# Publications by Type: Journal Article

2017
A. J. Moy and Tunnell, J. W., “Combinatorial immunotherapy and nanoparticle mediated hyperthermia,” Advanced Drug Delivery Reviews, vol. 114, pp. 175 - 183, 2017. Publisher's VersionAbstract
Immune checkpoint therapy has become the first widely adopted immunotherapy for patients with late stage malignant melanoma, with potential for a wide range of cancers. While some patients can experience long term disease remission, this is limited only to a subset of patients and tumor types. The path forward to expand this therapy to more patients and tumor types is currently thought to be combinatorial treatments, the combination of immunotherapy with other treatments. In this review, the combinatorial approach of immune checkpoint therapy combined with nanoparticle-assisted localized hyperthermia is discussed, starting with an overview of the different nanoparticle hyperthermia approaches in development, an overview of the state of immune checkpoint therapy, recent reports of immune checkpoint therapy and nanoparticle-assisted hyperthermia in a combinatorial approach, and finally a discussion of future research topics and areas to be explored in this new combinatorial approach to cancer treatment.
X. Feng, Moy, A. J., Nguyen, H. T. M., Zhang, J., Fox, M. C., Sebastian, K. R., Reichenberg, J. S., Markey, M. K., and Tunnell, J. W., “Raman active components of skin cancer,” Biomed. Opt. Express, vol. 8, pp. 2835–2850, 2017. Publisher's VersionAbstract
Raman spectroscopy (RS) has shown great potential in noninvasive cancer screening. Statistically based algorithms, such as principal component analysis, are commonly employed to provide tissue classification; however, they are difficult to relate to the chemical and morphological basis of the spectroscopic features and underlying disease. As a result, we propose the first Raman biophysical model applied to in vivo skin cancer screening data. We expand upon previous models by utilizing in situ skin constituents as the building blocks, and validate the model using previous clinical screening data collected from a Raman optical fiber probe. We built an 830nm confocal Raman microscope integrated with a confocal laser-scanning microscope. Raman imaging was performed on skin sections spanning various disease states, and multivariate curve resolution (MCR) analysis was used to resolve the Raman spectra of individual in situ skin constituents. The basis spectra of the most relevant skin constituents were combined linearly to fit in vivo human skin spectra. Our results suggest collagen, elastin, keratin, cell nucleus, triolein, ceramide, melanin and water are the most important model components. We make available for download (see supplemental information) a database of Raman spectra for these eight components for others to use as a reference. Our model reveals the biochemical and structural makeup of normal, nonmelanoma and melanoma skin cancers, and precancers and paves the way for future development of this approach to noninvasive skin cancer diagnosis.
T. Cantu, Walsh, K., Pattani, V. P., Moy, A. J., Tunnell, J. W., Irvin, J. A., and Betancourt, T., “Conductive polymer-based nanoparticles for laser-mediated photothermal ablation of cancer: synthesis, characterization, and in vitro evaluation,” Int J Nanomedicine, vol. 12, pp. 615-632, 2017.Abstract
Laser-mediated photothermal ablation of cancer cells aided by photothermal agents is a promising strategy for localized, externally controlled cancer treatment. We report the synthesis, characterization, and in vitro evaluation of conductive polymeric nanoparticles (CPNPs) of poly(diethyl-4,4'-[2,5-bis(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)-1,4-phenyle ne] bis(oxy)dibutanoate) (P1) and poly(3,4-ethylenedioxythiophene) (PEDOT) stabilized with 4-dodecylbenzenesulfonic acid and poly(4-styrenesulfonic acid-co-maleic acid) as photothermal ablation agents. The nanoparticles were prepared by oxidative-emulsion polymerization, yielding stable aqueous suspensions of spherical particles of <100 nm diameter as determined by dynamic light scattering and electron microscopy. Both types of nanoparticles show strong absorption of light in the near infrared region, with absorption peaks at 780 nm for P1 and 750 nm for PEDOT, as well as high photothermal conversion efficiencies ( 50%), that is higher than commercially available gold-based photothermal ablation agents. The nanoparticles show significant photostability as determined by their ability to achieve consistent temperatures and to maintain their morphology upon repeated cycles of laser irradiation. In vitro studies in MDA-MB-231 breast cancer cells demonstrate the cytocompatibility of the CPNPs and their ability to mediate complete cancer cell ablation upon irradiation with an 808-nm laser, thereby establishing the potential of these systems as agents for laser-induced photothermal therapy.
2016
W. Goth, Lesicko, J., Sacks, M. S., and Tunnell, J. W., “Optical-Based Analysis of Soft Tissue Structures,” Annual Review of Biomedical Engineering, vol. 18, pp. 357-385, 2016. Publisher's Version
B. Yang, Lesicko, J., Moy, A., Reichenberg, J., Sacks, M., and Tunnell, J. W., “Color structured light imaging of skin,” Journal of Biomedical Optics, vol. 21, pp. 050503, 2016. Publisher's VersionAbstract
Abstract.  We illustrate wide-field imaging of skin using a structured light (SL) approach that highlights the contrast from superficial tissue scattering. Setting the spatial frequency of the SL in a regime that limits the penetration depth effectively gates the image for photons that originate from the skin surface. Further, rendering the SL images in a color format provides an intuitive format for viewing skin pathologies. We demonstrate this approach in skin pathologies using a custom-built handheld SL imaging system.
T. Cantu, Rodier, B., Iszard, Z., Kilian, A., Pattani, V., Walsh, K., Weber, K., Tunnell, J., Betancourt, T., and Irvin, J., “Electroactive Polymer Nanoparticles Exhibiting Photothermal Properties,” no. 107, pp. e53631, 2016. Publisher's VersionAbstract
A method for the synthesis of electroactive polymers is demonstrated, starting with the synthesis of extended conjugation monomers using a three-step process that finishes with Negishi coupling. Negishi coupling is a cross-coupling process in which a chemical precursor is first lithiated, followed by transmetallation with ZnCl2. The resultant organozinc compound can be coupled to a dibrominated aromatic precursor to give the conjugated monomer. Polymer films can be prepared via electropolymerization of the monomer and characterized using cyclic voltammetry and ultraviolet-visible-near infrared (UV-Vis-NIR) spectroscopy. Nanoparticles (NPs) are prepared via emulsion polymerization of the monomer using a two-surfactant system to yield an aqueous dispersion of the polymer NPs. The NPs are characterized using dynamic light scattering, electron microscopy, and UV-Vis-NIR-spectroscopy. Cytocompatibility of NPs is investigated using the cell viability assay. Finally, the NP suspensions are irradiated with a NIR laser to determine their effectiveness as potential materials for photothermal therapy (PTT).
2015
B. Yang, Lesicko, J., Sharma, M., Hill, M., Sacks, M. S., and Tunnell, J. W., “Polarized light spatial frequency domain imaging for non-destructive quantification of soft tissue fibrous structures,” Biomed. Opt. Express, vol. 6, pp. 1520–1533, 2015. Publisher's VersionAbstract
The measurement of soft tissue fiber orientation is fundamental to pathophysiology and biomechanical function in a multitude of biomedical applications. However, many existing techniques for quantifying fiber structure rely on transmitted light, limiting general applicability and often requiring tissue processing. Herein, we present a novel wide-field reflectance-based imaging modality, which combines polarized light imaging (PLI) and spatial frequency domain imaging (SFDI) to rapidly quantify preferred fiber orientation on soft collagenous tissues. PLI utilizes the polarization dependent scattering property of fibers to determine preferred fiber orientation; SFDI imaging at high spatial frequency is introduced to reject the highly diffuse photons and to control imaging depth. As a result, photons scattered from the superficial layer of a multi-layered sample are highlighted. Thus, fiber orientation quantification can be achieved for the superficial layer with optical sectioning. We demonstrated on aortic heart valve leaflet that, at spatial frequency of f $=$ 1mm&\#x2212;1, the diffuse background can be effectively rejected and the imaging depth can be limited, thus improving quantification accuracy.
R. Hennessy, Markey, M. K., and Tunnell, J. W., “Impact of one-layer assumption on diffuse reflectance spectroscopy of skin,” Journal of Biomedical Optics, vol. 20, pp. 027001, 2015. Publisher's VersionAbstract
Abstract.  Diffuse reflectance spectroscopy (DRS) can be used to noninvasively measure skin properties. To extract skin properties from DRS spectra, you need a model that relates the reflectance to the tissue properties. Most models are based on the assumption that skin is homogenous. In reality, skin is composed of multiple layers, and the homogeneity assumption can lead to errors. In this study, we analyze the errors caused by the homogeneity assumption. This is accomplished by creating realistic skin spectra using a computational model, then extracting properties from those spectra using a one-layer model. The extracted parameters are then compared to the parameters used to create the modeled spectra. We used a wavelength range of 400 to 750 nm and a source detector separation of 250  μm. Our results show that use of a one-layer skin model causes underestimation of hemoglobin concentration [Hb] and melanin concentration [mel]. Additionally, the magnitude of the error is dependent on epidermal thickness. The one-layer assumption also causes [Hb] and [mel] to be correlated. Oxygen saturation is overestimated when it is below 50% and underestimated when it is above 50%. We also found that the vessel radius factor used to account for pigment packaging is correlated with epidermal thickness.
V. P. Pattani, Shah, J., Atalis, A., Sharma, A., and Tunnell, J. W., “

Role of apoptosis and necrosis in cell death induced by nanoparticle-mediated photothermal therapy

,” Journal of Nanoparticle Research, vol. 17, 2015. Publisher's VersionAbstract
Current cancer therapies can cause significant collateral damage due to a lack of specificity and sensitivity. Therefore, we explored the cell death pathway response to gold nanorod (GNR)-mediated photothermal therapy as a highly specific cancer therapeutic to understand the role of apoptosis and necrosis during intense localized heating. By developing this, we can optimize photothermal therapy to induce a maximum of ‘clean’ cell death pathways, namely apoptosis, thereby reducing external damage. GNRs were targeted to several subcellular localizations within colorectal tumor cells in vitro, and the cell death pathways were quantitatively analyzed after photothermal therapy using flow cytometry. In this study, we found that the cell death response to photothermal therapy was dependent on the GNR localization. Furthermore, we demonstrated that nanorods targeted to the perinuclear region irradiated at 37.5 W/cm2 laser fluence rate led to maximum cell destruction with the ‘cleaner’ method of apoptosis, at similar percentages as other anti-cancer targeted therapies. We believe that this indicates the therapeutic potential for GNR-mediated photothermal therapy to treat cancer effectively without causing damage to surrounding tissue.
2014
L. Lim, Nichols, B., Migden, M. R., Rajaram, N., Reichenberg, J. S., Markey, M. K., Ross, M. I., and Tunnell, J. W., “Clinical study of noninvasive in vivo melanoma and nonmelanoma skin cancers using multimodal spectral diagnosis,” Journal of Biomedical Optics, vol. 19, pp. 117003, 2014. Publisher's VersionAbstract
Abstract.  The goal of this study was to determine the diagnostic capability of a multimodal spectral diagnosis (SD) for in vivo noninvasive disease diagnosis of melanoma and nonmelanoma skin cancers. We acquired reflectance, fluorescence, and Raman spectra from 137 lesions in 76 patients using custom-built optical fiber-based clinical systems. Biopsies of lesions were classified using standard histopathology as malignant melanoma (MM), nonmelanoma pigmented lesion (PL), basal cell carcinoma (BCC), actinic keratosis (AK), and squamous cell carcinoma (SCC). Spectral data were analyzed using principal component analysis. Using multiple diagnostically relevant principal components, we built leave-one-out logistic regression classifiers. Classification results were compared with histopathology of the lesion. Sensitivity/specificity for classifying MM versus PL (12 versus 17 lesions) was 100%/100%, for SCC and BCC versus AK (57 versus 14 lesions) was 95%/71%, and for AK and SCC and BCC versus normal skin (71 versus 71 lesions) was 90%/85%. The best classification for nonmelanoma skin cancers required multiple modalities; however, the best melanoma classification occurred with Raman spectroscopy alone. The high diagnostic accuracy for classifying both melanoma and nonmelanoma skin cancer lesions demonstrates the potential for SD as a clinical diagnostic device.
R. Hennessy, Goth, W., Sharma, M., Markey, M. K., and Tunnell, J. W., “Effect of probe geometry and optical properties on the sampling depth for diffuse reflectance spectroscopy,” Journal of Biomedical Optics, vol. 19, pp. 107002, 2014. Publisher's VersionAbstract
Abstract.  The sampling depth of light for diffuse reflectance spectroscopy is analyzed both experimentally and computationally. A Monte Carlo (MC) model was used to investigate the effect of optical properties and probe geometry on sampling depth. MC model estimates of sampling depth show an excellent agreement with experimental measurements over a wide range of optical properties and probe geometries. The MC data are used to define a mathematical expression for sampling depth that is expressed in terms of optical properties and probe geometry parameters.
B. Yang and Tunnell, J. W., “Real-time absorption reduced surface fluorescence imaging,” Journal of Biomedical Optics, vol. 19, pp. 090505, 2014. Publisher's VersionAbstract
Abstract.  We introduce a technique that limits absorption effects in fluorescence imaging and does not require extensive imaging processing, thus allowing for video rate imaging. The absorption minimization is achieved using spatial frequency domain imaging at a single high spatial frequency with standard three-phase demodulation. At a spatial frequency f=0.5  mm−1, we demonstrated in both in-vitro phantoms and ex-vivo tissue that the absorption can be significantly reduced. In the real-time implementation, we achieved a video rate of 19  frames/s. This technique has potential in cancer visualization and tumor margin detection.
M. Sharma, Marple, E., Reichenberg, J., and Tunnell, J. W., “Design and characterization of a novel multimodal fiber-optic probe and spectroscopy system for skin cancer applications,” Review of Scientific Instruments, vol. 85, pp. -, 2014. Publisher's Version
I. K. Ilev, Tunnell, J., Chen, Y., Hildebrandt, N., Dholakia, K., and Vo-Dinh, T., “Introduction to the Issue on Nanobiophotonics,” Selected Topics in Quantum Electronics, IEEE Journal of, vol. 20, pp. 3-6, 2014.
S. F. Bish, Sharma, M., Wang, Y., Triesault, N. J., Reichenberg, J. S., Zhang, J. X. J., and Tunnell, J. W., “Handheld Diffuse Reflectance Spectral Imaging (DRSi) for in-vivo characterization of skin.,” Biomed Opt Express, vol. 5, no. 2, pp. 573-86, 2014.Abstract
Diffuse reflectance spectroscopy provides a noninvasive means to measure optical and physiological properties of tissues. To expand on these measurements, we have developed a handheld diffuse reflectance spectral imaging (DRSi) system capable of acquiring wide field hyperspectral images of tissue. The image acquisition time was approximately 50 seconds for a 50x50 pixel image. A transport model was used to fit each spectra for reduced scattering coefficient, hemoglobin concentration and melanin concentration resulting in optical property maps. The system was validated across biologically relevant levels of reduced scattering (5.14% error) and absorption (8.34% error) using tissue simulating phantoms. DRSi optical property maps of a pigmented skin lesion were acquired in vivo. These trends in optical properties were consistent with previous observations using point probe devices.
M. Fitzmaurice, Pogue, B. W., Tearney, G. J., Tunnell, J. W., and Yang, C., “Advances in optics for biotechnology, medicine and surgery.,” Biomed Opt Express, vol. 5, no. 2, pp. 560-1, 2014.Abstract
The guest editors introduce a Biomedical Optics Express feature issue that includes contributions from participants at the 2013 conference on Advances in Optics for Biotechnology, Medicine and Surgery XIII.
2013
M. Sharma, Hennessy, R., Markey, M. K., and Tunnell, J. W., “Verification of a two-layer inverse Monte Carlo absorption model using multiple source-detector separation diffuse reflectance spectroscopy.,” Biomed Opt Express, vol. 5, no. 1, pp. 40-53, 2013.Abstract
A two-layer Monte Carlo lookup table-based inverse model is validated with two-layered phantoms across physiologically relevant optical property ranges. Reflectance data for source-detector separations of 370 μm and 740 μm were collected from these two-layered phantoms and top layer thickness, reduced scattering coefficient and the top and bottom layer absorption coefficients were extracted using the inverse model and compared to the known values. The results of the phantom verification show that this method is able to accurately extract top layer thickness and scattering when the top layer thickness ranges from 0 to 550 μm. In this range, top layer thicknesses were measured with an average error of 10% and the reduced scattering coefficient was measured with an average error of 15%. The accuracy of top and bottom layer absorption coefficient measurements was found to be highly dependent on top layer thickness, which agrees with physical expectation; however, within appropriate thickness ranges, the error for absorption properties varies from 12-25%.
B. Yang, Sharma, M., and Tunnell, J. W., “Attenuation-corrected fluorescence extraction for image-guided surgery in spatial frequency domain.,” J Biomed Opt, vol. 18, no. 8, pp. 80503, 2013.Abstract
A new approach to retrieve the attenuation-corrected fluorescence using spatial frequency-domain imaging is demonstrated. Both in vitro and ex vivo experiments showed the technique can compensate for the fluorescence attenuation from tissue absorption and scattering. This approach has potential in molecular image-guided surgery.
R. Hennessy, Lim, S. L., Markey, M. K., and Tunnell, J. W., “Monte Carlo lookup table-based inverse model for extracting optical properties from tissue-simulating phantoms using diffuse reflectance spectroscopy.,” J Biomed Opt, vol. 18, no. 3, pp. 037003, 2013.Abstract
We present a Monte Carlo lookup table (MCLUT)-based inverse model for extracting optical properties from tissue-simulating phantoms. This model is valid for close source-detector separation and highly absorbing tissues. The MCLUT is based entirely on Monte Carlo simulation, which was implemented using a graphics processing unit. We used tissue-simulating phantoms to determine the accuracy of the MCLUT inverse model. Our results show strong agreement between extracted and expected optical properties, with errors rate of 1.74% for extracted reduced scattering values, 0.74% for extracted absorption values, and 2.42% for extracted hemoglobin concentration values.
2012
V. P. Pattani and Tunnell, J. W., “Nanoparticle-mediated photothermal therapy: a comparative study of heating for different particle types.,” Lasers Surg Med, vol. 44, no. 8, pp. 675-84, 2012.Abstract
INTRODUCTION: Near-infrared (NIR) absorbing plasmonic nanoparticles enhance photothermal therapy of tumors. In this procedure, systemically delivered gold nanoparticles preferentially accumulate at the tumor site and when irradiated using laser light, produce localized heat sufficient to damage tumor cells. Gold nanoshells and nanorods have been widely studied for this purpose, and while both exhibit strong NIR absorption, their overall absorption and scattering properties differ widely due to their geometry. In this paper, we compared the photothermal response of both nanoparticle types including the heat generation and photothermal efficiency. METHODS: Tissue simulating phantoms, with varying concentrations of gold nanoparticles, were irradiated with a near-infrared diode laser while concurrently monitoring the surface temperature with an infrared camera. We calculated nanoshell and nanorod optical properties using the Mie solution and the discrete dipole approximation, respectively. In addition, we measured the heat generation of nanoshells and nanorods at the same optical density to determine the photothermal transduction efficiency for both nanoparticle types. RESULTS: We found that the gold nanoshells produced more heat than gold nanorods at equivalent number densities (# of nanoparticles/ml), whereas the nanorods generated more heat than nanoshells at equivalent extinction values at the irradiance wavelength. To reach an equivalent heat generation, we found that it was necessary to have ∼36× more nanorods than nanoshells. However, the gold nanorods were found to have two times the photothermal transduction efficiency than the gold nanoshells. CONCLUSION: For the nanoparticles tested, the nanoshells generated more heat, per nanoparticle, than nanorods, primarily due to their overall larger geometric cross-section. Conversely, we found that the gold nanorods had a higher photothermal efficiency than the gold nanoshells. In conclusion, the ideal choice of plasmonic nanoparticle requires not only per particle efficiency, but also the in vivo particle targeting ability under study.