Development of a MUC16-Targeted Near-Infrared Fluorescent Antibody Conjugate for Intraoperative Imaging of Pancreatic Cancer.

Surgical resection is currently the only potentially curative option for patients with pancreatic cancer. However, the 5-year survival rate after resection is only 25%, due in part to high rates of R1 resections, in which cells are left behind at the surgical margin, resulting in disease recurrence. Fluorescence-guided surgery (FGS) has emerged as a method to reduce incomplete resections and improve intraoperative assessment of cancer. Mucin-16 (MUC16), a protein biomarker highly overexpressed in pancreatic cancer, is a potential target for FGS. In this study, we developed a fluorescent MUC16-targeted antibody probe, AR9.6-IRDye800, for image-guided resection of pancreatic cancer. We demonstrated the efficacy of this probe to bind human pancreatic cancer cell lines in vitro and in vivo In an orthotopic xenograft model, AR9.6-IRDye800 exhibited superior fluorescence enhancement of tumors and lower signal in critical background organs in comparison to a nonspecific IgG control. The results of this study suggest that AR9.6-IRDye800 has potential for success as a probe for FGS in pancreatic cancer patients, and MUC16 is a feasible target for intraoperative imaging.

Tuned near infrared fluorescent hyaluronic acid conjugates for delivery to pancreatic cancer for intraoperative imaging.

The prognosis of pancreatic cancer remains poor. Intraoperative fluorescence imaging of tumors could improve staging and surgical resection, thereby improving prognosis. However, imaging pancreatic cancer with macromolecular delivery systems, is often hampered by nonspecific organ accumulation. Methods: We describe the rational development of hyaluronic acid (HA) conjugates that vary in molecular weight and are conjugated to near infrared fluorescent (NIRF) dyes that have differences in hydrophilicity, serum protein binding affinity, and clearance mechanism. We systematically investigated the roles of each of these properties on tumor accumulation, relative biodistribution, and the impact of intraoperative imaging of orthotopic, syngeneic pancreatic cancer. Results: Each HA-NIRF conjugate displayed intrapancreatic tumor enhancement. Regardless of HA molecular weight, Cy7.5 conjugation directed biodistribution to the liver, spleen, and bowels. Conjugation of IRDye800 to 5 and 20 kDa HA resulted in low liver and spleen signal while enhancing the tumor up to 14-fold compared to healthy pancreas, while 100 kDa HA conjugated to IRDye800 resulting in liver and spleen accumulation. Conclusion: These studies demonstrate that by tuning HA molecular weight and the physicochemical properties of the conjugated moiety, in this case a NIRF probe, peritoneal biodistribution can be substantially altered to achieve optimized delivery to tumors intraoperative abdominal imaging.

Image-guided tumor surgery: The emerging role of nanotechnology.

Surgical resection is a mainstay treatment for solid tumors. Yet, methods to distinguish malignant from healthy tissue are primarily limited to tactile and visual cues as well as the surgeon's experience. As a result, there is a possibility that a positive surgical margin (PSM) or the presence of residual tumor left behind after resection may occur. It is well-documented that PSMs can negatively impact treatment outcomes and survival, as well as pose an economic burden. Therefore, surgical tumor imaging techniques have emerged as a promising method to decrease PSM rates. Nanoparticles (NPs) have unique characteristics to serve as optical contrast agents during image-guided surgery (IGS). Recently, there has been tremendous growth in the volume and types of NPs used for IGS, including clinical trials. Herein, we describe the most recent contributions of nanotechnology for surgical tumor identification. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Implantable Materials and Surgical Technologies > Nanoscale Tools and Techniques in Surgery Diagnostic Tools > in vivo Nanodiagnostics and Imaging.

Nanoparticle Formulation of Indocyanine Green Improves Image-Guided Surgery in a Murine Model of Breast Cancer.

PURPOSE: Negative surgical margins (NSMs) have favorable prognostic implications in breast tumor resection surgery. Fluorescence image-guided surgery (FIGS) has the ability to delineate surgical margins in real time, potentially improving the completeness of tumor resection. We have recently developed indocyanine green (ICG)-loaded self-assembled hyaluronic acid (HA) nanoparticles (NanoICG) for solid tumor imaging, which were shown to enhance intraoperative contrast. PROCEDURES: This study sought to assess the efficacy of NanoICG on completeness of breast tumor resection and post-surgical survival. BALB/c mice bearing iRFP+/luciferase+ 4T1 syngeneic breast tumors were administered NanoICG or ICG, underwent FIGS, and were compared to bright light surgery (BLS) and sham controls. RESULTS: NanoICG increased the number of complete resections and improved tumor-free survival. This was a product of improved intraoperative contrast enhancement and the identification of a greater number of small, occult lesions than ICG and BLS. Additionally, NanoICG identified chest wall invasion and predicted recurrence in a model of late-stage breast cancer. CONCLUSIONS: NanoICG is an efficacious intraoperative contrast agent and could potentially improve surgical outcomes in breast cancer.

Hyaluronic acid formulation of near infrared fluorophores optimizes surgical imaging in a prostate tumor xenograft.

The presence of positive surgical margins confers an increased risk of biochemical relapse and need for salvage therapy in men undergoing radical prostatectomy. Image-guided surgery using near-infrared (NIR) fluorescent contrast agents is a potential method to detect remaining cancerous tissue. The objective of this study was to evaluate three hyaluronic acid (HA) nanoparticle (NP) formulations loaded with NIR fluorophore for their ability to contrast-enhance prostate cancer. HA was modified by conjugation with the hydrophobic ligand, aminopropyl-1-pyrenebutanamide to drive nanoparticle self-assembly. Indocyanine green (ICG) was physicochemically entrapped in the HA-NP, termed NanoICG. Alternatively, Cy7.5 was directly conjugated to amphiphilic HA, termed NanoCy7.5. NanoCy7.5 was synthesized with two HA molecular weights to determine the HA size contribution to delivery to PC3 prostate tumor xenografts. Contrast-enhancement of the tumors and relative biodistribution were assessed by a series of fluorescence imaging, image-guided surgery with spectroscopy, and microscopic techniques. Intravenously administered NanoICG improved tumor signal-to-noise ratio (SNR) at 24 h over ICG by 2.9-fold. NanoCy7.5 with 10 kDa and 100 kDa HA improved tumor SNR by 6.6- and 3.1-fold over Cy7.5 alone, respectively. The PC3 xenograft was clearly identified with the image-guided system providing increased contrast enhancement compared to surrounding tissue for NanoICG and NanoCy7.5 with 10 kDa HA. NIR fluorescence microscopy showed that Cy7.5 in NPs with 10 kDa HA were distributed throughout the tumor, while NanoCy7.5 with 100 kDa HA or NanoICG delivered dye mainly to the edge of the tumor. CD31 staining suggested that PC3 tumors are poorly vascularized. These studies demonstrate the efficacy of a panel of HA-derived NPs in identifying prostate tumors in vivo, and suggest that by tuning the structural properties of these NPs, optimized delivery can be achieved to poorly vascularized tumors. STATEMENT OF SIGNIFICANCE: We have demonstrated the potential of a panel of near-infrared fluorescent (NIRF) nanoparticles (NPs) for image-guided surgery in a prostate cancer xenograft model. Image-guided surgery and imaging of organs ex vivo showed greater tumor signal and contrast when mice were administered NIRF dyes that were covalently conjugated to (NanoCy7.510k-PBA) or physicochemically entrapped in (NanoICGPBA) hyaluronic acid (HA) NPs, compared to free dyes. These results show the potential to use these NPs as tools to detect the margins of tumors and to differentiate healthy and tumor tissue intraoperatively. Moreover, this project provides insight into selecting optimal formulation strategies for poorly vascularized tumors.

Indocyanine green loaded hyaluronan-derived nanoparticles for fluorescence-enhanced surgical imaging of pancreatic cancer.

Pancreatic ductal adenocarcinoma is highly lethal and surgical resection is the only potential curative treatment for the disease. In this study, hyaluronic acid derived nanoparticles with physico-chemically entrapped indocyanine green, termed NanoICG, were utilized for intraoperative near infrared fluorescence detection of pancreatic cancer. NanoICG was not cytotoxic to healthy pancreatic epithelial cells and did not induce chemotaxis or phagocytosis, it accumulated significantly within the pancreas in an orthotopic pancreatic ductal adenocarcinoma model, and demonstrated contrast-enhancement for pancreatic lesions relative to non-diseased portions of the pancreas. Fluorescence microscopy showed higher fluorescence intensity in pancreatic lesions and splenic metastases due to NanoICG compared to ICG alone. The in vivo safety profile of NanoICG, including, biochemical, hematological, and pathological analysis of NanoICG-treated healthy mice, indicates negligible toxicity. These results suggest that NanoICG is a promising contrast agent for intraoperative detection of pancreatic tumors.

Near Infrared Fluorescent Nanoparticles Derived from Hyaluronic Acid Improve Tumor Contrast for Image-Guided Surgery.

Tumor tissue that remains undetected at the primary surgical site can cause tumor recurrence, repeat surgery, and treatment strategy alterations that impose a significant patient and healthcare burden. Intraoperative near infrared fluorescence (NIRF) imaging is one potential method to identify remaining tumor by visualization of NIR fluorophores that are preferentially localized to the tumor. This requires development of fluorophores that consistently identify tumor tissue in different patients and tumor types. In this study we examined a panel of NIRF contrast agents consisting of polymeric nanoparticle (NP) formulations derived from hyaluronic acid (HA), with either physically entrapped indocyanine green (ICG) or covalently conjugated Cy7.5. Using orthotopic human breast cancer MDA-MB-231 xenografts in nude mice we identified two lead formulations. One, NanoICGPBA, with physicochemically entrapped ICG, showed 2.3-fold greater tumor contrast than ICG alone at 24 h (p < 0.01), and another, NanoCy7.5100-H, with covalently conjugated Cy7.5, showed 74-fold greater tumor contrast than Cy7.5 alone at 24 h (p < 0.0001). These two lead formulations were then tested in immune competent BALB/c mice bearing orthotopic 4T1 breast cancer tumors. NanoICGPBA showed 2.2-fold greater contrast than ICG alone (p < 0.0001), and NanoCy7.5100-H showed 14.8-fold greater contrast than Cy7.5 alone (p < 0.0001). Furthermore, both NanoICGPBA and NanoCy7.5100-H provided strong tumor enhancement using image-guided surgery in mice bearing 4T1 tumors. These studies demonstrate the efficacy of a panel of HA-derived NPs in delineating tumors in vivo, and identifies promising formulations that can be used for future in vivo tumor removal efficacy studies.