Direct Fabrication of the Graphene-Based Composite for Cancer Phototherapy through Graphite Exfoliation with a Photosensitizer

We report on the application of pristine graphene as a drug carrier for phototherapy (PT). The loading of a photosensitizer, chlorin e6 (Ce6), was achieved simply by sonication of Ce6 and graphite in an aqueous solution. During the loading process, graphite was gradually exfoliated to graphene to give its composite with Ce6 (G–Ce6). This one-step approach is considered to be superior to the graphene oxide (GO)-based composites, which required pretreatment of graphite by strong oxidation. Additionally, the directly exfoliated graphene ensured a high drug loading capacity, 160 wt %, which is about 10 times larger than that of the functionalized GO. Furthermore, the Ce6 concentration for killing cells by G–Ce6 is 6–75 times less than that of the other Ce6 composites including GO–Ce6

Fluorescence-Guided Surgery of Liver Metastasis in Orthotopic Nude-Mouse Models

<div><p>We report here the development of fluorescence-guided surgery of liver metastasis. HT29 human colon cancer cells expressing green fluorescent protein (GFP) were initially injected in the spleen of nude mice. Three weeks later, established liver metastases were harvested and implanted on the left lobe of the liver in other nude mice in order to make an orthotopic liver metastasis model. Fourteen mice with a single liver metastasis were randomized into bright-light surgery (BLS) or fluorescence-guided surgery (FGS) groups. Seven mice were treated with BLS, seven were treated with FGS. Three weeks after implantation, the left lobe of the liver with a single metastasis was exposed through a median abdominal incision. BLS was performed under white light. FGS was performed using a hand-held portable fluorescence imaging system (Dino-Lite). Post-surgical residual tumor fluorescence was visualized with the OV100 Small Animal Imaging System. Residual tumor fluorescence after BLS was clearly visualized at high magnification with the OV100. In contrast, residual tumor fluorescence after FGS was not detected even at high magnification with the OV100. These results demonstrate the feasibility of FGS for liver metastasis.</p></div

Pre-operative and post-operative images from the orthotopic liver metastasis model treated with FGS.

<p><b>(A)—(C)</b> Upper panels show bright field images, and lower panels are images of tumor fluorescence obtained with the OV100. Residual tumor fluorescence could not be detected even at high magnification <b>(C). (D,F,H)</b> Pre-FGS tumor fluorescence was clearly visualized with the Dino-Lite imaging system. <b>(E,G,I)</b> Dino-Lite imaging showed no evidence of tumor after FGS. <b>(J-K)</b> Dino-Lite settings. <b>(J)</b> After exposing the left lobe of the liver, the mouse was put under the Dino-Lite. <b>(K)</b> Connection between the Dino-Lite and computer. Tumor fluorescence was imaged on the monitor during FGS. Magnifications are indicated above the columns.</p

Pre-operative and post-operative images from the orthotopic liver metastasis model treated with BLS.

<p><b>(A)—(C)</b> Upper panels show bright field images, and lower panels are images of tumor fluorescence obtained with the OV100. At low magnification, residual tumor fluorescence was marginally detected. <b>(B)</b> However, at high magnification, residual tumor fluorescence was clearly visualized (arrows) <b>(C)</b>. Arrowheads show residual tumor fluorescence in <b>B</b> and <b>C</b>. <b>(D)</b> Resected specimen. Magnifications are indicated above the columns.</p

Imaging of IGF-1R targeting of liver metastasis.

<p>Anti-IGF-1R conjugated to PEGylated 650 nm dye selectively labeled the HCT 116-GFP metastatic tumors. Both in vivo (A) and ex vivo (B) imaging show that the 650 nm fluorophore-conjugated IGF-1R antibodies co-localized with HCT 116-GFP fluorescence and more accurately demarked the tumor compared to bright light imaging (white arrowheads). H & E staining of tissue sample (x200) expressing fluorescence confirms the presence of metastatic tumor in the mouse liver.</p

Imaging of IGF-1R targeting of orthotopically-transplanted HCT 116 colon tumors in vivo.

<p>Fluorescence from orthotopically transplanted colon tumors at the cecum was detected before and after abdominal laparotomy. Also, fluorescence from the resected tumor was detected. Weak fluorescence was also detected from the skin and, bladder and intestinal contents, but at much lower intensity than the tumor. White arrows indicate colon tumor.</p

Evaluation of tumor fluorescence at day 28 after surgery.

<p><b>(A)</b> Upper panel shows the bright field image, and lower panel shows the GFP tumor fluorescence image obtained with the OV100 at a magnification of 0.56. Laparotomy was performed at the 28^th postoperative day. Bright field image shows tumor in the resection site in the liver (arrows). Strong GFP fluorescence from the tumor is seen in the lower panel. Arrows show recurrent tumor in the resection site. Arrowheads show operative scar on the liver. <b>(B)</b> The GFP tumor fluorescence area was significantly larger in the BLS group compared to the FGS group, where only autofluorescence was detected. Error bars show SD. *<i>P</i><0.05.</p

Imaging of 650 nm fluorophore-conjugated IGF-1R antibody targeting of subcutaneous colon tumors in vivo.

<p>(A) The mouse that is imaged under both white light and fluorescence illumination. The intensity of fluorescence from the HCT 116 and HT-29 subcutaneous tumors is much greater than background. (B) Hematoxylin & eosin staining (x200, left). Fluorescence immunostaining for IGF-1R (x20 regular objective, IV-100 scanning laser microscope (Olympus) with a 633 nm laser, right) of frozen HCT 116 tumor samples shows fluorescence on the membrane of the cancer cells.</p

Characterization of colon cancer cell lines.

<p>(A) Western blot analysis shows pro-IGF-1R and IGF-1Rα expression at 200 and 130 kDa in colon cancer cell lines, respectively (HT-29 and HCT 116). (B) Labeling of live HCT 116 and HT-29 cells with 550 nm fluorophore-conjugated antibodies shows multiple fluorescent foci on the surface. Representative fluorescence images merged with corresponding DIC (differential interference contrast) images (x60 water immersion objective with the FV1000, using the 559 nm laser).</p

Rates of chronic endometritis in each stage of endometriosis and comparison between early stages (I+II) and late stages (III+IV).

<p>Rates of chronic endometritis in each stage of endometriosis and comparison between early stages (I+II) and late stages (III+IV).</p