Sandra Pinto

High-density antimicrobial peptide coating with broad activity and low cytotoxicity against human cells

UNLABELLED Medical device-associated infections are a multi-billion dollar burden for the worldwide healthcare systems. The modification of medical devices with non-leaching coatings capable of killing microorganisms on contact is one of the strategies being investigated to prevent microorganism colonization. Here we developed a robust antimicrobial coating based on the chemical immobilization of the antimicrobial peptide (AMP), cecropin-melittin (CM), on gold nanoparticles coated surfaces. The concentration of AMP immobilized (110 μg/cm(2)) was higher than most of the studies reported so far (<10 μg/cm(2)). This translated onto a coating with high antimicrobial activity against Gram positive and negative bacteria sp., as well as multi-drug resistant bacteria. Studies with E. coli reporter bacteria showed that these coatings induced the permeability of the outer membrane of bacteria in less than 5 min and the inner membrane in approximately 20 min. Importantly, the antimicrobial properties of the coating are maintained in the presence of 20% (v/v) human serum, and have low probability to induce bacteria resistance. We further show that coatings have low toxicity against human endothelial and fibroblast cells and is hemocompatible since it does not induce platelet and complement activation. The antimicrobial coating described here may be promising to prevent medical device-associated infections. STATEMENT OF SIGNIFICANCE In recent years, antimicrobial peptides (AMPs) have been chemically immobilized on surfaces of medical devices to render them with antimicrobial properties. Surfaces having immobilized cationic peptides are susceptible to be adsorbed by plasma proteins with the subsequent loss of antimicrobial activity. Furthermore, with the exception of very few studies that have determined the cytotoxicity of surfaces in mammalian cells, the effect of the immobilized AMP on human cells is relatively unknown. Here we report a coating based on cecropin-melittin peptide (CM) that maintains its antimicrobial activity against Gram-positive and negative bacteria including multi-drugs resistance bacteria in the presence of serum and has relatively low cytotoxicity against human cells. The reported coatings may be translated on to variety of substrates (glass and titanium) and medical devices to prevent device-associated microbial infection.

High Antimicrobial Activity and Low Human Cell Cytotoxicity of Core–Shell Magnetic Nanoparticles Functionalized with an Antimicrobial Peptide

Superparamagnetic iron oxide nanoparticles (SPIONs) functionalized with antimicrobial agents are promising infection-targeted therapeutic platforms when coupled with external magnetic stimuli. These antimicrobial nanoparticles (NPs) may offer advantages in fighting intracellular pathogens as well as biomaterial-associated infections. This requires the development of NPs with high antimicrobial activity without interfering with the biology of mammalian cells. Here, we report the preparation of biocompatible antimicrobial SPION@gold core-shell NPs based on covalent immobilization of the antimicrobial peptide (AMP) cecropin melittin (CM) (the conjugate is named AMP-NP). The minimal inhibitory concentration (MIC) of the AMP-NP for Escherichia coli was 0.4 μg/mL, 10-times lower than the MIC of soluble CM. The antimicrobial activity of CM depends on the length of the spacer between the CM and the NP. AMP-NPs are taken up by endothelial (between 60 and 170 pg of NPs per cell) and macrophage (between 18 and 36 pg of NPs per cell) cells and accumulate preferentially in endolysosomes. These NPs have no significant cytotoxic and pro-inflammatory activities for concentrations up to 200 μg/mL (at least 100 times higher than the MIC of soluble CM). Our results in membrane models suggest that the selectivity of AMP-NPs for bacteria and not eukaryotic membranes is due to their membrane compositions. The AMP-NPs developed here open new opportunities for infection-site targeting.

Intracellular coexpression of CXC- and CC- chemokine receptors and their ligands in human melanoma cell lines and dynamic variations after xenotransplantation.

BackgroundChemokines have been implicated in tumor progression and metastasis. In melanoma, chemokine receptors have been implicated in organ selective metastasis by regulating processes such as chemoattraction, adhesion and survival.MethodsIn this study we have analyzed, using flow cytometry, the systems formed by the chemokine receptors CXCR3, CXCR4, CXCR7, CCR7 and CCR10 and their ligands in thirteen human melanoma cell lines (five established from primary tumors and eight established from metastasis from different tissues). WM-115 and WM-266.4 melanoma cell lines (obtained from a primary and a metastatic melanoma respectively) were xenografted in nude mice and the tumors and cell lines derived from them were also analyzed.ResultsOur results show that the melanoma cell lines do not express or express in a low degree the chemokine receptors on their cell surface. However, melanoma cell lines show intracellular expression of all the aforementioned receptors and most of their respective ligands. When analyzing the xenografts and the cell lines obtained from them we found variations in the intracellular expression of chemokines and chemokine receptors that differed between the primary and metastatic cell lines. However, as well as in the original cell lines, minute or no expression of the chemokine receptors was observed at the cell surface.ConclusionsCoexpression of chemokine receptors and their ligands was found in human melanoma cell lines. However, this expression is intracellular and receptors are not found at the cell membrane nor chemokines are secreted to the cell medium. The levels of expressed chemokine receptors and their ligands show dynamic variations after xenotransplantation that differ depending on the origin of the cell line (from primary tumor or from metastasis).

Characterization of a new human melanoma cell line with CD133 expression

A novel human malignant melanoma cell line, designated MEL-RC08, was established from a pericranial metastasis of a malignant melanoma of the skin. The cell line has been subcultured for more than 150 passages and is tumorigenic in nude mice. Growth kinetics, cytogenetics, flow cytometry, and molecular techniques for analysis of the genes implicated in cell cycle control; mutations in BRAF, NRAS, C-KiT, RB, and TP53 genes; and amplification of MDM2, CDK4, and cyclin D1 have been studied. Cytogenetically, the tumor and the cell line showed a hypertriploid karyotype with many clonal numeric and structural abnormalities. DNA flow cytometry showed an aneuploid peak with a DNA index value of 1.5. Mutations in TP53 and BRAF genes were demonstrated in both tumor and cell line. Furthermore, stem cell marker CD133 expression was detected in most cells, together with other stem cell markers, suggesting the presence of cells with tumor-initiating potential in this cell line.

Prolonged intracellular accumulation of light-inducible nanoparticles in leukemia cells allows their remote activation

Leukaemia cells that are resistant to conventional therapies are thought to reside in protective niches. Here, we describe light-inducible polymeric retinoic acid (RA)-containing nanoparticles (NPs) with the capacity to accumulate in the cytoplasm of leukaemia cells for several days and release their RA payloads within a few minutes upon exposure to blue/UV light. Compared to NPs that are not activated by light exposure, these NPs more efficiently reduce the clonogenicity of bone marrow cancer cells from patients with acute myeloid leukaemia (AML) and induce the differentiation of RA-low sensitive leukaemia cells. Importantly, we show that leukaemia cells transfected with light-inducible NPs containing RA can engraft into bone marrow in vivo in the proximity of other leukaemic cells, differentiate upon exposure to blue light and release paracrine factors that modulate nearby cells. The NPs described here offer a promising strategy for controlling distant cell populations and remotely modulating leukaemic niches.

Antimicrobial peptide-gold nanoscale therapeutic formulation with high skin regenerative potential

Chronic skin wounds affect ≈3% of persons aged >60years (Davies et al., 2007) [1]. These wounds are typically difficult to heal by conventional therapies and in many cases they get infected making even harder the regeneration process. The antimicrobial peptide (AMP) LL37 combines antimicrobial with pro-regenerative properties and thus represents a promising topical therapy to address both problems. Here, we investigated the wound healing potential of soluble and immobilized LL37 (LL37-conjugated gold nanoparticles, LL37-Au NPs), both in vitro (migration of keratinocytes) and in vivo (skin wound healing). Our results show that LL37-Au NPs, but not LL37 peptide, have the capacity to prolong the phosphorylation of EGFR and ERK1/2 and enhance the migratory properties of keratinocytes in a large in vitro wound model. We further report that both LL37 and LL37-Au NPs promote keratinocyte migration by the transactivation of EGFR, a process that seems to be initiated at the P2X7 receptor, as confirmed by chemical and genetic inhibition studies. Finally, we show in vivo that LL37-Au NPs have higher wound healing activity than LL37 peptide in a splinted mouse full thickness excisional model. Animal wounds treated by LL37-Au NPs have higher expression of collagen, IL6 and VEGF than the ones treated with LL37 peptide or NPs without LL37. Altogether, the conjugation of AMPs to NPs offers a promising platform to enhance their pro-regenerative properties.

CONGRUENCES ON DYNAMIC ALGEBRAS

The lattice CongD of all dynamic congruences on a given dynamic algebra D is presented. Whenever D is separable with zero we define dynamic ideal on D, given rise to the lattice IdeD. The notions of kernel of a dynamic congruence and the congruence generated by a dynamic ideal are introduced to describe a Galois connection between CongD and IdeD. We study conditions under which a dynamic congruence is determined by its kernel.

CONGRUENCES AND IDEALS ON BOOLEAN MODULES: A HETEROGENEOUS POINT OF VIEW

Definitions for heterogeneous congruences and heterogeneous ideals on a Boolean module are given and the respective lattices and are presented. A characterization of the simple bijective Boolean modules is achieved differing from that given by Brink in a homogeneous approach. We construct the smallest and the greatest modular congruence having the same Boolean part. The same is established for modular ideals. The notions of kernel of a modular congruence and the congruence induced by a modular ideal are introduced to describe an isomorphism between and . This isomorphism leads us to conclude that the class of the Boolean module is ideal determined.

Nanoparticles Conjugated with Photocleavable Linkers for the Intracellular Delivery of Biomolecules

We report the synthesis and characterization of phototriggerable polymeric nanoparticles (NPs) for the intracellular delivery of small molecules and proteins to modulate cell activity. For that purpose, several photocleavable linkers have been prepared providing diverse functional groups as anchoring points for biomolecules.

Expression of the Chemokine Receptors CXCR3, CXCR4, CXCR7 and Their Ligands in Rhabdomyosarcoma.

Rhabdomyosarcomas (RMS) are soft tissue malignant tumors of childhood and adolescents. The mechanisms underlying their aggressiveness are still poorly understood. Chemokines are chemotactic proteins involved in pathological processes that have been intensely studied in several types of cancers because of their influence in migration, angiogenesis, or metastases. We analyzed the expression of the chemokine receptors CXCR3, CXCR4 and CXCR7 and their ligands CXCL9, CXCL10, CXCL11 and CXCL12, in 15 RMS samples derived from nine patients. Expression was measured in tumors and primary cultures of RMS by Real-Time Polymerase Chain Reaction, immunostaining and flow cytometry. Our results show that these receptors are widely expressed in RMS. A significant difference between CXCL12/CXCR4, CXCL12/CXCR7, CXCL11/CXCR7 expression ratios was found in alveolar versus embryonal RMS and similarly between CXCL12/CXCR4 and CXCL11/CXCR3 ratios in primary versus recurrent tumors. These findings suggest a possible association between the interrelation of chemokine/chemokine-receptor and an aggressive biological behavior in RMS.