Undergraduate Researcher University of Alabama at Birmingham Mobile, Alabama, United States
Introduction::
Close to 50,000 Americans will be diagnosed with oral cancer this year, killing 1 person every hour. Most patients diagnosed with early-stage oral cancer undergo tumor resection surgery, but it does not come without its own complications. Often, surgery consists of the removal of a significant amount of healthy tissue leading to a compromise in facial structure and function with the possibility of microscopic residual disease being left behind, which leads to cancer recurrence. This is in part due to the inability of white- light imaging, the current standard, to accurately determine tumor dimensions, specifically tumor depths. While there are limitations associated with single mode imaging, such as fluorescence imaging, multimodal imaging probes that enable target visualization through complementary imaging technologies provide an attractive alternative. Studies from Hasan Lab at Massachusetts General Hospital3 and others have performed pre-clinical studies combining fluorescence imaging with photoacoustic imaging to generate 3- dimensional tumor images in real time during tumor resection surgery. Photoacoustic (PA) imaging is a novel biomedical imaging modality that combines the high-contrast and spectroscopic-based specificity of optical imaging with the 3-dimensional resolution of ultrasound imaging.
Materials and Methods::
For the synthesis of DFAC and nanoliposomal constructs, DFAC will be synthesized through routine EDC-NHS based conjugation of the two dyes (IRDye800 and AF647) in ratios previously determined in Hasan lab. Nanoliposomes, with either surface conjugation of DFAC or encapsulated with DFAC, are prepared by the thin film hydration method. The synthesized liposomes will be characterized by spectrophotometry and DLS to identify the conjugation efficiency or encapsulation efficiency of DFAC. Photophysical characterization will be performed by photoacoustic and fluorescence imaging. To characterizing and test the nanoliposomes in 2D, the surface conjugation of DFAC on nanoconstructs will be validated by studying their interaction with an EGFR overexpressing cell line (A431). Cellular binding and localization patterns will be assessed by FACS and confocal microscopy. To mimic stages in the receptor-mediated endocytosis process, DFAC-NAL and NAL-DFAC were prepared by mixing DPPC, DOTAP, Cholesterol, DSPE-mPEG2000, and DSPE-PEG2000-DIBO, followed by drying to form a film. Once the liposomes are synthesized, several photophysical characterizations are performed to ensure conjugation/encapsulation. Using DLS, we measured the hydrodynamic diameter (nm), polydispersity index (PDI), and surface charge of the artificial cells. Further experiments will test their interaction with EGFR-expressing cells in 2D culture by FACS and confocal microscopy.
Results, Conclusions, and Discussions::
I found that the liposomal formulations of DFAC, NAL-DFAC, and DFAC-NAL produced similar absorbance spectra for both dyes: AF647 and IRDye800 (Figure 2). I interpreted from these results that the liposomes preserved the photophysical properties of the dyes and could serve as an accurate representative of cells in the RME pathway.
To assess DFAC location in the nanoliposomes, I measured the binding specificity to the different cell lines using flow cytometry and confocal imaging. After analyzing the data, one could clearly see that DFAC-NAL bound abundantly to A431, the EGFR overexpressing line, while NAL-DFAC bound significantly less (Figure 3 and 4). There was no binding with CHO-WT, the EGFR negative cell line. From the confocal imaging of the DFAC treated cells (Figure 4), an abundant interaction of DFAC-NAL with A431 cells could be observed with little to no interaction of NAL-DFAC with A431 cells. The confocal imaging results followed a similar pattern as the results derived from flow cytometry data.
Finally, I observed a clear shift in PA and fluorescence signal intensity of the different formulations with NAL-DFAC producing the weakest signals (Figure 5). Photoacoustic imaging will be an important imaging tool for tumor surgeries, especially when used in conjunction with fluorescence imaging. Through my experiments, I can assess that fluorescence and PA signals tend to decrease when the DFAC is encapsulated within the liposomes as opposed to when it is conjugated on the nanoliposomal surface. With regards to the RME pathway, this would suggest that the signal intensity from the DFACs would peak early in the RME pathway (when DFAC is bound to the cell surface) as compared to later stages in the RME pathway (when DFAC is internalized). Furthermore, I conclude that my experiments confirm that the timing of fluorescence and PA imaging are essential factors to be considered when using multimodal imaging probes for measuring tumor margins.
Acknowledgements (Optional): : Funding for this project was provided by the National Science Foundation under Award No 1852430 to the REU Site: Wellman-HST Summer Institute for Biomedical Optics and The National Institute of Health grant for Project No 1R01CA231606-01A1: Dual function theranostic constructs for photoacoustic image-guided surgery and photodynamic therapy.
References (Optional): :
1 Oral cancer facts. The Oral Cancer Foundation. (n.d.) 2 Oral cavity (mouth) cancer treatment options, by stage. American Cancer Society. (n.d.) 3 Saad, M. A., et al. (2021) Dual function antibody conjugates for multimodal imaging and photoimmunotherapy of cancer cells. Photochemistry & Photobiology, 98(1) 220–231. 4 Beard, P. (2011). Biomedical photoacoustic imaging. Interface Focus, 1(4), 602–631. 5 What are Antibody-drug conjugates? Journal of Antibody-Drug Conjugates (n.d.) 6 Nakhaei, P., et al (2021). Liposomes. Frontiers in Bioengineering and Biotechnology, 9.
