Karla Juarez-Moreno

Targeting Serous Epithelial Ovarian Cancer with Designer Zinc Finger Transcription Factors

Background: There is a need for novel targeted therapies for metastatic ovarian cancers. Results: We reactivated the tumor suppressor Maspin in ovarian carcinoma cells by delivering tumor-specific nanoparticles encapsulating a chemically modified ATF-mRNA. Conclusion: LPR nanoparticles encapsulating the ATF mRNA inhibited ovarian cancer tumor growth in a mouse model. Significance: We report the first non-viral delivery of an ATF in vivo and the discovery of novel anti-metastatic targets for ovarian cancer. Ovarian cancer is the leading cause of death among gynecological malignancies. It is detected at late stages when the disease is spread through the abdominal cavity in a condition known as peritoneal carcinomatosis. Thus, there is an urgent need to develop novel therapeutic interventions to target advanced stages of ovarian cancer. Mammary serine protease inhibitor (Maspin) represents an important metastasis suppressor initially identified in breast cancer. Herein we have generated a sequence-specific zinc finger artificial transcription factor (ATF) to up-regulate the Maspin promoter in aggressive ovarian cancer cell lines and to interrogate the therapeutic potential of Maspin in ovarian cancer. We found that although Maspin was expressed in some primary ovarian tumors, the promoter was epigenetically silenced in cell lines derived from ascites. Transduction of the ATF in MOVCAR 5009 cells derived from ascitic cultures of a TgMISIIR-TAg mouse model of ovarian cancer resulted in tumor cell growth inhibition, impaired cell invasion, and severe disruption of actin cytoskeleton. Systemic delivery of lipid-protamine-RNA nanoparticles encapsulating a chemically modified ATF mRNA resulted in inhibition of ovarian cancer cell growth in nude mice accompanied with Maspin re-expression in the treated tumors. Gene expression microarrays of ATF-transduced cells revealed an exceptional specificity for the Maspin promoter. These analyses identified novel targets co-regulated with Maspin in human short-term cultures derived from ascites, such as TSPAN12, that could mediate the anti-metastatic phenotype of the ATF. Our work outlined the first targeted, non-viral delivery of ATFs into tumors with potential clinical applications for metastatic ovarian cancers.

Toxicity of silver nanoparticles in biological systems: Does the complexity of biological systems matter?

Currently, nanomaterials are more frequently in our daily life, specifically in biomedicine, electronics, food, textiles and catalysis just to name a few. Although nanomaterials provide many benefits, recently their toxicity profiles have begun to be explored. In this work, the toxic effects of silver nanoparticles (35nm-average diameter and Polyvinyl-Pyrrolidone-coated) on biological systems of different levels of complexity was assessed in a comprehensive and comparatively way, through a variety of viability and toxicological assays. The studied organisms included viruses, bacteria, microalgae, fungi, animal and human cells (including cancer cell lines). It was found that biological systems of different taxonomical groups are inhibited at concentrations of silver nanoparticles within the same order of magnitude. Thus, the toxicity of nanomaterials on biological/living systems, constrained by their complexity, e.g. taxonomic groups, resulted contrary to the expected. The fact that cells and virus are inhibited with a concentration of silver nanoparticles within the same order of magnitude could be explained considering that silver nanoparticles affects very primitive cellular mechanisms by interacting with fundamental structures for cells and virus alike.

Aminosilane Functionalization and Cytotoxicity Effects of Upconversion Nanoparticles Y2O3 and Gd2O3 Co-doped with Yb3+and Er3+

In this study, luminescent upconversion nanoparticles (UCNPs) Y2O3 and Gd2O3 co-doped with Yb3+ and Er3+ were prepared by the sol-gel method (SG). These NPs are able to absorb near infrared photons and upconvert them into visible radiation with a direct application in bioimaging, as an important tool to diagnose and visualize cancer cells. The UCNPs were coated with a thin silica shell and functionalized with amino groups for further folic acid conjugation to allow their interaction with folate ligands on the cell surface. Their physical properties were analysed by Transmission Electron Microscopy (TEM), Fourier transform infrared spectroscopy (FTIR) and photoluminescence (PL) measurements. The PL results revealed excellent luminescence properties on all core-shell UCNPs. Cytotoxicity experiments with concentrations of bare and aminosilane coated/functionalized UCNPs between 0.001 μg/mL to 1 μg/mL were tested on two different cell lines from human cervix carcinoma (HeLa) and human colorectal adenocarcinoma (DLD-1) with a colorimetric assay based on the reduction of MTT reagent (methy-134-thiazolyltetrazolium). The assays show that some concentrations of bare UCNPs were cytotoxic for cervical adenocarcinoma cells (HeLa); however, for human colorectal adenocarcinoma all UCNPs are non-cytotoxic. After UCNPs functionalization with silica-aminosilane (APTES/TEOS), all of the nanoparticles tested were found to be non-cytotoxic for both cell lines. The UCNPs functionalized in this work can be further conjugated with specific ligands and used as biolabels for detection of cancer cells.

EPR and LC-MS studies on the mechanism of industrial dye decolorization by versatile peroxidase from Bjerkandera adusta

The mechanisms of industrial dye transformation by versatile peroxidase were elucidated. Purified versatile peroxidase from Bjerkandera adusta was able to decolorize different classes of dyes including azo and phthalocyanines, but unable to transform any of the anthraquinones tested. Kinetic constants for selected dyes were determined and the transformation products were analyzed by EPR spectroscopy and mass spectrometry. The EPR and MS analyses of the enzymatic decolorization products showed the cleavage of the azo bond in azo dyes and the total disruption of the phthalocyaninic ring in phthalocyanine dyes. The EPR analysis on two copper-containing dyes, reactive violet 5 (azo) and reactive blue 72 (phthalocyanine), showed that the transformation can or not break the metal-ion coordination bond according the dye nature. The role of the catalytic Trp172 in the dye transformation by a long-range electron transfer pathway was confirmed and the oxidation mechanisms are proposed and discussed.

Cytochrome P450 Bioconjugate as a Nanovehicle for Improved Chemotherapy Treatment.

Cancer is still a growing public health problem, especially breast cancer that is one of the most important cancers in women. Chemotherapy, even though a successful treatment, is accompanied by severe side effects. Moreover, most of the drugs used for chemotherapy are administered as prodrugs and need to be transformed to the active form by cytochromes P450 (CYPs). In addition, increasing numbers of cancer tissues show lower CYP activity than the surrounding healthy tissues in which prodrugs are preferentially activated causing cytotoxicity. Here, the design of a functionalized cytochrome P450 bioconjugate is reported as nanovehicle for the enzyme direct delivery to the tumor tissue in order to improve the local drug activation. MCF-7 breast cancer cells are treated with CYP-polyethylene glycol bioconjugate functionalized folic acid, where it activates the prodrug tamoxifen and significantly reduces the dose of tamoxifen needed to kill the tumor cells. The CYP bioconjugate covered with polyethylene glycol shows no immunogenic activity. The advantages of increasing the site-specific CYP activity in tumor tissues are discussed.

Biocatalytic virus capsid as nanovehicle for enzymatic activation of Tamoxifen in tumor cells

Most of the drugs used in chemotherapy should be activated by a transformation catalyzed by cytochrome P450 (CYP) enzymes. In this work, bacteriophage P22 virus-like particles (VLPs) containing CYP activity, immunologically inert and functionalized in order to be recognized by human cervix carcinoma cells and human breast adenocarcinoma cells were designed. The CYP was encapsulated inside the virus capsid obtained from the bacteriophage P22. CYP and coat protein were both heterologously expressed in E. coli. The VLPs with enzymatic activity were covered with polyethylene glycol that was functionalized in its distal end with folic acid in order to be recognized by folate receptors exhibited on tumor cells. The capacity of biocatalytic VLPs to be recognized and internalized into tumor cells is demonstrated. The VLP-treated cells showed enhanced capacity for the transformation of the pro-drug tamoxifen, which resulted in an increase of the cell sensitivity to this oncological drug. In this work, the potential use of biocatalytic VLPs vehicles as a delivery system of medical relevant enzymes is clearly demonstrated. In addition to cancer treatment, this technology also offers an interesting platform as nano-bioreactors for intracellular delivery of enzymatic activity for other diseases originated by the lack of enzymatic activity.

Synthesis and cytotoxic effects of SrAl 2 O 4 persistent luminescence nanoparticles co-doped with Eu 2+ /Dy 3+ ions

Persistent luminescence strontium aluminate nanoparticles co-doped with Eu2+ and Dy3+ were prepared by urea-assisted combustion synthesis. Different percentages of co-dopants were evaluated in order to optimize luminescence of the nanophosphor. Luminescence measurements showed that excitation of this green-emitting phosphor occurred within a wide range of wavelengths (254 – 460 nm) while the half-life time of persistent luminescence laid within the seconds regime. Presence of Dy3+ as the co-dopant enhanced the green emission in this interval of time, and the entire decay time occurred in minutes. Crystallinity and morphology were evaluated by X-ray diffraction (XRD) and transmission electron microscopy (TEM), respectively. Strontium aluminate co-doped with 1%Eu, and 1%Dy, and 1%Eu, and 3%Dy emitted an intense green signal and long decay time. These crystal nanophosphors displayed sizes of 18 nm and 22 nm, respectively. The cytotoxic effect of nanoparticles was determined by a cell viability test where the tri-methyl-tetrazolium reagent (MTT) was reduced only by metabolically active cells. Different concentrations of bare nanoparticles were tested in a 96-well plate containing 10, 000 cells per well of a human cervix carcinoma cell line (HeLa). Evaluation of cell viability by this cytotoxic assay showed that in most of the cases cell viability was higher than 60% after incubation with bare nanoparticles. Since our bare nanoparticles were not cytotoxic, these results open a broad field of biomedical applications for phosphorescent materials as cell biolabels and imaging research area.

Silver Nanoparticles for the Rapid Healing of Diabetic Foot Ulcers

Diabetic foot ulcers are one of the major complications of patients with diabetes mellitus. And due to their high susceptibility to microbial infections, are the leading cause of hospitalization and amputation of lower limbs. It has been well studied the antimicrobial properties of silver nanoparticles (AgNPs), therefore their use in biomedicine is a trend. Herein we present for the first time the use of AgNPs for the treatment of diabetic foot ulcers of grade II and III of Wagner classification. Ulcers were treated by topical administration of AgNPs (at 1.8 mg/mL of metallic silver) in addition to conventional antibiotics. In all the cases presented in this study, a significant improvement in the evolution of ulcers was observed upon AgNPs administration. The edges of the lesion reached the point of closure. These results constituted the basis for further studies on the use of AgNPs for the treatment of chronic ulcers from different origins.

Synthesis and cytotoxic effects of SrAl2O4 persistent luminescence nanoparticles co-doped with Eu2+/Dy3+ ions: publisher’s note

This publisher’s note amends the affiliations in the author list of [Opt. Mater. Express6, 1488–1499, (2016)].