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Supplemental Figure S4: (A) S2013 mouse demonstrate localization of V7-750 probe, but not K7-750 to the region of the tumor bed, signal intensity in the pancreas tumor of S2013 mice with injection of V7-750 is 578.3 MSOT a.u., compared to 5.1 for K7-750 with p=0.0005. K7-750 signal was largely localized in the kidney.(B) Orthogonal views of the pancreatic tumor bed at 4 hours demonstrate distribution of V7-750 probe signal throughout the tumor bed at 4 hours, while minimal accumulation is observed with K7-750.

Supplemental Figure Legend from 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

Supplemental Figure S1. Quantification of EGF-750 probe MSOT signal accumulation within S2VP10L tumor xenografts at various time points.

Supplemental Figure Legend. Legend for Supplemental Figure S1.

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.

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

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. Cancer Res; 74(21); 6271–9. ©2014 AACR.

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.

Kinetic folding experiments by pulsed hydrogen/deuterium exchange (HDX) mass spectrometry (MS) are a well-established tool for water-soluble proteins. To the best of our knowledge, the current study is the first that applies this approach to an integral membrane protein. The native state of bacteriorhodopsin (BR) comprises seven transmembrane helices and a covalently bound retinal cofactor. BR exposure to sodium dodecyl sulfate (SDS) induces partial unfolding and retinal loss. We employ a custom-built three-stage mixing device for pulsed-HDX/MS investigations of BR refolding. The reaction is triggered by mixing SDS-denatured protein with bicelles. After a variable folding time (10 ms to 24 h), the protein is exposed to excess D2O buffer under rapid exchange conditions. The HDX pulse is terminated by acid quenching after 24 ms. Subsequent off-line analysis is performed by size exclusion chromatography and electrospray MS. These measurements yield the number of protected backbone N–H sites as a function of folding time, reflecting the recovery of secondary structure. Our results indicate that much of the BR secondary structure is formed quite late during the reaction, on a time scale of 10 s and beyond. It is hoped that in the future it will be possible to extend the pulsed-HDX/MS approach employed here to membrane proteins other than BR. Copyright © 2012 John Wiley & Sons, Ltd.

The effect of sodium dodecyl sulfate (SDS) on the micellization and aggregation behavior of a poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (PEO-PPO-PEO) amphiphilic copolymer (Pluronic L64: EO13 PO30 EO13) have been investigated by various techniques like, cloud point, viscosity, isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), fluorescence spectroscopy, room temperature phosphorescence (RTP), and small angle neutron scattering (SANS). Addition of SDS in L64 solutions shows mark alteration of different properties. We observed synergistic interaction between SDS and Pluronic L64. The changes in the critical micelle concentration (CMC), critical micelle temperature (CMT), cloud point (CP), micelle size, and shape has been correlated and reported in terms of structure dynamics and mechanics. The ITC titrations have been used to explore the different stages of binding and interactions of SDS with L64. The enthalpies of aggregation for copolymer-SD...

A triple hydrophilic block copolymer comprised of poly(ethylene oxide), poly(sodium 2-acrylamido-2-methylpropanesulfonate), and poly(methacrylic acid) (PEO–PAMPS–PMAA) does not form a micelle by itself when it is dissolved in water. However, in the previous paper, we fabricated the nanoaggregates of PEO–PAMPS–PMAA and cationic surfactant, such as cetyltrimethylammonium chloride (CTAC), by insolubilizing the anionic PAMPS and/or PMAA blocks of the polymer with CTAC only at high pH. In this paper, we fabricated the nanoaggregates of dodecyltrimethylammonium chloride (DTAC) and PEO–PAMPS–PMAA in a wide range of pH to examine the effect of ionization of the PMAA blocks of the polymer on the aggregates formation of PEO–PAMPS–PMAA. The properties of the nanoaggregates are affected by the ionization of PMAA block of the polymer. DTAC (C12 alkyl chain) was employed instead of CTAC (C16 alkyl chain) to reveal the effect of alkyl chain length of surfactant on the aggregate formation of PEO–PAMPS–PMAA. The properties of PEO–PAMPS–PMAA nanoaggregates also depend on the structure of surfactant. The binding of DTAC to PEO–PAMPS–PMAA was monitored by electrophoresis measurements, while the formation of DTAC/PEO–PAMPS–PMAA nanoaggregates was confirmed by scanning electron microscopy, dynamic light scattering measurements and fluorescence spectroscopy.

The effects of phenol on the micellization, micellar growth, and phase separation of a poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (PEO-PPO-PEO) amphiphilic copolymer (Pluronic P65: EO19 PO30 EO19) in aqueous solution have been studied by cloud point, viscosity, dynamic light scattering (DLS), differential scanning calorimetry (DSC), fluorescence spectroscopy, and small-angle neutron scattering (SANS). Various concentrations of P65 have been chosen to estimate the effect of phenol on different concentration regions of P65. Phenol interacts quite differently at low concentrations (0-2%) than at high concentrations (2-10%) of P65, as per the observation that phenol is more predominant at smaller concentrations of P65. A marked decrease in the cloud points of the P65 solutions is observed in presence of phenol. The critical micelle temperature (CMT) of P65 shows a synergistic effect of phenol on P65 aggregates. Micellar transitions, phase separation, and aggregation behaviours like micellization and micellar growth in the presence of phenol have been observed by combining viscometry, DLS, DSC, and CP. DLS shows that the effect of phenol is predominant at high temperatures. SANS shows a high increase in axial ratio and aggregation numbers in the presence of phenol at fixed concentrations of P65. Fluorescence data illustrate that addition of phenol makes micelles polar but at the same time its favours aggregation. Water-soluble phenol (present in low concentrations) forms aggregates with P65, which can be separated by cloud point extraction, making this study interesting for separation of phenol from the phenol-water system.

Symmetrical poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide), PEO-PPO-PEO, triblock copolymers with 80% polyethylene oxide (PEO, the hydrophilic end blocks) and polypropylene oxide (PPO, the hydrophobic middle block) usually remain as molecularly dissolved at ambient temperature even at fairly high-concentrations (2 wt.% or more). However, the micellization is induced at lower concentration/temperature in the presence of salts. The results on salt induced micellization from four such hydrophilic copolymers Pluronic (R) F38, F68, F88 and F108 obtained from several independent techniques are described. FTIR and fluorescence results provide essentially identical critical micelle temperatures (CMTs) showing marked decrease with increase in PPO molecular weight and in the presence of salt. These copolymers were weakly surface active and did not show a clear break point in surface tension concentration plot typical of surfactants. While addition of salt decreases the cloud point, no significant micelle growth was observed even at temperature close to cloud point (CP). Marked increased in solubilization of an oil dye was observed in presence of KC1. Different methods showed good agreement in temperature/salt-induced micellization of these hydrophilic copolymers. (C) 2007 Elsevier Ltd. All rights reserved.

Synchrophasor technology is fast being deployed in electric power grids all over the world and is fast changing the way the grids are managed. This trend is to continue until the entire power grids are fully connected so they can be monitored and controlled in real-time. Much achievement has been made in the synchrophasor technology development and deployment, and there are still much more to be achieved. For instance, real-time power grid control and protection potentials of synchrophasor are yet to be explored. It is of necessity that researchers keep in view the various challenges that still need to be overcome in expanding the frontiers of synchrophasor technology. This paper outlines the major challenges that should be dealt with in order to achieve the goal of total power grid visualization, monitoring, and control using synchrophasor technology., {"references":["MISO, \"Synchrophasor Integration Approach Whitepaper -\nSynchrophasor Integration into Planning and Operational Reliability\nProcesses, December 3, 2010,\" https://www.misoenergy.org/_layouts/\nMISO/ECM/Redirect.aspx?ID","US-DOE, \"Synchrophasor Technology and their Deployment in the\nRecovery Act Smart Grid Programs,\" Electricity Delivery and Energy\nReliability, August, 2013.","California-ISO, \"Five Years Synchrophasor Plan,\" www.caiso.com/\nDocuments/FiveYearSynchrophasorPlan.pdf , Nov. 2011.","R. O. Burnett, Jr., M. M. Butts, T. W. Cease, V. Centeno, G. Michel, R.\nJ. Murphy, et al., \"Synchronized phasor measurements of a power\nsystem event,\" Power Systems, IEEE Transactions on, vol. 9, pp. 1643-\n1650, 1994.","A.G. Phadke, \"Synchronized phasor measurements-a historical\noverview,\" in Transmission and Distribution Conference and Exhibition\n2002: Asia Pacific. IEEE/PES, 2002, pp. 476-479.","A.Kattan and R. Poli, \"Evolutionary synthesis of lossless compression\nalgorithms with GP-zip3,\" in Evolutionary Computation (CEC), 2010\nIEEE Congress on, 2010, pp. 1-8.","R. Klump, P. Agarwal, J. E. Tate, and H. Khurana, \"Lossless\ncompression of synchronized phasor measurements,\" in Power and\nEnergy Society General Meeting, 2010 IEEE, 2010, pp. 1-7.","NASPI, \"Synchrophasor Data Quality,\" North American Synchrophasor\nInitiative (NASPI) Working Group Meeting, Forth Work, Texas;\nFebruary 24, 2011.","K. Reinhard, \"On data quality and availability modeling of power grid\nphasor measurements,\" in North American Power Symposium (NAPS),\n2012, 2012, pp. 1-5.\n[10] J. Stewart, T. Maufer, R. Smith, C. Anderson, and E. Ersonmez,\n\"Synchrophasor security practices,\" Schweitzer Engineering\nLaboratories, Pullman, Washington (www.selinc.com/WorkArea/\nDownloadAsset.aspx, 2010.\n[11] B. Walker, \"Synchrophasor Cost Overview \"PMU Subgroup meeting,\nPJM, Nov. 2, 2012.\n[12] A.Khanal, E. Oleka, A. Osareh, and G. Lebby, \"Optimal Placement of\nPhasor Measurement Units for Maximum Network Observability Using\nPython-Gurobi,\" Proceedings of The 2014 IAJC/ISAM International\nConference ISBN 978-1-60643-379-9, ed, 2014."]}

Nanoaggregate formation of poly(ethylene oxide)-b-polymethacrylate (PEO-b PMA) is induced by chitosan (Ch) or methylglycolchitosan (MGC). The nanoaggregates are basically obtained by electrical charge neutralization of the anionic PMA block of the PEO-b-PMA polymer with cationic Ch or MGC, which results in insolubilization of the PMA block to form the core of the aggregates. Formation of the nanoaggregates was confirmed by dynamic light scattering, fluorescence spectroscopy, atomic force microscopy and zeta-potential measurements. The properties of the nanoaggregates depend upon the concentration of the polymer as well as the concentration of Ch or MGC. The significance of these aggregates is their ability to incorporate ionic species, leading them to potential applications as drug carriers and nanoreactors.

New organic–inorganic nano-complexes were produced from a micelle of tri-block polymers; poly(styrene)-b-poly(2-vinylpyridine)- b-poly (ethylene oxide) (PS-PVP-PEO) and tungsten compounds such as tungstate (W 1 2− ), undecatungstophospate (PW 11 7− ) and undecatungstosilicate (SiW 11 8− ) in acidic aqueous solutions. The size and morphology of the complexes were characterized by measurements of dynamic light scattering, atomic force microscopy, and scanning electron microscopy. This complex is assembled mainly by the charge interaction between the inorganic polyanions and the positively charged PVP block in the PS-PVP-PEO molecule, which was confirmed by zeta-potential and fluorescence spectroscopic studies. In the absence of the inorganic anions, the zeta-potential of the micelle was +11 mV at pH 3 due to the positive charge of the PVP block. When the inorganic anion was mixed with the PS-PVP-PEO micelle, decrease in the zeta-potential due to charge neutralization occurred with incorporation of inorganic anions into the PS-PVP-PEO micelle. The minimum zeta-potential was 0, −33, and −35 mV for W 1 2− /PS-PVP-PEO, PW 11 7− /PS-PVP-PEO, and SiW 11 8− /PS-PVP-PEO complexes, respectively. Excess negative charge which occurred in the latter two complexes indicates that PS-PVP-PEO molecules bound PW 11 7− and SiW 11 8− by forces other than charge interaction. In addition, the incorporation of an inorganic polyanion into the micelle gave a new morphology to the micelle. In the absence of the polyanion, the PS-PVP-PEO micelles showed an extended conformation due to repulsive forces working among the positively charged PVP blocks. Addition of the polyanion caused the formation of shrunken forms of the micelles, since the charge repulsion was cancelled by the polyanion. This feature may be useful in developing a new type of functioning micelle.

Micelles of poly(styrene-b-2-vinylpyridine-b-ethylene oxide) (PS-b-P2VP-b-PEO) triblock copolymer were prepared in aqueous solutions. The physicochemical properties of the micelles were investigate...

Morphological change of a micelle of poly(styrene)-b-poly(2-vinylpyridine)-b-poly(ethylene oxide) (PS-PVP-PEO) polymer was induced by binding sodium dodecyl sulfate (SDS) to the PVP block in acidic aqueous solutions. The change in the size of SDS/PS-PVP-PEO complexes was detected by dynamic light scattering measurements and atomic force microscopy, and the binding of SDS was confirmed by zeta-potential measurements. When the micelle was free from SDS in acidic aqueous solutions, the hydrodynamic diameter of the micelle was 216 nm, reflecting the extended conformation of the PVP block due to the repulsion between protonated pyridine units. As the cationic PVP block was electrically neutralized with anionic SDS, the diameter was gradually reduced concomitant with the decrease in zeta-potential and finally reached 175 nm when the PVP block was completely neutralized. The decrease in the diameter shows the morphological change of the PVP block from extended to shrunken forms. Further addition of SDS did not cause the changes of the diameter nor zeta-potential. This indicates that SDS was not bound to the PS-PVP-PEO polymer after the PVP block was fully neutralized and that the hydrophobic binding of SDS to the polymer was negligible due to the low concentration of SDS.

A triple hydrophilic block copolymer composed of poly(ethylene oxide), poly(sodium 2-acrylamido-2-methylpropanesulfonate), and poly(methacrylic acid) (PEO-PAMPS-PMAA) does not form a micelle by itself when it is dissolved in water. However, if the anionic PAMPS and/or PMAA blocks are electrically neutralized with a cationic surfactant, such as cetyltrimethylammonium chloride (CTAC), micelle-like nanoaggregates are obtained, where the core is formed by the insolubilized PAMPS and/or PMAA blocks. Formation of the nanoaggregates was confirmed by dynamic light scattering (DLS) measurements and scanning electron microscopy (SEM), while the binding of CTAC to PEO-PAMPS-PMAA was monitored by electrophoresis measurements. The aggregates were characterized by fluorescence spectroscopy as well as DLS and SEM. It was found that the nanoaggregates have a spherical structure, and the hydrodynamic diameter ranges from 125 to 193 nm depending on the concentrations of the PEO-PAMPS-PMAA and CTAC. The critical aggregate concentration is on the order of 10-4 g L-1 when the ionic blocks of PEO-PAMPS-PMAA are fully neutralized with CTAC.

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

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

Purpose: Modern methods of pancreatic cancer diagnosis and treatment are severely lacking and have failed to provide effectual treatment options for patients. The root cause of this inadequacy stems from the hypovascularized nature of pancreatic cancer, making traditional chemotherapeutics and cancer detecting contrast agents nearly obsolete. A potential solution for tumor-targeting difficulties is through the implementation of nanotechnology, specifically targeting ligand capped, theranostic nanoparticles. We hypothesize that silica based, mesoporous nanoparticles capped with the targeting ligand, urokinase plasminogen activator (UPA) will serve as theranostic agents for treatment and diagnosis of pancreatic cancer. Methods: Mesoporous, silica nanoparticles (MSNs) were synthesized through the covering of acetyl trimethylammonium bromide (CTAB) scaffolding with tetraethyl orthosilicate (TEOS) in an aqueous environment. Refluxing was later used to remove the CTAB scaffolding and construct the desired mesoporous structure of the nanoparticles. MSNs were chemically engineered to be sensitive to acidic pH, contain fluorescent dye, and utilize a protein targeting ligand UPA. Multiple characterization techniques were employed to analyze the synthesized MSNs including UV-Vis spectroscopy, Zeta Potential, and Transmission Electron Microscopy (TEM). Pancreatic cancer tumor cells, S2CP9 and S2VP10, were treated with UPA-targeted or no-ligand encapsulated MSNs at pH 7.4 and 6.5. Following treatment, cells were washed to remove unbound particles followed by particle visualization based upon fluorescent dyes rhodamine or indocyanine green. Alternatively, cells treated with indocyanine green (ICG) loaded MSNs were placed into a tissue phantom and viewed using the Multispectral Optoacoustic Tomography system (MSOT). Mice with S2CP9 orthotopic pancreatic tumors were i.v. injected with 100 μL of UPA-ICG loaded MSNs. Particle localization was conducted using MSOT after 4h post injection. Results: Zeta potential and UV-Vis analysis confirmed the MSNs chemical conjugation, attributing the MSNs properties of pH sensitivity and fluorescence. pH sensitivity tests on the MSNs displayed drug release quantities at pH 6.5 to be ∼x2 the quantities of pH 6.8 and 7.4. Fluorescence microscopy showed the MSN uptake into S2VP10 and S2CP9 cell lines, while NIH3T3 cells (negative control) showed less binding. Use of UPAR antibody on S2VP10 and S2CP9 cell lines (positive control) blocked cellular binding. In tissue phantoms, UPA-ICG loaded MSNs at pH 6.5 demonstrated 20X and 7X more cell signal than without ligand or at pH 7.4, respectively. In-vivo, UPA-ICG loaded MSNs were detected within the orthotopic pancreatic tumor. Conclusion: UPA targeted, pH sensitive MSNs were found to possess substantial tumor cell binding against multiple cell lines and demonstrate potential as a theranostic nanoparticle for pancreatic cancer. Citation Format: Dillon S. Pender, Anil Khanal, Michael E. Egger, Lacey R. McNally. Development of theranostic mesoporous silica nanoparticles for pancreatic cancer. [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 1481. doi:10.1158/1538-7445.AM2015-1481

A novel drug carrier system was constructed from anionic drug cloxacillin sodium (CLX) and micelle of poly(styrene-b-2-vinyl pyridine-b-ethylene oxide) (PS-PVP-PEO) by incorporating the former into the micelle of the latter. The incorporation of CLX into the micelle of PS-PVP-PEO was confirmed by zeta-potential measurements, dynamic light scattering (DLS), and fluorescence spectroscopy. In the absence of the anionic drug, CLX, the zeta-potential of neat PS-PVP-PEO micelle was + 13 mV under aqueous acidic condition due to the presence of positive charges in the PVP unit. The addition of CLX into the micelle of PS-PVP-PEO decreased the zeta-potential of the micelle smoothly and finally led to a minimum zeta-potential around 0 mV. This fact shows that the added CLX is effectively incorporated into the PS-PVP-PEO micelle by electrostatic attraction. In concomitant with the decrease in zeta-potential, a decrease in hydrodynamic diameter from 94 to 69 nm was observed on addition of CLX to the PS-PVP-PEO micellar solution. This fact also indicates the incorporation of CLX into the PS-PVP-PEO micelles because the binding of CLX to the PVP block of the micelles induces a conformational change from an extended to a shrunken form due to the cancellation of the repulsive force in the PVP blocks by CLX. Fluorescence quenching of pyrene by CLX gave additional evidence for the effective bindings of CLX to the PS-PVP-PEO micelles. Further, release of CLX from the nanoaggregates of CLX/PS-PVP-PEO was investigated in vitro. It was found that the release kinetics of the CLX is conformed to a model based on the consecutive chemical kinetics.

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

Hollow silica nanospheres have been successfully prepared by templating the polymeric micelles of poly(styrene-b-2-vinylpyridine-b-ethylene oxide) (PS–PVP–PEO), which shows a core–shell–corona stru...

The micelle of an ABC triblock copolymer having a core−shell−corona architecture was used as a template for the fabrication of a hollow silica nanosphere. Silica was deposited on a poly(styrene-b-2-vinyl pyridine-b-ethylene oxide) (PS−PVP−PEO) micelle by the sol−gel method. At pH 4, the PVP block of the PS−PVP−PEO acts as an acid catalyst site for the hydrolysis of tetramethoxysilane (TMOS), while the PEO block of the micelle shows a weak interaction with the silanol groups of the inorganic polymer. Therefore, silica networks were mostly grown on the PVP block of the PS−PVP−PEO micelle in comparison to the PEO block. Hollow silica has almost the same diameter as that of the PS core of the PS−PVP−PEO neat micelle, and the thickness of the silica shell wall can be controlled by changing the relative concentration of TMOS with respect to the PVP block length of the micelle.

Purpose Photodynamic therapy (PDT) is gaining increasing recognition for breast cancer treatment because it offers local selectivity and reduced toxic side effects compared to radiotherapy and chemotherapy. In PDT, photosensitizer drugs are loaded in different nanomaterials and used in combination with light exposure. However, the most representative issue with PDT is the difficulty of nanomaterials to encapsulate anticancer drugs at high doses, which results in low efficacy of the PDT treatment. Here, we proposed the development of the poly(N-isopropylacrylamide) (PNIPAM) microgel for the encapsulation of methylene blue, an anticancer drug, for its use as breast cancer treatment in MCF-7 cell line. Methods We developed biocompatible microgels based on nonfunctionalized PNIPAM and its corresponding anionically functionalized PNIPAM and polyacrylic acid (PNIPAM-co-PAA) microgel. Methylene blue was used as the photosensitizer drug because of its ability to generate toxic reactive oxygen species upon exposure to light at 664 nm. Core PNIPAM and core/shell PNIPAM-co-PAA microgels were synthesized and characterized using ultraviolet-visible spectroscopy and dynamic light scattering. The effect of methylene blue was evaluated using the MCF-7 cell line. Results Loading of methylene blue in core PNIPAM microgel was higher than that in the core/shell PNIPAM-co-PAA microgel, indicating that electrostatic interactions did not play an important role in loading a cationic drug. This behavior is probably due to the skin layer inhibiting the high uptake of drugs in the PNIPAM-co-PAA microgel. Core PNIPAM microgel effectively retained the cationic drug (i.e., methylene blue) for several hours compared to core/shell PNIPAM-co-PAA and enhanced its photodynamic efficacy in vitro more than that of free methylene blue. Conclusion Our results showed that the employment of core PNIPAM and core/shell PNIPAM-co-PAA microgels enhanced the encapsulation of methylene blue. Core PNIPAM microgel released the drug more slowly than did core/shell PNIPAM-co-PAA, and it effectively inhibited the growth of MCF-7 cells.