Quantifying number of AS1411 strands on AuNS Cy5-labeled aptamer 5’-(C6-S-S-C6)- Cy5-TTG GTG GTG GTG GTT GTG GTG GTG GTG G-3’ (Cy5-Apt) was used to estimate the number of aptamers on each particle. Attachment of Cy5-Apt to the AuNS followed the same procedure as described previously. We centrifuged 250 µL of Cy5-labeled Apt-AuNS (Cy5-Apt-AuNS) or Cy5-labeled Apt-AuNS-PEG at 15,000 rpm for 11 min. The supernatant was removed, and the nanoconstructs were suspended in 1 mL of 50 mM HEPES buffer. This process was repeated twice to eliminate unbound Cy5-Apt. The pellets of nanoconstructs were treated with 100 µL of 20 mM potassium cyanide (KCN) overnight to dissolve the Au core of the nanoconstruct and release Cy5 aptamer into the solution. The Cy5 fluorescence intensity of the KCN solution was measured using a NanoDrop Spectrophotometer, and the concentration of AS1411 was determined based on the intensity of the Cy5 signal. This fluorescence assay indicated approximately 110 strands of AS1411 per Apt-AuNS construct and 95 ± 4 strands of AS1411 per Apt-AuNS-PEG construct. Quantifying number of PEG on PEG-AuNS or Apt-AuNS-PEG FITC-labeled PEG (2 kDa) was used to estimate the number of PEG on nanoconstructs using a similar procedure for quantification of AS1411. The FITC fluorescence intensity was measured using a NanoDrop Spectrophotometer at 482 nm, and the concentration of PEG was determined based on the intensity of the FITC signal. This fluorescent assay indicated approximately 1100 strands of PEG on a single Apt-AuNS-PEG and a full coverage of PEG (~2200 strands) per single PEG-AuNS. Preparation of nanoconstructs for injection in vivo Nanoconstructs were sterilized by filtration using a 2-μm filter to remove contaminants or particle aggregates. The nanoconstructs were then pelleted and concentrated by centrifugation
Quantifying number of AS1411 strands on AuNS Cy5-labeled aptamer 5’-(C6-S-S-C6)- Cy5-TTG GTG GTG GTG GTT GTG GTG GTG GTG G-3’ (Cy5-Apt) was used to estimate the number of aptamers on each particle. Attachment of Cy5-Apt to the AuNS followed the same procedure as described previously. We centrifuged 250 µL of Cy5-labeled Apt-AuNS (Cy5-Apt-AuNS) or Cy5-labeled Apt-AuNS-PEG at 15,000 rpm for 11 min. The supernatant was removed, and the nanoconstructs were suspended in 1 mL of 50 mM HEPES buffer. This process was repeated twice to eliminate unbound Cy5-Apt. The pellets of nanoconstructs were treated with 100 µL of 20 mM potassium cyanide (KCN) overnight to dissolve the Au core of the nanoconstruct and release Cy5 aptamer into the solution. The Cy5 fluorescence intensity of the KCN solution was measured using a NanoDrop Spectrophotometer, and the concentration of AS1411 was determined based on the intensity of the Cy5 signal. This fluorescence assay indicated approximately 110 strands of AS1411 per Apt-AuNS construct and 95 ± 4 strands of AS1411 per Apt-AuNS-PEG construct. Quantifying number of PEG on PEG-AuNS or Apt-AuNS-PEG FITC-labeled PEG (2 kDa) was used to estimate the number of PEG on nanoconstructs using a similar procedure for quantification of AS1411. The FITC fluorescence intensity was measured using a NanoDrop Spectrophotometer at 482 nm, and the concentration of PEG was determined based on the intensity of the FITC signal. This fluorescent assay indicated approximately 1100 strands of PEG on a single Apt-AuNS-PEG and a full coverage of PEG (~2200 strands) per single PEG-AuNS. Preparation of nanoconstructs for in vivo injection Nanoconstructs were sterilized by filtration using a 2-μm filter to remove contaminants or particle aggregates. The nanoconstructs were then pelleted and concentrated by centrifugation
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