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Hybrid quantum dot−fatty ester stealth nanoparticles: toward clinically
relevant in vivo optical imaging of deep tissue
Shuhendler, Adam J.; Prasad, Preethy; Chan, Ho-Ka Carol; Gordijo, Claudia
R.; Saroushian, Behrouz; Kolios, Michael; Yu, Kui; O’Brien, Peter J.; Rauth,
Andrew Michael; Wu, Xiao Yu
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https://publications-cnrc.canada.ca/fra/voir/objet/?id=cffc700c-eb51-4d91-86d0-31cc0a110398
1
Hybrid Quantum Dot-Fatty Ester
2
Stealth Nanoparticles: Toward
3
Clinically Relevant in Vivo Optical
4
Imaging of Deep Tissue
5
Adam J. Shuhendler,
†Preethy Prasad,
†Ho-Ka Carol Chan,
†Claudia R. Gordijo,
†Behrouz Soroushian,
‡ 6Michael Kolios,
‡Kui Yu,
§Peter J. O’Brien,
†Andrew Michael Rauth,
^,* and Xiao Yu Wu
†,*
7 †
Leslie L. Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario, Canada, M5S 3M2,
‡Department of Physics, Ryerson University, 350
8
Victoria Street, Toronto, Ontario, Canada, M5B 2K3,
§NRC Steacie Institute for Molecular Sciences, National Research Council of Canada, 100 Sussex Drive, Ottawa,
9
Ontario, Canada, K1A 0R6, and
^Department of Medical Biophysics, University Health Network, Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario,
10
Canada, M5G 2M9
11
12
M
edical imaging has become a
sta-13
ple in a clinician's arsenal for the
14detection and treatment of
hu-15
man disease, especially for cancers.
1Current
16
imaging modalities applied to oncology,
17such as magnetic resonance imaging and
18positron emission tomography (PET),
re-19quire expensive instrumentation and long
20subject exposure times. In the case of PET,
21potentially genotoxic radioisotope contrast
22
agents are employed.
2In contrast,
fluores-23cent imaging methods utilize nonionizing
24radiation are relatively less expensive and
25less hazardous, and can be performed
26quickly with brief exposure times. However,
27traditional fluorescent imaging in medicine
28has been severely limited by light scattering
29and autofluorescence of tissue in the
ultra-30violet and visible light range.
3,4Hence images
31have only been acquired at depths of a few
32millimeters below the surface of the skin.
3,4In
33the near-infrared (NIR) wavelength range of
34
650-900 nm, the light scattering,
absor-35bance, and autofluorescene of tissue are at
36a minimum, providing an opportunity for
37deeper optical interrogations in vivo with a
38predicted tissue penetration depth of 1-2
39cm (Figure 1
F1
).
4,5Whether or not fluorescent
40medical imaging can become of broad use
41
in clinical practice now depends on the
42
development of a safe, biocompatible
fluoro-43phore with excitation and emission
wave-44lengths in the NIR.
45
Ideally, an NIR fluorophore for medical
46applications should have a large absorption
47exctinction coefficient, high NIR quantum
48yield, low photobleaching, and no toxicity.
6 49Organic fluorophores are poor candidates for
50deep fluorescent medical imaging because of
51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 7172
their poor circulation half-lives, poor NIR
73
quantum yields, rapid photobleaching, and
74spectral shifting due to rapid plasma protein
75
binding, metabolism, and/or chemical
76
degradation.
6-8For this reason, NIR-tuned
77
semiconductor quantum dots (QD) have
78
been developed and applied to medical
imag-79
ing for sentinal lymph node mapping
9-11 80and tumor imaging.
12,13The properties of
81QDs that render them effective
fluoropho-82res for medical fluorescence imaging stem
83
*Address correspondence to xywu@phm.utoronto.ca.
Received for review November 8, 2010 and accepted December 27, 2010. Published online
10.1021/nn103024b
CXXXX American Chemical Society
ABSTRACT
Despite broad applications of quantum dots (QDs) in vitro, severe toxicity and
dominant liver uptake have limited their clinical application. QDs that excite and emit in the
ultraviolet and visible regions have limited in vivo applicability due to significant optical interference
exerted by biological fluids and tissues. Hence we devised a new biocompatible hybrid fluorophore
composed of near-infrared-emitting PbSe quantum dots encapsulated in solid fatty ester
nanoparticles (QD-FEN) for in vivo imaging. The quantum yield and tissue penetration depth of
the QD-FEN were characterized, and their biological fate was examined in a breast tumor-bearing
animal model. It was found for the first time that chemical modification of the headgroup of
QD-encapsulating organic fatty acids was a must as these groups quenched the photoluminescence of
PbSe nanocrystals. The use of fatty esters enhanced aqueous quantum yields of PbSe QDs up to
∼45%, which was 50% higher than that of water-soluble PbSe nanocrystals in an aqueous medium.
As a result, a greater than previously reported tissue penetration depth of fluorescence was recorded
at 710 nm/860 nm excitation/emission wavelengths. The QD-FEN had much lower short-term
cytotoxicity compared to nonencapsulated water-soluble QDs. More importantly, reduced liver
uptake, tumor retention, lack of toxic response, and nearly complete clearance of QD-FEN from the
tested animals was demonstrated. With a combination of near-infrared spectral properties,
enhanced optical properties,and significantly improved biosafety profile, this novel hybrid
nanoparticulate fluorophore system demonstrably provides real-time, deep-tissue fluorescent
imaging of live animals, laying a foundation for further development toward clinical application.
KEYWORDS: hybrid quantum dot-fatty ester nanoparticles• high biocompatibility •deep tissue penetration • near-infrared whole-body imaging • breast tumor
model
ARTICLE
ACS Nano | 3b2 | ver.9 | 5/1/011 | 10:53 | Msc: nn-2010-03024b | TEID: tes00 | BATID: 00000 | Pages: 8.16
84
from their high quantum yields with NIR excitation and
85their large effective Stoke's shifts, resulting in the
86potential for optical image acquisition at greater tissue
87depths.
3,14,15Currently, however, the major hurdle in
88the clinical application of QDs as medical-grade
fluor-89ophores is their toxicity,
16attributable to the heavy
90
metal precursors required for their synthesis,
17the
91
ligands employed for their surface functionalization,
1892
and their extensive liver uptake and poor clearance
93
rates that result in unsafe pharmacokinetic profiles.
19,20 94By mitigating the toxicity issues of QDs through
im-95
provement of their pharmacokinetic profiles while
96maintaining their superior spectral properties in the
97NIR region, an inorganic QD-organic fatty ester
nano-98particle hybrid fluorophore has been formulated.
99These particles may be well suited for medical imaging
100applications, advancing the field of fluorescence
med-101ical imaging.
102
RESULTS AND DISCUSSION
103
This paper describes the formulation and
character-104
ization of quantum dots encapsulated in fatty ester
105
nanoparticles. The novel nanocarrier system can
effec-106tively encapsulate NIR PbSe QDs resulting in lower in
107
vitro
cytotoxicity, improved in vivo biodistribution, and
108
clearance of the semiconductor nanocrystals, and
en-109hanced aqueous QD optical properties resulting in
110deep-tissue imaging capabilities. While QDs emitting
111in the visible range have previously been encapsulated
112in lipid-based nanoparticles,
21this paper presents the
113first encapsulation of QDs with clinically relevant
op-114
tical properties in stealth, fatty ester-based
nanoparti-115
cles, the first demonstration of improved aqueous
116quantum yield of QDs, and a vast improvement of
117
imaging depth, resulting in significant progress toward
118enhanced fluorophore biocompatibility and utility.
119Formulation of QD-FEN.
Lead-selenide (PbSe)
nano-120
crystals were employed for the formulation of QD-FENs
121due to their relative ease of synthesis, the tunability of
122their emisson within the NIR range, their high resultant
123
quantum yields with NIR excitation, and the demonstrated
124
photostability of this semiconductor system.
14,15,22The
125
synthesis of PbSe QDs was based on a room
tempera-126
ture scheme under ambient atmospheric conditions
127
previously published, resulting in hydrophobic, oleic
128
acid-capped nanocrystals, ∼7 nm in diameter that
129
were suspended in hexanes.
15The formulated
QD-130
loaded fatty acid or fatty ester nanoparticles were
131
110 ( 15 nm in hydrodynamic diameter, as measured
132
by dynamic light scattering, with the PbSe QD being
133
trapped within the now solid matrix (Figure 2
A). The
F2 134measured zeta potential (ζ = -20 ( 1.3 mV) was
135
favorable to both a stable emulsion and to good
136
biocompatibility. The Myrj block copolymers used in
137
the formulation are essential to both stabilize the
138
molten nanoemulsion droplets and coat the surface
139
of the solidified nanoparticles with poly(ethylene
140
glycol) chains, imparting enhanced biocompatilibity
141
to the QD-FENs (Figure 2A). The coating of
nanoparti-142
cles with a poly(ethylene glycol) corona has previously
143
been shown to impart a stealth property to liposomal
144
formulations, evading the recognition and uptake by
145
the reticuloendothelial system thus extending the
146
circulation time of the particles in the body.
23Under
147
electron microscopic imaging, the PbSe QDs were
148
clearly seen as black spots within the matrix of the
149
QD-FENs (Figure 2B). The electron density
character-150
istic of metal particles was absent in electron
micro-151
graphs of QD-free fatty ester nanoparticles (Figure 2C).
152
Spectral Characteristics of QD-FEN.
The PbSe QDs in our
153
hands showed an emission maximum at 860 nm
154
(Figure 3
A) and a quantum yield in hexanes of ∼15%
F3 155at an excitation wavelength (λ
ex) of 710 nm, as
deter-156
mined using 1,1
0-diethyl-4,4
0-carbocyanine iodide as
157
the quantum yield standard (λ
ex= 710 nm, λ
em= 735
158
nm, quantum yield =3.6%
24). Suprisingly, when PbSe
159
QDs were encapsulated in myristic acid-based
nano-160
particles, near complete quenching of the QD emission
161
was observed (Figure 3A). Initially, this novel QD
162
quenching phenomenon was attributed to either the
163
physical transition of the QD environment from a liquid
164
to a solid phase as the nanoemulsion was cooled and
165
the fatty droplets were solidified, or to the presence of
166
the carboxylic acid headgroups of the fatty acid chains.
167
To investigate each potential cause separately, PbSe
168
QDs were synthesized and suspended in n-octanol or
169
1-octanoic acid, and the fluorescence emission spectra
170
were recorded (Figure 3B). Since both dispersing
me-171
dia were liquid at room temperature, any fluorescence
172
quenching would only be due to the presence of the
173
carboxylate of the octanoic acid and not the physical
174
phase of the medium itself. The photoluminescence
175
curves obtained provided strong evidence that the
176
fluorescence quenching of the QDs by the myristic acid
177
nanoparticles was due to the carboxylate groups of the
178
fatty acid matrix. To mask the carboxylate groups of the
179
fatty acid matrix, fatty acid ethyl esters of eicosanoic acid
Figure 1. Schematic representation of the utility of fluoro-phores in the visible (green, yellow) versus the near-infrared (dark red) for deep fluorescence imaging. In the visible region, due to light scattering and absorption by living tissue, poor penetration of the excitation photon into the skin (green) or the penetration of the emission photon out of the skin (yellow) prevents the detection of the fluoro-phore signal in vivo. Photons in the near-infrared are cap-able of deep tissue penetration.
ARTICLE
180
were synthesized according to previously published
181methods
25and QD-FENs were formulated. The return
182
of the strong photoluminescence at 860 nm was
183observed (Figure 3A), providing further evidence that
184
the previously observed QD fluorescence quenching
185
was due to the interaction of the carboxylate groups of
186
the fatty acid with the surface of the PbSe QDs and not
187
due to the liquid-to-solid phase transition of the
188
nanoparticulate carrier matrix.
189
Unexpectedly with the use of ethyl eicosanoate as
190
the carrier matrix, the quantum yield of QD-FENs
191
suspended in water increased 3-fold over free PbSe
192
QDs suspended in hexanes, from ∼15% to ∼45%,
193
determined at λ
ex= 710 nm with 1,1
0-diethyl-4,4
0-194
carbocyanine iodide as the quantum yield standard.
195
The previously published quantum yield for
water-196
soluble PbSe QDs stabilized by
(1-mercaptoundec-11-197
yl)tetra(ethylene glycol) (MTPEG) was 30%,
15making
198
our QD-FEN formulation 50% more efficient, spectrally,
199
than the free water-soluble QDs. The enhanced
fluor-200
escence efficiency of QD-FENs can be ascribed to the
201
protection of QDs in a hydrophobic nanoenvironment
202
of fatty ester matrix. The preparation of water-soluble
203
QDs involves exchange of hydrophobic ligand with
204
hydrophilic ones and direct dispersion of QDs in
aqu-205
eous medium, which have previously been shown to
206
decrease the quantum yield of PbSe QDs.
15In our
207
formulation the PbSe QDs are maintained in their oleic
208
acid cap and encapsulated in the hydrophobic matrix
209
of QD-FENs. Thus they are never in direct contact with
210
the aqueous medium, leading to high quantum yields.
211
Imaging Depth of Penetration of QD-FEN.
One limitation
212
of medical fluorescence imaging is the shallow
dis-213
tance of image acquisition, often to below 10 mm from
214
the surface of the skin.
6To quantitatively measure the
Figure 2. PbSe QDs are dispersed throughout the matrix of QD-FENs, which bear a corona of poly(ethylene glycol), as shown in the schematic A. Transmission electron micro-graphs of QD-FENs (B) and fatty ester nanoparticles (C) clearly demonstrate the encapsulation of the metal QDs within the fatty matrix. Transmission electron micrograph of PbSe QDs on their own is shown in the top right inset of panel B. Scale bars in panels B and C are 100 nm, scales bars in insets of panel B are 10 nm.
Figure 3. (A) The photoluminescence quenching of PbSe QDs encapsulated in solid fatty acid nanoparticles is clear by comparing the emission spectra of free PbSe QDs (red) to the emission of QD-loaded myristate nanoparticles (blue), as is the reversal of this quenching effect with QD-FENs (green). (B) The appearance of this quenching effect when QDs were dispersed in 1-octanoic acid (blue) but not in n-octanol (red) suggests that the hydrophobic fatty acid moiety of the fatty carrier matrix as the cause of the QD signal attenuation.
ARTICLE
215
depth of image acquisition possible using QD-FENs, a
216
novel system for the continuous measurement of
217biologically relevant imaging depth was developed
218and implemented (Figure 4
F4
). Gelatin tissue phantoms
219were created, mimicking the absorbance and
scatter-220ing characterisitcs of living tissue as previously
report-221ed.
26Gelatin, hemoglobin, sodium azide, and
Intrali-222pid were mixed at elevated temperatures and were
223poured into a prechilled plastic mold tilted at an ∼45
224angle (Figure 4A). A 1-mm thick gelatin slab was cast
225
containing the desired dilution of QD-FENs, and was
226
laid on top of the bottom portion of the set
gela-227
tin-hemoglobin-Intralipid phantom. The mold
con-228taining the diagonal phantom bottom and the
229
encapsulated QD slab was set flat on a surface and
230
was slowly filled with more
gelatin-hemoglobin-Int-231ralipid mixture heated to 40 C. The addition of warm
232phantom mixture slightly melted the hardened
phan-233tom, preventing the formation of a phantom-phantom
234
interface to yield a single solid phantom mass (Figure 4A).
235
Measures of the distance a photon could travel in the
236
phantom before the photon was absorbed (μ
a) or
237
scattered (μ
s) were performed on 1-mm thick samples
238
of the tissue phantom using two integrating spheres in
239
series to prevent cross talk between absorption and
240
scattering measurements (Figure 4B), and were found
241
to be in agreement with the same parameters of real
242
tissue.
26The 1-mm thick slab containing the QD-FENs
243
continuously and nearly linearly decreased in depth
244
from one side of the phantom to the other, permitting
245
the quantification of fluorescence signal strength as a
246
function of phantom depth from a single fluorescent
247
image acquired with a Xenogen IVIS Spectrum whole
248
animal imager (Figure 4C). Quantification of the signal
249
intensity versus depth was performed with ImageJ,
27250
yielding the plot of signal intensity versus penetration
251
depth in Figure 4D. From this plot, it can be seen that the
252
QD-FENs showed strong signal penetration beyond 20
Figure 4. Quantitative, continuous measurement of fluorophore signal tissue depth penetration in a tissue phantom model. (A) Schematic diagram of the tissue phantom constructed for depth penetration measurements, showing the 1-mm thick gel (red) containing QD-FENs encased at an angle within the tissue-like phantom (brown). The depth measurements account for excitation photon (λex, orange) and emission photon (λem, red) penetration. (B) The light scattering and light absorption
characteristics of the tissue phantom characterized by the scattering (μs, dotted) and absorption (μa, solid) coefficients,
respectively, as measured by two integrating spheres in series. (C) Fluorescent images taken from the top of a 1.5 cm (1 mg QD-FEN/mL, top) and 2.7 cm (5 mg QD-FEN/mL, bottom) maximal QD-FEN gel depth phantom, illustrating
disappearance of detectable signal as a function of gel depth. (D) A plot of the pixel intensity versus tissue phantom depth of the phantoms depicted in Figure 4C, quantifying the maximal penetration of the 1 mg QD-FEN/mL gel to ∼1.3 cm (solid) and of the 5 mg QD-FEN/mL gel to ∼2.4 cm (dotted). Backgound fluorescence levels are indicated by a solid red line for the 1 mg QD-FEN/mL phantom and a dotted red line for the 5 mg QD-FEN/mL phantom.
ARTICLE
253
mm, reaching nearly 25 mm in depth with 5 mg
QD-254FENs/mL phantom containing a total of 1.6 mg of PbSe
255QDs. Previous quantitative investigations of the
pene-256tration depth of fluorescence of NIR fluorophores have
257reported limits up to 9.7 mm,
26less than half of the
258penetration depth of the QD-FENs. By increasing the
259
quantum yield of the encapsulated QDs, it is
reason-260
able to postulate that this depth of penetration may be
261
significantly extended.
262
Toxicity Assessment of QD-FEN.
The main limitation
263
preventing the application of semiconductor
nano-264
crystals to medical fluorescent imaging is the toxicity
265
arising from their heavy metal constituents,
17their
266surface capping and functionalizing agents,
18and their
267poor pharmacokinetic performance,
19for example,
268accumulating mainly in the liver without being
269cleared.
20In the case of PbSe QDs, the well-known
270toxicity of Pb
28and the potential toxicity of Se at high
271doses
29limit the biomedical applications of this
nano-272crystal in its free form. As a preliminary assessment of
273the safety of FEN for in vivo use, the toxicity of
QD-274
FENs was assessed against water-soluble
MTPEG-275
capped PbSe QDs using isolated rat hepatocytes. This
276
model has been used for rapid toxicity screening and
277
has demonstrated in vitro-in vivo toxicity
extrapola-278tion.
30Water-soluble, MTPEG-PbSe QDs showed
sig-279
nificant hepatocyte toxicity, resulting in less than 40%
280hepatocyte survival upon exposure to 6 μg/mL of PbSe
281QDs after 120 min (Figure 5
F5
). Exposure of hepatocytes
282to the same dose of PbSe QDs in QD-FENs for 120 min
283resulted in the survival of 80% of hepatocytes. Even
284
after exposure to 30 μg/mL of QD-FENs for 120 min,
285
approximately 70% of hepatocytes still survived. The
286
fatty ester nanoparticle itself has been shown to be
287
biocompatible in vitro
31and in vivo,
32a result that was
288
confirmed with the hepatocyte toxicity screening assay
289
presented herein. The reduced toxicity of QD-FENs
290
may arise from the mitigation of two processes
in-291
volved in QD toxicity: the loss of free heavy metal ions
292
from QD degradation
17and the aggregation of the
293
QDs in tissue due to loss of the water-soluble capping
294
ligand from the nanocrystal surface.
6,18The
degrada-295
tion of the nanocrystal into its free metal ions is
296
thought to occur in the endolysosomal compartment
297
of the cells following QD uptake, a compartment that is
298
acidic and promotes metal ion loss. Since PbSe QDs are
299
trapped within the solid matrix core of the fatty ester
300
carrier (Figure 2), the endolysosomal acidification is
301
unlikely to reach the fatty ester encased QDs, resulting
302
in maintenance of structural integrity and prevention
303
of free Pb
2þleaching. Since the PbSe QDs in their
304
hydrophobic, oleic acid capped state favorably interact
305
with the hydrophobic core of the fatty ester
nanopar-306
ticles that are stabilized by PEG and surfactant, their
307
aggregation becomes moot. As a result, the
encapsula-308
tion of PbSe QDs in the unique hydrophobic,
biocom-309
patible matrix of fatty ester nanoparticles significantly
310
reduces the toxicity of PbSe QDs in vitro. The low acute
311
toxicity of QD-FEN observed in this preliminary test is
312
encouraging suggesting the enhanced
biocompatibil-313
ity of PbSe QDs. However further assessment of in vivo
314
end points of PbSe QD toxicity in more detail is
315
required to establish the safety profile of QD-FEN.
316
In Vivo
Whole Animal Imaging With QD-FEN.
To both
317
demonstrate the utility of QD-FENs for deep tissue,
318
whole animal fluorescent imaging, and preliminarily
319
assess the in vivo fate and tolerability of this
formula-320
tion, 200 μL of QD-FENs in saline suspension (64 μg of
321
PbSe QD total) was injected intravenously in the tail
322
vein of nude mice (Figure 6
A). The mice were inocu-
F6 323lated in the inguinal mammary fat pad with a human
324
breast tumor cell line (MDA435) stably expressing
325
green fluorescent protein. The subiliac lymph node
326
was used as a landmark for tumor inoculation in the
327
mammary fat pad. The tumor can be seen in the
328
leftmost panel of Figure 6A after 3 weeks of growth
329
using whole animal fluorescent imaging. At this point
330
the QD-FENs were injected intravenously. Mice were
331
imaged intact for 7 days to determine both the
dis-332
tribution of the fluorescent hybrid nanoparticle, as well
333
as the clearance of the fluorophore over time.
Back-334
ground fluorescence can be seen in the “background”
335
panel of Figure 6A, with mostly intestinal background
336
due to porphyrins in the mouse chow. Following the
337
intravenous injection of QD-FENs, clear, bright
fluores-338
cence can be seen. Within the first 2 h of
administra-339
tion, broad fluorescence was observed throughout the
340
animal with bright spots in the abdominal region.
Figure 5. The toxicity of QD-FENs or PbSe QDs in their free, water-soluble form was assessed in vitro in a rat hepatocyte assay for accelerated assessment of cytotoxicity. Saline, fat-ty ester nanoparticles, QD-FENS, and water-soluble QDs (M-TPEG-PbSe QD) were incubated at a low (60μg) and high (300μg) dose in 10 mL of cell suspension, and cytotoxicity was assessed at 30 min (black), 60 min (dark gray), and 120 min (light gray). Statistical comparisons of survival data of QD-FEN and MTPEG-PbSe QD against fatty ester nanopar-ticles for each dosing level were performed using a Nested ANOVA and posthoc Tukey's Test. (†) Significant differences (p < 0.05) in survival rates between low dose fatty ester nano-particles, and QD-FEN or MTPEG-PbSe QD. (††) Significant differences (p < 0.05) in survival rates between low dose QD-FEN and MTPEG-PbSe QD. (/) Significant differences (p < 0.05) in survival rates between high dose fatty ester nano-particles and QD-FEN or MTPEG-PbSe QD. (//) Significant differences (p < 0.05) in survival rates between high dose QD-FEN and MTPEG-PbSe QD. Data represent the mean and standard deviation of three trials.
ARTICLE
341
Suprisingly, none of the abdominal fluorescence arose
342from the liver, which is the main site of uptake of free,
343water-soluble QDs and the main site of QD retention
344that results in the poor biocompatibility of the
flu-345orophore.
20From 4 to 8 h after injection, the QD-FENs
346
were seen to accumulate in the spleen, and this
acc-347umulation could be seen to decrease to nearly
pre-348injection levels by 96 h after injection. Within 7 days
349(168 h), the whole animal fluorescence decreased to
350nearly background levels, providing preliminary
evi-351dence that the encapsulation of PbSe QDs in fatty ester
352nanoparticles can enhance the biocompatibility of QDs
353
by enabling near complete clearance of the
fluoro-354phore from the body. This is in contrast to the
applica-355tion of free QDs in vivo, as long-term retention and
356poor clearance have been noted to be the major cause
357
of concern regarding the application of semiconductor
358nanocrystals in a clinical setting.
19,20,33By zooming in
359on the mammary fat pad area of the animal, tumor
360fluorescence above tissue background could be noted
361by 1 h (60 min) after QD-FEN injection, indicating the
362passive accumulation of QD-FENs within the tumor
363tissue (Figure 6B). A post-mortem examination of nude
364mice 3 h after injection with saline, 3 h after QD-FEN
in-365jection, and 7 days following QD-FEN injection
re-366vealed accumulation of the QD-FENs primarily in the
367spleen and gall bladder at 3 h, with some accumulation
368
in the kidneys (Figure 6C). The renal accumulation
de-369creased to background and the QD-FENs were almost
370completely cleared from the spleen and gall bladder by
371
7 days. No hepatic, heart, or lung accumulation was
372
noted even at 3 h after injection, further demonstrating
373
that the danger of hepatic uptake of the PbSe QDs was
374
mitigated by encapsulation in the solid fatty ester
375
nanoparticles. Of note is confirmation of the passive
376
accumulation of the QD-FENs in the tumor by 3 h after
377
injection and the retention of some of the QD-FENs
378
even at 7 days after administration. This preliminary
379
assessment of the novel nanocomposite QD-FEN
de-380
monstrates the synergy of enhanced biocompatibility
381
through optimized tissue distribution and clearance
382
with enhanced aqueous optical efficiency, resulting in
383
an advancement in the applicability of QDs to deep
384
tissue, macroscopic, fluorescent imaging. A
shortcom-385
ing of nontomographic fluorescence imaging
em-386
ployed in this work is the inability to distinguish the
387
precise depth profile of the QD-FEN in vivo. To precisely
388
locate the QD-FEN in three-dimensional space in an
389
animal, further studies are in progress using
fluores-390
cence molecular tomography. In addition, an in depth
391
assessment of the retention of Pb
2þin body tissues and
392
longer term tissue toxicity following intravenous
ad-393
ministration of QD-FENs will need to follow. The results
394
of this study will have a large impact on the possible
395
application of NIR QDs to medical fluorescent imaging
396
applications.
397
CONCLUSIONS
398
Noninvasive, nonionizing, and low cost deep
fluor-399
escent imaging can provide rapid, high spatial and
400
temporal resolution images and may present a new
Figure 6. The formulation of QD-FENs provides for deep, real-time, in vivo fluorescence imaging following intravenous administration, providing favorable biodistribution and clearance of the nanocrystal cargo. (A) The biodistribution of QD-FENs in a green fluorescent protein-expressing MDA435 model of human breast cancer was followed over 7 days with whole animal fluorescent imaging: (right) fluorescent image of the breast tumor; (left) longitudinal biodistribution and elimination of the QD-FENs. (B) Tumor accumulation of the QD-FENs was noted by 30 min after intravenous administration. (C) Necropsy analysis of the tissue distribution showed significant accumulation of QD-FENs in the spleen and gall bladder, without accumulation in the liver after 3 h. Near complete elimination of the QD-FENs was observed 7 days after
administration: GB, gall bladder; H, heart, K, kidney; Li, liver; Lu, lung; S, spleen; T, tumor.
ARTICLE
401
frontier in medical imaging modalities. While the work
402
presented here uses single photon excitation for whole
403
animal fluorescence imaging, innovation in
multipho-404ton imaging instrumentation continues to advance the
405possible applicability of this modality. Similar
innova-406tive strides need to be made in the development of
407biocompatible NIR fluorophores for deep tissue
ima-408ging so that, in combination with instrumentation
409innovation, fluorescent medical imaging may become
410a reality. Here we present the formulation of a novel
411nanocomposite fluorophore of PbSe QDs
encapsu-412
lated in a stealth fatty ester nanoparticle carrier. Along
413
with demonstrated enhanced spectral efficiency and
414
deeper tissue imaging capability, this system boasts an
415
improved safety profile that contributes to the
mitiga-416
tion of concerns regarding the in vivo use of
water-417
soluble semiconductor nanocrystals. While the
liver-418
sparing biodistribution and more complete
elimina-419
tion results are preliminary, work is in progress to
420
quantify these important characteristics of this NIR
421
nanocomposite fluorophore.
422
423
424
METHODS
425 Myristic acid, eicosanoic acid, poly(ethylene
oxide)-40-stea-426 rate (Myrj 52), poly(ethylene glycol)-100-stearate (Myrj 56), lead
427 oxide, selenium, trioctylphosphine, oleic acid, octadecene,
hex-428 anes, 1,10
-diethyl-4,40
-carbocyanine iodide, methanol, ethanol,
429 chloroform, octanoic acid, octanol,
(1-mercaptoundec-11-yl)tet-430 ra(ethylene glycol), hemoglobin, Intralipid, Trizma base, sodium
431 chloride, cupric acetate, butanol, and acetyl chloride were
432 purchased from Sigma-Aldrich and used without further
pur-433 ification. Gelatin was purchased from J. T. Baker, and sodium
434 azide was purchased from Fisher Scientific. Pluronic F-68 was
435 purchased from BASF. Type II Collagenase was purchased from
436 Worthington. CDCl3was purchased from CIL. Human breast
437 cancer MDA435 cells were purchased from ATCC. Nude mice
438 (NCR-NU-F) were purchased from Taconic, Inc.
439 Synthesis of Fatty Esters. Fatty esters were synthesized as
440 previously reported.25Briefly, eicosanoic acid was dissolved in
441 chloroform, to which an ethanolic solution of cupric acetate, as
442 well as ethanolic HCl, was added. This solution was stirred for 1
443 h, after which time the reaction was quenched with distilled
444 deionized water, and the fatty ester was extracted with
chloro-445 form. The chloroform was evaporated under nitrogen gas,
446 yielding a white solid. The solid was dissolved in CDCl3, and
447 the structure was confirmed by standard1H NMR and13C NMR
448 performed on a Varian Mercury 300 MHz instrument. The proton
449 and carbon NMR spectra of the fatty ester and the fatty acid are
450 given in the Supporting Information. No residual eicosanoic
451 acid can be seen in either NMR spectrum, indicating its
com-452 plete conversion to ester.
453 Synthesis of PbSe Quantum Dots. PbSe quantum dots were
454 synthesized according to previously published methods.15
455 Briefly, lead oxide was dissolved in octadecene and oleic acid
456 under a nitrogen atmosphere at 150 C. Once dissolved, the
457 solution was cooled, opened to ambient air, and a solution of
458 selenium in trioctylphosphine was added. This solution was
459 stirred for 4 h, after which time the quantum dots were isolated
460 through the addition of butanol and methanol. For preparation
461 of water-soluble QD for toxicity assessment, the exchange of
462 oleic acid for MTPEG on the PbSe QD surface was accomplished
463 as previously described.15The fluorescence spectra of the PbSe
464 QDs were recorded on an Ocean Optics spectrofluorimeter.
465 Formulation of QD-FENs. The fatty esters prepared in this work,
466 Myrj 52, and Myrj 56, were heated to 80 C until melted. The
467 molten mixture was stirred and PbSe QD suspended in hexanes
468 was added. The mixture was stirred for 20 min to allow for the
469 evaporation of hexanes and dispersion of the QDs in the molten
470 fatty ester. An aqueous solution of Pluronic F68 was then added
471 to the molten mixture, followed by distilled deionized water
472 preheated to 80 C. This emulsion was ultrasonicated using a
473 Hielscher UP-100S probe sonicator (100 W, 30 kHz) for 5 min.
474 Immediately following sonication, the entire emulsion was
475 dispersed in cold 0.9% saline. Particle size and zeta potential
476 were measured on a Nicomp 380 Zetasizer by dynamic light
477 scattering and electrophoretic light scattering, respectively.
Parti-478 cle morphology and QD loading were assessed by transmission
479
electron microscopy of unstained samples using a Hitachi
480
H-7000 microscope.
481
Preparation of Tissue Phantom.Tissue phantoms were prepared
482
based on the compositions reported previously.26Tris-buffered
483
saline was poured on top of a known amount of sodium azide
484
and gelatin to make a final concentration of 15 mM and 10w/v%
485
respectively. The mixture was heated to 50 C using a water
486
bath (Haake D8 Immersion heater) with constant stirring by a
487
Caframo BDC 1850 overhead stirrer at 650-800 rpm. After the
488
gelatin had dissolved, the mixture was cooled to 37 C with
489
constant stirring. Hemoglobin and intralipid were added to
490
reach concentrations of 170 μM and 1 v/v%, respectively.
491
A gel cassette intended for electrophoresis was used as a
492
mold to form a 1 mm thick slab of tissue phantom containing
493
QD-FEN. QD-FEN in saline was mixed with a warm phantom
494
mixture at 1 mg of QD-FE/mL or 5 mg of QD-FEN/mL
concen-495
trations. For the 5 mg QD-FEN/mL slab, extra gelatin was added
496
to make a final gelatin solution of 10 wt % in order to ensure
497
proper slab solidification. All glassware used was warmed to 37 C
498
to prevent premature solidification of the phantom mixture.
499
The warm QD-FEN mixture was then pipetted into the space
500
between the two glass plates almost to the brim. The entire set
501
up was then placed in refrigeration (4 C) until it solidified.
502
The construction of the tissue phantom is outlined in
503
Supporting Information, Figure S2. Before phantom
construc-504
tion, the mold was kept under refrigeration to ensure rapid
505
solidification of the phantom and prevent intralipid separation.
506
To construct the bottom layer of the phantom, the mold was
507
tilted such that the bottom was inclined to 45. Warm tissue
508
phantom mixture was poured to a desired depth and tapped to
509
remove any air bubbles. The bottom layer was refrigerated until
510
solid. The remainder of the tissue phantom mixture was kept
511
warm at 37 C with constant stirring. The QD-FEN slab was
512
removed from the electrophoresis gel cassette and trimmed to
513
fit the mold. The slab was then laid midline across the length of
514
the bottom tissue phantom layer, and any air bubbles were
515
smoothed out. Warm tissue phantom mixture was poured over
516
top to fill the mold, and the mold was refrigerated until
517
solidified. For depths approximately 3 cm or deeper, warm
518
tissue phantom was poured and solidified layer-by-layer to
519
prevent dissolution of the QD-FEN slab layer. Solidified tissue
520
phantom was removed from the mold by running a thin spatula
521
around the edge. Once complete, the tissue phantom was
522
stored at 4 C until needed. Excess tissue phantom mixture or
523
QD-FEN slab mixtures were also stored at 4 C, to be melted at
524
37 C for reuse.
525
Optical Characterization of Tissue Phantom. To ensure that the
526
composition of the tissue phantom produced a gel with optical
527
properties comparable to that of human tissue, the scattering
528
coefficient (μs) and absorption coefficient (μa) were quantified
529
using integrating spheres. The sample was mounted between 2
530
BK7, 1 mm thick optical windows (Esco Products), which were
531
separated by a 1 mm thick spacer. The spacer had a cutout at the
532
top so that the phantom mixture could be poured in before it
533
solidified. A halogen white light source (HL-2000, Ocean Optics)
534
was used, via focusing lenses, to illuminate the sample when it
ARTICLE
535 was mounted in a reflectance-transmittance (RT) integrating
536 sphere system. The 6 in. diameter integrating spheres
(Sphere-537 Optic) were mounted one at a time to eliminate cross talk. The R
538 sphere was mounted in front of the sample position and
refle-539 ctance, background, and reference signal spectra were
re-540 corded. The R sphere was then removed and the T sphere
541 was mounted behind the sample. The transmittance,
back-542 ground, and reference spectra were recorded. A Monte Carlo
543 simulation using 106photons and an asymmetry parameter =
544 0.9 generated a lookup table which matched the R and T values
545 with μaand μsvalues.
546 Tissue Phantom Image Acquisition and Data Analysis. Tissue
phan-547 toms were imaged using a Xenogen IVIS Spectrum instrument
548 in epifluorescence mode. The phantom was placed on the stage
549 and images were taken with automatic exposure and aperture
550 settings in field of view C. One image was taken from the top of the
551 gel phantom, and a second image was taken of the cross section of
552 the gel. Intensity scales were set to a monochromatic (red) type
553 with units of radiant efficiency and the range was set to full.
554 ImageJ27was used for image analysis, which comprised four
555 steps:
556 (1) Using the monochromatic scale, a calibration of pixel
557 intensity versus the intensity value in radiant efficiency
558 was performed. The dimensions of the intensity scale
559 were determined in pixels, allowing the dimensions of
560 one pixel to be determined in terms of the intensity
561 scale dimensions. ImageJ was then used to determine
562 the pixel-by-pixel intensity of the intensity scale bar,
563 allowing for a plot of pixel intensity versus radiant
564 efficiency to be generated.
565 (2) Using the top view image of the gel phantom, the pixel
566 intensity versus the distance in millimeters from the
567 origin (selected to be the topmost point of the QD-FEN
568 slab in the gel phantom) was determined. The
determi-569 nation of the dimensions of a pixel allowed for a plot of
570 pixel intensity versus distance in millimeters from the
571 origin to be generated.
572 (3) Using the cross-sectional image, the depth of the
QD-573 FEN gel slab was determined from the top of the gel
574 phantom, resulting in a plot of distance from the origin
575 versus QD-FEN slab depth.
576 (4) Combining the plot of pixel intensity versus distance
577 from the origin (Step 2) with the plot of distance from
578 the origin versus QD-FEN slab depth (Step 3), a plot of
579 pixel intensity in radiant efficiency versus QD-FEN slab
580 depth was generated, as shown in Figure 4D.
581 Determination of Cytotoxicity of QDs and QD-FENs. Hepatocytes
582 were isolated from rats by perfusion of the liver with
collage-583 nase as described previously.34Isolated hepatocytes (106cells/
584 mL) (10 mL) were suspended in Krebs-Henseleit buffer (pH 7.4)
585 containing 12.5 mM HEPES in continually rotating 50 mL
round-586 bottomed flasks, under an atmosphere of 95% O2and 5% CO2in
587 a water bath of 37 C for 30 min. Hepatocyte viability was
588 assessed microscopically by plasma membrane disruption as
589 determined by the trypan blue (0.1% w/v) exclusion test.35
590 Hepatocyte viability was determined at 30, 60, and 120 min,
591 and the cells were at least 80-90% viable before use.
592 Transfection of Tumor Cell.Human MDA435 breast
adenocar-593 cinoma cells were stably transfected to express enhanced green
594 fluorescent protein (EGFP). The EGFP plasmid (pEGFP-N1,
595 Clonetech) was linearized by digestion with the restriction
596 endonuclease Dra III (New England Biolabs), and was
trans-597 fected into the cells by incubation with CaPO4. After 24 h of
598 incubation, cells were grown in selection media containing
599 minimal essential medium supplemented with 10% fetal calf
600 serum and 600 μg/mL of the selection antibiotic G418. Cell
601 colonies that formed and that were fluorescent were lifted and
602 plated in selection medium in individual wells of a 24-well
603 microplate. The three best colony expansions, determined by
604 fluorescent intensity and number of fluorescent cells, were
605 selected for sorting by fluorescence activated cell sorting
606 (FACS). The brightest 20% of each of the three expanded
607 colonies were selected by FACS and were further expanded in
608 regular growth medium.
609
In VivoWhole Animal Imaging in Tumor-Bearing Mice. To establish
610
orthotopic xenograft breast tumors, 1 million human MDA435
611
cells expressing EGFP were injected into the inguinal mammary
612
fat pads of nude mice using the subiliac lymph node as an
613
injection landmark. This ensured greater mouse-to-mouse
re-614
producibility of the tumor model. Tumors were monitored for
615
growth with fluorescent imaging during the course of the
three-616
week growth period. Mice were injected with QD-FEN
formula-617
tion into the lateral tail veins and anaesthetized with isoflurane.
618
Images were acquired using a Xenogen IVIS Spectrum whole
619
animal imager with λex= 710 nm and λem= 840 nm. All animal
620
work was approved by the animal care committee at the
621
University Health Network, and all experiments were performed
622
in accordance with all guidelines and regulations put forth by
623
the Canadian Council on Animal Care.
624
Acknowledgment. The authors gratefully thank the
Cana-625
dian Breast Cancer Foundation-Ontario Region for funding the
626
work herein, the National Science and Engineering Council of
627
Canada, the University of Toronto and the Ben Cohen Fund for
628
providing scholarships to A. Shuhendler. The authors also
629
acknowledge A. Worthington for performing the
measure-630
ments of the optical properties of the tissue phantoms, J. T.
631
Henderson for providing the enhanced green fluorescent
pro-632
tein plasmid used to label the tumor cells, M. Monroy for
633
technical assistance with the mammary fat pad mouse model,
634
and STTARR for technical assistance with fluorescence imaging.
635
Supporting Information Available:Further experimental
de-636
tails regarding the NMR characterization of fatty acid ester
637
structure and the construction of the tissue phantom for
638
quantification of optical probe fluorescent depth penetration.
639
This material is available free of charge via the Internet at http://
640
pubs.acs.org.
641
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