Angelo Gaitas

Extracorporeal Photo-Immunotherapy for Circulating Tumor Cells

It is well established that metastasis through the circulatory system is primarily caused by circulating tumor cells (CTCs). In this preliminary effort, we report an approach to eliminate circulating tumor cells from the blood stream by flowing the blood though an extracorporeal tube and applying photodynamic therapy (PDT). Chlorin e6 (Ce6), a photosensitizer, was conjugated to CD44 antibody in order to target PC-3, a prostate cancer cell line. PC-3 cells were successfully stained by the Ce6-CD44 antibody conjugate. PDT was performed on whole blood spiked with stained PC-3 cells. As the blood circulated through a thin transparent medical tube, it was exposed to light of 660 nm wavelength generated by an LED array. An exposure of two minutes was sufficient to achieve selective cancer cell necrosis. In comparison, to PDT of cells growing inside a tissue culture, the PDT on thin tube exhibited significantly enhanced efficiency in cell killing, by minimizing light attenuation by blood. It suggests a new extracorporeal methodology of PDT for treating CTCs as well as other hematological pathogens.

Chemically Modified Plastic Tube for High Volume Removal and Collection of Circulating Tumor Cells

In this preliminary effort, we use a commercially available and chemically modified tube to selectively capture circulating tumor cells (CTCs) from the blood stream by immobilizing human anti-EpCAM antibodies on the tubes interior surface. We describe the requisite and critical steps required to modify a tube into a cancer cell-capturing device. Using these simple modifications, we were able to capture or entrap about 85% of cancer cells from suspension and 44% of cancer cells from spiked whole blood. We also found that the percentage of cells captured was dependent on the tubes length and also the number of cancer cells present. It is our strong belief that with the utilization of appropriate tube lengths and procedures, we can ensure capture and removal of nearly the entire CTC population in whole blood. Importantly after a patient’s entire blood volume has circulated through the tube, the tube can then be trypsinized to release the captured live CTCs for further analysis and testing.

A device for rapid and quantitative measurement of cardiac myocyte contractility

Cardiac contractility is the hallmark of cardiac function and is a predictor of healthy or diseased cardiac muscle. Despite advancements over the last two decades, the techniques and tools available to cardiovascular scientists are limited in their utility to accurately and reliably measure the amplitude and frequency of cardiomyocyte contractions. Isometric force measurements in the past have entailed cumbersome attachment of isolated and permeabilized cardiomyocytes to a force transducer followed by measurements of sarcomere lengths under conditions of submaximal and maximal Ca(2+) activation. These techniques have the inherent disadvantages of being labor intensive and costly. We have engineered a micro-machined cantilever sensor with an embedded deflection-sensing element that, in preliminary experiments, has demonstrated to reliably measure cardiac cell contractions in real-time. Here, we describe this new bioengineering tool with applicability in the cardiovascular research field to effectively and reliably measure cardiac cell contractility in a quantitative manner. We measured contractility in both primary neonatal rat heart cardiomyocyte monolayers that demonstrated a beat frequency of 3 Hz as well as human embryonic stem cell-derived cardiomyocytes with a contractile frequency of about 1 Hz. We also employed the β-adrenergic agonist isoproterenol (100 nmol l(-1)) and observed that our cantilever demonstrated high sensitivity in detecting subtle changes in both chronotropic and inotropic responses of monolayers. This report describes the utility of our micro-device in both basic cardiovascular research as well as in small molecule drug discovery to monitor cardiac cell contractions.

A Novel Pathogen Capturing Device for Removal and Detection

A simple technique that employs an antibody coated polydimethylsiloxane tube is used for effective capturing of bloodborne and foodborne pathogens. By recirculating the entire sample through the antibody coated tube, accumulation of target pathogens is achieved, thereby delivering a higher concentration of pathogens in a small volume. The described method can provide an effective and economical solution to microbiology techniques that rely on enrichment, thereby expediting diagnostics. Using this method 80.3 ± 5.6% of Staphylococcus aureus with a starting concentration of ~107 CFU/mL and 95.4 ± 1.0% of Methicillin-resistant Staphylococcus aureus with starting concentration of ~104 CFU/mL were removed from 5 mL blood in a few hours. This concept was extended to live rats with an induced bloodstream S. aureus infection. A reduction of two orders of magnitude in the bacterial load of the rats was observed within a few hours. The same technique was used to capture a food pathogen, Salmonella typhimurium, with starting concentrations as low as ~100 CFU, from 100 or 250 mL of culture broth within similar timeframes as above. The feasibility for food pathogen testing applications was additionally confirmed by capturing and detecting S. typhimurium in ground chicken and ground beef.

SU-8 microcantilever with an aperture, fluidic channel, and sensing mechanisms for biological and other applications

We describe a method for fabricating an aperture on a fluidic cantilever device using SU-8 as a structural material. The device can ultimately be used for patch clamping, microinjections, fluidic delivery, fluidic deposition, and micromaterial removal. In the first generation of this device, the initial aperture diameter is 10 μm and is fabricated on a silicon-on-insulator (SOI) wafer that is structurally used to define the aperture. The aperture can be reduced in size through mask design. This self-aligned process allows for patterning on the sharp tip projecting out of the fluidic plane on the cantilever and is batch fabricated, reducing the cost and time for manufacture. The initial mask, SOI device layer thickness, and the width of the base of the tip define the size of the aperture. The SU-8 micromachined cantilever includes an electrode and a force sensing mechanism. The cantilever can be easily integrated with an atomic force microscope or an optical microscope.

Light-based methods for whole blood bacterial inactivation enabled by a recirculating flow system

Light of certain wavelengths can be used to inactivate pathogens. Whole blood is opaque; thus, the penetration of light is reduced. Here, we overcame this limitation using a thin transparent tube that is illuminated from all angles. Three light‐based techniques were evaluated: photodynamic therapy (PDT) using a 660‐nm light and antibody‐photosensitizer conjugates, ultraviolet, and violet light. We observed a reduction of 55–71% of Staphylococcus aureus after 5 h of exposure (starting concentration 107 CFU mL−1) and an 88–97% reduction in methicillin‐resistant Staphylococcus aureus (MRSA) (starting 104 CFU mL−1). An 83–92% decrease for S. aureus and 98–99.9% decrease for MRSA were observed when combined with an immunocapture approach. Complete blood count with differential analysis did not reveal any significant changes in the blood cell numbers. Genotoxicity studies showed that violet and ultraviolet did not induce any significant level of single strand breaks and alkali labile sites in the peripheral blood mononuclear cells (PBMC). In contrast, ultraviolet did induce a very low level of cyclobutane pyrimidine dimers, a UV damage indicator. PDT generated a significant level of single strand breaks and 8‐oxoGua in these cells. The approaches showed promise for whole blood pathogen inactivation with minimal collateral damage to PBMC.

Exchange protein directly activated by cAMP plays a critical role in regulation of vascular fibrinolysis

Rationale To maintain vascular patency, endothelial cells (ECs) actively regulate hemostasis. Among the myriad of pathways by which they control both fibrin formation and fibrinolysis is EC expression of annexin A2 (ANXA2) in a heterotetrameric complex with S100A10 [(ANXA2-S100A10)2]. This complex is a well-recognized endothelial surface platform for the activation of plasminogen by tissue plasminogen activator. A noteworthy advance in this field came about when it was shown that the cAMP pathway is linked to the regulation of (ANXA2-S100A10)2 in ECs. Objective These findings prompted us to determine whether a druggable target, namely the exchange protein directly activated by cAMP (EPAC) pathway, plays a role in vascular luminal fibrinolysis. Methods and Results Taking advantage of our Epac1-null mouse model, we found that depletion of Epac1 results in fibrin deposition, fibrinolytic dysfunction, and decreased endothelial surface ANXA2 in mice, which are similar to phenomena discovered in ANXA2-null and S100A10-null mice. We observed upregulation of EPAC1 and downregulation of fibrin in endocardial tissues beneath atrial mural thrombi in humans. Of note, our thrombosis model revealed that dysfunction of fibrinolysis in EPAC1-null mice can be ameliorated by recombinant ANXA2. Furthermore, we demonstrated that suppression of EPAC1 using a small-molecule inhibitor (ESI09) reduces the expression of ANXA2 in lipid rafts and impedes ANXA2 association with S100A10. Endothelial apical surface expression of both ANXA2 and S100A10 were markedly decreased in ESI09-treated ECs, which was corroborated by results from a nanoforce spectroscopy study. Moreover, inactivation of EPAC1 decreases tyrosine 23 phosphorylation of ANXA2 in the cell membrane compartment. Conclusions Our data reveal a novel role for EPAC1 in vascular fibrinolysis, by showing that EPAC1 is responsible for the translocation of ANXA2 to the EC surface. This process promotes conversion of plasminogen to plasmin, thereby enhancing local fibrinolytic activity.

Extracorporeal Photoimmunotherapy for Circulating Tumor Cells

It is believed that metastasis through the cardiovascular system is primarily caused by circulating tumor cells (CTCs). We report an approach to eliminate circulating tumor cells from the blood stream by flowing the blood though an extracorporeal tube and applying photodynamic therapy (PDT). Chlorin e6 (Ce6), a photosensizer, was conjugated to antibody CD44 in order to target PC-3, a prostate cancer cell line. PC-3 cells were successfully stained by the Ce6-CD44 antibody conjugate. PDT was performed on whole blood spiked with stained PC-3 cells. As the blood circulated through a transparent medical tube, it was exposed to light of a specific wavelength generated by an LED array. A 2 minute exposure was sufficient to achieve selective cancer cell necrosis. Control studies showed no cell death.

An introduction to antibiotic-free techniques to eliminate Staphylococcus aureus from blood

Here, we describe the implementation of three techniques for capturing and killing Staphylococcus aureus in blood in vitro inside a medical tube. The first technique involves capturing and removing pathogens using antibodies that are coated, via a simple chemical process, on the inner walls of a modified medical tube (tube capturing technique). In the second technique, a photosensitizer-antibody conjugate adheres to the pathogens while in circulation. When blood flows through the same kind of tube, the conjugate is activated by near-infrared (NIR) light to kill pathogens (photodynamic therapy technique). For the third technique, pathogens are exposed to light in the ultraviolet (UV) range while circulating through a similar tube (UV technique), which kills the pathogens. We spiked blood with S. aureus, starting with about 107 CFU/mL and ending at 108 CFU/mL after 5 hours. While the spiked bacteria rapidly grew in nutrition-rich whole blood, each of the three techniques were able to independently remove between 61% and 84% more S. aureus in the experimental blood sample compared to the controls groups. When combined, these techniques demonstrated a removal rate between 87% and 92%.