Somatostatin Decorated Quantum Dots for Targeting of Somatostatin Receptors

Ahmed Abdelfattah Hafez Abdellatif, Wael Abdellah Abdelhafez, Hatem Abdelmunsef Sarhan, Ahmed Abdelfattah Hafez Abdellatif, Wael Abdellah Abdelhafez, Hatem Abdelmunsef Sarhan

Abstract

Due to the unique optical properties like high brightness and narrow emission bands of Quantum dots, it is used as simple fluorescence materials in bio-imaging, immunoassays, microarrays, and other applications. To easy invistigate cell lines that overexpressed somtostatin receptors, somatostatin (SST) was conjugated with Quantum dots carrying PEG amine (Qdots-PEG-NH2). The conjugation of SST to Qdots-PEG-NH2 started with the thiolation of SST using Traut's reagent. Moreover, the Qdots-PEG-NH2 were subsequently activated by 500-fold molar excess of sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) dissolved in phosphate buffer. The Qdots-PEG-NH2-sulfo-SMCC was conjugated to the thiolated-SST to form Qdots-SST. The number of sulfhydryl groups can be controlled by the molar ratio of Traut´s reagent to SST. Thiolation was necessary for the conjugation of SST to Qdots-PEG-NH2. This was achieved by reacting the SST with Traut's reagent in a 1:1 molar ratio. Ellman's reagent was used to determine the number of sulfhydryle groups. Furthermore, cellular uptake study on triple negative breast cancer cells (HCC-1806) showed that the numbers of Qdots-SST per cell were significantly higher compared to unmodified Qdots-PEG-NH2 when quantified using inductively coupled plasma optical emission spectroscopy (ICP-OES). Moreover, the binding of Qdots-SST to cells can be suppressed by addition of free SST, indicating that the binding of Qdots-SST to cells is due to receptor-specific binding.

Keywords: Cellular uptake; Quantum dots; Receptor targeting; Somatostatin; Somatostatin receptors.

Figures

Figure 1
Figure 1
Thiolation of SST with Traut’s reagent. Running through an equilibrated sephadex G-25 mini-column purified the reaction mixture.
Figure 2
Figure 2
Bio-conjugation of SST to Qdots-PEG-NH2. Qdots-PEG-NH2 activated with sulfo-SMCC. The activated Qdots-PEG-NH2-sulfo-SMCC was conjugated to the thiolated-SST to form Qdots-SST. The obtained bio-conjugate was purified by using sephadex G-25.
Figure 3
Figure 3
Determination of the number of free sulfhydryl groups. SST was reduced with TCEP and then thiolated with Traut´s reagent.
Figure 4
Figure 4
Identification of thiol groups of thiolated-SST. The thiolated-SST labeled with eosin-5-maliemide. Fluorescence detection detected the peaks at wavelengths of 524 nm / 545 nm.
Figure 5
Figure 5
Calibration curve of cysteine for estimation of sulfhydryle groups using Ellman´s reagent. The calibration curve of cysteine was linear from 0 mM to 1.5 mM.
Figure 6
Figure 6
Quantitating the sulfhydryl groups of SST, SST reduced with TCEP and seglitide using a cysteine calibration.
Figure 7
Figure 7
HPLC chromatographs of SST reacted with Traut´s reagent. Signals were detected at 274 nm. Single peak eluted at about 19 min corresponded to SST. Thiolated-SST eluted at 18.6 min.
Figure 8
Figure 8
HPLC chromatograph of SST, SST thiolated with Traut´s reagent and labeled with eosin-5-maliemide. Peak (3-a) corresponds to thiolated-SST. Peak (3-b) represents the thiolated-SST labeled with eosin-5-maleimide, which shows a conversion of about 59.1%, confirming that the SST was thiolated by Traut´s reagent. The thiol-reactive eosin-5-maleimide signals were detected using fluorescence detection at 545nm.
Figure 9
Figure 9
The bio-distribution of Qdots-PEG-NH2 in HCC1806 cells from mice as determined by ICP-OES.

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