Two-step synthesis of galactosylated human serum albumin as a targeted optical imaging agent for peritoneal carcinomatosis

Abstract

An optical probe, RG-(gal)(28)GSA, was synthesized to improve the detection of peritoneal implants by targeting the beta-d-galactose receptors highly expressed on the cell surface of a wide variety of cancers arising from the ovary, pancreas, colon, and stomach. Evaluation of RG-(gal)(28)GSA, RG-(gal)(20)GSA, glucose-analogue RG-(glu)(28)GSA, and control RG-HSA demonstrates specificity for the galactose, binding to several human adenocarcinoma cell lines, and cellular internalization. Studies using peritoneally disseminated SHIN3 xenografts in mice also confirmed a preference for galactose with the ability to detect submillimeter size lesions. Preliminary toxicity study for RG-(gal)(28)GSA using Balb/c mice reveal no toxic effects up to 100x of the standard imaging dose of 1 mg/kg administered either intraperitoneally or intravenously. These data indicate that RG-(gal)(28)GSA can selectively target a variety of human adenocarcinomas, can improve intraoperative or endoscopic tumor detection and resection, and may have little or no toxic in vivo effects; hence, it may be clinically translatable.

Figure 1

Mass spectral data showing the difference in m/z of 5017.34 from the starting material human serum albumin (HSA) to the glucosylated GSA ((glu)₂₈GSA) (top portion) indicating a modification of ~28 glucosamine to HSA. Lower portion demonstrates the Δ m/z of 1166.19 between the galactosylated GSA’s at different reaction time estimating the difference in the number of galactosamine to ~8.

Figure 2

Flow cytometry studies from different human adenocarcinoma cell lines after incubation with 3 μg/mL of RG-(gal)₂₈GSA (green), RG-(gal)₂₀GSA (blue), RG-(glu)₂₈GSA (pink), RG-HSA (orange), and buffer (black). The percentage fluorescently-gated cells and mean fluorescence intensity is highest with RG-(gal)₂₈GSA incubation for all cell lines tested.

Figure 3

Fluorescence microscopy images (right panel) and differential interference contrast imaging (left panel) of SHIN3 cells 6 hr after incubation with 3 μg/mL of the Rhodamine dye conjugates of the glycosylated GSA and non-glycosylated HSA. Cells incubated with the glycosylated GSA demonstrated internalization of the agent with the (gal)₂₈GSA showing the largest number of fluorescent dots within the cytoplasm under the same exposure time (200 ms) and with RG-HSA showing no fluorescence within the SHIN3 cells even at longer exposure time (1 s).

Figure 4

Spectral fluorescence imaging of the peritoneal cavities of SHIN3-xenografted mice 4 hr after intraperitoneal injection of 20 μg of the 1:RG-(gal)₂₈GSA, 2:RG-(gal)₂₀GSA, 3:RG-(glu)₂₈GSA, 4:RG-HSA, and a 5:nontreated mouse. Spectral unmixed Rhodamine Green fluorescence images (upper), composite (with autofluorescence) (middle) images, and white light images (lower) are shown. Aggregated large tumor foci are pointed by the arrows.

Figure 5

Small SHIN3 implants are detected using RG-(gal)₂₈GSA on the peritoneal membranes as shown by the spectral fluorescence images with the spectral unmixed Rhodamine green fluorescence (right most panel), composite (with autofluorescence) (middle panel), and white light (left most panel) images.

Figure 6

Sensitivity and specificity of the RG-(gal)₂₈GSA was validated using RFP-transfected SHIN3 ovarian cancer-bearing mice. The spectral fluorescence images were unmixed based on the spectral patterns of Rhodamine Green (lower right panel), RFP (DsRed) (lower left panel), and autofluorescence.

Figure 7

Receptor-mediated uptake was demonstrated by the decrease in the uptake of RG-(gal)₂₈GSA upon co-adminstration of 10 mg of unlabeled (gal)₂₈GSA with 20 μg of RG-(gal)₂₈GSA.