Raquel Mejías

Dimercaptosuccinic acid-coated magnetite nanoparticles for magnetically guided in vivo delivery of interferon gamma for cancer immunotherapy

As radio- and chemotherapy-based cancer treatments affect both tumors and healthy tissue, cancer immunotherapy attempts to specifically enhance the natural immune response to tumor cells. In mouse models of cancer, we tested uniform dimercaptosuccinic acid (DMSA)-coated monodisperse magnetic nanoparticles as a delivery system for the anti-tumorigenic cytokine IFN-γ. IFN-γ-adsorbed DMSA-coated magnetic nanoparticles were targeted to the tumor site by application of an external magnetic field. We analyzed nanoparticle biodistribution before and after IFN-γ conjugation, as well as the efficiency of nanoparticle accumulation in tumors, IFN-γ release in the area of interest, and the effects of both on tumor development. At the tumor site, we observed a high degree of nanoparticle accumulation and of cytokine delivery, which led to increased T cell and macrophage infiltration and promoted an anti-angiogenic effect. The combined action led to a notable reduction in tumor size. Our findings indicate that IFN-γ-adsorbed DMSA-coated magnetite nanoparticles can be used as an efficient in vivo drug delivery system for tumor immunotherapy.

Long term biotransformation and toxicity of dimercaptosuccinic acid-coated magnetic nanoparticles support their use in biomedical applications

Although iron oxide magnetic nanoparticles (MNP) have been proposed for numerous biomedical applications, little is known about their biotransformation and long-term toxicity in the body. Dimercaptosuccinic acid (DMSA)-coated magnetic nanoparticles have been proven efficient for in vivo drug delivery, but these results must nonetheless be sustained by comprehensive studies of long-term distribution, degradation and toxicity. We studied DMSA-coated magnetic nanoparticle effects in vitro on NCTC 1469 non-parenchymal hepatocytes, and analyzed their biodistribution and biotransformation in vivo in C57BL/6 mice. Our results indicate that DMSA-coated magnetic nanoparticles have little effect on cell viability, oxidative stress, cell cycle or apoptosis on NCTC 1469 cells in vitro. In vivo distribution and transformation were studied by alternating current magnetic susceptibility measurements, a technique that permits distinction of MNP from other iron species. Our results show that DMSA-coated MNP accumulate in spleen, liver and lung tissues for extended periods of time, in which nanoparticles undergo a process of conversion from superparamagnetic iron oxide nanoparticles to other non-superparamagnetic iron forms, with no significant signs of toxicity. This work provides the first evidence of DMSA-coated magnetite nanoparticle biotransformation in vivo.

Liver and brain imaging through dimercaptosuccinic acid-coated iron oxide nanoparticles

BACKGROUND & AIM Uptake, cytotoxicity and interaction of improved superparamagnetic iron oxide nanoparticles were studied in cells, tissues and organs after single and multiple exposures. MATERIAL & METHOD We prepared dimercaptosuccinic acid-coated iron oxide nanoparticles by thermal decomposition in organic medium, resulting in aqueous suspensions with a small hydrodynamic size (< 100 nm), high saturation magnetization and susceptibility, high nuclear magnetic resonance contrast and low cytotoxicity. RESULTS In vitro and in vivo behavior showed that these nanoparticles are efficient carriers for drug delivery to the liver and brain that can be combined with MRI detection.

Cytokine adsorption/release on uniform magnetic nanoparticles for localized drug delivery.

Attachment of cytokines to magnetic nanoparticles has been developed as a system for controlled local drug release in cancer therapy. We studied the adsorption/release of murine interferon gamma (IFN-gamma) on negatively charged magnetic nanoparticles prepared by three different methods, including coprecipitation, decomposition in organic media, and laser pyrolysis. To facilitate IFN-gamma adsorption, magnetic nanoparticles were surface modified by distinct molecules to achieve high negative charge at pH 7, maintaining small aggregate size and stability in biological media. We analyzed carboxylate-based coatings and studied the colloidal properties of the resulting dispersions. Finally, we incubated the magnetic dispersions with IFN-gamma and determined optimal conditions for protein adsorption onto the particles, as well as the release capacity at different pH and as a function of time. Particles prepared by decomposition in organic media and further modified with dimercaptosuccinic acid showed the most efficient adsorption/release capacity. IFN-gamma adsorbed on these nanoparticles would allow concentration of this protein or other biomolecules at specific sites for treatment of cancer or other diseases.

Ac magnetic susceptibility study of in vivo nanoparticle biodistribution

We analysed magnetic nanoparticle biodistribution, before and after cytokine conjugation, in a mouse model by ac susceptibility measurements of the corresponding resected tissues. Mice received repeated intravenous injections of nanoparticle suspension for two weeks and they were euthanized 1 h after the last injection. In general, only 10% of the total injected nanoparticles after multiple exposures were found in tissues. The rest of the particles may probably be metabolized or excreted by the organism. Our findings indicate that the adsorption of interferon to DMSA-coated magnetic nanoparticles changes their biodistribution, reducing the presence of nanoparticles in lungs and therefore their possible toxicity. The specific targeting of the particles to tumour tissues by the use of an external magnetic field has also been studied. Magnetic nanoparticles were observed by transmission electron microscopy in the targeted tissue and quantified by ac magnetic susceptibility.

Effect of Anesthesia on Magnetic Nanoparticle Biodistribution After Intravenous Injection

The role of anesthesia on magnetic nanoparticle biodistribution among different organs after intravenous injection has been studied in a murine model. Animals were anesthetized by inhalation with isoflurane (0.5% in oxygen) or by intraperitoneal injection with a mixture of ketamine and xylazine. Then, monodisperse dimercaptosuccinic acid coated magnetic nanoparticles (diameter of 9.2 nm 1.2 nm) were administered intravenously to the animals. Lung and liver tissues were collected after the particle administration and the amount of particles in each tissue was determined by alternating current magnetic susceptibility measurements. Whereas the amount of particles that reaches the liver seems not to be affected by the anesthesia used, the amount of particles that reaches the lungs for inhaled isoflurane is three times less than for the intraperitoneally injected anesthetic.

Ral GTPases Regulate Cell-Mediated Cytotoxicity in NK Cells

NK cells are key components of the immune response to virally infected and tumor cells. Recognition of target cells initiates a series of events in NK cells that culminates in target destruction via directed secretion of lytic granules. Ral proteins are members of the Ras superfamily of small GTPases; they regulate vesicular trafficking and polarized granule secretion in several cell types. In this study, we address the role of Ral GTPases in cell-mediated cytotoxicity. Using a human NK cell line and human primary NK cells, we show that both Ral isoforms, RalA and RalB, are activated rapidly after target cell recognition. Furthermore, silencing of RalA and RalB impaired NK cell cytotoxicity. RalA regulated granule polarization toward the immunological synapse and the subsequent process of degranulation, whereas RalB regulated degranulation but not polarization of lytic granules. Analysis of the molecular mechanism indicated that Ral activation in NK cells leads to assembly of the exocyst, a protein complex involved in polarized secretion. This assembly is required for degranulation, as interference with expression of the exocyst component Sec5 led to reduced degranulation and impaired cytotoxicity in NK cells. Our results thus identify a role for Ral in cell-mediated cytotoxicity, implicating these GTPases in lymphocyte function.

Lipid-oligonucleotide conjugates improve cellular uptake and efficiency of TCTP-antisense in castration-resistant prostate cancer

ABSTRACT Translationally controlled tumor protein (TCTP) has been implicated in a plethora of important cellular processes related to cell growth, cell cycle progression, malignant transformation and inhibition of apoptosis. Therefore, TCTP is now recognized as a potential therapeutic target in several cancers including prostate, breast and lung cancers. We previously showed that TCTP is overexpressed in castration‐resistant prostate cancer (CRPC), and it has been implicated resistance to treatment. Recently, we developed TCTP antisense oligonucleotides (ASOs) to inhibit TCTP expression. However, the intracellular delivery and silencing activity of these oligonucleotides remains a challenge, and depend on the use of transfection agents and delivery systems. Here we show that lipid‐modified ASO (LASOs) has improved penetration and efficiency in inhibiting TCTP expression in the absence of additional transfection agents, both in vitro and in vivo. Transfection with TCTP‐LASO led to rapid and prolonged internalization via macropinocytosis, TCTP downregulation and significant decreased cell viability. We also show that lipid‐modification led to delayed tumor progression in CRPC xenografts models, with no significant toxic effects observed.

PI3K p85 β regulatory subunit deficiency does not affect NK cell differentiation and increases NKG2D-mediated activation

Activation of NK cells depends on a balance between activating and inhibitory signals. Class Ia PI3K are heterodimeric proteins with a catalytic and a regulatory subunit and have a central role in cell signaling by associating with tyrosine kinase receptors to trigger signaling cascades. The regulatory p85 subunit participates in signaling through NKG2D, one of the main activating receptors on NK cells, via its interaction with the adaptor protein DAP10. Although the effects of inhibiting catalytic subunits or deleting the regulatory p85α subunit have been studied, little attention has focused on the role of the p85β subunit in NK cells. Using p85β knockout mice, we found that p85β deficiency does not alter NK cell differentiation and maturation in spleen or bone marrow. NK cells from p85β−/− mice nonetheless produced more IFN‐γ and degranulated more effectively when stimulated with anti‐NKG2D antibody. These cells also degranulated and killed NKG2D ligand‐expressing target cells more efficiently. We show that p85β deficiency impaired NKG2D internalization, which could contribute to the activated phenotype. Decreasing p85β subunit protein levels might thus constitute a therapeutic target to promote NK cell activity toward NKG2D ligand‐expressing cells.

Magnetic Nanoparticle Location and Quantification in Mice Tissues after Intravenous Injection

DMSA‐coated magnetic nanoparticles have been used for drug delivery, in particular to transport cytokines towards an induced tumour in a murine model. In this work, the use of transmission electron microscopy and AC magnetic susceptibility measurements of the tissue have allowed the detection of the particles within the target tissue.