions. The lyophilizing can
include freezing the polymer dots in aqueous solutions that include
a variety of constituents described herein. Freezing can be
performed at a variety of temperatures. For example, the polymer
dots compositions can be lyophilized by freezing at temperatures at
or below about -10.degree. C., at or below about -20.degree. C., at
or below about -30.degree. C., at or below about -40.degree. C., at
or below about -50.degree. C. at or below about -60.degree. C., at
or below about -70.degree. C. or at or below about -80.degree.
C.
[0039] The concentrations of the polymer dots in the solutions
prior to lyophilization can also vary over a wide range. In certain
aspects, the concentration of the polymer dots in the solutions
prior to lyophilization can depend on the size of the polymer dots.
Smaller polymer dots can have a higher concentration than larger
polymer dots. In some embodiments, the polymer dots can be present
in the solution prior to lyophilization in the millimolar,
micromolar, nanomolar, or picomolar range. In some embodiments, the
polymer dots can be present between about 1 nM-100 .mu.M, between
about 100 nM-1 .mu.M, between about 100 nM-750 nM, between about
100 nM-500 nM, between about 1 nM-500 nM, 1-100 nM, between about
1-75 nM, between about 1-50 nM, between about 1-25 nM, between
about 1-20 nM, between about 1-15 nM, between about 1-10 nM, or
between about 1-5 nM.
[0040] One useful aspect of the lyophilized compositions includes
the ability to store the polymer dots for long periods of time.
After the storage of the polymer dots over long periods of time as
lyophilized polymer dot compositions, the polymer dots can be
redispersed in solution and used for a variety of purposes.
Advantageously, the polymer dots, e.g., can be redispersed without
aggregation, thereby having the same size (e.g., particle diameter)
characteristics of the polymer dots prior to lyophilization.
Suitable storage periods can include, but are not limited to,
longer than one day, longer than one week, longer than one month,
longer than two months, longer than three months, longer than six
months, or longer than one year. In some embodiments, the storage
period can range from about one day to about one year, from about
one day to about six months, from about one day to about three
months, from about one day to about two months, or from about one
day to about one month.
[0041] Another useful aspect of the lyophilized compositions
includes the ability to improve the photophysical properties of the
Pdots through a lyophilization process. Pdots can contain
hydrophobic core and thus the chain conformation may change if
water is driven from the system. In case of using lyoprotectants
during lyophilization, lyoprotectant molecules can form a surface
layer, and diffuse into the Pdot shell as water is driven out,
therefore reducing polymer chain-chain interactions. As a result,
the photophysical properties of the Pdots can be improved after
lyophilization and being reconstituted into a solution after
storage. In some embodiments, fluorescence quantum yield can be
increased. In some embodiments, the fluorescence emission bandwidth
can be reduced. In some embodiments, the cell labeling brightness
can be increased.
[0042] In one aspect, the present invention further includes
methods for preparing lyophilized polymer dot compositions. The
methods can include lyophilizing the polymer dots in a variety of
solutions described herein. Lyophilizing the polymer dots in
solution can include freezing the polymer dot solutions at any
suitable temperature to produce a lyophilized polymer dot
compositions. For example, the polymer dots compositions can be
lyophilized by freezing at temperatures at or below about
-10.degree. C., at or below about -20.degree. C., at or below about
-30.degree. C., at or below about -40.degree. C., at or below about
-50.degree. C. at or below about -60.degree. C., at or below about
-70.degree. C. or at or below about -80.degree. C. In some aspects,
the methods, e.g., can include providing a solution including
polymer dots in combination with other constituents, and freezing
the solution at a desired temperature, e.g., at about -80.degree.
C. or -20.degree. C. for a period of time. The lyophilized polymer
dots can be redispersed by combining the lyophilized polymer dots
with a solution.
[0043] In some embodiments, the present invention includes method
of producing a lyophilized composition including lyophilizing a
suspension of polymer dots, thereby forming the lyophilized
composition of polymer dots, wherein the polymer dots are
fluorescent nanoparticles including at least one condensed
conjugated polymer. In some embodiments, the methods can include,
e.g., (a) combining (i) a liquid including polymer dots with (ii) a
first aqueous solution, thereby forming a first suspension
comprising the polymer dots; and (b) lyophilizing the suspension,
thereby forming the lyophilized composition of polymer dots,
wherein the polymer dots are fluorescent nanoparticles including at
least one condensed conjugated polymer.
[0044] In some embodiments, the present invention includes method
of producing a lyophilized composition including polymer dot with
reactive functional groups. The reactive functional groups can
include amine reactive functional group such as succinimidyl ester,
sulfhydryl reactive functional group such as maleimide, or reactive
functional group for click chemistry such as alkyne, azide,
strained alkyne, cyclooctyne, and phosphine groups. In some
embodiments, the methods can include, e.g., (a) combining (i) a
solution of conjugated polymer with reactive functional groups in
good solvent with (ii) a poor solvent, followed by evaporation of
the poor solvent, thereby forming a first suspension comprising the
polymer dots; and (b) lyophilizing the suspension, thereby forming
the lyophilized composition of polymer dots with reactive
functional groups, wherein the polymer dots are fluorescent
nanoparticles including at least one condensed conjugated polymer.
Good solvents can include, e.g., a solvent in which the conjugated
polymer is soluble without forming a polymer dot. Poor solvents can
include, e.g., a solvent in which the conjugated polymer is poorly
soluble and thereby forms polymer dots after introduction into the
poor solvent.
[0045] The lyophilization methods above can be used, e.g., to
produce amine-reactive Pdots such as Pdots with succinimidyl ester.
These Pdots, e.g., can be directly mixed with a biomolecule (e.g.,
a protein) to form polymer dot bioconjugates. In some embodiments,
EDC/NHS can be used to activate a Pdot-COOH to form Pdot-NHS, after
which lyophilization can be performed. In certain embodiments,
NHS-terminated conjugated polymer can be synthesized, injected into
a solution of methanol or ethanol to form Pdots. The Pdots can then
be lyophilized to produce lyophilized Pdot-NHS.
[0046] In another aspect, the present invention provides kits
including the lyophilized polymer dot compositions. A typical kit
of the invention includes a unit dosage form of a lyophilized
polymer dot composition of the present invention, e.g., in a sealed
container. In one embodiment, the kit further comprises a sealed
container of a suitable vehicle in which the polymer dot
composition can be dissolved to form a particulate-free sterile
solution that is suitable for administration or use.
EXAMPLES
Example 1
[0047] This example describes an example lyophilization, a
freeze-drying/dehydration technique, that can be used to prepare
Pdot bioconjugates for long-term storage or shipping, provided the
right conditions are used. Lyophilization is an important practical
advance for making Pdots practical to use in biomedical
research.
[0048] Materials.
Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-(2,10,3)-thiadiazole)]
(PFBT; MW, 157 000 Da; polydispersity, 3.0),
Poly(9,9-dioctylfluorenyl-2,7-diyl) end capped with dimethyl phenyl
(PFO, MW 120000 Da),
poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-(2,1',3)-thiadiazole)]-
10% benzothiadiazole (PF.sub.10BT, MW 100000 Da) and
poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylene-1,4-phenylene)]
(CNPPV, MW 15000 Da) were purchased from American Dye Source Inc
(Quebec, Canada). PFBT directly functionalized with carboxylic acid
(PFBT-COOH) groups was synthesized in our lab. Polystyrene-grafted
ethylene oxide functionalized with carboxyl groups (PS-PEG-COOH; MW
21,700 Da of PS moiety; 1200 Da of PEG-COOH; polydispersity, 1.25)
were purchased from Polymer Source Inc. (Quebec, Canada). Sucrose
was ordered from Avantor Performance Materials (Phillipsburg, N.J.,
USA). Streptavidin was purchased from Invitrogen (Eugene, Oreg.,
USA). Bovine serum albumin (BSA) and ethylcarbodiimide
hydrochloride (EDC) were bought from Sigma (St. Louis, Mo.,
USA).
[0049] Streptavidin conjugation of Pdots. Pdots were prepared using
a nano-reprecipitation method as reported earlier. Wu et al., J.
American Chem. Soc. 132: 15410-15417 (2010). Briefly, a
tetrahydrofuran (THF) solution containing 50 .mu.g/mL of
semiconducting polymer (PFBT, CNPPV, PFO or PF.sub.10BT) and 16
.mu.g/mL of PS-PEG-COOH was prepared. A 5-mL aliquot of the mixture
was quickly injected into 10 mL of water under vigorous sonication.
THF was removed by blowing nitrogen gas into the solution at
90.degree. C. The THF-free Pdot solution was sonicated for 1-2
minutes and filtrated through a 0.2-.mu.m cellulose membrane
filter. For PFBT-COOH Pdots, no additional PS-PEG-COOH was added.
THF solution containing only 50 .mu.g/mL PFBT-COOH was injected
into water directly. In a typical conjugation reaction, 80 .mu.L of
polyethylene glycol (5% w/v PEG, MW 3350) and 80 .mu.L of HEPES
buffer (1M, PH 7.3) was added to 4 mL of Pdot solution.
Streptavidin (1 mg/mL, 30 .mu.L) was then added to the solution and
mixed well. Finally, 80 .mu.L of freshly-prepared EDC solution (5
mg/mL in MilliQ water) was added to the solution, and the mixture
was magnetically stirred for 4 hr at room temperature. The
resulting Pdot conjugates were finally concentrated in a spin
column (100K MW) and were purified with a Bio-Rad Econo-Pac 10DG
column (Hercules, Calif., USA). After purification, the proper
amount of BSA was added to reach a final concentration of 1% (w/v).
The hydrodynamic sizes of Pdots were measured with a dynamic light
scattering (DLS) spectrometer (Malvern Zetasizer Nano ZS,
Worcestershire, United Kingdom). Fluorescence quantum yields were
collected using an integrating sphere (model C9920-02, Hamamatsu
Photonics) with proper wavelength excitation. Fluorescence spectra
of Pdots were taken with a Fluorolog-3 fluorospectrometer (HORIBA
JobinYvon, NJ, USA).
[0050] Lyophilization. Solutions of streptavidin-conjugated Pdots
or unconjugated Pdots were prepared at two different concentrations
(4 nM and 20 nM) by diluting the Pdot solution with a buffer that
was composed of 20 mM HEPES (pH 7.3), 0.1% (w/v) PEG and 0.05%
(w/v) BSA. For PFBT, Pdots at 100 nM concentration were also
prepared. Sucrose was added to reach the desired final
concentrations (10%, w/v). The stock Pdot solutions were aliquoted
into several vials. Half of them were rapidly frozen in liquid
nitrogen for 2 minutes, and were immediately placed under vacuum on
a Labconco Freezone 6 freeze-dryer (Kansas City, Mo., USA). After
.about.18 hr, lyophilized samples were removed from the
freeze-dryer and were labeled as "lyophilized Pdots" and stored at
-80.degree. C. for a desired amount of time (from one day to 6
months). The other half of the aliquots were labeled
"unlyophilized" and were placed in a 4.degree. C. refrigerator.
[0051] Cell culture. The breast cancer cell line, MCF-7, was
ordered from American Type Culture Collection (ATCC, Manassas, Va.,
USA). Cells were cultured at 37.degree. C., 5% CO.sub.2 in Eagles
minimum essential medium supplemented with 10% Fetal Bovine Serum
(FBS) and 1% Pen Strep (5000 units/mL penicillin G, 50 .mu.g/mL
streptomycin sulfate in 0.85% NaCl). Cells were cultured prior to
experiments until confluence was reached. The cells were harvested
from the culture flask by briefly rinsing with culture media
followed by incubation with proper amount of Trypsin-EDTA solution
(0.25% w/v Trypsin, 0.53 mM EDTA) at 37.degree. C. for 5 minutes.
After complete detachment, cells were rinsed, centrifuged, and
re-suspended in the culture media. Their concentration was
determined by microscopy using a hemacytometer.
[0052] Immunofluorescence. For labeling cell-surface markers with
IgG conjugates, a million MCF-7 cells in 100 .mu.L labeling buffer
(1.times.PBS, 2 mM EDTA, 1% BSA) were incubated with 0.3 .mu.L of
0.5 mg/mL biotinylated primary anti-human CD326 EpCAM antibody
(eBioScience, San Diego, Calif., USA) on a rotary shaker in the
dark and at room temperature for 30 minutes. This was followed by a
washing step using the labeling buffer. The cells were incubated
with 4 nM streptavidin-conjugated Pdots (diluted from the 20 nM
Pdots solution) in BlockAid.TM. blocking buffer (Invitrogen,
Eugene, Oreg., USA) for 30 minutes on a shaker in the dark and at
room temperature, followed by two washing steps with the labeling
buffer. Negative controls were obtained by incubating cells with
streptavidin-conjugated Pdots without any previous incubation with
the primary biotinylated antibody. Cell fixation was performed
afterwards by dissolving the cell pellet obtained by centrifugation
in 500 .mu.L of fixing buffer (1.times.PBS, 2 mM EDTA, 1% BSA, 1%
paraformaldehyde).
[0053] Flow cytometry experiments. Measurements were performed on
labeled cell samples containing 10.sup.6 cells/0.5 ml and prepared
as previously described. Wu et al., Angewandte Chemie-Intl. Ed.,
49:9436-9440 (2010). The flow cytometer BD FACSCanto II (BD
Bioscience, San Jose, Calif. USA) was used. Cells flowing in the
detection chamber were excited by a 488-nm laser light. Side- and
forward-scattered light were collected and filtered by a 488/10 nm
band-pass filter, while fluorescence emission was collected and
filtered by a 502-nm long-pass and a 530/30 nm (for PFBT and
PFBT-COOH) or a 582/42 nm (for CNPPV) band-pass filter. All signals
were detected by photomultiplier tubes. For all flow experiments,
representative populations of detected cells were chosen by
selecting an appropriate gate. Detection of cell fluorescence was
continued until at least 10.sup.4 events had been collected in the
active gate.
[0054] FIG. 1 shows an example lyophilization procedure. After
being stored at -80.degree. C. for the desired amount of time (from
1 day to 6 months), a lyophilized aliquot was taken out of the
freezer and the appropriate amount of water was added for
re-dispersion. The final volume of the reconstituted solution was
kept the same as that prior to lyophilization. For comparison, the
unlyophilized aliquot stored at 4.degree. C. was used. Both samples
had the same composition and were measured with the same
instrumentation. We tested various lyophilization conditions and
chose the optimized procedure. We then made comparisons between the
lyophilized and unlyophilized Pdot bioconjugates for hydrodynamic
size, absorption spectra, emission spectra, quantum yield, and
labeling efficiency. We also measured the hydrodynamic size,
absorption spectra, emission spectra, quantum yield, and labeling
efficiency of freshly prepared Pdots versus Pdots stored at
4.degree. C. for one day (unlyophilized 1-day), and found them to
be similar in all properties that we measured.
[0055] Size. We chose streptavidin conjugated PFBT Pdots
(Strep-PFBT) to optimize the lyophilization recipe. The particular
Strep-PFBT Pdots we prepared had a hydrodynamic diameter of 32 nm
(FIG. 2A), which was measured immediately after they were prepared.
We then lyophilized the same Pdot-streptavidin conjugates without
adding any reagents, and then re-hydrated the Pdots with water.
Even after vigorous sonication, the hydrodynamic diameter of the
rehydrated Pdots had increased to 220 nm, indicating that the
lyophilization process had caused severe aggregation of the Pdots.
Aggregated Pdot bioconjugates are not suitable for biological
studies.
[0056] We modified our lyophilization recipe. First, we added 1%
(w/v) sucrose. Here, we found the hydrodynamic diameter of the
rehydrated Pdots was 50 nm (FIG. 2C), which was much smaller than
the 220 nm we had obtained without sucrose but it was still
significantly larger than the original size of 32 nm. Sonication
did not help further shift the size distribution of rehydrated
Pdots to that before lyophilization. Therefore, the
Pdot-streptavidin bioconjugates still were partially aggregated,
albeit much less severely than without sucrose. We increased the
concentration of sucrose to 10% (w/v). FIG. 2D shows the size of
the rehydrated Pdots returned to 32 nm. It should be noted that
additional sonication was not needed after the rehydration
procedure. The lyophilized Strep-PFBT Pdots used in the above
measurements were stored at -80.degree. C. for one day after
lyophilization. We also tested lyophilized Strep-PFBT Pdots at two
different concentrations (4 nM and 20 nM) that were stored for
longer (1-6 months) at -80.degree. C. As shown in table 1, the size
remained at around 32 nm independent of concentrations after 6
months. 100 nM concentration was also tested and the results were
similar to that of 4 and 20 nM. We also applied this lyophilization
recipe to Pdots made of other semiconducting polymers (CNPPV, PFO,
PF.sub.10BT), including both streptavidin conjugated and
unconjugated Pdots. The structures of the tested polymers are
included in FIG. 5. We first measured the hydrodynamic sizes of
unlyophilized Pdots stored over different durations (1-6 months).
We then measured the hydrodynamic sizes of the different
lyophilized Pdots stored for up to 6 months and compared them with
that of their unlyophilized counterparts. As shown in table 1, all
the lyophilized Pdots possessed similar size to the unlyophilized
Pdots after rehydration. To facilitate the bioconjugation reaction
and dispersion in aqueous solution, the aforementioned Pdots were
functionalized with carboxylic acid groups by doping PS-PEG-COOH
during preparation. A new type of semiconducting polymers directly
functionalized with low density carboxylic acid groups in the same
polymer chain was also synthesized in our lab (PFBT-COOH, FIG. 5).
With the directly incorporated carboxylic acid groups, PFBT-COOH
Pdots offer a number of significant advantages, such as higher
brightness and better colloidal stability. The size of such
prepared streptavidin conjugated PFBT-COOH Pdots after
lyophilization was measured; we found the sizes of lyophilized
Strep-PFBT-COOH Pdots stored for 1 month were the same as that of
unlyophilized Pdots. The results in table 1 show that lyophilized
Pdots were easily re-dispersed back to their single particle form,
independent of the concentration of the initial Pdot solution, even
after being stored for up to 6 months. This indicates that the
colloidal stability of streptavidin conjugated and unconjugated
Pdots did not change after the lyophilization process with 10%
sucrose. Therefore, the lyophilization procedure described in the
following sections was carried out with 10% sucrose.
[0057] Table 1. The hydrodynamic diameter of lyophilized and
unlyophilized streptavidin conjugated and unconjugated Pdots after
long term storage. Strep-PFBT-COOH Pdots were stored at -80.degree.
C. for 1 month. All the other Pdots were stored for 6 months. All
lyophilized Pdots were rehydrated with water. Both lyophilized and
unlyophilized Pdots were measured with dynamic light scattering
without sonication. (Lyoph.: lyophilized; Unlyoph.:
unlyophilized.)
TABLE-US-00001 Concentration Lyoph./ Diamter Pdot (mM) Unlyoph.
(nM) Strep-PFBT 4 Lyoph. 32 .+-. 2 Unlyoph. 32 .+-. 2 20 Lyoph. 30
.+-. 2 Unlyoph. 32 .+-. 1 100 Lyoph. 32 .+-. 1 Unlyoph. 32 .+-. 1
Strep-CNPPV 4 Lyoph. 28 .+-. 2 Unlyoph. 28 .+-. 2 20 Lyoph. 29 .+-.
2 Unlyoph. 28 .+-. 2 Strep-PFBT- 4 Lyoph. 26 .+-. 2 COOH Unlyoph.
26 .+-. 2 20 Lyoph. 24 .+-. 2 Unlyoph. 24 .+-. 2 Unconjugated 4
Lyoph. 23 .+-. 2 PFBT Unlyoph. 21 .+-. 2 20 Lyoph. 22 .+-. 2
Unlyoph. 20 .+-. 2 Unconjugated 4 Lyoph. 21 .+-. 2 CNPPV Unlyoph.
20 .+-. 2 20 Lyoph. 20 .+-. 2 Unlyoph. 20 .+-. 2 Unconjugated 4
Lyoph. 21 .+-. 2 PFO Unlyoph. 21 .+-. 2 20 Lyoph. 22 .+-. 2
Unlyoph. 21 .+-. 2 Unconjuagted 4 Lyoph. 24 .+-. 2 PF10BT Unlyoph.
21 .+-. 2 20 Lyoph. 24 .+-. 2 Unlyoph. 22 .+-. 2
[0058] Optical property. The optical properties of both lyophilized
and unlyophilized Pdots were measured and compared. We focused on
the absorption spectra, emission spectra, and quantum yield. We
measured the absorption and emission spectra of lyophilized Pdots
after 6 months storage and compared them with that of their
unlyophilized counterparts. As shown in FIGS. 3A-3C, the absorption
and emission spectra of lyophilized streptavidin conjugated Pdots
(Strep-PFBT, Strep-CNPPV and Strep-PFBT-COOH) were identical to
their unlyophilized counterparts after being stored for up to 6
months. The same phenomenon was also observed in Pdots made of PFO
(FIG. 3D) and PF.sub.10BT (FIG. 3E). These results indicate
lyophilization did not change the absorption and emission spectra
of these conjugated and unconjugated Pdots.
[0059] We next studied the brightness of Pdots to determine if
lyophilization had a negative effect on their fluorescence
intensity. To facilitate the brightness comparison between
lyophilized and unlyophilized Pdots of different concentrations, we
measured their quantum yield (QY), because quantum yield is less
dependent on concentration than fluorescence intensity as shown
below:
F=.alpha.*I*Q*n (1)
[0060] Where F is fluorescence emission intensity; .alpha. is the
instrument factor; I is the excitation intensity; Q is the quantum
yield; n is the concentration of Pdots. Table 2 shows the quantum
yield values of various Pdots that had and had not undergone
lyophilization. First, we found that the conjugated streptavidin
molecules did not affect the quantum yield of Pdots. The quantum
yield values of streptavidin conjugated CNPPV and PFBT Pdots stayed
at similar levels as their corresponding unconjugated Pdots. For
example, the QY values of 4 nM lyophilized Strep-PFBT and
unconjugated PFBT Pdots after 6 months storage were both 33%.
Second, the quantum yield of lyophilized Pdots did not fluctuate
much among different concentrations of Pdots. For example, the QY
values of 4 nM lyophilized and 20 nM lyophilized PFO Pdots after 6
months storage were both 47%.
[0061] More importantly, the quantum yield of most unlyophilized
Pdots decreased after long term storage, but the quantum yield of
lyophilized Pdots remained at the same level for the duration of
the storage time. For example, the quantum yield of 20 nM
lyophilized Strep-PFBT Pdots was 37% after stored for 1 day and 36%
after 6 months storage, respectively. In contrast, the quantum
yield of most unlyophilized Pdots showed a small but consistent
decrease: when the same Pdots (20 nM Strep-PFBT Pdots) were stored
unlyophilized, the quantum yield decreased from 33% to 30% after 6
months storage. Similar quantum yield changes were also found in
unconjugated PFBT and PFO Pdots. It is likely that oxidation of the
semiconducting polymer, which would reduce the quantum yield, was
minimized when the Pdots were lyophilized and stored at -80.degree.
C.
[0062] These results demonstrate that the brightness of Pdots
certainly was not adversely affected by the lyophilization process,
and remarkably, there could even be an enhancement in the optical
performance of Pdots by going through the lyophilization procedure.
For example, the quantum yield enhancement of 4 nM lyophilized
Strep-PFBT Pdots over unlyophilized Strep-PFBT Pdots after 1 day
storage was (0.35-0.32)/0.32=9.4%. Although the mechanism that
underlies this increase in quantum yield caused by lyophilization
is unclear, we think the lyophilization process caused the internal
rearrangement of the backbone or internal packing of the
semiconducting polymer.
[0063] Table 2. The quantum yield values of lyophilized and
unlyophilized Pdots stored for up to 6 months. For each Pdot,
samples with two concentrations (4 nM and 20 nM) were tested. (1 D:
1 day; 1 M: 1 month; 6 M: 6 months.)
TABLE-US-00002 Lyoph./ Storage Concentration Quantum Pdot Unlyoph.
Time (nM) Yield (%) Strep-PFBT Lyoph. 1 D 4 35 .+-. 1 20 37 .+-. 1
100 35 .+-. 1 6 M 4 35 .+-. 1 20 36 .+-. 1 100 34 .+-. 1 Unlyoph. 1
D 4 32 .+-. 1 20 33 .+-. 1 100 32 .+-. 1 6 M 4 29 .+-. 1 20 30 .+-.
1 100 28 .+-. 1 Strep-CNPPV Lyoph. 1 D 4 46 .+-. 1 20 49 .+-. 1 6 M
4 49 .+-. 1 20 48 .+-. 1 Unlyoph. 1 D 4 48 .+-. 1 20 48 .+-. 1 6 M
4 48 .+-. 1 20 48 .+-. 1 Strep-PFBT- Lyoph. 1 D 4 29 .+-. 1 COOH 20
30 .+-. 1 1 M 4 28 .+-. 1 20 29 .+-. 1 Unlyoph. 1 D 4 27 .+-. 1 20
29 .+-. 1 1 M 4 26 .+-. 1 20 27 .+-. 1 Unconjugated Lyoph. 1 D 4 36
.+-. 1 PFBT 20 34 .+-. 1 6 M 4 34 .+-. 1 20 32 .+-. 1 Unlyoph. 1 D
4 34 .+-. 1 20 33 .+-. 1 6 M 4 31 .+-. 1 20 29 .+-. 1 Unconjugated
Lyoph. 1 D 4 46 .+-. 1 CNPPV 20 49 .+-. 1 6 M 4 49 .+-. 1 20 50
.+-. 1 Unlyoph. 1 D 4 47 .+-. 1 20 48 .+-. 1 6 M 4 47 .+-. 1 20 49
.+-. 1 Unconjugated Lyoph. 1 D 4 47 .+-. 1 PFO 20 49 .+-. 1 6 M 4
47 .+-. 1 20 47 .+-. 1 Unlyoph. 1 D 4 48 .+-. 1 20 48 .+-. 1 6 M 4
44 .+-. 1 20 43 .+-. 1 Unconjugated Lyoph. 1 D 4 71 .+-. 2
PF.sub.10BT 20 70 .+-. 2 6 M 4 70 .+-. 2 20 71 .+-. 2 Unlyoph. 1 D
4 68 .+-. 2 20 66 .+-. 2 6 M 4 67 .+-. 2 20 70 .+-. 2
[0064] Labeling efficiency. Once we found the conditions where
Pdots retained their colloidal stability after lyophilization and
confirmed that the brightness of lyophilized Pdots did not
decrease, we tested the cell targeting capability of the Pdot
bioconjugates to ensure they maintained their biological
specificity. We used streptavidin-conjugated Pdots to label the
cell surface receptor, EpCAM, which is an epithelial cell adhesion
marker currently used for the detection of circulating tumor cells.
We used flow cytometry to quantify the brightness of the cell
labeling and the degree of non-specific absorption. For comparison
between lyophilized and unlyophilized Pdot-streptavidin, we used 20
nM streptavidin conjugated Pdots (Strep-CNPPV, Strep-PFBT and
Strep-PFBT-COOH) with 10% sucrose. The labeling efficiency of both
lyophilized and unlyophilized Pdot-streptavidin conjugates stored
for up to 6 months was tested.
[0065] We then compared the non-specific absorption and positive
cell labeling by Pdot-streptadvidin that had been lyophilized and
stored over various time versus Pdots that were not lyophilized.
Samples stored for 1 day after lyophilization were first tested.
This experiment reports on any potential effect caused by
undergoing the lyophilization process. As shown in the top panels
in FIG. 4, when cells were incubated with Pdot-streptavidin in the
absence of the primary antibody (negative), the intensity peaks of
both lyophilized (green curve) and unlyophilized (black curve)
samples were low and comparable. This result confirmed that both
lyophilized and unlyophilized Pdots produced very low amounts of
non-specific binding in the absence of the primary antibody.
[0066] The data also show that the intensity peaks for cells
labeled with both lyophilized (red curve) and unlyophilized (green
curve) Pdots in the presence of primary antibody (positive) were
well separated from that of the negative control samples.
Specifically, for Strep-CNPPV and Strep-PFBT-COOH Pdots, the
positive peak intensity values of lyophilized Pdots were similar to
that of unlyophilized Pdots. For Strep-PFBT Pdots, the peak
intensity value of lyophilized Pdots was a little larger than that
of unlyophilized Pdots. The labeling brightness difference is
consistent with our measured quantum yield values: for 20 nM
Strep-CNPPV and Strep-PFBT-COOH, which were stored for 1 day, the
QY values of lyophilized Pdots were similar to that of
unlyophilized Pdots (table 2); for Strep-PFBT, the cell labeling
enhancement is 10%, which is similar to our measured quantum yield
enhancement (9%). This result indicates that the lyophilization
process did not impair the performance of Pdot bioconjugates, and
for some semiconducting polymers, even enhanced their
performance.
[0067] We further tested the cell labeling efficiency of
lyophilized and unlyophilized Pdots after long term storage. As
displayed in the bottom panels in FIG. 4, the intensity peaks for
cells labeled with either lyophilized or unlyophilized Pdots stored
for up to 6 months in the presence of primary antibody (positive)
were still well separated from that of negative control samples.
Specifically, for Strep-CNPPV Pdots, positive peaks of lyophilized
and unlyophilized samples overlapped. For Strep-PFBT-COOH Pdots,
positive peak of lyophilized sample was slightly higher than that
of unlyophilized sample. For Strep-PFBT Pdots, the lyophilized
sample showed a more noticeable brightness enhancement of 22%,
which is consistent with our measured quantum yield enhancement of
20%. This result again indicates that the lyophilization process
effectively maintained the performance of Pdot bioconjugates. Our
data suggest that lyophilization is a good strategy for the
long-term storage of Pdot bioconjugates.
[0068] Control Experiment. Here, we used flow cytometry to quantify
the brightness of Pdot-tagged MCF-7 cells, where the cells were
labeled using Pdot solutions that contained either 10% or 0%
sucrose. FIG. 6 shows the resulting flow-cytometry data, which
clearly indicates the presence of 10% sucrose had no effect on the
brightness of the labeled cells and thus did not affect cell
labeling. The negative controls (performed under identical
conditions except in the absence of primary antibody) were also
similar between these two samples, which shows the presence of
sucrose in the Pdot solution also had no effect on the non-specific
binding properties of the Pdot bioconjugates.
[0069] The flow data was collected with a commercial BD FACSCanto
II cytometer (BD Bioscience, San Jose, Calif. USA). Cells were
illuminated by 488-nm laser light and fluorescence emission was
filtered by a 502-nm long pass filter before being detected by an
array of photomultiplier tubes (PMTs).
[0070] We studied the effect of lyophilization on the properties of
Pdot-streptavidin bioconjugates, including colloidal stability,
spectral properties, brightness, and labeling efficiency. Samples
of various concentrations were stored for up to 6 months. We found
that lyophilization with 10% sucrose was a good strategy to
preserve Pdot bioconjugates. The rehydrated Pdots after
lyophilization had the same size as that before lyophilization,
even in the absence of sonication to help re-disperse the Pdots.
The lyophilization procedure did not negatively affect the optical
properties of Pdots. The quantum yield values of lyophilized Pdots
using sucrose showed a consistent, albeit small, improvement in
quantum yield after lyophilization; this phenomenon is likely
caused by the rearrangement of the polymer backbone or internal
packing during lyophilization. The use of other lyoprotectant
molecules other than sucrose can result in much more significant
increase in quantum yield (see Example 2), which is an important
finding because Pdots with high quantum is highly desired for
improving the brightness of the probe for a wide range of
application. In addition to improving quantum yield, the use of
appropriate lyoprotectant molecules can also result in a narrowing
of the emission spectrum of the Pdot (FIG. 7), which is also
desired because narrow-band emission Pdots are valuable for their
multiplexing capability.
[0071] The brightness of cells labeled with lyophilized Strep-PFBT
Pdots stored for 6 months showed a 22% enhancement over the
unlyophilized counterpart, likely because oxidation of the
semiconducting polymer was minimized when the Pdots were
lyophilized and stored at -80.degree. C. We believe lyophilization
will be a preferred route for the long-term storage of Pdots, which
makes it an important practical consideration for the wide-spread
adoption of bioconjugated Pdots in biomedical research.
Example 2
[0072] This example describes the effect of sucrose concentration
on the lyophilization of Pdots. We used a series of sucrose
concentrations (0%, 1%, 10%, 20%, 50%) (w/v) to lyophilize Pdot.
Two types of Pdots were used. One is PFBT (Mw=73 k)+30% (w/w)
PS-PEG-COOH, and the other is the directly functionalized PFBT-COOH
2%. The Pdots were prepared using nanoprecipitation as described in
Example 1. The samples had a size of 21 nm at 20 nM concentration
in aqueous solution. Different concentration of sucrose was added
to the Pdot aqueous solution. The sample was then lyophilized.
After lyophilization, the sample was stored in -80.degree. C.
freezer for 1 day and then re-dispersed in aqueous solution.
[0073] Size and quantum yield were measured to describe whether
there is any change of the Pdot after lyophilization (FIG. 8).
Without the addition of sucrose (0%), a size as large as 220 nm for
lyophilized PFBT/PS-PEG-COOH Pdot and 230 nm for lyophilized
PFBT-COOH2% (their unlyophilized counterparts have the size of
.about.21 nm) was obtained, indicating serious aggregation during
lyophilization. Compared to 0% sucrose, the lyophilization with 1%
sucrose resulted in relatively smaller Pdot, i.e. .about.50 nm
(FIG. 8). However, it was still significantly larger than the
original size of 21 nm. At a sucrose concentration of 10% or 20%,
we found that the lyophilized Pdot showed exactly the same size as
that of the original Pdot, both having a size of 21 nm. However,
when the sucrose concentration was at 50%, the sample could not be
completely dried under vacuum during lyophilization and the
lyophilization at this sucrose concentration was not
successful.
Example 3
[0074] This example describes the use of several lyophilization
agents in the application of Pdot lyophilization. The direct
functionalized PFBT-COOH 2% Pdots and BODIPY-690 were prepared
using nanoprecipitation method as described in Example 1. Both the
as-prepared Pdots had a size of 20 nm and at a concentration of 20
nM in aqueous solution. Different lyophilization agents with 5-20%
(w/v) were added to the Pdot aqueous solution. In the combination
of lyophilization agents, the total agents concentration was 10%
(w/v) in the solution. The sample with lyophilization agent was
then lyophilized. After lyophilization, the sample was stored in
-80.degree. C. freezer for 1 day and then re-dispersed to aqueous
solution.
[0075] FIG. 9 in the upper panel shows the chemical structures of
the lyophilization agents used for the Pdots; they are sucrose,
glucose, mannitol, trehalose, maltose, hydroxypropyl-cyclodextrin,
and bovine serum albumin (BSA). In addition, two combination agents
were used; they are 5% sucrose+5% trehalose and 5% sucrose+5%
maltose.
[0076] Size and quantum yield were measured to describe whether
there was any change of the Pdots after lyophilization. The results
were shown in the lower panel in FIG. 9. The results indicate that
all these agents we used here are able to lyophilize the Pdots. For
example, the size and quantum yield of lyophilized Pdots were
similar to its unlyophilized counterpart for many of these
lyophilization agents. Remarkably, several agents showed an ability
to increase significantly the Pdots' quantum yield after
lyophilization. For example, when mannitol was used, the size of
the lyophilized PFBT-COOH 2% Pdot did not change as compared to its
unlyophilized Pdot, but its quantum yield increased .about.50% for
the Pdots after lyophilization. For BODIPY-690 Pdot, its quantum
yield showed .about.20% increase as compared to its unlyophilized
counterpart. When hydroxypropyl-cyclodextrin was used, the quantum
yield of lyophilized PFBT-COOH 2% Pdot showed .about.75% increase
and lyophilized BODIPY-690 showed .about.30% increase as compared
to their unlyophilized counterparts, respectively.
[0077] We also lyophilized the Pdots with BSA, and the result
showed that the quantum yield of the lyophilized Pdots was also
generally higher than that of unlyophilized Pdots. The size of the
lyophilized Pdots by DLS was not recorded due at least in-part to
the presence of large amounts of BSA in the solution, which can
interfere with DLS measurements. There was a strong peak at around
3 nm that corresponded to the size of BSA molecules. But both
lyophilized and unlyophilized Pdots solution were very clear,
indicating no obvious aggregation was formed during the
lyophilization process with BSA.
Example 4
[0078] This example describes the lyophilization of three types of
BODIPY based Pdots with centered emission at 570 nm, 590 nm and 690
nm. It shows the lyophilized BODIPY Pdots can be stored for at
least 6 months.
[0079] FIG. 10 in the upper panel shows the structures of the three
BODIPY based polymer structures. The Pdots were prepared by mixing
the BODIPY based conjugated polymer with 30% PS-PEG-COOH (w/w)
using the nanoprecipitation method. The three types of Pdots had a
size of 23 nm at 20 nM concentration in aqueous solution. We also
did bioconjugation to the BODIPY-690 Pdot by covalently linking
streptavidin to the Pdot, which gave a .about.4 nm increase to the
final Pdot size. All the Pdots (including the BODIPY-690 Pdot
conjugated to streptavidin) were lyophilized with 10% sucrose
(w/v). After lyophilization, the sample was stored in -80.degree.
C. freezer for up to 6 months and then was re-dispersed in aqueous
solution, after which we measured the size and quantum yield.
[0080] FIG. 10 in the middle panel shows the size and quantum yield
values of various BODIPY based Pdots that had and had not undergone
lyophilization. First, we found that the conjugated streptavidin
molecules did not affect the quantum yield of BODIPY based Pdots.
The quantum yield values of streptavidin conjugated BODIPY-690
stayed at similar levels as their corresponding unconjugated Pdots.
Second, compared to their unlyophilized counterparts, the size and
quantum yield of lyophilized Pdots did not change much.
[0081] The optical properties of both lyophilized and unlyophilized
Pdots were measured and compared. We measured the absorption and
emission spectra of lyophilized Pdots after 6 months storage and
compared them with that of their unlyophilized counterparts. As
shown in FIG. 10 lower panel, the absorption and emission spectra
of lyophilized streptavidin conjugated BODIPY-690 Pdots were
similar to their unlyophilized counterparts after being stored for
up to 6 months.
[0082] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
* * * * *