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grafts exhibited fast clearance from the blood pool through
hepatobiliary excretion and low tumor uptake. Metabolite anal-
ysis uncovered a higher stability of 7 nm AuNPs in the blood
and a 4–5 times higher bone uptake compared with 14 nm
AuNPs. The high affinity for bone mineral was confirmed with
in vitro experiments with hydroxyapatite.
the precipitate was isolated by centrifugation. The product as
a black solid was washed three times with methanol and dried in
vacuo.
Functionalization of QDs and AuNPs
The photoinduced phase transfer was adapted from a reported
In future, the major challenge consists in the design of
probes with decent blood retention for tumor accumulation.
The conjugation of molecules with tunable surface charges to
the HS-PEG-DAP building block will induce prolonged blood
retention time of DAP-based coating ligands. Efforts in this di-
rection are currently underway in our laboratories.
[25]
procedure with modifications. Briefly, a mixture of HS-PEG-DAP
7.4 mg, 25 mmol) and HS-PEG-DAP-TF (17.1 mg, 25 mmol) were dis-
(
solved in methanol (0.8 mL) and transferred in a nitrogen-flushed
glass vial with a magnetic stir bar, sealed with an aluminum crimp
cap with a butyl septa. NPs (ca. 1.0 mg) were dispersed in hexane
(0.8 mL) and overlaid on the methanol phase. The glass vial was
placed in front of a UV lamp (365 nm) and irradiated under vigo-
rous stirring for 50 min. During this procedure, the originally
purple hexane phase became completely transparent, whereas the
as-functionalized NPs precipitated and the free ligands stayed dis-
persed in the solvent. The supernatant with excess ligands was re-
moved by decantation. The precipitated NPs were washed with
methanol and dispersed in PBS pH 7.4 (0.5 mL). A further purifica-
tion step was performed with a PD-10 size exclusion and PBS
pH 7.4 as a mobile phase. The colored fraction was collected and
TEM, UV/Vis, IR, DLS, and z-potential analyses were carried out.
Experimental Section
Materials, characterization, and synthesis of HS-PEG-DAP
(
12) and HS-PEG-DAP-TF (20)
A detailed description can be found in the Supporting Information.
Synthesis of CdSe/ZnS core/shell QDs
CdSe/ZnS core/shell nanocrystals were prepared according to a re-
[23b]
99m
+
ported procedure.
Briefly, trioctylphosphine oxide (5 g), hexade-
Labeling of functionalized QDs with [ Tc(OH
)
2
(CO)
3
]
3
cylamine (2.5 g), and trioctylphosphine (1.25 mL) were kept under
vacuum at 1308C for 1 h. Under atmospheric pressure the temper-
ature was elevated to 3508C. In a separate flask, cadmium acetyla-
cetonate (155 mg), 1,2-hexadecanediol (300 mg), and trioctylphos-
phine (2.5 mL) were heated under vacuum to 1008C until the solu-
tion became homogeneous. After cooling to 808C under atmos-
pheric pressure, 2.5 mL of a 1m solution of selenium powder in tri-
octylphosphine (2.5 mL) was added. This mixture with the
cadmium and selenium precursors was immediately injected into
the 3508C hot flask and then cooled to 2708C. At this temperature
the mixture was stirred for 60 min and after cooling to room tem-
perature, butanol (50 mL) was added to precipitate the QDs. After
centrifugation and removal of the supernatant, the QDs were dis-
persed in hexane (5 mL) and the precipitation procedure was re-
peated twice. For the overcoating, trioctylphosphine oxide (30 g)
was kept under vacuum at 1208C for 2 h. The purified CdSe QDs
from step 1 were dispersed in hexane (10 mL) and 1 mL of this so-
lution was added to the trioctylphosphine oxide at atmospheric
pressure. The solvent was removed under vacuum. At atmospheric
pressure, the mixture was heated to 1808C. In a separate two-neck
round-bottom flask trioctylphosphine (4 mL), diethyl zinc (1m in
hexane; 1.0 mL), and hexamethyldisilathiane (180 mg) were mixed
and loaded into a syringe. This solution was slowly added to the
QD solution and afterwards the temperature was lowered to 808C.
The mixture was stirred at 808C for 2 h and the overcoated QDs
were isolated by precipitation with butanol (30 mL) and
centrifugation.
9
9m
+
An aqueous solution of [ Tc(OH ) (CO) ] (0.5 mL, pH 7–8) was
2
3
3
added to QDs in PBS pH 7.4 (0.5 mL). The mixture was stirred using
a temperature gradient (50–608C) over 60 min and additional stir-
ring at 608C for 60 min. Radiolabeled QDs were purified with a PD-
0 column, whereas only the reddish-colored fraction was collect-
ed and analyzed with size-exclusion HPLC.
1
9
9m
+
Labeling of functionalized AuNPs with [ Tc(OH ) (CO) ]
2
3
3
99m
+
An aqueous solution of [ Tc(OH ) (CO) ] (0.5 mL, pH 7–8) was
2 3
3
added to purified AuNPs in PBS pH 7.4 (0.5 mL). The mixture was
stirred using a temperature gradient (50–708C) over 60 min and
additional stirring at 708C for 60 min. The radiolabeled AuNPs
were purified with a PD-10 column, whereas only the purple-col-
ored fraction was collected and analyzed with size-exclusion HPLC.
Cellular uptake studies
LNCaP (ATCC CRL-1740) and Cos-7 (ATCC CRL-1651) cell lines were
purchased from ATCC (France). Both cell lines were cultured ac-
cording to the ATCC guidelines and seeded on glass coverslips,
which were placed in 24-well plates. The cells were incubated with
400 mL AuNP solution (PBS pH 7.4/medium 1:4) for 30 s and 90 min
at 378C under 5% CO2 atmosphere. The concentration of 7 nm
À1
À1
AuNPs was 179 mgmL and of 14 nm AuNPs it was 189 mgmL
(determined by ICP-MS). Control experiments for both incubation
times were carried out under the same conditions, but without
using AuNP solution. After incubation, the AuNP solutions were re-
moved, the glass coverslips were washed with PBS and transferred
into well plates containing 2.5% glutaraldehyde in 0.1m PB solu-
tion. After fixation at 48C for 1 h the samples were washed with
PBS and incubated in 1% osmium tetroxide in 0.1m PB at room
temperature for 30 min. After washing with water, the cells were
dehydrated in an increasing concentration of ethanol (70, 96, and
100%). The cells were infiltrated in a mixture of Epon 812 resin
and ethanol (2:1) and finally embedded in pure resin at 608C for
60 h. Ultrathin sections (65 nm) were cut with a Reichert Ultracut
Synthesis of AuNPs
[23a]
AuNPs were prepared according to a reported procedure.
Brief-
ly, a solution of toluene (49 mL) and oleylamine (2.9 mL) were
heated to 115 8C. In a separate flask tetrachloroauric acid trihydrate
(
60 mg) was dissolved in oleylamine (1.2 mL, 3.7 mmol) and tolu-
ene (1 mL). The gold precursor solution was injected in one por-
tion to the boiling solution and the mixture was heated at reflux
for 9 (6.6 nm AuNPs) or 130 min (13.8 nm AuNPs). The particle
growth was quenched by the addition of methanol (100 mL) and
Chem. Eur. J. 2015, 21, 6090 – 6099
6097
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