Concentration and time optimization of TSHR targeting bionanofluid to BCPAP cells.

<p>Included in these experimental conditions are, α-THSR-, Thyrogen-, and purified Thyrotropin-Thiol-PEG-CNT conjugates. Control conditions included PBS and CNT alone. <b>A.</b> Determination of optimal cell to conjugate BioNanofluid ratio to achieve specific maximum targeted BCPAP cell killing. Laser exposure time was 30 seconds for all conditions. Ratios are represented as volume:volume ratios, thus for a 1:1 ratio, 100 μL of cells (of 250,000–350,000 cells per ml) were mixed with 100 μL of Conjugated-BioNanofluid of 2 μg/mL concentration. <b>B.</b> Optimal exposure time determination experiment. BCPAP cells were exposed to laser treatment for 20, 30, and 40 seconds, at a 2:1 cell:conjugated- or unconjugated-Thiol-PEG CNT ratio.</p

Structure and coupling chemistry of Au-modified MWCNTs.

<p><b>A.</b> Scanning electron microscopy (SEM) images of COOH-functionalized Au-labeled Thiol-Carbon derived bionanofluids, at two different magnifications. Au particles have defined spherical structures, highlighted by the arrowhead. <b>B.</b> EDC-NHS coupling chemistry to attach bio-affinity molecules, whether antibody or protein/mitogen to the Thiol-PEG-CNT. PEGylation of the Thiol-CNT is described in the Materials and Methods.</p

Bionanofluid conjugate stability assessment.

<p><b>A</b>. α-TSHR- and Thyrogen-Thiol-PEG-CNT conjugates were prepared on day 1 and kept at 4°C for up to 21 days. Conjugates activity was assessed by cell killing assay of BCPAP cells (as described above). <b>B.</b> Similarly, α-TSHR- and Thyrogen-Thiol-PEG-CNT conjugates were prepared on day 1 and were kept at -20°C or -80°C for up to 6 weeks. Conjugates activity was assessed by cell killing assay at day 5, day 7, and every week for up to 6 weeks.</p

Selective cell killing of BCPAP TSHR-positive vs NSC-34 TSHR-negative cells with TSHR-targeted bionanofluid.

<p><b>A.</b> TSHR expression of BCPAP and NSC-34 cells was determined by Western blot analysis, using TSHR specific antibody. BCPAP were positive for TSHR expression, whereas NSC-34 cells were null. B-ACTIN was used as a loading control. <b>B.</b> BCPAP and NSC-34 cell were incubated α-THSR-, Thyrogen-, and purified Thyrotropin-Thiol-PEG-CNT conjugates. Control conditions included IgG-thiol-PEG-CNTs, PBS and CNT alone. All conditions were performed in 2:1 cell:bionanofluid ratio and 30 second laser exposure. The BCPAP cells showed ~60% to ~73% cell killing with all TSHR targeted bionanofluid conjugates, whereas minimal cell death was observed with the control other conditions. The NSC-34 cell line showed negligible cell death in all conditions.</p