Akilan Palanisami

PDT Dose Parameters Impact Tumoricidal Durability and Cell Death Pathways in a 3D Ovarian Cancer Model

The successful implementation of photodynamic therapy (PDT)‐based regimens depends on an improved understanding of the dosimetric and biological factors that govern therapeutic variability. Here, the kinetics of tumor destruction and regrowth are characterized by systematically varying benzoporphyrin derivative (BPD)‐light combinations to achieve fixed PDT doses (M × J cm−2). Three endpoints were used to evaluate treatment response: (1) Viability evaluated every 24 h for 5 days post‐PDT; (2) Photobleaching assessed immediately post‐PDT; and (3) Caspase‐3 activation determined 24 h post‐PDT. The specific BPD‐light parameters used to construct a given PDT dose significantly impact not only acute cytotoxic efficacy, but also treatment durability. For each dose, PDT with 0.25 μM BPD produces the most significant and sustained reduction in normalized viability compared to 1 and 10 μM BPD. Percent photobleaching correlates with normalized viability for a range of PDT doses achieved within BPD concentrations. To produce a cytotoxic response with 10 μM BPD that is comparable to 0.25 and 1 μM BPD a reduction in irradiance from 150 to 0.5 mW cm−2 is required. Activated caspase‐3 does not correlate with normalized viability. The parameter‐dependent durability of outcomes within fixed PDT doses provides opportunities for treatment customization and improved therapeutic planning.

Selective treatment and monitoring of disseminated cancer micrometastases in vivo using dual-function, activatable immunoconjugates

Significance Residual micrometastases following standard therapies limit our ability to cure many cancers. This article demonstrates a new therapy and visualization platform that targets residual cancer micrometastases with enhanced sensitivity and selectivity based on “tumor-targeted activation.” This targeted activation feature not only provides a potent therapeutic arm that is effective against chemoresistant disease while minimizing side effects due to nonspecific toxicities but also enables micrometastasis imaging in common sites of disease recurrence to screen patients harboring residual tumor deposits. This approach offers promise for treating and monitoring drug-resistant micrometastases presently “invisible” to clinicians. Drug-resistant micrometastases that escape standard therapies often go undetected until the emergence of lethal recurrent disease. Here, we show that it is possible to treat microscopic tumors selectively using an activatable immunoconjugate. The immunoconjugate is composed of self-quenching, near-infrared chromophores loaded onto a cancer cell-targeting antibody. Chromophore phototoxicity and fluorescence are activated by lysosomal proteolysis, and light, after cancer cell internalization, enabling tumor-confined photocytotoxicity and resolution of individual micrometastases. This unique approach not only introduces a therapeutic strategy to help destroy residual drug-resistant cells but also provides a sensitive imaging method to monitor micrometastatic disease in common sites of recurrence. Using fluorescence microendoscopy to monitor immunoconjugate activation and micrometastatic disease, we demonstrate these concepts of “tumor-targeted, activatable photoimmunotherapy” in a mouse model of peritoneal carcinomatosis. By introducing targeted activation to enhance tumor selectively in complex anatomical sites, this study offers prospects for catching early recurrent micrometastases and for treating occult disease.

Simultaneous delivery of cytotoxic and biologic therapeutics using nanophotoactivatable liposomes enhances treatment efficacy in a mouse model of pancreatic cancer

A lack of intracellular delivery systems has limited the use of biologics such as monoclonal antibodies (mAb) that abrogate molecular signaling pathways activated to promote escape from cancer treatment. We hypothesized that intracellular co-delivery of the photocytotoxic chromophore benzoporphyrin derivative monoacid A (BPD) and the anti-VEGF mAb bevacizumab in a nanophotoactivatable liposome (nanoPAL) might enhance the efficacy of photodynamic therapy (PDT) combined with suppression of VEGF-mediated signaling pathways. As a proof-of-concept we found that nanoPAL-PDT induced enhanced extra- and intracellular bevacizumab delivery and enhanced acute cytotoxicity in vitro. In an in vivo subcutaneous mouse model of pancreatic ductal adenocarcinoma, nanoPAL-PDT achieved significantly enhanced tumor reduction. We attribute this to the optimal incorporation of insoluble BPD into the lipid bilayer, enhancing photocytotoxicity, and the simultaneous spatiotemporal delivery of bevacizumab, ensuring efficient neutralization of the rapid but transient burst of VEGF following PDT. From the Clinical Editor: Most patients with pancreatic ductal adenocarcinoma (PDAC) by the time present the disease it is very advanced, which unavoidably translates to poor survival. For these patients, use of traditional chemotherapy often becomes ineffective due to tumor resistance to drugs. Photodynamic therapy (PDT) can be an effective modality against chemo-resistant cancers. In this article, the authors investigated the co-delivery of a photocytotoxic agent and anti-VEGF mAb using liposomes. This combination was shown to results in enhanced tumor killing. This method should be applicable to other combination of treatments.

Torque-induced slip of the rotary motor F1-ATPase.

F(1)-ATPase plays an essential role in cellular metabolism by linking rotational motion to ATP hydrolysis/synthesis. We measure the torque profile of F(1) in both ATP hydrolysis and synthesis directions using a novel magnetic nanorod assay. F(1) is found to decouple ATP synthesis from rotary motion at a surprisingly low torque. This low-torque slip mechanism protects the enzyme from excessive load and may play a broader biological role by reducing the production of reactive oxygen species.

Simultaneous sizing and electrophoretic mobility measurement of sub‐micron particles using Brownian motion

The size and surface chemistry of micron scale particles are of fundamental importance in studies of biology and air particulate pollution. However, typical electrophoretic measurements of these and other sub‐micron scale particles (300 nm–1 μm) cannot resolve size information within heterogeneous mixtures unambiguously. Using optical microscopy, we monitor electrophoretic motion together with the Brownian velocity fluctuations – using the latter to measure size by either the Green–Kubo relation or by calibration from known size standards. Particle diameters are resolved to ±12% with 95% confidence. Strikingly, the size resolution improves as the particle size decreases due to the increased Brownian motion. The sizing ability of the Brownian assessed electrophoresis method described here complements the electrophoretic mobility resolution of the traditional CE.

Rapid, low-cost fluorescent assay of β-lactamase-derived antibiotic resistance and related antibiotic susceptibility

Abstract. Antibiotic resistance (AR) is increasingly prevalent in low and middle income countries (LMICs), but the extent of the problem is poorly understood. This lack of knowledge is a critical deficiency, leaving local health authorities essentially blind to AR outbreaks and crippling their ability to provide effective treatment guidelines. The crux of the problem is the lack of microbiology laboratory capacity available in LMICs. To address this unmet need, we demonstrate a rapid and simple test of β-lactamase resistance (the most common form of AR) that uses a modified β-lactam structure decorated with two fluorophores quenched due to their close proximity. When the β-lactam core is cleaved by β-lactamase, the fluorophores dequench, allowing assay speeds of 20 min to be obtained with a simple, streamlined protocol. Furthermore, by testing in competition with antibiotics, the β-lactamase-associated antibiotic susceptibility can also be extracted. This assay can be easily implemented into standard lab work flows to provide near real-time information of β-lactamase resistance, both for epidemiological purposes as well as individualized patient care.

Efficient measurement of total tumor microvascularity ex vivo using a mathematical model to optimize volume subsampling

Abstract. We introduce immunofluorescence and automated image processing protocols for serial tumor sections to objectively and efficiently quantify tumor microvasculature following antivascular therapy. To determine the trade-off between tumor subsampling and throughput versus microvessel quantification accuracy, we provide a mathematical model that accounts for tumor-specific vascular heterogeneity. This mathematical model can be applied broadly to define tumor volume samplings needed to reach statistical significance, depending on the biomarker in question and the number of subjects. Here, we demonstrate these concepts for tumor microvessel density and total microvascularity (TMV) quantification in whole pancreatic ductal adenocarcinoma tumors ex vivo. The results suggest that TMV is a more sensitive biomarker for detecting reductions in tumor vasculature following antivascular treatment. TMV imaging is a broadly accessible technique that offers robust assessment of antivascular therapies, and it offers promise as a tool for developing high-throughput assays to quantify treatment-induced microvascular alterations for therapeutic screening and development.

Nonlinear Impedance of Whole Cells Near an Electrode as a Probe of Mitochondrial Activity

By simultaneously measuring the bulk media and electrode interface voltages of a yeast (Saccharomyces cerevisiae) suspension subjected to an AC voltage, a yeast-dependent nonlinear response was found only near the current injection electrodes. Computer simulation of yeast near a current injection electrode found an enhanced voltage drop across the yeast near the electrode due to slowed charging of the electrode interfacial capacitance. This voltage drop is sufficient to induce conformation change in membrane proteins. Disruption of the mitochondrial electron transport chain is found to significantly change the measured nonlinear current response, suggesting nonlinear impedance can be used as a non-invasive probe of cellular metabolic activity.

Nonlinear dielectric spectroscopy for label-free detection of respiratory activity in whole cells

We report on a novel electromagnetic biosensing technique for detecting respiratory activity in whole cells suspended in aqueous solution. Application of a pure sinusoidal voltage between two outer electrodes applies an oscillatory electric field to the aqueous cell suspension at frequencies in the range of one to several kHz. The fundamental and higher order harmonic responses are measured across two inner electrodes using a dynamic signal analyzer. Aqueous suspensions of S. cerevisiae (budding yeast), with both active and inactive mitochondrial electron transport (respiratory) chains are employed for this study. We find that the measured third harmonic for certain frequency ranges shows significant temporal changes in actively respiring yeast, while little significant changes are observed in yeast with suppressed respiratory activity, i.e. mutant yeast strains or yeast in the presence of respiratory inhibitors. The method holds potential for further development to detect respiratory activity in live tissue in vitro and perhaps in vivo for clinical applications.

Rapid morphological characterization of isolated mitochondria using Brownian motion.

Mitochondrial morphology has been associated with numerous pathologies including cancer, diabetes, obesity and heart disease. However, the connection is poorly understood-in part due to the difficulty of characterizing the morphology. This impedes the use of morphology as a tool for disease detection/monitoring. Here, we use the Brownian motion of isolated mitochondria to characterize their size and shape in a high throughput fashion. By using treadmill exercise training, mitochondria from heart and gastrocnemius of Balb/c mice were modulated in size and used to investigate the protocol. Consistent with previous reports, the heart mitochondria of untrained mice increased 5% in diameter immediately after a single bout of moderate exercise (1.091 ± 0.004 μm) as compared to completely sedentary controls (1.040 ± 0.022 μm). In addition, no change was observed in the size of gastrocnemius mitochondria (1.025 ± 0.018 μm), which was also in agreement with previous studies. The method was also successfully applied to smaller Saccharomyces cerevisiae mitochondria.