Anil Khanal

Targeted Noninvasive Imaging of EGFR-Expressing Orthotopic Pancreatic Cancer Using Multispectral Optoacoustic Tomography

Detection of orthotopic xenograft tumors is difficult due to poor spatial resolution and reduced image fidelity with traditional optical imaging modalities. In particular, light scattering and attenuation in tissue at depths beyond subcutaneous implantation hinder adequate visualization. We evaluate the use of multispectral optoacoustic tomography (MSOT) to detect upregulated epidermal growth factor (EGF) receptor in orthotopic pancreatic xenografts using a near-infrared EGF-conjugated CF-750 fluorescent probe. MSOT is based on the photoacoustic effect and thus not limited by photon scattering, resulting in high-resolution tomographic images. Pancreatic tumor-bearing mice with luciferase-transduced S2VP10L tumors were intravenously injected with EGF-750 probe before MSOT imaging. We characterized probe specificity and bioactivity via immunoblotting, immunocytochemistry, and flow cytometric analysis. In vitro data along with optical bioluminescence/fluorescence imaging were used to validate acquired MSOT in vivo images of probe biodistribution. Indocyanine green dye was used as a nonspecific control to define specificity of EGF-probe accumulation. Maximum accumulation occurred at 6 hours postinjection, demonstrating specific intratumoral probe uptake and minimal liver and kidney off-target accumulation. Optical bioluminescence and fluorescence imaging confirmed tumor-specific probe accumulation consistent with MSOT images. These studies demonstrate the utility of MSOT to obtain volumetric images of ligand probe biodistribution in vivo to detect orthotopic pancreatic tumor lesions through active targeting of the EGF receptor.

Targeting Acidity in Pancreatic Adenocarcinoma: Multispectral Optoacoustic Tomography Detects pH-Low Insertion Peptide Probes In Vivo

Background: pH-low insertion peptides (pHLIP) can serve as a targeting moiety that enables pH-sensitive probes to detect solid tumors. Using these probes in conjunction with multispectral optoacoustic tomography (MSOT) is a promising approach to improve imaging for pancreatic cancer. Methods: A pH-sensitive pHLIP (V7) was conjugated to 750 NIR fluorescent dye and evaluated as a targeted probe for pancreatic adenocarcinoma. The pH-insensitive K7 pHLIP served as an untargeted control. Probe binding was assessed in vitro at pH 7.4, 6.8, and 6.6 using human pancreatic cell lines S2VP10 and S2013. Using MSOT, semiquantitative probe accumulation was then assessed in vivo with a murine orthotopic pancreatic adenocarcinoma model. Results: In vitro, the V7-750 probe demonstrated significantly higher fluorescence at pH 6.6 compared with pH 7.4 (S2VP10, P = 0.0119; S2013, P = 0.0160), whereas no difference was observed with the K7-750 control (S2VP10, P = 0.8783; S2013, P = 0.921). In the in vivo S2VP10 model, V7-750 probe resulted in 782.5 MSOT a.u. signal compared with 5.3 MSOT a.u. in K7-750 control in tumor (P = 0.0001). Similarly, V7-750 probe signal was 578.3 MSOT a.u. in the S2013 model compared with K7-750 signal at 5.1 MSOT a.u. (P = 0.0005). There was minimal off-target accumulation of the V7-750 probe within the liver or kidney, and probe distribution was confirmed with ex vivo imaging. Conclusions: Compared with pH-insensitive controls, V7-750 pH-sensitive probe specifically targets pancreatic adenocarcinoma and has minimal off-target accumulation. The noninvasive detection of pH-targeted probes by means of MSOT represents a promising modality to improve the detection and monitoring of pancreatic cancer. Clin Cancer Res; 21(20); 4576–85. ©2015 AACR. See related commentary by Reshetnyak, p. 4502

Tumor targeted mesoporous silica-coated gold nanorods facilitate detection of pancreatic tumors using Multispectral optoacoustic tomography

Multispectral optoacoustic tomography (MSOT) is an emerging imaging technology that offers several advantages over traditional modalities, particularly in its ability to resolve optical contrast at depth on the microscopic scale. While potential applications include the early detection of tumors below clinical thresholds set by current technology, the lack of tumor-specific contrast agents limits the use of MSOT imaging. Therefore, we constructed highly stable nano-contrast agents by coating gold nanorods (GNRs) with either polyacrylic acid (PAA) or aminefunctionalized mesoporous silica (MS). Syndecan-1, which has been shown to target insulin-like growth factor 1 receptor (IGF1-R) (upregulated in pancreatic tumors), was conjugated on the surface of PAA-coated GNRs (PAA-GNRs) or MS-coated GNRs (MS-GNRs) to create tumor-targeted nanoparticles. In vitro, tumor targeting of nanoparticles was assessed with flow cytometry. In S2VP10L cells (positive for IGF1-R), the syndecan-1 MS-GNRs (Syndecan-MS-GNRs) demonstrated an increase in OA signal, 10x, compared to syndecan-1 PAAGNRs (Syndecan-PAA-GNRs). Minimal binding was observed in MiaPaca-2 cells (negative for IGF1-R). In vivo, tumor specific targeting of Syndecan-MS-GNRs was evaluated using a murine orthotopic pancreatic cancer model. The Syndecan- MS-GNRs demonstrated significantly greater accumulation within pancreatic tumors than in off-target organs such as the liver. Mice implanted with the IGF1-R negative MiaPaca-2 cells did not demonstrate specific tumor targeting. In summary, we report that targeted nano-contrast agents (Syndecan-MS-GNRs) can successfully detect orthotopic pancreatic tumors with minimum off-target binding in vivo using MSOT.

Preparation and optimisation of anionic liposomes for delivery of small peptides and cDNA to human corneal epithelial cells

Abstract Drug delivery to corneal epithelial cells is challenging due to the intrinsic mechanisms that protect the eye. Here, we report a novel liposomal formulation to encapsulate and deliver a short sequence peptide into human corneal epithelial cells (hTCEpi). Using a mixture of Phosphatidylcholine/Caproylamine/Dioleoylphosphatidylethanolamine (PC/CAP/DOPE), we encapsulated a fluorescent peptide, resulting in anionic liposomes with an average size of 138.8 ± 34 nm and a charge of −18.2 ± 1.3 mV. After 2 h incubation with the peptide-encapsulated liposomes, 66% of corneal epithelial (hTCEpi) cells internalised the FITC-labelled peptide, demonstrating the ability of this formulation to effectively deliver peptide to hTCEpi cells. Additionally, lipoplexes (liposomes complexed with plasmid DNA) were also able to transfect hTCEpi cells, albeit at a modest level (8% of the cells). Here, we describe this novel anionic liposomal formulation intended to enhance the delivery of small cargo molecules in situ.

Abstract 1500: Syndecan-1 targeted mesoporous silica-coated gold nanorods act as theranostic agents for in vivo detection of orthotopic pancreatic tumors using multispectral optoacoustic tomography

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Detection of pancreatic cancer is especially challenging in comparison to many cancers due to severe limitations of both contrast agent delivery to pancreatic tumors and limitations of traditional imaging techniques such as poor resolution and low depth penetration. Theranostic nanoparticles encompassing both therapeutic and diagnostic capabilities can overcome limitations associated with conventional cancer diagnosis and therapy. In this study, we compared mesoporous silica coated gold nanorods (MS-GNR) and polyacrylic acid coated gold nanorods (PAA-GNR) targeted to tumor cells via Syndecan-1 ligand for detection of orthotopic pancreatic cancer in vivo. Because one of the major difficulties for translation of theranostic nanoparticles to the clinic is an inability track performance of nanoparticles in vivo, especially at depths required of orthotopic tumors, we will overcome this impediment by utilizing multispectral optoacoustic tomography (MSOT). Multispectral optoacoustic tomography provides high optical contrast images at a microscale resolution and reasonable penetration depth by combining the advantages of optical (high sensitivity) and ultrasound (increased depth of penetration). Our results indicate that the Syndecan-1 MS-GNR were superior to Syndecan-1 PAA-GNR, untargeted MS-GNR, or untargeted PAA-GNR as contrast agents to identify pancreatic cancer in vivo via MSOT. The Syndecan-1 MS-GNR particles also resulted in reduced off-target accumulation compared to Syndecan-1 PAA-GNR. This study is among the first to evaluate biodistribution of ligand-targeted nanoparticles in the context of orthotopic pancreatic cancer using multispectral optoacoustic tomography. The MS GNR can be utilized as theranostic platform due to ability of mesoporous silica to encapsulate drug and easy surface modification with cancer recognizing motif peptide. Citation Format: Anil Khanal, Charles W. Kimbrough, Nichola C. Garbett, Joseph A. Burlison, William E. Grizzle, Lacey R. McNally. Syndecan-1 targeted mesoporous silica-coated gold nanorods act as theranostic agents for in vivo detection of orthotopic pancreatic tumors using multispectral optoacoustic tomography. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1500. doi:10.1158/1538-7445.AM2015-1500

Abstract 5116: Detection of pancreatic cancer using acidic pH targeted probes detected using multispectral optoacoustic tomography

Introduction: The use of theranostic nanoparticles for simultaneous tumor imaging and drug delivery is of great research interest. The tumor extracellular environment is significantly more acidic than physiologic pH, often in the range of pH 6.0-6.8. Exploitation the acidic extracellular tumor environment is a promising method of tumor localization with pH sensitive probes and targets. Further, the use of multispectral optoacoustic tomography (MSOT) allows in vivo 3D tumor imaging via gold nanoparticles or near-infrared dyes conjugated to a targeting molecule. Gemzar is used for first-line treatment of pancreatic adenocarcinoma, a malignancy with less than 5% survival at 5 years. We have created chitosan-capped mesoporous silica gold nanorods (C-MS-GNRs) to serve as pH sensitive theranostic nanoparticles for in vivo tumor imaging and targeted drug delivery. Methods: The chemical nature of all particles were characterized by electron microscopy, UV-Vis, and zeta-potential. Acidic pH specific Gemzar release was measured from C-MS-GNRs by UV-Vis. Cell viability assays on S2VP10 pancreatic adenocarcinoma cells with G-C-MS-GNR and Gemzar alone were performed at pH 6.5 and 7.4. S2VP10 cells were treated in pH 6.5 or 7.4 media with C-MS-GNR loaded with Indocyanin green (ICG) to demonstrate pH-sensitive cell targeting. ICG uptake was read on an Odyssey imaging system. Results: Particle characterization demonstrated appropriate chemical composition. A drug release assay of G-C-MS-GNR demonstrated 1/10 as much drug release at pH7.4 compared to in pH 6.4 or 6.0 media. Cell viability assays demonstrated enhanced cytotoxicity with Gemzar loaded C-MS-GNR at pH 6.5 and 7.4 compared to Gemzar alone. In vitro treatment of S2VP10 cells with C-MS-ICG in pH 6.5 media demonstrated 8X greater ICG delivery by C-MS-ICG than observed at pH 7.4. Conclusions: C-MS-GNR provides an efficacious means of targeting the acidic tumor environment for in vivo tumor imaging and drug delivery. C-MS-GNRs may be a suitable vehicle for tumor-targeted, pH-sensitive drug delivery to this currently untreatable tumor. Citation Format: Matthew Zeiderman, Anil Khanal, Charles W. Kimbrough, Jorge Gomez, William E. Grizzle, Kelly M. McMasters, Lacey R. McNally. Detection of pancreatic cancer using acidic pH targeted probes detected using multispectral optoacoustic tomography. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5116. doi:10.1158/1538-7445.AM2015-5116

Abstract 4301: Detection and characterization of regions of hypoxia within orthotopic pancreatic tumors using multispectral optoacoustic tomography

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Optical imaging is a vital tool in cancer research for visualization of tumors in preclinical models. However, resolution for bioluminescence and fluorescence modalities is restricted to a depth of five millimeters, as light is attenuated at increased depth and autofluorescence can create background noise. Light propagation becomes diffuse at only a few millimeters, resulting in light scattering and absorption. To improve imaging resolution and acquisition in real-time, multispectral optoacoustic tomography (MSOT) was utilized to visualize molecular phenomena such as the tissue oxygenation of characteristically hypoxic aggressive pancreatic tumors. SCID mice were orthotopically implanted with S2VP10L and MIA PaCa-2 pancreatic cancer cells that highly express the epidermal growth factor receptor (EGFR), which exhibits extracellular domain binding of the epidermal growth factor (EGF) ligand. Cohorts were then intravenously injected with fluorescent EGF ligand probes and imaged semiweekly to follow tumor growth and morphology. Medical Grade Air levels were set to 0.9 L and oxygen at 0.1 L to assess the correlation of increased hypoxia with tumor growth and progression. Pancreatic tumor cells were imaged concurrently with evaluating levels of oxygenated and deoxygenated hemoglobin, providing a distinct picture of the tumor and its vascularization. Use of MSOT to detect regions of hypoxia within tumor was validated using pimonidazole immunohistochemical staining of tumor tissue. For the first time, our data demonstrate regions of tumor hypoxia in living subjects, at depth, and noninvasively, via optoacoustic tomography. Dynamic assessment of tumor microenvironment, especially regions of tumor hypoxia, is essential to effectively target and treat tumors that are otherwise resistant to treatment. Citation Format: Shanice V. Hudson, Charles Kimbrough, Michael Egger, Anil Khanal, Michelle E. Smith, William E. Grizzle, Lacey R. McNally. Detection and characterization of regions of hypoxia within orthotopic pancreatic tumors using multispectral optoacoustic tomography. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4301. doi:10.1158/1538-7445.AM2014-4301