(M1230-10-68) Ultrasmall Nanoprobes for Fluorescence-Guided Surgical Resection of Peritoneally Disseminated Tumors

Purpose: Peritoneal metastasis (PM) is a rare end stage malignancy that arises from gynecologic and gastrointestinal origin [1]. Current treatment approaches are palliative cytoreductive surgery followed by administration of chemotherapy. In PM, the completeness of tumor resection directly correlates with the overall survival of patients and thus it is critical to visualize and resect microscopic residual tumors. FDA approved agents such as Indocyanine Green (ICG) and Methylene Blue (MB) for cytoreductive surgery are the gold standard for image guided surgery [2]. These probes show non-specific tissue uptake whereas fluorescently labelled antibodies restrict pan-cancer application of the agents, demonstrate tumor accumulation and poor tumor to background signal [3]. Thus, there is a need for the development of new imaging techniques and probes to improve cytoreductive surgery and patient outcomes. Silica nanoparticles have emerged as promising platform for drug delivery and diagnostic imaging as silica is considered as ‘Generally Recognized as Safe’ (GRAS) by the FDA. However, nanotechnology-based probes suffer from limited tumor accumulation. To this end, we have designed ultrasmall porous silica nanoparticles (UPSNs) (hydrodynamic diameter < 15 nm) with tumor selective uptake (17.7% ID/g), higher than any reported nanoparticle till date and rapid hepatobiliary clearance [4]. Here, we demonstrate the superiority of UPSNs as fluorescent surgical guides over gold standard ICG in surgical resection of micro- and macro- PMs of colorectal cancer.

Methods: We synthesized and characterized the physicochemical properties of UPSNs. The UPSN were conjugated with fluorescent dyes Cy3 (λ_abs ~ 550 nm, λ_em~ 570 nm) and Cy5 (λ_abs ~640 nm, λ_em ~ 670 nm) for surgical resection and biodistribution studies, respectively. PM tumor model was generated by intraperitoneal (i.p.) injection of colorectal cancer (CT26-GFP-Luc) cells in female Balb/c mice. Tumor growth was monitored by measuring bioluminescence imaging (BLI). For image-guided surgery, UPSN-Cy3 was administered i.p., and tumors were resected under a fluorescence stereomicroscope. A separate cohort of mice injected with ICG was used as a control. The biodistribution of UPSN-Cy5 and ICG was assessed, 24 h post-injection by ex vivo fluorescence in tumors and organs of interest using IVIS and sensitivity and specificity were determined.

Results: Transmission electron microscopy (TEM) images and dynamic light scattering (DLS) measurements indicate uniform morphology and narrow size distribution of UPSNs (~ 13.72 ± 0.24) and UPSN-dye conjugates (Figure 1 (a, b)). There was gradual increase in the uptake of the particles by CT26 cells as indicated by confocal microscopy (Figure 1c). UPSN-Cy3 facilitated near-complete surgical resection in both high and low-tumor-burden models (Figure 2a). The signal in the resected tumors was selectively higher than residual intestine tissues. Further, ex vivo biodistribution indicated that UPSN-Cy5 accumulation was 18-fold higher in resected tumors compared to the intestine and other organs. The tumor-to-background ratio for UPSN-Cy5 (17.8) was significantly higher compared to ICG (2.19), confirming the tumor-selective uptake of our probes, resulting in a specificity of ~ 93% in comparison to 65% for ICG. Both UPSN-Cy5 and ICG demonstrated similar sensitivity, > 93% for tumor detection (Figure 2b). Ex vivo imaging of tumor uptake of UPSN-Cy5 was further validated by confocal microscopy (Figure 3). Thus, UPSN-based fluorescent probes exhibit strong potential for precise and complete resection of tumors and preventing excess tissue removal in metastatic disease, which can impact overall survival.

Conclusion: Our data establishes the potential of UPSNs to serve as tumor-selective fluorescent surgical guides, with 8 – fold enhanced specificity over the current clinical probe.

References: 1. Cortés-Guiral, D., Hübner, M., Alyami, M., Bhatt, A., Ceelen, W., Glehen, O., ... & Chand, M. (2021). Primary and metastatic peritoneal surface malignancies. Nature Reviews Disease Primers, 7(1), 1-23.
2. Wang, K., Du, Y., Zhang, Z., He, K., Cheng, Z., Yin, L., ... & Tian, J. (2023). Fluorescence image-guided tumour surgery. Nature Reviews Bioengineering, 1-19.
3. Ito, R., Kamiya, M., & Urano, Y. (2022). Molecular probes for fluorescence image-guided cancer surgery. Current Opinion in Chemical Biology, 67, 102112.
4. Goel, S., Ferreira, C. A., Dogra, P., Yu, B., Kutyreff, C. J., Siamof, C. M., ... & Cai, W. (2019). Size‐optimized Ultrasmall porous silica nanoparticles depict vasculature‐based differential targeting in triple-negative breast cancer. Small, 15(46), 1903747.

Acknowledgements: This work is supported, in part, by the University of Utah, the University of Utah Immunology, Inflammation and Infectious Diseases (3i) Initiative, the 5 For the Fight Fellowship, and the Elsa U Pardee Foundation. We acknowledge direct financial support provided by the Huntsman Cancer Foundation and the Experimental Therapeutics Program at Huntsman Cancer Institute; we also acknowledge support by the National Cancer Institute of the National Institutes of Health under Award Number P30CA042014. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Figure 1: Physiochemical properties of UPSNs (a)TEM of UPSN depicting uniform morphology b) Hydrodynamic diameter of UPSNs and UPSN-dye conjugates via DLS and c) Cell uptake of UPSN-Cy5 (red) in CT26 cells. DAPI represents cell nuclei. Scale bar: 10 μm

Figure 2: UPSNs for fluorescence image guided surgery in PM (a) Representative images of surgical resection of low and high tumor burden model, 24 h p.i UPSN-Cy3 indicates complete resection with no residual tumor margins. (b) Representative ex vivo IVIS images of tumor and intestine depicting tumor specific uptake of UPSN-Cy5 compared to clinically used ICG.

Figure 3: Representative confocal microscopy images showing uptake of UPSN-Cy5 (red) in CT26 peritoneally disseminated tumor nodules at 40X magnification. Control represents resected CT26 nodules after PBS injection. Scale bar: 45 μm