Trafficking of a Single Photosensitizing Molecule to Different Intracellular Organelles Demonstrates Effective Hydroxyl Radical-Mediated Photodynamic Therapy in the Endoplasmic Reticulum.

Photodynamic therapy (PDT) is often used in preclinical and clinical treatment regimens. Reactive oxygen species (ROS) generated by photosensitizers (PSs) upon exposure to light induce cell death via diverse mechanisms. PSs can exert therapeutic effects in different cellular organelles, although the efficacy of organelle-specific PDT has yet to be determined as most previous studies use different PSs in different organelles. Here, we explored how a single PS, chlorin e6 (Ce6), targeted to different organelles altered the effectiveness of PDT. Ce6 intrinsically localizes to the ER after 4 h of incubation. Modification of Ce6 via conjugation with an octapeptide (LS765), a monosubstituted triphenylphosphonium (TPP) derivative (LS897), or a disubstituted TPP derivative (LS909) altered the intrinsic localization. We determined that LS765 and LS9897 predominantly accumulated in the lysosomes, but LS909 trafficked equally to both the mitochondria and the lysosomes. Moreover, the conjugation altered the type of ROS produced by Ce6, increasing the ratio of hydrogen peroxide to hydroxyl radicals. Irradiation of identical concentrations of the PSs in solution with 650 nm, 0.84 mW/cm2 light for 10 min showed that the TPP conjugates nearly doubled the hydrogen peroxide production from ∼0.2 µM for Ce6 and LS765 to ∼0.37 µM for LS897 and LS909. In contrast, Ce6 produced ∼1.5-fold higher hydroxyl radicals than its conjugates. To compare the effect of each PS on cell death, we normalized the intracellular concentration of each PS. Hydrogen peroxide-producing PSs are effective PDT agents in the lysosomes while the hydroxyl-generating PSs are very effective in the ER. Compared to the PSs that accumulated in the lysosomes, only the ER-targeted Ce6 exerted >50% cell death at either low light power or low intracellular concentration. By delineating the contributions of cellular organelles and types of ROS produced, our work suggests that targeting hydroxyl radical-producing PSs to the ER is an exciting strategy to improve the therapeutic outcome of PDT.

Growth of Plasmodium falciparum in response to a rotating magnetic field.

BACKGROUND: Plasmodium falciparum is the deadliest strain of malaria and the mortality rate is increasing because of pathogen drug resistance. Increasing knowledge of the parasite life cycle and mechanism of infection may provide new models for improved treatment paradigms. This study sought to investigate the paramagnetic nature of the parasite's haemozoin to inhibit parasite viability. RESULTS: Paramagnetic haemozoin crystals, a byproduct of the parasite's haemoglobin digestion, interact with a rotating magnetic field, which prevents their complete formation, causing the accumulation of free haem, which is lethal to the parasites. Plasmodium falciparum cultures of different stages of intraerythrocytic growth (rings, trophozoites, and schizonts) were exposed to a magnetic field of 0.46 T at frequencies of 0 Hz (static), 1, 5, and 10 Hz for 48 h. The numbers of parasites were counted over the course of one intraerythrocytic life cycle via flow cytometry. At 10 Hz the schizont life stage was most affected by the rotating magnetic fields (p = 0.0075) as compared to a static magnetic field of the same strength. Parasite growth in the presence of a static magnetic field appears to aid parasite growth. CONCLUSIONS: Sequestration of the toxic haem resulting from haemoglobin digestion is key for the parasites' survival and the focus of almost all existing anti-malarial drugs. Understanding how the parasites create the haemozoin molecule and the disruption of its creation aids in the development of drugs to combat this disease.

Hybrid TiO2 -Ruthenium Nano-photosensitizer Synergistically Produces Reactive Oxygen Species in both Hypoxic and Normoxic Conditions.

Photodynamic therapy (PDT) is widely used to treat diverse diseases, but its dependence on oxygen to produce cytotoxic reactive oxygen species (ROS) diminishes the therapeutic effect in a hypoxic environment, such as solid tumors. Herein, we developed a ROS-producing hybrid nanoparticle-based photosensitizer capable of maintaining high levels of ROS under both normoxic and hypoxic conditions. Conjugation of a ruthenium complex (N3) to a TiO2 nanoparticle afforded TiO2 -N3. Upon exposure of TiO2 -N3 to light, the N3 injected electrons into TiO2 to produce three- and four-fold more hydroxyl radicals and hydrogen peroxide, respectively, than TiO2 at 160 mmHg. TiO2 -N3 maintained three-fold higher hydroxyl radicals than TiO2 under hypoxic conditions via N3-facilitated electron-hole reduction of adsorbed water molecules. The incorporation of N3 transformed TiO2 from a dual type I and II PDT agent to a predominantly type I photosensitizer, irrespective of the oxygen content.

Protonation and Trapping of a Small pH-Sensitive Near-Infrared Fluorescent Molecule in the Acidic Tumor Environment Delineate Diverse Tumors in Vivo.

Enhanced glycolysis and poor perfusion in most solid malignant tumors create an acidic extracellular environment, which enhances tumor growth, invasion, and metastasis. Complex molecular systems have been explored for imaging and treating these tumors. Here, we report the development of a small molecule, LS662, that emits near-infrared (NIR) fluorescence upon protonation by the extracellular acidic pH environment of diverse solid tumors. Protonation of LS662 induces selective internalization into tumor cells and retention in the tumor microenvironment. Noninvasive NIR imaging demonstrates selective retention of the pH sensor in diverse tumors, and two-photon microscopy of ex vivo tumors reveals significant retention of LS662 in tumor cells and the acid tumor microenvironment. Passive and active internalization processes combine to enhance NIR fluorescence in tumors over time. The low background fluorescence allows tumors to be detected with high sensitivity, as well as dead or dying cells to be delineated from healthy cells. In addition to demonstrating the feasibility of using small molecule pH sensors to image multiple aggressive solid tumor types via a protonation-induced internalization and retention pathway, the study reveals the potential of using LS662 to monitor treatment response and tumor-targeted drug delivery.

Galectin-3 modulation of T-cell activation: mechanisms of membrane remodelling.

Galectin-3 (Gal3) is a multifaceted protein which belongs to a family of lectins and binds ß-galactosides. Gal3 expression is altered in many types of cancer, with increased expression generally associated with poor prognosis. Although the mechanisms remain unknown, Gal3 has been implicated in several biological processes involved in cancer progression, including suppression of T cell-mediated immune responses. Extracellular Gal3 binding to the plasma membrane of T cells alters membrane organization and the formation of an immunological synapse. Its multivalent capacity allows Gal3 to interact specifically with different membrane proteins and lipids, influencing endocytosis, trafficking and T cell receptor signalling. The ability of Gal3 to inhibit T cell responses may provide a mechanism by which Gal3 aids in cancer progression. In this review, we seek to give an overview of the mechanisms by which Gal3 alters the spatial organization of cell membranes and how these processes impact on T cell activation.

Enhancing proteasome-inhibitory activity and specificity of bortezomib by CD38 targeted nanoparticles in multiple myeloma.

The establishment of more effective treatments that can circumvent chemoresistance in Multiple Myeloma (MM) is a priority. Although bortezomib (BTZ) is one of the most potent proteasome inhibitors available, still possesses limitations related to dose limiting side effects. Several strategies have been developed to improve the delivery of chemotherapies to MM by targeting different moieties expressed on MM cells to nanoparticle delivery systems (NPs), which have failed mainly due to their heterogeneous expression on these cells. Our goal was to test CD38 targeted chitosan NPs as novel targeting moiety for MM to improve the potency and efficacy of BTZ in MM cells and reduce the side effects in healthy tissue. We have showed preferential BTZ release in tumor-microenvironment, specific binding to MM cells, and an improved drug cellular uptake through BTZ diffusion from the surface and endocytosed NPs, which translated in enhanced proteasome inhibition and robust cytotoxic effect on MM cells when BTZ was administered through anti-CD38 chitosan NPs. Furthermore, the anti-CD38 chitosan NPs specifically delivered therapeutic agents to MM cells improving therapeutic efficacy and reducing side effects in vivo. The anti-CD38 chitosan NPs showed low toxicity profile allowing enhancement of proteasome-inhibitory activity and specificity of BTZ by endocytosis-mediated uptake of CD38 representing a promising therapy in MM.

3D tissue-engineered bone marrow as a novel model to study pathophysiology and drug resistance in multiple myeloma.

PURPOSE: Multiple myeloma (MM) is the second most prevalent hematological malignancy and it remains incurable despite the introduction of several novel drugs. The discrepancy between preclinical and clinical outcomes can be attributed to the failure of classic two-dimensional (2D) culture models to accurately recapitulate the complex biology of MM and drug responses observed in patients. EXPERIMENTAL DESIGN: We developed 3D tissue engineered bone marrow (3DTEBM) cultures derived from the BM supernatant of MM patients to incorporate different BM components including MM cells, stromal cells, and endothelial cells. Distribution and growth were analyzed by confocal imaging, and cell proliferation of cell lines and primary MM cells was tested by flow cytometry. Oxygen and drug gradients were evaluated by immunohistochemistry and flow cytometry, and drug resistance was studied by flow cytometry. RESULTS: 3DTEBM cultures allowed proliferation of MM cells, recapitulated their interaction with the microenvironment, recreated 3D aspects observed in the bone marrow niche (such as oxygen and drug gradients), and induced drug resistance in MM cells more than 2D or commercial 3D tissue culture systems. CONCLUSIONS: 3DTEBM cultures not only provide a better model for investigating the pathophysiology of MM, but also serve as a tool for drug development and screening in MM. In the future, we will use the 3DTEBM cultures for developing personalized therapeutic strategies for individual MM patients.

Calibration-free structured-illumination photoacoustic flowgraphy of transverse flow in scattering media.

We propose a calibration-free photoacoustic (PA) method for transverse flow measurements. In this method, a pulsed periodically structured (i.e., grating patterned) optical beam is used to illuminate flowing absorptive particles in an optically scattering medium. The PA signal amplitudes measured over consecutive laser pulses carry an imprint of the illumination structure. The modulation frequency of the imprint is proportional to the component of the flow speed projected onto the normal axis of the striped illumination pattern. This method can tolerate high particle density, and is insensitive to the particle size, thus calibration-free. Bovine blood and microsphere phantoms were used to validate the proposed method. Blood flow in a mouse ear was measured in vivo as well.

Cloning, Purification, and Partial Characterization of the Halobacterium sp. NRC-1 Minichromosome Maintenance (MCM) Helicase.

The MCM gene from the archaeon Halobacterium, with and without its intein, was cloned into an Escherichia coli expression vector, overexpressed and the protein was purified and antibodies were generated. The antibodies were used to demonstrate that in vivo only the processed enzyme, without the intein, could be detected.