Catarina Rodrigues

Development of functionalized nanoparticles for vaccine delivery to dendritic cells: a mechanistic approach

AIM Produce biodegradable nanoparticles to target antigen-presenting cells (APCs) and evaluate their potential to be used as a vaccine delivery system. MATERIALS & METHODS Untargeted PEGylated poly(d,l-lactic-co-glycolide)-based nanoparticles and mannose-grafted nanoparticles were formulated and physicochemically characterized. Immortalized and primary APCs were used to study nanoparticle internalization patterns. The endocytic pathways and intracellular trafficking followed by nanoparticles were also investigated. RESULTS & DISCUSSION Nanoparticles displayed mannose residues available for binding at the nanoparticle surface. Different nanoparticle internalization patterns by immortalized and primary APCs were verified. Macropinocytosis, clathrin-mediated endocytosis, caveolin- and lipid raft-dependent endocytosis are involved in nanoparticles internalization. Nanoparticles demonstrate both endolysosomal and cytosolic localizations and a tendency to accumulate nearby the endoplasmic reticulum. CONCLUSION The developed nanoparticles might drive antigens to be presented through MHC class I and II molecules to both CD8(+) and CD4(+) T cells, favoring a complete and coordinated immune response.

Targeting the Erythrocytic and Liver Stages of Malaria Parasites with s‐Triazine‐Based Hybrids

A diversity‐oriented library of s‐triazine‐based hybrids was screened for activity against the chloroquine‐resistant Plasmodium falciparum W2 strain. The most striking result was sub‐micromolar activity against cultured erythrocytic‐stage parasites of hybrid molecules containing one or two 8‐aminoquinoline moieties. These compounds were not clearly toxic to human cells. The most effective blood‐schizontocidal s‐triazine derivatives were then screened for activity against the liver stage of malaria parasites. The s‐triazine hybrid containing two 8‐aminoquinoline moieties and one chlorine atom emerged as the most potent against P. berghei liver‐stage infection, active in the low nanomolar region, combined with good metabolic stability in rat liver microsomes. These results indicate that s‐triazine‐8‐aminoquinoline‐based hybrids are excellent starting points for lead optimization as dual‐stage antimalarials.

Rational design of nanoparticles towards targeting antigen-presenting cells and improved T cell priming

ABSTRACT Vaccination is a promising strategy to trigger and boost immune responses against cancer or infectious disease. We have designed, synthesized and characterized aliphatic‐polyester (poly(lactic‐co‐glycolic acid) (PLGA) nanoparticles (NP) to investigate how the nature of protein association (adsorbed versus entrapped) and polymer/surfactant concentrations impact on the generation and modulation of antigen‐specific immune responses. The ability of the NP formulations to target dendritic cells (DC), be internalized and activate the T cells was characterized and optimized in vitro and in vivo using markers of DC activation and co‐stimulatory molecules. Ovalbumin (OVA) was used as a model antigen in combination with the engraftment of CD4+ and CD8+ T cells, carrying a transgenic OVA‐responding T cell receptor (TCR), to trace and characterize the activation of antigen‐specific CD4+ and CD8+ lymph node T cells upon NP vaccination. Accordingly, the phenotype and frequency of immune cell stimulation induced by the NP loaded with OVA, isolated or in combination with synthetic unmethylated cytosine‐phosphate‐guanine (CpG) oligodeoxynucleotide (ODN) motifs, were characterized. DC‐NP interactions increased with incubation time, presenting internalization values between 50 and 60% and 30–40%, in vitro and in vivo, respectively. Interestingly, animal immunization with antigen‐adsorbed NP up‐regulated major histocompatibility complex (MHC) class II (MHCII), while NP entrapping the antigen up‐regulated MHCI, suggesting a more efficient cross‐presentation. On the other hand, rather surprisingly, the surfactant used in the NP formulation had a major impact on the activation of antigen presenting cells (APC). In fact, DC collected from lymph nodes of animals immunized with NP prepared using poly(vinil alcohol) (PVA), as a surfactant, expressed significantly higher levels of CD86, MHCI and MHCII. In addition, those NP prepared with PVA and co‐entrapping OVA and the toll‐like receptor (TLR) ligand CpG, induced the most profound antigen‐specific T cell response, by both CD4+ and CD8+ T cells, in vivo. Overall, our data reveal the impact of NP composition and surface properties on the type and extension of induced immune responses. Deeper understanding on the NP‐immune cell crosstalk can guide the rational development of nano‐immunotherapeutic systems with improved and specific therapeutic efficacy and avoiding off‐target effects.

A 1,3,5-triazine based polymer as a nonlinear near-infrared antenna for two-photon activated volumetric optical memory devices

The ability of a 1,3,5-triazine based polymer to work as a nonlinear near-infrared (NIR) antenna in functional optical materials is discussed. The push–pull polymer is composed of alternating electron acceptor cores of 2,4,6-tris(thiophen-2-yl)-1,3,5-triazine, bridged by electron donor groups of 9,9-dihexyl-9H-fluorene. It has been tailored for high two-photon absorption and efficient up-conversion of the excitation energy into fluorescence emission. These properties are here explored in the operation of prototypical storage material for volumetric nonlinear optical memory devices. Multilayer data storage is demonstrated in composite films of photochromic (PC) molecules doped with the polymer. The two-photon excitation of the polymer triggers a unidirectional isomerization of the PC molecules (a cycloreversion process from the closed to the open isomer) via Forster resonant energy transfer. Data recording with as little as 1 ms of exposure time and 1 mW of 740 nm radiation is accomplished.

Excited-state proton transfer of fluorescein anion as an ionic liquid component.

Fluorescent ionic liquids (FILs) incorporating the fluorescein anion have been prepared by anion exchange of the parent quaternary ammonium chloride (Quat(+)Cl(-)) ionic liquid. By controlling the molar ratio of fluorescein to Quat(+)Cl(-), ionic liquids incorporating different prototropic forms of fluorescein were prepared. The 1:1 molar ratio ionic liquid (FIL1) is essentially composed of monoanionic fluorescein, while dianionic fluorecein is predominant in the FIL with a 1:2 molar ratio (FIL2). The fluorescence excitation spectrum of FIL2 is markedly different from its absorption spectrum. Absorption features the fluorescein dianion, while the excitation spectrum is exclusively due to the monoanion. In FIL1, the absorption and excitation spectra are both characteristic of the monoanion. In both FILs, emission of the dianion is observed upon excitation of the monoanion. This unusual behavior is interpreted in the context of a fast deprotonation of the monoanion in the excited state. The presence of residual water in the ionic liquid is important for the proton transfer process. By lowering the pH of FIL1, the transient proton transfer is inhibited, and the emission of the monoanion could be observed. The FILs have completely different spectroscopic properties from solvated fluorescein in Quat(+)Cl(-), where the prototropic equilibrium is shifted toward the neutral forms.

Valorization of Oleuropein via Tunable Acid-Promoted Methanolysis

The acid-promoted methanolysis of oleuropein was studied using a variety of homogeneous and heterogeneous acid catalysts. Exclusive cleavage of the acetal bond between the glucoside and the monoterpene subunits or further hydrolysis of the hydroxytyrosol ester and subsequent intramolecular rearrangement were observed upon identification of the most efficient catalyst and experimental conditions. Furthermore, selected conditions were tested using oleuropein under continuous flow and using a crude mixture extracted from olive leaves under batch. Formation of (-)-methyl elenolate was also observed in this study, which is a reported precursor for the synthesis of the antihypertensive drug (-)-ajmalicine.

Phenotypic screening identifies a new oxazolone inhibitor of necroptosis and neuroinflammation

Necroptosis is a regulated form of necrosis, which may be critical in the pathogenesis of neurodegenerative diseases. Neuroinflammation, characterized by the activation of glial cells such as microglia, is closely linked with neurodegenerative pathways and constitutes a major mechanism of neural damage and disease progression. Importantly, inhibition of necroptosis results in disease improvement, unveiling an alternative approach for therapeutic intervention. In the present study, we screened a small library of new molecules, potentially inhibitors of necroptosis, using two cellular models of necroptosis. A new oxazolone, Oxa12, reduced tumour necrosis factor α (TNF-α)-induced necroptosis in mouse L929 fibrosarcoma cells. Notably, Oxa12 strongly inhibited zVAD-fmk-induced necroptosis in murine BV2 microglial cells. Moreover, Oxa12 blocked phosphorylation of mixed-lineage kinase domain-like protein (MLKL), and interfered with necrosome complex formation, indicating that Oxa12 targets components upstream of MLKL. In fact, in silico molecular docking studies revealed that Oxa12 is occupying a region similar to the 1-aminoisoquinoline type II kinase inhibitor inside the receptor-interacting protein 1 (RIP1) kinase domain. Finally, in microglial cells, Oxa12 attenuated zVAD-fmk- and lipopolysaccharide (LPS)-induced inflammatory processes, as revealed by a marked decrease of TNF-α and/or IL-1β expression. More specifically, Oxa12 negatively targeted c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) pathways, as well as NF-κB activation. Overall, we identified a strong lead inhibitor of necroptosis that is also effective at reducing inflammation-associated events. Oxa12 is a promising candidate molecule for further development to target disease states dependent on RIP kinase activity.

Optimization of protein loaded PLGA nanoparticle manufacturing parameters following a quality-by-design approach

This paper discusses the development of a multivariate-based regression model for estimating the critical attributes to establish a design-space for poly(lactic-co-glycolic acid) (PLGA) nanoparticles formulated by a double emulsion–solvent evaporation method. A three-level, full factorial experimental design is used to assess the impact of three different manufacturing conditions (polymer viscosity, surfactant concentration and amount of model antigen ovalbumin) on five critical particle attributes (zeta potential, polydispersity index, hydrodynamic diameter, loading capacity and entrapment efficiency). The optimized formulation was achieved with a viscosity of 0.6 dl g−1, a surfactant concentration of 11% (w/v) in the internal phase and 2.5% (w/w) of ovalbumin. The design-space that is satisfied for nanoparticles with the targeted attributes was obtained with a polymer viscosity between 0.4 and 0.9 dl g−1, a surfactant concentration ranging from 8 to 15% (w/v) and 2.5% (w/w) of ovalbumin. The nanoparticles were spherical and homogenous and were extensively taken up by JAWS II murine immature dendritic cells without affecting the viability of these phagocytic cells. Better understanding was achieved by multivariate regression to control process manufacturing to optimize PLGA nanoparticle formulation. Utilization of multivariate regression with a defined control space is a good tool to meet product specifications, particularly over a narrow variation range.

Acquisition of the fexA and cfr genes in Staphylococcus pseudintermedius during florfenicol treatment of canine pyoderma

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) isolates are usually multidrug-resistant, posing a serious therapeutic challenge in small animal veterinary practice [1]. Chloramphenicol has been recommended for the treatment of MRSP infections as a second-line agent; however, severe adverse effects have been described in pets [1]. A report described that 57% of MRSP isolated from dogs were resistant to chloramphenicol owing to the presence of the catpC221 gene [2]. Florfenicol, however, has less adverse effects and cannot be inactivated by the chloramphenicol acetyltransferases (CAT enzymes) produced by chloramphenicol-resistant strains [3]. In staphylococci, florfenicol resistance is mediated either by the fexA gene (coding for a phenicol-specific efflux pump), the cfr gene (coding for an rRNA methyltransferase) or the optrA gene (coding for an ABC transporter), all of which mediate combined resistance to phenicols [3,4]. The fexA gene has been detected either as part of the small non-conjugative transposon Tn558 or in combination with the cfr gene in transposition-deficient Tn558 variants in several staphylococcal species [3,4]. In S. pseudintermedius, a variant of the fexA gene (fexAv) has been described that confers resistance only to chloramphenicol owing to two amino acid substitutions (Gly33Ala and Ala37Val) in the region responsible for substrate binding [3]. In 2011 and 2013, two dogs with severe pyoderma were presented at the teaching hospital of the Faculty of Veterinary Medicine, University of Lisbon (FMV-UL) (Lisbon, Portugal). The diagnosis of pyoderma was achieved shortly after the dogs entered the dermatology consultation by compatible cytology (neutrophils, free cocci and intracellular cocci in inflammatory neutrophils) and clinical evaluation. Both animals had a previous history of multiple courses of amoxicillin/clavulanic acid treatment. Skin swabs were taken and were sent to the laboratory. Susceptibility to a range of antimicrobial agents was determined by broth microdilution, and resistance genes were detected by PCR [3–5]. The two isolates were identified as MRSP and were multidrugresistant but were susceptible to florfenicol (Table 1). Treatment of both dogs was initiated with florfenicol (Nuflor; 30 mg/kg subcutaneously every 12 h). The dogs were frequently observed by the attending veterinarian and after a treatment course of 3–6 months the dogs returned for one more follow-up consultation and the remaining skin lesions were swabbed again. At this point, Dog 1 still had an MRSP, whilst Dog 2 had a methicillin-susceptible S. pseudintermedius strain. Resistance to chloramphenicol and