Rods GNPs (shape 15X55 nm, peak wavelength at 785 nm) were synthesized using the seed mediated growth method38 . This method, charges the rods GNPs with a positive charge, which prevent their aggregation within a biological sample. A solution of GNRs suspended in cetyltrimethylammonium bromide (CTAB) (Sigma-Aldrich, USA) was centrifuged at 11 000 g for 10 minutes, decanted and resuspended in water to remove excess CTAB. In order to stabilize the particles in physiological solutions, a layer of polyethylene glycol (mPEG-SH, MW 5000 g/mol) (creative PEGWorks, Winston Salem, USA) was adsorbed onto the GNRs. This layer also provided the chemical groups that are required for antibody conjugation (SH–PEG–COOH, MW 3400g/mol). A 200 ml mixture of mPEG–SH (5mM) (85%) and SH–PEG–COOH (1mM) (15%) was added to 1 ml of GNR solution. The mixture was stirred for 24 hours at room temperature. The absorption spectrum of PEGylated GNR solution presented the same absorption peak at 785 nm. The heterofunctional PEG was covalently conjugated to a CC49 monoclonal antibody, which is specific to the TAG-72 antigen39 (link).
Mpeg sh
Manufactured by Creative PEGWorks
Sourced in United States
The MPEG-SH is a laboratory equipment designed for the compression and conversion of audio and video data. It supports the MPEG-1, MPEG-2, and MPEG-4 compression standards. The core function of the MPEG-SH is to efficiently encode and decode multimedia content for various applications.
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5 protocols using mpeg sh
1
Synthesis of Targeted Plasmonic Nanoparticles
2
Synthesis and PEGylation of Gold Nanoparticles
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Ac-DWK-amide was prepared
on Rink resin (Advanced Chemtech) using the standard solid-phase peptide
synthesis method (detailed description inFigure S2 ).
GNPs stabilized in citrate buffer were purchased
from Cytodiagnostic Inc., at three different sizes: 10, 30, 100 nm.
A protective layer of PEG was absorbed on the surface of the GNPs
at different molar ratios and different lengths consisting of a mixture
of thiol-polyethylene-glycol (mPEG-SH) and a heterofunctional thiol-
polyethylene-acid (SH-PEG-COOH) (Creative PEGWorks, Winston Salem,
NC). In a typical process, 11 μL of PEG mixture solution was
added to 1.0 mL of GNP solution (OD 1) at different concentrations
to obtain the following final molar concentrations of PEG: 0.01, 0.1,
1, 10 mM. The reaction mixture was vortexed immediately and then incubated
at room temperature for 2 h. After PEGylation, each solution was centrifuged
at 14000, 9400, 8600 rpm (10, 30, 100 nm, respectively) for 20 min
and redispersed with Milli-Q water by gentle shaking. This process
was repeated three times. From the resulting solution, a small part
was analyzed by measuring absorption spectra, hydrodynamic size, and
zeta potential. The mixture was stored at 4 °C. PEGylation at
0.01 mM final concentration was found to be most efficient and stable.
on Rink resin (Advanced Chemtech) using the standard solid-phase peptide
synthesis method (detailed description in
GNPs stabilized in citrate buffer were purchased
from Cytodiagnostic Inc., at three different sizes: 10, 30, 100 nm.
A protective layer of PEG was absorbed on the surface of the GNPs
at different molar ratios and different lengths consisting of a mixture
of thiol-polyethylene-glycol (mPEG-SH) and a heterofunctional thiol-
polyethylene-acid (SH-PEG-COOH) (Creative PEGWorks, Winston Salem,
NC). In a typical process, 11 μL of PEG mixture solution was
added to 1.0 mL of GNP solution (OD 1) at different concentrations
to obtain the following final molar concentrations of PEG: 0.01, 0.1,
1, 10 mM. The reaction mixture was vortexed immediately and then incubated
at room temperature for 2 h. After PEGylation, each solution was centrifuged
at 14000, 9400, 8600 rpm (10, 30, 100 nm, respectively) for 20 min
and redispersed with Milli-Q water by gentle shaking. This process
was repeated three times. From the resulting solution, a small part
was analyzed by measuring absorption spectra, hydrodynamic size, and
zeta potential. The mixture was stored at 4 °C. PEGylation at
0.01 mM final concentration was found to be most efficient and stable.
3
PEGylation of Gold Nanorods
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The GNRs were synthesized using the
seed-mediated growth method.53 (link) A solution
of GNRs suspended in cetyltrimethylammonium bromide (CTAB) (Sigma-Aldrich,
USA) was centrifuged at 11 000g for 10 min,
decanted, and resuspended in water to remove excess CTAB. To prevent
aggregation and to stabilize the particles in physiological solutions,
a layer of polyethylene glycol (mPEG-SH, MW 5000 g/mol) (creative
PEGWorks, Winston Salem, USA) was adsorbed onto the GNRs. A 200 μL
mixture of mPEG-SH (5 mM) (85%) and SH-PEG-COOH (1 mM) (15%) was added
to 1 mL of GNR solution. The mixture was stirred for 24 h at room
temperature. The GNR extinction coefficient spectrum was determined
using a spectrophotometer, and the resultant extinction peak was 640
nm (seeFigure 8 ). The absorption spectrum of PEGylated GNR
solution presented the same absorption peak at 640 nm (data not shown).
seed-mediated growth method.53 (link) A solution
of GNRs suspended in cetyltrimethylammonium bromide (CTAB) (Sigma-Aldrich,
USA) was centrifuged at 11 000g for 10 min,
decanted, and resuspended in water to remove excess CTAB. To prevent
aggregation and to stabilize the particles in physiological solutions,
a layer of polyethylene glycol (mPEG-SH, MW 5000 g/mol) (creative
PEGWorks, Winston Salem, USA) was adsorbed onto the GNRs. A 200 μL
mixture of mPEG-SH (5 mM) (85%) and SH-PEG-COOH (1 mM) (15%) was added
to 1 mL of GNR solution. The mixture was stirred for 24 h at room
temperature. The GNR extinction coefficient spectrum was determined
using a spectrophotometer, and the resultant extinction peak was 640
nm (see
solution presented the same absorption peak at 640 nm (data not shown).
4
Synthesis and Characterization of PEGylated Gold Nanorods
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GNRs were synthesized using the seed mediated growth method.37 Their size, shape and uniformity were characterized using transmission electron microscopy (TEM) and the resultant shape was 25×65 nm, with narrow size distribution (10%), TEM images are presented in Figure 3A .2 (link),3 ,38 (link) In order to prevent aggregation, to stabilize the particles in physiological solutions and to improve blood circulation time, a layer of polyethylene glycol (mPEG-SH, MW 5,000 g/mol) (creative PEGWorks, Winston Salem, USA) was conjugated to the GNR. A solution of GNRs suspended in cetyltrimethylammonium bromide (CTAB) (Sigma-Aldrich Co., St Louis, MO, USA) was centrifuged at 7,000×g for 30 minutes, decanted and resuspended in water to remove excess CTAB. mPEG (200 ml of 5 mM) was added to 1 mL of GNR solution. The mixture was stirred for a few hours at room temperature and was dialyzed for three days. The absorption spectrum of PEGylated GNR solution presented a strong peak at 645 nm, as can be seen in Figure 3 .
5
Synthesis and Functionalization of Gold Nanorods
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GNRs
were synthesized using the seed-mediated growth method (Figure1 a shows a schematic presentation of the process).26 The resultant shape was 25 nm × 65 nm (see
Figure1 c). Subsequently the GNRs were coated
by 5% PEG7 (MW 458.56 g/mol) (Creative PEGWorks) and 95% mPEG-SH (MW
5000 g/mol) (Creative PEGWorks). The coating was done by adding to
the GNRs mPEG and PEG7 and leaving it to stir for 2 h. Once the GNRs
were coated with mPEG, mPEG-SH, and PEG7, they were divided into three
samples before binding the fluorescein: the first sample contained
500 μL of GNRs (∼0.4 mg/mL) and 1.2 μL of fluorescein
solution (50 μM), the second sample contained 500 μL of
GNRs (∼0.4 mg/mL) and 1.2 μL of fluorescein solution
(100 μM), and the third sample contained 1000 μL of GNRs
(∼0.4 mg/mL) and 1.2 μL of fluorescein solution (50 μM).
The samples were left to stir overnight in order to facilitate creation
of hydrogen bonds between the PEG carbon chain and the fluorescein
carbon chain. Finally the GNRs were purified from total solution after
the solution was centrifuged for 3 min at 13 400 rpm.
were synthesized using the seed-mediated growth method (Figure
Figure
by 5% PEG7 (MW 458.56 g/mol) (Creative PEGWorks) and 95% mPEG-SH (MW
5000 g/mol) (Creative PEGWorks). The coating was done by adding to
the GNRs mPEG and PEG7 and leaving it to stir for 2 h. Once the GNRs
were coated with mPEG, mPEG-SH, and PEG7, they were divided into three
samples before binding the fluorescein: the first sample contained
500 μL of GNRs (∼0.4 mg/mL) and 1.2 μL of fluorescein
solution (50 μM), the second sample contained 500 μL of
GNRs (∼0.4 mg/mL) and 1.2 μL of fluorescein solution
(100 μM), and the third sample contained 1000 μL of GNRs
(∼0.4 mg/mL) and 1.2 μL of fluorescein solution (50 μM).
The samples were left to stir overnight in order to facilitate creation
of hydrogen bonds between the PEG carbon chain and the fluorescein
carbon chain. Finally the GNRs were purified from total solution after
the solution was centrifuged for 3 min at 13 400 rpm.
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