Intraoperative fluorescence imaging of peritoneal seedings using intraperitoneal staining with folate-human serum albumin-indocyanine green conjugates in a mouse tumor model

Objectives The aim is to demonstrate the feasibility of intraoperative fluorescence imaging with intraperitoneally administrated folate-human serum albumin(HSA)-indocyanine green (ICG) conjugates to detect peritoneal seedings in a mouse tumor model instead of conventional intravenous administration.

Methods Folate was conjugated with HSA, and mixed with ICG in a light-shield tube. We made a portable intraoperative fluorescence imaging system comprising a 740-nm LED and a CCD camera with an 820-nm bandpass filter. MDAH-2774 ovarian cancer cells expressing luciferase were cultured in DMEM. 2×10⁷ MDAH-2774 ovarian cancer cells were intraperitoneally administrated in female BALB/c-nu/nu mice 6 to 10 weeks old. After 2 to 3 weeks when multifocal bioluminescent signals were detected by IVIS® Lumina, 200 µl of folate-HSA-ICG conjugates mixed with 200 µl of normal saline (NS) was intraperitoneally administrated. HSA-ICG conjugates without folate, and pure ICG diluted with NS were also administrated for comparison. On the next day, laparotomy was conducted and bioluminescence/fluorescence images were taken using IVIS® Lumina, and our custom-made system. Specimens were confirmed by pathologic examination.

Results Multiple peritoneal seedings with increased fluorescent signal were successfully detected with IVIS® Lumina, and our custom-made portable intraoperative fluorescence imaging system. Submillimeter-sized seedings were able to be identified. In comparison, when HSA-ICG conjugates without folate or pure ICG diluted with NS was administrated, fluorescence imaging showed diffuse non-specific uptake with much less intensity.

Conclusions Peritoneal seedings were successfully stained with folate-HSA-ICG conjugates. This new peritoneal staining method with folate-HSA-ICG conjugates could find applications in intraoperative detection and photodynamic therapy using a handheld camera or surgical laparoscopic apparatus.

Research Support This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MEST) (2011-0019794)