Phosphorylated silk fibroin matrix for methotrexate release

Silk-based matrix was produced for delivery of a model anticancer drug, methotrexate (MTX). The calculation of net charge of silk fibroin and MTX was performed to better understand the electrostatic interactions during matrix formation upon casting. Silk fibroin films were cast at pH 7.2 and pH 3.5. Protein kinase A was used to prepare phosphorylated silk fibroin. The phosphorylation content of matrix was controlled by mixing at specific ratios the phosphorylated and unphosphorylated solutions. In vitro release profiling data suggest that the observed interactions are mainly structural and not electrostatical. The release of MTX is facilitated by use of proteolytic enzymes and higher pHs. The elevated -sheet content and crystallinity of the acidified-cast fibroin solution seem not to favor drug retention. All the acquired data underline the prevalence of structural interactions over electrostatical interactions between methotrexate and silk fibroin.The authors would like to acknowledge the support, granted by European NOVO Project, Contract No. FP7-HEALTH 2011-two-stage 278402. This work was partially supported by FEDER through POFC-COMPETE and by national funds from FCT through the projects PEst-C/BIA/UI4050/2011 (CBMA). V.V. also wants to thank Dr. Claudia Botelho for her helpful discussion and comments made during the critical reading of the manuscript

Enzymatic phosphorylation of silk fibroins : a platform for the production of biocompatible, cell-static, materials

Silks are natural protein polymers produced by insects. Silk heavy chain of B.mori is primarily composed of hydrophobic, –(–Ala–Gly–)n– -sheet crystalline domains. Based on silk biocompatibility, biodegradability and strength, different materials were developed. Silk offers a stabilizing environment for incorporated proteins and molecules. Silk properties can be controlled via structure manipulation, by coupling molecules of biological significance; its Tyr and Ser residues can be modified. Once incorporated into a protein, the phosphate group establishes hydrogen bonds that affect intra- and inter-molecular interactions16. Phosphorylation is stable under physiological conditions, thus directing the formation and reorganization of protein networks. Curiously, using phosphorylation for protein functionalization is largely unexplored. Significant research is devoted to bio-inspired materials with various cell-differentiating and cell-supporting features. However, little attention is paid to develop cell-static bio-materials. Such materials do not promote cell growth. That can be achieved by lowering the probability of cell attachment to the material, via creation of negatively charged material surface. The goal of this study was to produce bio-compatible materials with the cell-static properties by phosphorylation. Silk solutions were made to cast films of variable pH and phosphorylated content. Obtained materials were tested and a dependency between amount of phosphorylation and bio-chemical properties confirmed

Novel liposomes for Alzheimers disease treatment

This work was supported by the strategic programme UID/BIA/04050/2019, funded by national funds through the FCT IP, and project FUN2CYT: Harnessing the potential for biomedical applications of pleiotropic cytokines LIF and oncostatin M (PTDC/BTM-MAT/30568/2017, POCI-01-0145-FEDER-030568) supported by POCI through FEDER and FCT IP.info:eu-repo/semantics/publishedVersio

Functionalized protein nanoemulsions by incorporation of chemically modified BSA

The incorporation of bioactive compounds in stealth nanoparticles or nanoemulsions enhances their half-life in systemic circulation and can overcome the problems associated with the free drug. Bovine serum albumin (BSA)-drug conjugates were produced with either methotrexate (MTX), a potent anticancer agent, or vancomycin (VCM), a potent antibiotic. Those conjugates were used to produce functionalized BSA nanoemulsions in a formulation composed by aqueous phase and organic phase. BSA-Folic acid (FA) conjugates were also produced allowing specific folate receptor (FR) mediated targeting of cancer cells (KB cell line). All conjugates had similar effects either in solution or in the form of nanoemulsions: BSA-MTX as anti-proliferative over Caco-2 cell line and BSA-VCM as lower minimum inhibitory concentration (MIC) comparatively to VCM solution on Staphylococcus aureus strain Newman. The production of nanoemulsions using BSA-drug conjugates for obtaining vectors loaded with stabilized drugs offers a good, flexible template for a wide range of medical applications.Ana Loureiro (SFRH/BD/81479/2011) holds a scholarship from Fundacao para a Ciencia e a Tecnologia (FCT). Goncalo J. L. Bernardes is a Royal Society University Research Fellow at the Department of Chemistry, University of Cambridge and an Investigador FCT at the Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa. This work has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. This work was supported by FEDER through POFC - COMPETE and by Portuguese funds from FCT through the project PEst-OE/BIA/UI4050/2014

Delivery of cytarabine by pegylated liposomes for efficient, long-term anticancer effects

The cytosine arabinoside cytarabine is an effective marine-derived antineoplastic agent for the treatment of acute myelogrnous and lymphocytic leukemias. As this nucleoside antimetabolite is an S-phase-specific drug, prolonged exposure of cells to toxic concentrations is critical to achieve maximum biological effect. The activity of cytarabine is nevertheless decreased by its rapid deamination to the biologically inactive metabolite uracil arabinoside. This rapid degradation process is the reason for the ongoing search for efficient formulations and derivatives of cytarabine that cannot be deaminated and exhibited better pharmacokinetic parameters. In the present study, pegylated liposomes were modified for intended prolonged delivery of cytarabine and tested for improved cytotoxic and cytostatic effect in different human cancer lines

Monoolein-based nanocarriers for enhanced folate receptor-mediated RNA delivery to cancer cells

We report the development and characterization of a novel nanometric system for specific delivery of therapeutic siRNA for cancer treatment. This vector is based on a binary mixture of the cationic surfactant dioctadecyldimethylammonium chloride (DODAC) and the helper lipid monoolein (MO). These liposomes were previously validated by our research group as promising non-viral vectors for nucleic acid delivery. In this work, the DODAC:MO vesicles were for the first time functionalized with polyethylene glycol and PEG-folate conjugates to achieve both maximal stability in biological fluids and increase selectivity toward folate receptor α expressing cells. The produced DODAC:MO:PEG liposomes were highly effective in RNA complexation (close to 100%), and the resulting lipoplexes also demonstrated high stability in conditions simulating their administration by intravenous injection (physiological pH, high NaCl, heparin and fetal bovine serum concentrations). In addition, cell uptake of the PEG-folate-coated lipoplexes was significantly greater in folate receptor α positive breast cancer cells (39% for 25 µg/mL of lipid and 31% for 40 µg/mL) when compared with folate receptor α negative cells (31% for 25 µg/mL of lipid and 23% for 40 µg/mL) and to systems without PEG-folate (≈13% to 16% for all tested conditions), supporting their selectivity towards the receptor. Overall, the results support these systems as appealing vectors for selective delivery of siRNA to cancer cells by folate receptor α-mediated internalization, aiming at future therapeutic applications of interest

Folic acid-tagged protein nanoemulsions loaded with CORM-2 enhance the survival of mice bearing subcutaneous A20 lymphoma tumors

Folic Acid (FA)-tagged protein nanoemulsions were found to be preferentially internalized on B-cell lymphoma cell line (A20 cell line), which, for the first time, are reported to express folate receptor (FR)-alpha. Carbon monoxide releasing molecule-2 (CORM-2) was incorporated in the oil phase of the initial formulation. FA-functionalized nanoemulsions loaded with CORM-2 exhibited a considerable antitumor effect and an increased survival of BALB/c mice bearing subcutaneous A20 lymphoma tumors. The developed nanoemulsions also demonstrated to be well tolerated by these immunocompetent mice. Thus, the results obtained in this study demonstrate that FA-tagged protein nanoemulsions can be successfully used in cancer therapy, with the important ability to delivery drugs intracellularly.SFRH/BD/81479/2011 and SFRH/BD/81269/2011 scholarships from Fundação para a Ciência e a Tecnologia (FCT). This work has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. This work was supported by FEDER through POFC-COMPETE and by Portuguese funds from FCT through the project PEst-OE/BIA/UI4050/2014

Fruit-based carbon dots as fluorescent probes: in vitro and in vivo toxicity evaluation

MNE2017 - 43rd International Conference on Micro and Nanoengineering (Conference Booklet)New solutions for biomedical purposes are a major focus of interest for the development of new nanomaterials. In comparison to traditional metal-based quantum dots, photoluminescent carbon-dots are greater in terms of aqueous solubility, chemical inertness, simple modification and fluorescent proprieties. In this work C-dots derived from kiwi and avocado and synthesized using a green method were evaluated for their toxicity and bioimaging in vitro and in vivo. Normal and cancer cells lines, and zebrafish embryos were used as in vitro an in vivo models, respectively, either for toxicological profile and confocal imaging. Both C-dots showed toxicity profiles in the range of mg/mL concentrations inducing delays in zebrafish embryos development. Concentrations of kiwi and avocado C-dots used for confocal bioimaging were adjusted below the NOAEL accordingly.info:eu-repo/semantics/publishedVersio

Rapid-behaviour responses as a reliable indicator of estrogenic chemical toxicity in zebrafish juveniles

a b s t r a c t Whereas biochemical and molecular parameters have been well recognised as important ''signposts'' of individual disturbance to endocrine disrupting chemical's (EDCs) exposure, behavioural endpoints are yet greatly overlooked as a routine tool in environmental risk assessment of EDCs. However, life histories are intimately associated with numerous inter-and intra-specific interactions, which invariably depend on the performance of effective behaviours. Within fish species, one of the most important factors influencing energy turnover earlier in the development is locomotor activity. This essential trait reflects the organism's ability to generate and coordinate the metabolic energy required for both reproductive and non-reproductive behaviours. Inappropriate movement responses due to toxic effects of contaminants may ultimately impact important ecological variables. Therefore, in the present study, the swimming bursts of zebrafish juveniles exposed for 40 d to the synthetic estrogen ethinylestradiol (EE 2 ), tested at environmentally relevant concentrations (nominal concentrations of 0.5, 1 and 2 ng L À1 ), were investigated in order to address the potential of rapid-behaviour patterns as an effective response indicator of estrogenic endocrine disrupting chemical's exposure. This synthetic estrogen was selected due to its high prevalence in aquatic ecosystems, ability to mimic natural estrogens and proven record of causing negative effects in fish reproduction. The behavioural responses were compared with established endpoints used in the screening of EE 2 effects at adulthood. Results indicate that zebrafish juveniles' swimming activity was significantly decreased upon EE 2 exposure. Since reduced locomotion of zebrafish may impact foraging, predator avoidance, drift and transport, and even interfere with social and reproductive behaviours, a fitness decline of wild fish populations can ultimately be hypothesized. Furthermore, behavioural endpoints were found to display higher sensitivity to EE 2 than either vitellogenin gene induction or reproductive parameters determined at adulthood. Overall, the findings of this work not only demonstrate the power of high-throughput behavioural responses, able to act as sensitive early warning signals of EDC exposure, but also highlight the potential of behavioural endpoints in providing a more comprehensive and non-invasive measure of EDC's exposure