Fischell Department of Bioengineering Research Works
Permanent URI for this collectionhttp://hdl.handle.net/1903/6627
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Item Test method for evaluating the photocytotoxic potential of fluorescence imaging products(Wiley, 2023-07-26) Vig, Shruti; Gaitan, Brandon; Frankle, Lucas; Chen, Yu; Elespuru, Rosalie; Pfefer, T. Joshua; Huang, Huang-ChiaoVarious fluorescence imaging agents are currently under clinical studies. Despite significant benefits, phototoxicity is a barrier to the clinical translation of fluorophores. Current regulatory guidelines on medication-based phototoxicity focus on skin effects during sun exposure. However, with systemic and local administration of fluorophores and targeted illumination, there is now possibility of photochemical damage to deeper tissues during intraoperative imaging procedures. Hence, independent knowledge regarding phototoxicity is required to facilitate the development of fluorescence imaging products. Previously, we studied a cell-free assay for initial screening of reactive molecular species generation from fluorophores. The current work addresses a safety test method based on cell viability as an adjunct and a comparator with the cell-free assay. Our goal is to modify and implement an approach based on the in vitro 3T3 neutral red uptake assay of the Organization for Economic Co-Operation and Development Test Guideline 432 (OECD TG432) to evaluate the photocytotoxicity of clinically relevant fluorophores. These included indocyanine green (ICG), proflavine, methylene blue (MB), and IRDye800, as well as control photosensitizers, benzoporphyrin derivative (BPD) and rose bengal (RB). We performed measurements at agent concentrations and illumination parameters used for clinic imaging. Our results aligned with prior studies, indicating photocytotoxicity in RB and BPD and an absence of reactivity for ICG and IRDye800. DNA interactive agents, proflavine and MB, exhibited drug/light dose–response curves like photosensitizers. This study provides evidence and insights into practices useful for testing the photochemical safety of fluorescence imaging products.Item Systematic Evaluation of Light-Activatable Biohybrids for Anti-Glioma Photodynamic Therapy(MDPI, 2019-08-21) Inglut, Collin T.; Baglo, Yan; Liang, Barry J.; Cheema, Yahya; Stabile, Jillian; Woodworth, Graeme F.; Huang, Huang-ChiaoPhotosensitizing biomolecules (PSBM) represent a new generation of light-absorbing compounds with improved optical and physicochemical properties for biomedical applications. Despite numerous advances in lipid-, polymer-, and protein-based PSBMs, their effective use requires a fundamental understanding of how macromolecular structure influences the physicochemical and biological properties of the photosensitizer. Here, we prepared and characterized three well-defined PSBMs based on a clinically used photosensitizer, benzoporphyrin derivative (BPD). The PSBMs include 16:0 lysophosphocholine-BPD (16:0 Lyso PC-BPD), distearoyl-phosphoethanolamine-polyethylene-glycol-BPD (DSPE-PEG-BPD), and anti-EGFR cetuximab-BPD (Cet-BPD). In two glioma cell lines, DSPE-PEG-BPD exhibited the highest singlet oxygen yield but was the least phototoxic due to low cellular uptake. The 16:0 Lyso PC-BPD was most efficient in promoting cellular uptake but redirected BPD’s subcellular localization from mitochondria to lysosomes. At 24 h after incubation, proteolyzed Cet-BPD was localized to mitochondria and effectively disrupted the mitochondrial membrane potential upon light activation. Our results revealed the variable trafficking and end effects of PSBMs, providing valuable insights into methods of PSBM evaluation, as well as strategies to select PSBMs based on subcellular targets and cytotoxic mechanisms. We demonstrated that biologically informed combinations of PSBMs to target lysosomes and mitochondria, concurrently, may lead to enhanced therapeutic effects against gliomas.Item Immunological and Toxicological Considerations for the Design of Liposomes(MDPI, 2020-01-22) Inglut, Collin T.; Sorrin, Aaron J.; Kuruppu, Thilinie; Vig, Shruti; Cicalo, Julia; Ahmad, Haroon; Huang, Huang-ChiaoLiposomes hold great potential as gene and drug delivery vehicles due to their biocompatibility and modular properties, coupled with the major advantage of attenuating the risk of systemic toxicity from the encapsulated therapeutic agent. Decades of research have been dedicated to studying and optimizing liposomal formulations for a variety of medical applications, ranging from cancer therapeutics to analgesics. Some effort has also been made to elucidate the toxicities and immune responses that these drug formulations may elicit. Notably, intravenously injected liposomes can interact with plasma proteins, leading to opsonization, thereby altering the healthy cells they come into contact with during circulation and removal. Additionally, due to the pharmacokinetics of liposomes in circulation, drugs can end up sequestered in organs of the mononuclear phagocyte system, affecting liver and spleen function. Importantly, liposomal agents can also stimulate or suppress the immune system depending on their physiochemical properties, such as size, lipid composition, pegylation, and surface charge. Despite the surge in the clinical use of liposomal agents since 1995, there are still several drawbacks that limit their range of applications. This review presents a focused analysis of these limitations, with an emphasis on toxicity to healthy tissues and unfavorable immune responses, to shed light on key considerations that should be factored into the design and clinical use of liposomal formulations.Item Flow-induced Shear Stress Confers Resistance to Carboplatin in an Adherent Three-Dimensional Model for Ovarian Cancer: A Role for EGFR-Targeted Photoimmunotherapy Informed by Physical Stress(MDPI, 2020-03-28) Nath, Shubhankar; Pigula, Michael; Khan, Amjad P.; Hanna, William; Ruhi, Mustafa Kemal; Dehkordy, Farzaneh Mahmoodpoor; Pushpavanam, Karthik; Rege, Kaushal; Moore, Kaitlin; Tsujita, Yujiro; Conrad, Christina; Inci, Faith; del Carmen, Marcela G.; Franco, Walfre; Celli, Jonathan P.; Demirci, Utkan; Hasan, Tayyaba; Huang, Huang-Chiao; Rizvi, ImranA key reason for the persistently grim statistics associated with metastatic ovarian cancer is resistance to conventional agents, including platinum-based chemotherapies. A major source of treatment failure is the high degree of genetic and molecular heterogeneity, which results from significant underlying genomic instability, as well as stromal and physical cues in the microenvironment. Ovarian cancer commonly disseminates via transcoelomic routes to distant sites, which is associated with the frequent production of malignant ascites, as well as the poorest prognosis. In addition to providing a cell and protein-rich environment for cancer growth and progression, ascitic fluid also confers physical stress on tumors. An understudied area in ovarian cancer research is the impact of fluid shear stress on treatment failure. Here, we investigate the effect of fluid shear stress on response to platinum-based chemotherapy and the modulation of molecular pathways associated with aggressive disease in a perfusion model for adherent 3D ovarian cancer nodules. Resistance to carboplatin is observed under flow with a concomitant increase in the expression and activation of the epidermal growth factor receptor (EGFR) as well as downstream signaling members mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) and extracellular signal-regulated kinase (ERK). The uptake of platinum by the 3D ovarian cancer nodules was significantly higher in flow cultures compared to static cultures. A downregulation of phospho-focal adhesion kinase (p-FAK), vinculin, and phospho-paxillin was observed following carboplatin treatment in both flow and static cultures. Interestingly, low-dose anti-EGFR photoimmunotherapy (PIT), a targeted photochemical modality, was found to be equally effective in ovarian tumors grown under flow and static conditions. These findings highlight the need to further develop PIT-based combinations that target the EGFR, and sensitize ovarian cancers to chemotherapy in the context of flow-induced shear stress.Item Photodynamic Priming Improves the Anti-Migratory Activity of Prostaglandin E Receptor 4 Antagonist in Cancer Cells In Vitro(MDPI, 2021-10-20) Sorrin, Aaron J.; Liu, Cindy; Cicalo, Julia; Reader, Jocelyn; Najafali, Daniel; Zhang, Yuji; Roque, Dana M.; Huang, Huang-ChiaoThe combination of photodynamic agents and biological inhibitors is rapidly gaining attention for its promise and approval in treating advanced cancer. The activity of photodynamic treatment is mainly governed by the formation of reactive oxygen species upon light activation of photosensitizers. Exposure to reactive oxygen species above a threshold dose can induce cellular damage and cancer cell death, while the surviving cancer cells are “photodynamically primed”, or sensitized, to respond better to other drugs and biological treatments. Here, we report a new combination regimen of photodynamic priming (PDP) and prostaglandin E2 receptor 4 (EP4) inhibition that reduces the migration and invasion of two human ovarian cancer cell lines (OVCAR-5 and CAOV3) in vitro. PDP is achieved by red light activation of the FDA-approved photosensitizer, benzoporphyrin derivative (BPD), or a chemical conjugate composed of the BPD linked to cetuximab, an anti-epithelial growth factor receptor (EGFR) antibody. Immunoblotting data identify co-inhibition of EGFR, cAMP-response element binding protein (CREB), and extracellular signal-regulated kinase 1/2 (ERK1/2) as key in the signaling cascades modulated by the combination of EGFR-targeted PDP and EP4 inhibition. This study provides valuable insights into the development of a molecular-targeted photochemical strategy to improve the anti-metastatic effects of EP4 receptor antagonists.Item Development of an Endoscopic Auto-Fluorescent Sensing Device to Aid in the Detection of Breast Cancer and Inform Photodynamic Therapy(MDPI, 2022-11-11) Gaitan, Brandon; Inglut, Collin; Kanniyappan, Udayakumar; Xu, He N.; Conant, Emily F.; Frankle, Lucas; Li, Lin Z.; Chen, Yu; Huang, Huang-ChiaoBreast cancer is the most diagnosed cancer type in women, with it being the second most deadly cancer in terms of total yearly mortality. Due to the prevalence of this disease, better methods are needed for both detection and treatment. Reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are autofluorescent biomarkers that lend insight into cell and tissue metabolism. As such, we developed an endoscopic device to measure these metabolites in tissue to differentiate between malignant tumors and normal tissue. We performed initial validations in liquid phantoms as well as compared to a previously validated redox imaging system. We also imaged ex vivo tissue samples after modulation with carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) and a combination of rotenone and antimycin A. We then imaged the rim and the core of MDA-MB-231 breast cancer tumors, with our results showing that the core of a cancerous lesion has a significantly higher optical redox ratio ([FAD]/([FAD] + [NADH])) than the rim, which agrees with previously published results. The mouse muscle tissues exhibited a significantly lower FAD, higher NADH, and lower redox ratio compared to the tumor core or rim. We also used the endoscope to measure NADH and FAD after photodynamic therapy treatment, a light-activated treatment methodology. Our results found that the NADH signal increases in the malignancy rim and core, while the core of cancers demonstrated a significant increase in the FAD signal.Item Nanoparticle-assisted, image-guided laser interstitialthermal therapy for cancer treatment(Wiley, 2022-06-23) Pang, Sumiao; Kapur, Anshika; Zhou, Keri; Anastasiadis, Pavlos; Ballirano, Nicholas; Kim, Anthony J.; Winkles, Jeffrey A.; Woodworth, Graeme F.; Huang, Huang-ChiaoLaser interstitial thermal therapy (LITT) guided by magnetic resonance imaging (MRI) is a new treatment option for patients with brain and non-central nervous system (non-CNS) tumors. MRI guidance allows for precise placement of optical fiber in the tumor, while MR thermometry provides real-time monitoring and assessment of thermal doses during the procedure. Despite promising clinical results, LITT complications relating to brain tumor procedures, such as hemorrhage, edema, seizures, and thermal injury to nearby healthy tissues, remain a significant concern. To address these complications, nanoparticles offer unique prospects for precise interstitial hyperthermia applications that increase heat transport within the tumor while reducing thermal impacts on neighboring healthy tissues. Furthermore, nanoparticles permit the co-delivery of therapeutic compounds that not only synergize with LITT, but can also improve overall effectiveness and safety. In addition, efficient heat-generating nanoparticles with unique optical properties can enhance LITT treatments through improved real-time imaging and thermal sensing. This review will focus on (1) types of inorganic and organic nanoparticles for LITT; (2) in vitro, in silico, and ex vivo studies that investigate nanoparticles' effect on light–tissue interactions; and (3) the role of nanoparticle formulations in advancing clinically relevant image-guided technologies for LITT.Item Quantifying the Photochemical Damage Potential of Contrast-Enhanced Fluorescence Imaging Products: Singlet Oxygen Production(Wiley, 2022-04-20) Gaitan, Brandon; Frankle, Lucas; Vig, Shruti; Oskoui, Ellen; Adwan, Miriam; Chen, Yu; Elespuru, Rosalie; Huang, Huang-Chiao; Pfefer, T. JoshuaThe benefits of contrast-enhancing imaging probes have become apparent over the past decade. However, there is a gap in the literature when it comes to the assessment of the phototoxic potential of imaging probes and systems emitting visible and/or near-infrared radiation. The primary mechanism of fluorescent agent phototoxicity is thought to involve the production of reactive molecular species (RMS), yet little has been published on the best practices for safety evaluation of RMS production levels for clinical products. We have proposed methods involving a cell-free assay to quantify singlet oxygen [(SO) a known RMS] generation of imaging probes, and performed testing of Indocyanine Green (ICG), Proflavine, Methylene Blue, IR700 and IR800 at clinically relevant concentrations and radiant exposures. Results indicated that SO production from IR800 and ICG were more than two orders of magnitude below that of the known SO generator Rose Bengal. Methylene Blue and IR700 produced much higher SO levels than ICG and IR800. These results were in good agreement with data from the literature. While agents that exhibit spectral overlap with the assay may be more prone to errors, our tests for one of these agents (Proflavine) appeared robust. Overall, our results indicate that this methodology shows promise for assessing the phototoxic potential of fluorophores due to SO production.Item Breaking the selectivity-uptake trade-off of photoimmunoconjugates with nanoliposomal irinotecan for synergistic multi-tier cancer targeting(Springer Nature, 2020-01-02) Liang, Barry J.; Pigula, Michael; Baglo, Yan; Najafali, Daniel; Hasan, Tayyaba; Huang, Huang-ChiaoPhotoimmunotherapy involves targeted delivery of photosensitizers via an antibody conjugate (i.e., photoimmunoconjugate, PIC) followed by light activation for selective tumor killing. The trade-off between PIC selectivity and PIC uptake is a major drawback limiting the efficacy of photoimmunotherapy. Despite ample evidence showing that photoimmunotherapy is most effective when combined with chemotherapy, the design of nanocarriers to co-deliver PICs and chemotherapy drugs remains an unmet need. To overcome these challenges, we developed a novel photoimmunoconjugate-nanoliposome (PIC-Nal) comprising of three clinically used agents: anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody cetuximab (Cet), benzoporphyrin derivative (BPD) photosensitizer, and irinotecan (IRI) chemotherapy.Item Quantitatively relating brain endothelial cell–cell junction phenotype to global and local barrier properties under varied culture conditions via the Junction Analyzer Program(Springer Nature, 2020-02-11) Gray, Kelsey M.; Jung, Jae W.; Inglut, Collin T.; Huang, Huang-Chiao; Stroka, Kimberly M.The endothelial cell–cell junctions of the blood–brain barrier (BBB) play a pivotal role in the barrier’s function. Altered cell–cell junctions can lead to barrier dysfunction and have been implicated in several diseases. Despite this, the driving forces regulating junctional protein presentation remain relatively understudied, largely due to the lack of efficient techniques to quantify their presentation at sites of cell–cell adhesion. Here, we used our novel Junction Analyzer Program (JAnaP) to quantify junction phenotype (i.e., continuous, punctate, or perpendicular) in response to various substrate compositions, cell culture times, and cAMP treatments in human brain microvascular endothelial cells (HBMECs). We then quantitatively correlated junction presentation with barrier permeability on both a “global” and “local” scale.