USO0RE43 944E
(19) United States (12) Reissued Patent
(10) Patent Number: US RE43,944 E (45) Date of Reissued Patent: Jan. 29, 2013
Zhang et a]. (54)
UPCONVERSION FLUORESCENT NANO-STRUCTURED MATERIAL AND USES THEREOF
(75) Inventors: Yong Zhang, Singapore (SG); Zhengguan Li, Singapore (SG) (73) Assignee: National University of Singapore,
Singapore (SG)
FOREIGN PATENT DOCUMENTS GB W0 W0 W0
2 413 334 A WO 2005/015213 Al WO 2006/131226 Al WO 2007/078262 Al
10/2005 2/2005 12/2006 7/2007
OTHER PUBLICATIONS P. Wang et al., “Luminescent Nanomaterials for Biological Label
ling,” Nanotechnology, vol. 17, pp. R1-R3, 2006. E. Beaurepaire, et al., “Functionalized Fluorescent Oxide Nanoparticles: Arti?cial Toxins for Sodium Channel Targeting and
(21) Appl.No.: 13/419,708
Imaging at the Single-Molecule Level,” Nano Letters, vol. 4, No. 11,
(22) Filed:
Mar. 14, 2012 Related US. Patent Documents
pp. 2079-2083, 2004. H. Sertchook et al., “Submicron Silica/Polystyrene Composite Par
Jan. 10, 2012
ticles Prepared by a One-Step Sol-Gel Process,” Chemistry of Mate rials, vol. 15, No. 8, pp. 1690-1694, 2003. F. van de Rijke, et al., “Up-Converting Phosphor Reporters for Nucleic Acid Microarrays,” Nature Biotechnology, vol. 19, pp. 273
Appl. No.:
12/445,904
276, Mar. 2001.
PCT Filed:
Oct. 17, 2007
PCT No.:
PCT/SG2007/000352
§ 371 (0)0), (2), (4) Date:
Apr. 16, 2009
Reissue of:
(64) Patent No.:
8,093,566
Issued:
(Continued) Primary Examiner * David Porta Assistant Examiner * Marcus Taningco
PCT Pub. No.: WO2008/048190
(74) Attorney, Agent, or Firm * Dickstein Shapiro LLP
PCT Pub. Date: Apr. 24, 2008
US. Applications:
(57)
ABSTRACT
(60)
Provisional application No. 60/829,768, ?led on Oct. 17, 2006.
Upconversion ?uorescent nano-structured material(s) com
(51)
Int. Cl.
(M3), and at least one polymer, Wherein: each X is the same or different and is selected from the group consisting of:
G01J1/58
(2006.01)
(52)
US. Cl. .................................................. .. 250/459.1
(58)
Field of Classi?cation Search .............. .. 250/459.1
See application ?le for complete search history. (56)
References Cited U.S. PATENT DOCUMENTS
2003/0030067 2007/0037215 2007/0087195 2009/0081461
Al Al Al Al
2/2003 2/2007 4/2007 3/2009
u
o
.
‘
'
D
e
I
same or different and is selected from the group consisting of:
Li, Na, K, Rb, Cs, Fr, Be, Mg, Ca, Sr, Ba, Ra, O and NH4; each M2 is the same or different and is a metal ion; each M3, independently, is the same or different and is selected from
the group consisting of Sc,Y, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu;j is OéjélO; k is lékélO; Wherein the polymer soluble in polar solvents. 26 Claims, 15 Drawing Sheets
.
‘ -
I
~
.\
Nu
halogen, O, S, Se, Te, N, P and As; each M1, ifpresent, is the
n is 1 Eng 10; and q is 1 éqé 10. In particular, the polymer is
Chen Patton Meyer et al. Yiet al.
I
prising at least one compound of formula (Ml)j(M2)kXn:
'
I
I
.
I
I U
I? .
v
.
9 l
a
I
O NIYFilYb-EDTM . S10: M:
I
(w),
US RE43,944 E Page 2 OTHER PUBLICATIONS
G.Yi, et al., “Synthesis, Characterization, and Biological Application of Size-Controlled Nanocrystalline NaYF4:Yb,Er Infrared-to-Vis ible Up-Conversion Phosphors,” Nano Letters, vol. 4, No. 11, pp. 2191-2196, 2004. J .F. Suyver et al., “Novel Materials Doped with Trivalent Lanthanides and Transition Metal Ions Showing Near-Infrared to
Visible Photon Upconversion,” Optical Materials, vol. 27, pp. 1111 1 130, 2005. K.W. Kramer, et al., “Hexagonal Sodium Yttrium Fluoride Based
Green and Bllue Emitting Upconversion Phosphors,” Chemistry of Materials, vol. 16, No. 7, pp. 1244-1251, 2004. S. Heer, et al., “Highly Ef?cient Multicolour Upconversion Emission
in Transparent Colloids of Lanthanide-Doped NaYF4 Nanocrystals,” Advanced Materials, vol. 16, No. 23-24, pp. 2102-2105, Dec. 2004. DR. Larson, et al., “Water-Soluble Quantum Dots for Multiphoton Fluorescence Imaging in Vivo,” Science, vol. 300, No. 5624, pp. 1434-1436, 2003. J-H Zeng, et al., “Synthesis and Upconversion Luminescence of
Hexagonal-Phase NaYF4zYb, Er3+Phosphors of Controlled Size and
Y. Gao, et al., “Silver Nanowires with Five-Fold Symmetric Cross Section,” Journal of Crystal Growth, vol. 276, pp. 606-612, 2005. M. Liu, et al., “An Investigation of the Interaction Between Polyvinylpyrrolidone and Metal Cations,” vol. 44, pp. 55-64, 2000. R. Si, et al., “Self-Organized Monolayer of Nano sized Ceria Colloids
Stabilized by Poly(vinylpyrrolidone),” Journal of Physical Chemis try B, vol. 110, pp. 5994-6000, 2006. C. Graf, et al., “A General Method for the Controlled Embedding of Nanoparticles in Silica Colloids,” Langmuir, vol. 22, No. 13, pp. 5604-5610, 2006. C. Graf, et al., “A General Method to Coat Colloidal Particles with
Silica,” Langmuir, vol. 19, No. 17, pp. 6693-6700, 2003. T. Nann, et al., “Single Quantum Dots in Spherical Silica Particles,” Angewandte Chemie International Edition, vol. 43, No. 40, pp. 5393 5396, 2004. D.K. Yi, et al., “ Silica-Coated Nanocomposites of Magnetic
Nanoparticles and Quantum Dots,” Journal of the American Chemi cal Society, vol. 127, No. 14, pp. 4990-4991, 2005.
T-J. Yoon, et al., “Speci?c Targeting, Cell Sorting and Bioimaging with Smart Magnetic Silica Core-Shell Nanomaterials,” Small, vol. 2, No. 2, pp. 209-215, 2006.
Morphology,” Advanced Materials, vol. 17, pp. 2119-2123, 2005. J-C Boyer, et al., “Synthesis of Colloidal Upconverting NaYF4
D. Chatterj ee et al., “Novel Nanodevice for Cancer Immunotherapy”
Nanocrystals Doped with Er3+, Yb3+and Tm3+, Yb3+via Thermal
p. 84, Oct. 8, 2006. Q. Li et al., “Luminescence of Europium(III) and Terbium(III) Com
Decomposition of Lanthanide Tri?uoroacetate Precursors,” Journal ofthe American Chemical Society, vol. 128, pp. 7444-7445, 2006. Y. Yang, et al., “A Gold Nanocomposite Made Soluble in Both Water and Oil by the Addition of a Second Adsorption Layer of Poly-N Vinyl-2-Pyrrolidone on Gold Nanoparticles that have been Made
Hydrophobic,” Nanotechnology, vol. 17, pp. 461-465, 2006. D.M.L. Goodgame, et al., “A Macrobicyclic Bimetallic Chain Poly
mer
Incorporating
Deprotonated
2-Pyrrolidone
Bridges,”
Angewandte Chemie-International Edition in English, vol. 27, No. 2, pp. 261-262, 1988.
Fourth International Nanomedicine and Drug Delivery Symposium,
pleXes Incorporated in Poly(Vinyl Pyrrolidone) Matrix” Journal if Physical Chemistry B, vol. 105, pp. 12293-12296, Nov. 16, 2001. H. Mai et al., “High-Quality Sodium Rare-Earth Fluoride
Nanocrystals: Controlled Synthesis and Optical Properties” Journal ofthe American Chemical Society, vol. 128, pp. 6426-6436, Apr. 20, 2006.
E. Ricci-Junior et al., “Zinc(II) Phthalocyanine Loaded PLGA Nanoparticles for Photodynamic Therapy Use” International Journal ofPharmaceutics, vol. 310, No. 1-2, pp. 187-195, Mar. 9, 2006.
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2
UPCONVERSION FLUORESCENT NANO-STRUCTURED MATERIAL AND USES THEREOF
hydrophobic and could only be dispersed in certain organic solvents such as hexane and dimethyl sulfoxide (DMSO) under ultrasound sonication (Boyer et al. 2006; Heer et al.
2004; Mai et al. 2006). Use of these nanocrystals directly for
Matter enclosed in heavy brackets [ ] appears in the original patent but forms no part of this reissue speci?ca
bio-applications is very limited due to their very small solu bility in water and unsuitable surface property. Accordingly, there is still a need in this ?eld of technology
tion; matter printed in italics indicates the additions made by reissue.
of improved upconversion nanoparticles. In fact, the synthe sis of monodisperse and water soluble ?uoride nanocrystals with upconversion ?uorescence is still very challenging. In particular, there is a need in developing suitable methods for
synthesizing up-conversion NaYF4 nanocrystals which are
This is a reissue 0fU.S. Pat. No. 8, 093, 566, issuedJan. 10, 2012, which is a 37] oflnternational Application No. PCT/
dispersible in water and organic solvents and have some functional chemical groups on their surfaces for conjugation
SG2007/OOO352,?led Oct. 1 7, 2007, which claims the bene?t 0fU.S. ProvisionalApplication No. 60/829, 768,?led Oct. 1 7,
of biomolecules (Larson et al. 2003).
2006. SUMMARY OF THE INVENTION FIELD OF THE INVENTION
The present invention relates to upconversion ?uorescent nano-structured material(s) and methods for their preparation and used thereof. In particular, the invention related to upcon version ?uorescent nanoparticles, methods for their prepara
20
The present invention addresses the problems above, and in particular to provide new and improved upconversion ?uo rescent nano-structured material(s). In particular, there are
provided new and improved nano-siZed phosphors with up conversion (UC) ?uorescence which have great potential for use in biological studies and clinical applications, as labeling
tion and uses thereof. 25
materials, imaging probes, and the like. According to a ?rst aspect, the present invention provides
BACKGROUND OF THE ART
at least one upconversion ?uorescent nano-structured mate
Nano-siZed ?uorescent labeling materials have been
widely used for biological studies and clinical applications. Conventional down-conversion ?uorescent labels require
rial comprising at least one compound of formula (Ml)j(M2)k Xn:(M3)q and at least one polymer, wherein 30
each X is the same or different and is selected from the
ultraviolet or blue excitation wavelength (Beaurepaire et al.
group consisting of: halogen, O, S, Se, Te, N, P and As;
2004; Wang et al. 2006). These single-photon ?uorescent
each Ml, if present, is the same or different and is selected
labels emit one lower energy photon after absorbing higher energy UV or visible photon. Their disadvantages include
from the group consisting of: Li, Na, K, Rb, Cs, Fr, Be, Mg, Ca, Sr, Ba, Ra, O and NH4;
low light penetration depth and possible severe photo-dam age to living organisms. Furthermore, many biological
35
samples show auto ?uorescence under short wavelength UV radiation, which decreases the sensitivity of detection. The
selected from the group consisting of Sc, Y, La, Ce, Pr,
Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm,Yb and Lu;
use of ?uorescent labels that can be excited in the near infra
red (NIR) region was suggested (Sertchook and Avnir 2003).
40
Yi et al. 2004 and van de Rijke et al. 2001, suggested the infrared-to-visible up-conversion nanocrystal. These nanoc rystals emit one higher energy photon after absorbing two or
more lower-energy photons. Different colors of visible light can be obtained from different up-conversion phosphors when excited by the same IR laser (van de Rijke et al. 2001). In comparison with down-conversion ?uorescent materials,
45
Accordingly to a particular embodiment (when j is Zero), M1 is not present and the compound of the invention has 50
formula (M2)kXn:(M3)q. In the upconversion ?uorescent nano-structured material according to the invention wherein the compound has for mula (M2)kXn:(M3)q or (Ml)j(M2)kXn:(M3)q, when q is 1, only M3 element is doped in the nano-structured material. A
55
co-doped NaYF4 nanocrystals have been prepared with
particular non-limiting example for this embodiment is NaYF4:Yb. When q is 2 (or a higher value), two (or more) preferably different M3 elements are co-doped into the nano
structured material. A particular non-limiting example for this embodiment (when q is 2) is NaYF4:Yb,Er 60
NaYF4 nanocrystals with controlled siZe and shape (Heer et al. 2004; Yi et al. 2004; Zeng et al. 2005). Ethylenediamine
With reference to the polymer, it may be a linear polymer or a branched polymer. It may be amphiphilic or hydrophilic. In
particular, the polymer is a polymer soluble and/or dispers ible in polar solvents, for example in water. The polymer may
tetraacetic acid (EDTA) was used as a chelating agent to
control the growth of NaYF4 nanocrystals, but the nanocrys tals as prepared tended to precipitate in solution (Yi et al. 2004; Zeng et al. 2005). Colloidal solutions of NaYF4 nanoc rystals were prepared. However, these nanocrystals were
selected from the group consisting of: Sc, Y, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu. Each M3, independently, is the same or different and may be selected
due to their unique ?uorescent properties. In addition, photo
strong up-conversion ?uorescence seven orders of magnitude higher than that of CdSeiZnS quantum dots (Heer et al. 2004; Larson et al. 2003). Some efforts have been made to produce up-conversion
j is OéjélO; k is lékélO; n is lénélO; and q is 1 i q; 10. Inparticular, M2 may be selected from the group consisting of: transition metal ions, inner transition metal ions, and Group I to Group VI metal ions. In particular, M2 may be
from the group consisting of: Yb, Er, Tm and Ho.
up-conversion nanocrystals show very low background light damage to biological tissues is minimal because these tissues are usually transparent to NIR light (Suyver et al. 2005). Among the most commonly used up-conversion nanocrys tals, Yb/Er or Yb/Tm co-doped NaYF4 nanocrystals have been reported as e?icient infrared-to-visible up-conversion material (Kramer et al. 2004). Colloidal Yb/Er and Yb/Tm
each M2 is the same or different and is a metal ion; each M3, independently, is the same or different and is
65
be a polymer having an amino group. The polymer may have an average molecular weight of about 5-50 kDa, for example,
about 10-40 kDa, about 15-25 kDa. In particular, the polymer has an average molecular weight of about 25 kDa. The poly
US RE43,944 E 3
4
mer may be selected from the group consisting of: polyeth
nolevulinic acid (ALA), methyl aminolevulinate, temopor?n,
ylenimine (PEI), poly-l-lysine (PLL), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), poly(ethylene glycol)
phtalocyanine, and the like. Any other photosensitiZer avail able in the art and suitable for the purpose of the present invention may also be used.
(PEG), poly(4 vinylpyridine) (P4VP), oleic acid, stearic acid, chitosan and mixtures thereof. In particular, the polymer is
The upconversion ?uorescent nano-structured material
PEI, PVP or a mixture thereof. More in particular, the poly mer is PEI. The nano-structured material according to the invention may comprise the polymer at a concentration of
according to the invention may further comprise at least one biomolecule. The biomolecule may be attached to the nano
structured material. The biomolecule may be selected from
the group consisting of: protein, nucleic acid, nucleosides,
about 5-50 Weight %, in particular, of about 10-25 Weight %. According to particular examples, the compound of for mula (Ml)j(M2)kXn:(M3)q may be selected from the group
nucleotides, DNA, hormone, amino acid, peptide, peptidomi metic, RNA, lipid, albumin, antibody, phospholipids, gly colipid, sterol, vitamins, neurotransmitter, carbohydrate, sugar, disaccharide, monosaccharide, oligopeptide, polypep
consisting of: NaM2F4:(M3)q, LiM2F4:(M3)q, KM2F4:(M3)q,
RbM2F4:(M3)q, CsM2F4:(M3)q, BeM2F5:(M3)q, Be(M2)2F8:
tide, oligosaccharide, polysaccharide and a mixture thereof.
(M3)q5 MgM2F53(M3)q> Mg(M2)2F8:(M3)q5 CaM2F5:(M3)q5 Ca(M2)2Fs3(M3)q> SrM2F53(M3)q> Sr(M2)2F8:(M3)q$ BaM2F5:(M3)q5 Ba(M2)2F8:(M3)q’ M2F3:(M3)q’ (M2)2O2SZ
There is also provided at least one article of manufacture
comprising the upconversion ?uorescent nano-structured material according to any aspect of the invention. The article of manufacture may be at least one of the folloWing: a display device, a solar cell, an optical data storage, a bio-probe, a
(M3)q, (M2)2O3S:(M3)q, (M2)2O3:(M3)q and a combination thereof, Wherein M2 and M3 are as de?ned through the Whole content of the present application. More in particular, When q is 2, the nano-structured material according to the invention may be selected from the group consisting of: PEI/NaYF4;
carrier for drug delivery, a lamp, a LED, a LCD, a Wear 20
obvious to a skilled person are also encompassed by the scope
PEI/NaYF4:Yb,Er; PEI/NaYF4:Yb,Tm; PEI/NaYF4:Yb,Ho; PVP/NaYF4; PVP/NaYF4:Yb,Er; PVP/NaYF4:Yb,Tm;
of the present invention.
PVP/NaYF4:Yb,Ho and a combination thereof.
The upconversion ?uorescent nano-structured material
25
according to the invention may have a structure selected from
the group consisting of: spherical, hexagonal, cubic, tetrago nal, rhombohedral, orthorhombic, monoclinic, triclinic and a combination thereof. For example, the nano-structured mate rial has a hexagonal structure. According to particular examples, the nano-structured material may be hexagonal
30
There is also provided a kit comprising at least one nano structured material or an article of manufacture according to
any one aspect of the invention. The kit may, optionally, comprise at least one biomolecule. There is also provided at least one bio-imaging and/or bio-detection apparatus comprising: at least one upconver sion ?uorescent nano-structured material according to any aspect of the invention; at least one biomolecule; at least one source of excitation; and at least one means for delivery of the source of excitation to the system. The source of excitation may be NIR. In particular, the NIR is at 980 nm. The means
phase NaYF4, hexagonal phase NaYF4:Yb,Er, hexagonal phase NaYF4:Yb,Tm or hexagonal phase NaYF4:Yb,Ho. The upconversion ?uorescent nano-structured material according to the invention may have at least one dimension of
resistance, a laser, optical ampli?er, and/or a device for bio imaging. HoWever, further article of manufacture knoW or
for delivery of the source of excitation to the apparatus may be 35
siZes 2100 nm. For example, 250 nm, 220 nm or 210 nm.
selected from the group consisting of: optical ?bres, endo scopes, external light and external laser.
The upconversion ?uorescent nano-structured material according to any preceding claim, Wherein the nano-struc
There is also provided a upconversion ?uorescent nano structured material according to the invention for use in medi
tured material is at least one nanoparticle and the average diameter of the nanoparticle(s) is 2100 nm. The nano-struc tured material according to the invention may be at least one
cine. In particular, there is provided a upconversion ?uores 40
cent nano-structured material according to the invention for use in photodynamic therapy or for use in non-invasive imag
nanoparticle and the average diameter of the nanoparticle(s)
ing. In particular, the photodynamic therapy is in cancer cells.
is 250 nm.
There is also provided the use of at least one upconversion
The upconversion ?uorescent nano-structured material according to the invention may be in the form of: nano particle(s), nano?lm or monolith. In particular, the nano structured material according to the invention may be a NIR
45
todynamic therapy.
to-visible upconversion ?uorescent nanoparticle. The upconversion ?uorescent nano-structured material according to the invention may further comprise at least one
?uorescent nano-structured material according to any aspect of the invention in the preparation of a medicament for pho
50
There is also provided a method for photodynamic therapy, the method comprising the step of administering to a subject the upconversion ?uorescent nano-structured material according to any aspect of the invention.
surfactant, lipid, polymer, inorganic material, or a mixture thereof Which is disposed about the nano-structured material
upconversion ?uorescent nano-structured material according
and modi?es the surface of the nano-structured material.
to any aspect of the invention, comprising: mixing ions of at
There is also provided a method of preparing at least one
The upconversion ?uorescent nano-structured material
layer of silica Which is disposed about the nano-structured
least one M3 and at least one M2 to obtain a mixture; adding at least one polymer to the mixture; and adding ions of at least one X. The method may further comprise adding the polymer
material and Which modi?es the surface of the nano-struc
in the presence of ions of at least one Ml.
tured material. According to this embodiment the layer of
There is also provided a method of controlling the siZe and/or shape of the upconversion ?uorescent nano-structured
according to the invention may further comprise at least one
silica is applied on the nano-structured material to form a core-shell structure.
55
60
The upconversion ?uorescent nano-structured material
material(s) comprising varying the amount of polymer in the upconversion ?uorescent nano-structured material.
according to the invention may further comprise at least one
photosensitiZer Which is disposed about the nano-structured
BRIEF DESCRIPTION OF THE FIGURES
material and modi?es the surface of the nano-structured
material. The photosensitiZer may be any suitable photosen sitiZer suitable for the purpose of the invention. In particular, the photosensitiZer may be Zinc phthalocyanine (ZnPC), ami
65
FIG. 1. TEM images of the PVP/NaYF4:Yb,Er nanocrys tals With different siZes (A-C, 30 nm; D, 48 nm; E, 65 nm; E,
87 nm).
US RE43,944 E 6
5 FIG. 3. Digital camera photos of the solutions of PVP stabilized NaYF4:Yb, Er NCs dispersed in different solvents.
to be approximately 97%. (c) Fluorescence emission spectra of PEI/NaYF4:Yb,Er nanoparticles When excited With 980 nm NIR laser (solid line) overlaps considerably With excita tion spectra of ZnPC (dashed line) (d). ADPA destruction
(DMF: N,N-dimethylformamide; DMSO: dimethyl sulfox
representing singlet oxygen production (measured by absorp
ide.)
tion intensity at 400 nm) as a function of exposure time to NIR
FIG. 2. XRD pattern (A) and FT-IR spectrum (B) of the
PVP/NaYF4:Yb,Er nanocrystals.
laser shoWs steady fall from original (100%) for the ZnPC PEI/NaYF4:Yb,Er nanoparticles (squares) While pure ADPA control undergoes slight bleaching on continuous exposure to laser (circles) (e). MTT assay to demonstrate the effective ness of the nanoparticles for photodynamic therapy, by mea
FIG. 4. Fluorescence spectra of the Yb,Er (A) and Yb,Tm
(B) doped PVP/NaYF4 nanocrystals in ethanol under excita tion of NIR laser (980 nm), and ?uorescence images of PVP/
NaYF4:Yb,Er (C, total ?uorescence; D, E, ?uorescence pass ing through ?lters for red and green light respectively) and FIG. 5. TEM images of silica coated PVP/NaYF4:Yb,Er
suring the viability of HT29 cells after exposed to 980 nm laser for 5 minutes, after incubation With different amount of
nanocrystals (A, B, C). TEM image of PVP/NaYF4:Yb,Er
ZnPC-PEI/NaYF4:Yb,Er nanoparticles for 24 hours (Bars
nanocrystals coated With a very thin silica layer is also given
shoW standard error, n:4). FIG. 16. imaging of mice and rats injected With quantum
PVP/NaYF4:Yb,Tm.
(D). FIG. 6. Fluorescence spectra of NaYF4:Yb,Er nanocrystals
dots and PEI/NaYF4:Yb,Er upconversion nanoparticles.
before (dot line) and after (solid line) silica coating (A) and
Intradermal injection of quantum dots (a) and PEI/NaYF4: Yb,Er nanoparticles (b) into mice both demonstrated visible ?uorescence. HoWever, only the latter shoWed luminescence When injected into deeper tissues such as heart (c), back muscles (d), groin muscles (e) and thigh muscles (f). Intrad
?uorescence intensity of the silica coated nanocrystals in Water as a function of time (B) and pH (C).
20
FIG. 7. TEM images of PEI/NaYF4:Yb3",Er3+ nanocrys tals With (a) 5 Wt %, (b,c) 10 Wt %, (d,e) 25 Wt %, (f) 50 Wt % PEI concentration. FIG. 8: XRD data of sample (a) With 10 Wt % (b) With 25 Wt % PEI concentration.
ermal injection of quantum dots into dorsal skin of rats 25
FIG. 9. NIR-to-visible upconversion ?uorescence spectra
shoWed no ?uorescence (g, back skin as compared to quan tum dot sample placed on dish) but some ?uorescence Was seen from thinner foot skin (h). In contrast, ?uorescence from
and photographs of the PEI/NaYF4:Yb3",Er3+ (a) and PEI/
upconversion nanoparticles injected into muscles of the groin
NaYF4:Yb3",Tm3+ (b) nanoparticles in aqueous solutions,
Was seen through intact skin (i) or When exposed (j). Intrad
ermal injection of quantum dots (to an approximate depth of
excited using a NIR laser.
FIG. 10: The up conversion ?uorescence spectra samples
30
With: 1) pure NaYF4:Yb3J', Er3+; 2) 5 Wt %; 3) 10 Wt %; 4) 25 Wt % PEI/NaYF4:Yb3+, Er3+
of upconversion nanoparticles shoWed ?uorescence upon
FIG. 11. FT-IR spectra of pure NaYF4:Yb,Er (a) and PEI/
excitation With a NIR laser (1). The middle square Was injected With PBS as control. These demonstrated the supe
NaYF4:Yb,Er (b) nanocrystals. FIG. 12. Scheme of synthesis of silica coated PVP/NaYF4
35
nanocrystals doped With lanthanide ions. FIG. 13. PEI/NaYF4 upconversion nanoparticles. (a) TEM 40
dispersity by the clear solution of the nanoparticles. (b) Fluo
EDAX analysis (i) of NaYF4:Yb,Er nanospheres.
and serum as a function of incubation time. Viability of bone marroW derived stem cells after incubated With the nanopar 45
different concentrations (d). (e,f) Biodistribution of the nano particles in organs of rat harvested at different time after
tail-vein injection of the nanoparticles. (All bars denote stan dard error, n:4). FIG. 14. Imaging of cells incubated With PEI/NaYF4:Yb, Er nanoparticles. Bright ?eld (a,c) and confocal ?uorescence (b,d) images of human colonic adenocarcinoma cells (HT29, a,b) and human breast cancer cells (SKBR3, c,d) incubated With folic acid functionaliZed PEI/NaYF4 nanoparticles for 1 hour.
centrifuged. Encapsulation e?iciency calculated from the standard curve of ?uorescence emission spectra of ZnPC before and after attachment to the nanoparticles is determined
FIG. 18. Coating of silica on nanocrystals. TEM images of silica coated NaYF4:Yb,Er nanospheres at different magni ?cations (a-c) and ?uorescence spectra of NaYF4:Yb,Tm (d) and NaYF4:Yb,Er (e) nanospheres With and Without silica
coating. FIG. 19. Confocal ?uorescence imaging of MCF-7 cells 50
using silica/NaYF4:Yb,Er nanospheres. a, Bright-?eld (left), confocal ?uorescence (middle) and superimposed (right) images of MCF-7 cells incubated With the nanospheres for 24 hours. b, Confocal ?uorescence images of MCF-7 cells With the nanospheres excited by 980 nm laser at different poWer
55
FIG. 15. Photodynamic therapy using PEI/NaYF4:Yb,Er nanoparticles attached With ZnPC. (a) Schematic draWing shoWing hoW photodynamic therapy Works using upconver sion nanoparticles. Upon exposure to NIR light, the nanopar ticles convert NIR light to visible light Which Will activate the photosensitiZer ZnPC to produce reactive oxygen species to kill cancer cells. (b) Fluorescence spectra of ZnPC attached to the nanoparticles and that in the supernatant, after ZnPC Was mixed With the nanoparticles and then the nanoparticles Were
NaYF4:Yb,Er nanospheres at different magni?cations. d, TEM images of NaYF4:Yb,Er nanoellipses. e,f, TEM images of NaYF4:Yb,Er nanoplates at different magni?cations. g, Fourier Transform of TEM image in f. XRD pattern (h) and
rescence intensity of PEI/NaYF4:Yb,Er nanoparticles in PBS
ticles for different time periods (c) and the nanoparticles With
rior luminescence depth of upconversion nanoparticles and the ability to image deeper tissues and/or to use photody namic therapy at these depths. FIG. 17. Control of nanocrystal shape. a-c. TEM images of
image of PEI/NaYF4 nanoparticles shoWing monodisperse nanoparticles With approximately 50 nm in siZe. Insert, Pho to graph of the nanoparticles in PBS demonstrating the mono
10 mm) on shaved rat abdominal skin did not shoW ?uores cence upon excitation With a UV lamp (k). Similar injection
intensities. FIG. 20. Multi-color NIR-to-visible upconversion nano
spheres. a, Schematic draWing of FRET based multi-color
silica/NaYF4 NIR-to-visible upconversion nanospheres. TEM images of FITC doped silica/NaYF4:Yb,Tm nano 60
spheres (b), TRITC doped silica/NaYF4:Yb,Er nanospheres (c), and QD605 doped silica/NaYF4:Yb,Tm nanospheres (d). e, ?uorescence spectra of pure silica/NaYF4:Yb,Tm nano
65
spheres (black line) and the nanospheres doped With FITC (green line) and QD605 (red line). f, ?uorescence spectra of pure silica/NaYF4:Yb,Er nanospheres (black line) and the nanospheres doped With TRITC (red line). g, ?uorescence spectra of silica/NaYF4:Yb,Er nanospheres (0.01 mmol)
