Phthalocyanine-Blue Nanoparticles for the Direct Visualization of Tumors with White Light Illumination

Abstract

The current standard of care for colon cancer surveillance relies heavily on white light endoscopy (WLE). However, dysplastic lesions that are not visible to the naked eye are often missed when conventional WLE equipment is used. Although dye-based chromoendoscopy shows promise, current dyes cannot delineate tumor tissues from surrounding healthy tissues accurately. The goal of the present study was to screen various phthalocyanine (PC) dye-loaded micelles for their ability to improve the direct visualization of tumor tissues under white light following intravenous administration. Zinc PC (tetra-tert-butyl)-loaded micelles were identified as the optimal formulation. Their accumulation within syngeneic breast tumors led the tumors to turn dark blue in color, making them clearly visible to the naked eye. These micelles were similarly able to turn spontaneous colorectal adenomas in Apc^+/Min mice a dark blue color for easy identification and could enable clinicians to more effectively detect and remove colonic polyps.

Keywords: chromoendoscopy; colorectal cancer; nanoparticles; phthalocyanine; white light endoscopy.

Figure 1.

(A) Scheme depicting the process of solubilizing hydrophobic PCs within PEG-PCL micelles using oil-in-water (O/W) emulsions at a weight-to-weight (w/w) ratio 0.5:1 of PCs: PEG-PCL. (B) The chemical structure of PC and photographs of PC-loaded micelle samples. A table summarizing the chemical differences of the various PCs evaluated, as well as the hydrodynamic size, PDI, and the average zeta potential of each PC-loaded micelle, is provided. (C) Photographs of subcutaneous 4T1 breast tumors in BALB/c mice 24 h following the intravenous (retro-orbital) injection of PC-loaded micelles (10 mg/kg) and MB (10 mg/kg) (n=3 per group). The signal-to-background ratio (SBR) was quantified for each tumor relative to the surrounding tissue.

Figure 2.

(A) Hydrodynamic size of the ZnPC(TB)-loaded micelles was monitored for 7 days in PBS at room temperature. (B) Viability of 4T1 cells (black bar) and HT-29 cells (gray bar) after incubation with increasing concentrations of ZnPC(TB)-loaded micelles for 24 h.

Figure 3.

(A) Illustration of a Apc^+/Min-FCCC mouse injected with ZnPC(TB)-loaded micelles and accumulation of blue dye in colon tumors (Left). Photographs of the excised colons (top) and small intestines (bottom) with polyps, injected (24 h, n=5) of 20 mg/kg ZnPC(TB)-loaded micelles or water (control). The signal-to-background between the tumor and surrounding healthy tissue was quantified. (B) Representative murine gastrointestinal tract. The area highlighted with a yellow square shows micelle-accumulated through lesions in the intact stomach. The stomach from a control mouse is also shown for comparison. SBR measurements of with and without injections of ZnPC(TB)-loaded micelles are presented. The unpaired t‐test was used for analyses. *P, **P, and ***P < 0.001. Statistical significance was defined as P < 0.05.

Figure 4.

Photograph, color, bright-field, and fluorescence images of (A) flank tumor (4T1 breast), (B) colon, and (C) small intestine tissues. Photographs were acquired with an RGB camera (scale bars= 5 mm). Microscope images were acquired with a 4x objective (scale bars= 550 μm). The white dashed lines indicate the approximate tumor margin. Cancer tissues encircled by the dashed lines appear blue-green, and in the fluorescence imaging can be easily distinguished from normal tissue.