Diffusion Reflection Method for Early Detection of Oral Squamous Cell Carcinoma Specifically Targeted by Circulating Gold-Nanorods Bio-Conjugated to Anti-Epidermal Growth Factor Receptor

Abstract

Background: Translation of nanomedical developments into clinical application is receiving an increasing interest. However, its use for oral squamous cell carcinoma (OSCC) diagnosis remains limited. We present an advanced nanophotonic method for oral cancer detection, based on diffusion reflection (DR) measurements of gold-nanorods bio-conjugated to anti-epidermal growth factor receptor (C-GNRs) specifically attached to OSCC cells.

Objective: To investigate in a rat model of oral carcinogenesis the targeting potential of C-GNRs to OSCC by using the DR optical method.

Materials and methods: OSCC was induced by the carcinogen 4-nitroquinoline-N-oxide (4NQO). C-GNRs were introduced locally and systemically and DR measurements were recorded from the surface of the rat tongue following illumination with red laser beam. Rats were divided into experimental and control groups. The results were compared with the histologic diagnosis.

Results: A total of 75 Wistar-derived rats were enrolled in the study. Local application did not reveal any statistical results. DR measurements following intravenous injection of C-GNRs revealed a significant increase in light absorption in rats with OSCC compare with rats without cancer (p<0.02, sensitivity 100%, specificity 89%). In addition, absorption of light increased significantly in cases of severe dysplasia and cancer (high risk) compared to rats without cancer and rats with mild dysplasia (low risk) (86% sensitivity and 89% specificity, AUC=0.79).

Conclusion: Combining nanotechnology and nanophotonics for in vivo diagnosis of OSCC serves as additional tier in the translation of advanced nanomedical developments into clinical applications. The presented method shows a promising potential of nanophotonics for oral cancer identification, and provides support for the use of C-GNRs as a selective drug delivery.

Keywords: anti-EGFR; cancer detection; gold nanoparticles; nanophotonic; oral cancer; squamous cell carcinoma.

Figure 1

Experimental setup. (A) Injection of GNRs to the rat’s tail vein. (B) GNRs are flowing in the rat’s circulation. (C, D) At the area of the tumor there Is higher penetration of the blood vessels, since the tumor over express EGFR on the cell membrane, GNRs are attached to the tumor in high concentration. (E) DR measurements are being recorded; the absorption of the red color laser beam is higher at the area of the tumor where there is high concentration of GNRs. It is also possible to see the illustration of the cell membrane with the attached GNRs (all images used in Figure 1 are original).

Figure 2

Example of logarithm representation of DR measurements. The logarithmic product between the distance and the reflectance (Γ) versus the distance (ρ). DR profile of a healthy rat (group A – black triangles) has a lower absolute slope compared to a rat with cancer (group F – blue circles).

Figure 3

(A) Cancer in the posterior dorsum of the tongue (arrow), (B) Low-power photomicrograph showing typical well differentiated squamous cell carcinoma exhibiting deep invasion (hematoxylin and eosin stain, original magnification ×40), (C) hyperspectral imaging, GNRs are shown as red dots, higher magnification of the marked area shown in B (original magnification ×200, scale bar is 500µm).

Figure 4

DR measurements of the study group. The highest absolute DR slope values were recorded in rats with cancer (group F) and in rats with moderate to severe dysplasia (group E). The lowest values were recorded in rats with hyperkeratosis and mild dysplasia and in all of the control rats reflecting the low concentration of EGFR. The oblique line represents the trend; a significant trend of the DR measurements in the posterior area correlated with the progression of the severity of the lesions (Cuzick trend test, P = 0.018).