Novel Strategies in Cancer Detection, Diagnosis, and Prognosis I
A Novel Fluorescence Imaging Approach to Aid Surgical Resection of Solid Tumors using Food Drug and Cosmetic Coloring Agents in Liposomal Nanoparticles
Associate Professor University of Southern California, United States
Introduction: Positive margins, indicating the presence of residual cancerous cells, serve as a prognostic factor associated with elevated cancer recurrence rates and poor patient outcomes. Achieving negative margins can be extremely difficult for surgeons as there is usually no visible difference between healthy adjacent tissue and cancerous sites. Physicians could benefit from various sensitive and safe imaging agents with increased tumor specificity to aid the accuracy of negative margins during surgical resection. The ideal diagnostic contrast agent should be biodegradable, non-toxic, and have high specificity and sensitivity. However, the clinical landscape lacks a diverse array of fluorescent imaging agents, as only three clinically approved fluorescent dyes exist. This limitation led to an investigation of 26 commonly used tattoo pigments and FDA-approved drug and cosmetic dyes, revealing their underutilized biomedical imaging potential, including excellent fluorescence and Raman scattering. Among these dyes, Green 8 (G8) emerged as the brightest and most promising candidate. Green 8 was loaded into a biodegradable liposome to take advantage of nanoparticle (NP)-specific properties such as passive tumor accumulation, retention, and a large surface area ideal for conjugation with tumor-targeting ligands. Due to the COVID-19 Moderna and Pfizer-BioNTech vaccines, there has been a resurgence of interest in using liposomal nanoparticles for many applications. Liposomal NPs mimic our cell’s bilayer lipid membrane and contain an aqueous core that can envelop solubilized Green 8. We believe that incorporating G8 into a biodegradable NP will serve as a more sensitive and optimal imaging technique for early cancer detection and surgical resection accuracy.
Materials and
Methods: Fabrication of G8 Liposomal NPs. Liposomal nanoparticles were prepared by creating a dry lipid film cake, rehydrating with G8, extruding to 150 nm sized nanoparticles, and washing to ensure no free dye is left with ultracentrifugation and columns. Characterization of G8 Liposomal NPs. Nanosight Tracking Analysis (NTA) instrument measured nanoparticle size and concentration. OBG was used to burst liposomes to ascertain encapsulation concentrations. Xenograft Models. Although several cancer types could benefit from this approach, oral squamous cell carcinoma (OSCC) is an ideal cancer to develop our new molecular imaging strategy due to its high post-surgery recurrence rate. Nude mice were inoculated subcutaneously with human OSCC cell lines (SCC 15 or SCC 25). In-vivo imaging. Tumors and organs were harvested at either 4 or 24 hr post IV injection of 4 nM liposomes and fluorescently analyzed at excitation/emission 405/530 with 5% power and 3 seconds. The Nikon AR1 HD upright multiphoton confocal microscope was used to image the in-vivo circulation of nude mice injected intravenously with G8 Liposomal NPs.
Results, Conclusions, and Discussions: Green 8 has superior fluorescence than all dyes investigated, including the three clinical dyes (Fig. 1A). A quenching effect is observed when liposomes are saturated with G8 at ~90 mg/mL. The highest fluorescent liposome occurred at 12.5 mg/mL encapsulation. (Fig. 1B). Preliminary studies in xenograft OSCC tumor-bearing mouse models confirm the superior fluorescence emission and effective tumor targeting of Green 8 dye-loaded liposomal NPs post 4 hr. (Fig. 1D & 1E). At 24 hours after injecting free dye and liposomes, liposomes demonstrate significant fluorescence over free dye. (Fig 1F). Green 8 liposomes have two-photon excitation imaging capabilities (Fig. 1G). Combining the exceptional fluorescence of G8 with the advantageous properties of NPs, our approach can offer a sensitive and optimized imaging technique with broad applications for early cancer detection and precise surgical intervention across diverse cancer types. We have demonstrated a new nano-based imaging strategy that can potentially improve cancer detection and localization. This marks a promising initial stride in demonstrating the potential of a drug & cosmetic dye loaded in a liposome to delineate tumor margins via fluorescence. Future studies will further assess their respective characteristics (ie. stability, imaging sensitivity, conjugation potential) for clinical translation. Multimodal imaging approaches will also be explored by further conjugating the fluorescent nanoparticle with Cu-64 for PET imaging or Gd for MRI imaging.
