Immobilization of Alcalase on Silica Supports Modified with Carbosilane and PAMAM Dendrimers.

Enzyme immobilization is a powerful strategy for enzyme stabilization and recyclability. Materials covered with multipoint molecules are very attractive for this goal, since the number of active moieties to attach the enzyme increases with respect to monofunctional linkers. This work evaluates different dendrimers supported on silica to immobilize a protease enzyme, Alcalase. Five different dendrimers were employed: two carbosilane (CBS) dendrimers of different generations (SiO2-G0Si-NH2 and SiO2-G1Si-NH2), a CBS dendrimer with a polyphenoxo core (SiO2-G1O3-NH2), and two commercial polyamidoamine (PAMAM) dendrimers of different generations (SiO2-G0PAMAM-NH2 and SiO2-G1PAMAM-NH2). The results were compared with a silica support modified with a monofunctional molecule (2-aminoethanethiol). The effect of the dendrimer generation, the immobilization conditions (immobilization time, Alcalase/SiO2 ratio, and presence of Ca2+ ions), and the digestion conditions (temperature, time, amount of support, and stirring speed) on Alcalase activity has been evaluated. Enzyme immobilization and its activity were highly affected by the kind of dendrimer and its generation, observing the most favorable behavior with SiO2-G0PAMAM-NH2. The enzyme immobilized on this support was used in two consecutive digestions and, unlike CBS supports, it did not retain peptides released in the digestion.

The Antibacterial Effect of PEGylated Carbosilane Dendrimers on P. aeruginosa Alone and in Combination with Phage-Derived Endolysin.

The search for new microbicide compounds is of an urgent need, especially against difficult-to-eradicate biofilm-forming bacteria. One attractive option is the application of cationic multivalent dendrimers as antibacterials and also as carriers of active molecules. These compounds require an adequate hydrophilic/hydrophobic structural balance to maximize the effect. Herein, we evaluated the antimicrobial activity of cationic carbosilane (CBS) dendrimers unmodified or modified with polyethylene glycol (PEG) units, against planktonic and biofilm-forming P. aeruginosa culture. Our study revealed that the presence of PEG destabilized the hydrophilic/hydrophobic balance but reduced the antibacterial activity measured by microbiological cultivation methods, laser interferometry and fluorescence microscopy. On the other hand, the activity can be improved by the combination of the CBS dendrimers with endolysin, a bacteriophage-encoded peptidoglycan hydrolase. This enzyme applied in the absence of the cationic CBS dendrimers is ineffective against Gram-negative bacteria because of the protective outer membrane shield. However, the endolysin-CBS dendrimer mixture enables the penetration through the membrane and then deterioration of the peptidoglycan layer, providing a synergic antimicrobial effect.

Immune cell arrival kinetics to peritoneum and role during murine-experimental trichomoniasis.

Trichomonas vaginalis causes trichomoniasis, an inflammatory process related to an increased rate of HIV transmission. In order to study T. vaginalis infection response in a microorganism-free environment, an infection model was established providing a host­parasite interaction system useful to study the interplay between immune cells and the parasite. Infected mice peritoneal cells were immunophenotyped at different times after infection using flow cytometry. Neutrophils and macrophages showed the most relevant increase from third to 12th day post-infection. A high number of B lymphocytes were present on 15th day post-infection, and an increase in memory T cells was observed on sixth day post-infection. The levels of NO increased at day 10 post-infection; no significant influence was observed on T. vaginalis clearance. Increased viability of T. vaginalis was observed when the NETs inhibitors, metformin and Cl− amidine, were administrated, highlighting the importance of this mechanism to control parasite infection (43 and 86%, respectively). This report presents a comprehensive cell count of the immune cells participating against trichomoniasis in an in vivo interaction system. These data highlight the relevance of innate mechanisms such as specific population changes of innate immune cells and their impact on the T. vaginalis viability.

Promising PEGylated cationic dendrimers for delivery of miRNAs as a possible therapy against HIV-1 infection.

BACKGROUND: The appearance of resistance against new treatments and the fact that HIV-1 can infect various cell types and develop reservoirs and sanctuaries makes it necessary to develop new therapeutic approaches to overcome those failures. RESULTS: Studies of cytotoxicity, genotoxicity, complexes formation, stability, resistance, release and particle size distribution confirmed that G2-SN15-PEG, G3-SN31-PEG, G2-SN15-PEG-FITC and G3-SN31-PEG-FITC dendrimers can form complexes with miRNAs being biocompatible, stable and conferring protection to these nucleic acids. Confocal microscopy and flow cytometry showed effective delivery of these four dendrimers into the target cells, confirming their applicability as delivery systems. Dendriplexes formed with the dendrimers and miRNAs significantly inhibited HIV-1 infection in PBMCs. CONCLUSIONS: These dendrimers are efficient delivery systems for miRNAs and they specifically and significantly improved the anti-R5-HIV-1 activity of these RNA molecules.

Cationic Carbosilane Dendritic Systems as Promising Anti-Amyloid Agents in Type†2 Diabetes.

The most common denominator of many of the neurodegenerative diseases is badly folded protein accumulation, which results in the formation of insoluble protein deposits located in different parts of the organism, causing cell death and tissue degeneration. Dendritic systems have turned out to be a promising new therapeutic approach for the treatment of these diseases due to their ability to modulate the folding of these proteins. With this perspective, and focused on type 2 diabetes (T2D), characterized by the presence of deposits containing the amyloidogenic islet amyloid polypeptide (IAPP), we demonstrate how different topologies of cationic carbosilane dendrimers inhibit the formation of insoluble protein deposits in pancreatic islets isolated from transgenic Tg-hIAPP mice. Also, the results obtained by the modification of dendritic carbosilane wedges with the chemical chaperone 4-phenylbutyric acid (4-PBA) at the focal point confirmed their potential as anti-amyloid agents with a concentration efficiency in their therapeutic action five orders of magnitude lower than that observed for free 4-PBA. Computational studies, which determined the main interaction between IAPP and dendrimers at the atomic level, support the experimental work.

Fine-Tuning the Interaction and Therapeutic Effect of Cu(II) Carbosilane Metallodendrimers in Cancer Cells: An In Vitro Electron Paramagnetic Resonance Study.

Copper(II) carbosilane metallodendrimers are promising nanosized anticancer metallodrugs. The precise control on their design enables an accurate structure-to-activity study. We hypothesized that different structural features, such as the dendrimer generation and metal counterion, modulate the interaction with tumor cells, and subsequently, the effectivity and selectivity of the therapy. A computer-aided analysis of the electron paramagnetic resonance (EPR) spectra allowed us to obtain dynamical and structural details on the interactions over time between the dendrimers and the cells, the myeloid U937 tumor cells and peripheral blood mononuclear cells (PBMC). The intracellular fate of the metallodendrimers was studied through a complete in vitro evaluation, including cytotoxicity, cytostaticity, and sublethal effects regarding mitochondria function, lysosomal compartments, and autophagic organelle involvement. EPR results confirmed a higher membrane stabilization for chloride dendrimers and low generation complexes, which ultimately influence the metallodrug uptake and intracellular fate. The in vitro evaluation revealed that Cu(II) metallodendrimers are cytostatic and moderate cytotoxic agents for U937 tumor cells, inducing death processes through the mitochondria-lysosome axis as well as autophagic vacuole formation, while barely affecting healthy monocytes. The study provided valuable insight into the mechanism of action of these nanosized metallodrugs and relevant structural parameters affecting the activity.

Silver Nanoparticles Surface-Modified with Carbosilane Dendrons as Carriers of Anticancer siRNA.

Gene therapy is a promising approach in cancer treatment; however, current methods have a number of limitations mainly due to the difficulty in delivering therapeutic nucleic acids to their sites of action. The application of non-viral carriers based on nanomaterials aims at protecting genetic material from degradation and enabling its effective intracellular transport. We proposed the use of silver nanoparticles (AgNPs) surface-modified with carbosilane dendrons as carriers of anticancer siRNA (siBcl-xl). Using gel electrophoresis, zeta potential and hydrodynamic diameter measurements, as well as transmission electron microscopy, we characterized AgNP:siRNA complexes and demonstrated the stability of nucleic acid in complexes in the presence of RNase. Hemolytic properties of free silver nanoparticles and complexes, their effect on lymphocyte proliferation and cytotoxic activity on HeLa cells were also examined. Confocal microscopy proved the effective cellular uptake of complexes, indicating the possible use of this type of silver nanoparticles as carriers of genetic material in gene therapy.

Carbosilane Dendron-Peptide Nanoconjugates as Antimicrobial Agents.

Over the last decades, multidrug-resistant bacteria have emerged and spread, increasing the number of bacteria, against which commonly used antibiotics are no longer effective. It has become a serious public health problem whose solution requires medical research in order to explore novel effective antimicrobial molecules. On the one hand, antimicrobial peptides (AMPs) are regarded as good alternatives because of their generally broad-spectrum activities, but sometimes they can be easily degraded by the organism or be toxic to animal cells. On the other hand, cationic carbosilane dendrons, whose focal point can be functionalized in many different ways, have also shown good antimicrobial activity. In this work, we synthetized first- and second-generation cationic carbosilane dendrons with a maleimide molecule on their focal point, enabling their functionalization with three different AMPs. After different microbiology studies, we found an additive effect between first-generation dendron and AMP3 whose study reveals three interesting effects: (i) bacteria aggregation due to AMP3, which could facilitate bacteria detection or even contribute to antibacterial activity by preventing host cell attack, (ii) bacteria disaggregation capability of second-generation cationic dendrons, and (iii) a higher AMP3 aggregation ability when dendrons were added previously to peptide treatment. These compounds and their different effects observed over bacteria constitute an interesting system for further mechanism studies.

Anticancer Activity of Dendriplexes against Advanced Prostate Cancer from Protumoral Peptides and Cationic Carbosilane Dendrimers.

The interaction of neuropeptides, vasoactive intestinal peptide (VIP), or growth hormone-releasing hormone (GHRH), with a cationic carbosilane dendrimer forms dendriplexes with antitumoral behavior in advanced prostate cancer cells PC3. At the concentrations used for dendriplexes formation, the free peptides were protumoral and prometastatic in advanced prostate cancer, while dendrimer only showed low cytotoxicity, but did not avoid the metastatic behavior of PC3 cells. However, these nanoplexes favored also cell adhesion and avoided cell migration. Also, the dendriplexes were not toxic for no tumoral prostate cells (RPWE-1) or fibroblasts. The use of labeled GHRH peptide (rhodamine labeled) and a dendrimer (fluorescein labeled) allowed us to observe that both systems reach the intracellular milieu after dendriplex formation. The treatment of PC3 cells with the nanoplexes reduced expression of vascular endothelial growth factor (VEGF) and cyclic adenosine monophosphate (cAMP). Molecular modeling analysis highlights the important contribution of the carbosilane framework in the stabilization of the dendriplex, since dendrimer interacts with a peptide region where hydrophobic amino acids are presented.

Synthesis and in vitro activity of new biguanide-containing dendrimers on pathogenic isolates of Acanthamoeba polyphaga and Acanthamoeba griffini.

The genus Acanthamoeba can cause Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). The treatment of these illnesses is hampered by the existence of a resistance stage that many times causes infection relapses. In an attempt to add new agents to our chemotherapeutic arsenal against acanthamebiasis, two Acanthamoeba isolates were treated in vitro with newly synthesized biguanide dendrimers. Trophozoite viability analysis and ultrastructural studies showed that dendrimers prevent encystment by lysing the cellular membrane of the amoeba. Moreover, one of the dendrimers showed low toxicity when tested on mammalian cell cultures, which suggest that it might be eventually used as an amoebicidal drug or as a disinfection compound in contact lens solutions.

Gold nanoparticles coated with carbosilane dendrons in protein sample preparation.

The feasibility of using carbosilane dendronized gold nanoparticles (GNPs) for protein sample preparation was evaluated. Three different dendrons with three different generations (1G, 2G, and 3G) were employed to modify the GNPs, viz. sulfonate terminated (STC-GNPs), carboxylate terminated (CTC-GNPs), and trimethylammonium terminated (ATC-GNPs) dendrons. The synthesis of the CTC-GNP is described. The other dendronized GNPs were synthesized using previously described routes. Bovine serum albumin, lysozyme, and myoglobin were employed to study the potential of GNPs to interact with proteins. The interaction between the GNPs and the proteins was evaluated using fluorescence spectroscopy and polyacrylamide gel electrophoresis. The CTC-GNPs and STC-GNPs under acidic and neutral conditions, respectively, promoted the establishment of electrostatic interactions with positively charged proteins. Proteins from 10 to 75 kDa molecular weights interacted with GNPs at protein: nanoparticle ratios of 1:0.25. The GNPs were applied to the extraction of proteins from a peach seed. In the authors' perception, the method is a clean alternative to established extraction methods based on the use of organic or polluting chemicals. Graphical abstract Schematic representation of the interaction of peach seeds proteins and carbosilane dendron coated gold nanoparticles, and the electrophoretic profiles of extracted proteins.

Brain substrates of unhealthy versus healthy food choices: influence of homeostatic status and body mass index.

BACKGROUND/OBJECTIVES: Unhealthy dietary choices are a major contributor to harmful weight gain and obesity. This study interrogated the brain substrates of unhealthy versus healthy food choices in vivo, and evaluated the influence of hunger state and body mass index (BMI) on brain activation and connectivity. SUBJECTS/METHODS: Thirty adults (BMI: 18-38 kg m-2) performed a food-choice task involving preference-based selection between beverage pairs consisting of high-calorie (unhealthy) or low-calorie (healthy) options, concurrent with functional magnetic resonance imaging (fMRI). Selected food stimuli were delivered to participants using an MRI-compatible gustometer. fMRI scans were performed both after 10-h fasting and when sated. Brain activation and hypothalamic functional connectivity were assessed when selecting between unhealthy-healthy beverage pairings, relative to unhealthy-unhealthy and healthy-healthy options. Results were considered significant at cluster-based family-wise error corrected P<0.05. RESULTS: Selecting between unhealthy and healthy foods elicited significant activation in the hypothalamus, the medial and dorsolateral prefrontal cortices, the anterior insula and the posterior cingulate. Hunger was associated with higher activation within the ventromedial and dorsolateral prefrontal cortices, as well as lower connectivity between the hypothalamus and both the ventromedial prefrontal cortex and dorsal striatum. Critically, people with higher BMI showed lower activation of the hypothalamus-regardless of hunger state-and higher activation of the ventromedial prefrontal cortex when hungry. CONCLUSIONS: People who are overweight and obese have weaker activation of brain regions involved in energy regulation and greater activation of reward valuation regions while making choices between unhealthy and healthy foods. These results provide evidence for a shift towards hedonic-based, and away from energy-based, food selection in obesity.

Prevalence and antifungal susceptibility of Candida species among pregnant women attending a school maternity at Natal, Brazil.

Vulvovaginal candidiasis (VVC) is an inflammatory disease of the vulva and vagina caused by different yeasts of the genus Candida which is responsible for infection in pregnant patients who attended Maternidade Escola Januário Cicco, Rio Grande do Norte, Brazil. From 41 samples, 19 yeasts were identified phenotypically as Candida albicans and one as Candida glabrata which is reported as the non-albicans species most frequently isolated from vulvovaginitis. The susceptibility to selected antifungal agents (flucytosine, fluconazole, voriconazole, amphotericin B, caspofungin and micafungin) was determined, and the association between patient-related signs and symptoms aided the construction of an epidemiological profile. Antifungal susceptibility testing performed by automated method showed that all strains were sensitive to the drugs tested, including the C. glabrata specimen despite its known resistance or dose-dependent susceptibility to azole derivatives. Regarding patient signs and symptoms, no statistically significant association between these and the establishment of VVC was found. It can be concluded that the laboratorial diagnosis of VVC is necessary prior to the administration of treatment, since only 48·78% of the patients had VVC but for all of them antifungal therapy were prescribed. SIGNIFICANCE AND IMPACT OF THE STUDY: Vulvovaginal candidiasis (VVC) is a problem that affects a significant number of pregnant women worldwide. This type of fungal infection generates great discomfort due to the symptomatology and difficulties of diagnosis and treatment. In view of the scarcity of data in the State of Rio Grande do Norte, Brazil, regarding studies carried out on fungal populations of the genus Candida associated with VVC in pregnant women, this study considered relevant, the phenotypic and genotypic identification of the species, to estimate the prevalence, to determine their susceptibility to the antifungal and to correlate with signs and symptoms.

Amphiphilic carbosilane dendrons as a novel synthetic platform toward micelle formation.

A novel family of amphiphilic ionic carbosilane dendrons containing fatty acids at the focal point were synthesized and characterized. They spontaneously self-assembled in aqueous solution into micelles both in the absence and presence of salt, as confirmed by surface tension, conductivity, and DLS measurements. Dendron based micelles have spherical shapes and increase in size on decreasing dendron generation. These dendritic micelles have been demonstrated to be able to form complexes with therapeutic macromolecules such as siRNA and show a high loading capacity for drugs such as procaine, suggesting their potential use as nanocarriers for therapeutics.

Dendronized Anionic Gold Nanoparticles: Synthesis, Characterization, and Antiviral Activity.

Anionic carbosilane dendrons decorated with sulfonate functions and one thiol moiety at the focal point have been used to synthesize water-soluble gold nanoparticles (AuNPs) through the direct reaction of dendrons, gold precursor, and reducing agent in water, and also through a place-exchange reaction. These nanoparticles have been characterized by NMR spectroscopy, TEM, thermogravimetric analysis, X-ray photoelectron spectroscopy (XPS), UV/Vis spectroscopy, elemental analysis, and zeta-potential measurements. The interacting ability of the anionic sulfonate functions was investigated by EPR spectroscopy with copper(II) as a probe. Different structures and conformations of the AuNPs modulate the availability of sulfonate and thiol groups for complexation by copper(II). Toxicity assays of AuNPs showed that those produced through direct reaction were less toxic than those obtained by ligand exchange. Inhibition of HIV-1 infection was higher in the case of dendronized AuNPs than in dendrons.

Improved Efficiency of Ibuprofen by Cationic Carbosilane Dendritic Conjugates.

In order to improve the efficiency of the anti-inflammatory drug ibuprofen, cationic carbosilane dendrimers and dendrons with ibuprofen at their periphery or at their focal point, respectively, have been synthesized, and the release of the drug was studied using HPLC. Macrophages were used to evaluate the anti-inflammatory effect of the ibuprofen-conjugated dendritic systems and compared with mixtures of non-ibuprofen dendritic systems in the presence of the drug. The cationic ibuprofen-conjugated dendron was the compound that showed higher anti-inflammatory properties. It reduces the LPS-induced COX-2 expression and decreases the release of several inflammatory cytokines such as TNFα, IL-1ß, IL-6, and CCL3. These results open new perspectives in the use of these compounds as drug carriers.

Novel Water-Soluble Mucoadhesive Carbosilane Dendrimers for Ocular Administration.

The purpose of this research was to determine the potential use of water-soluble anionic and cationic carbosilane dendrimers (generations 1-3) as mucoadhesive polymers in eyedrop formulations. Cationic carbosilane dendrimers decorated with ammonium -NH3(+) groups were prepared by hydrosylilation of Boc-protected allylamine and followed by deprotection with HCl. Anionic carbosilane dendrimers with terminal carboxylate groups were also employed in this study. In vitro and in vivo tolerance studies were performed in human ocular epithelial cell lines and rabbit eyes respectively. The interaction of dendrimers with transmembrane ocular mucins was evaluated with a surface biosensor. As proof of concept, the hypotensive effect of a carbosilane dendrimer eyedrop formulation containing acetazolamide (ACZ), a poorly water-soluble drug with limited ocular penetration, was tested after instillation in normotensive rabbits. The methodology used to synthesize cationic dendrimers avoids the difficulty of obtaining neutral -NH2 dendrimers that require harsher reaction conditions and also present high aggregation tendency. Tolerance studies demonstrated that both prototypes of water-soluble anionic and cationic carbosilane dendrimers were well tolerated in a range of concentrations between 5 and 10 µM. Permanent interactions between cationic carbosilane dendrimers and ocular mucins were observed using biosensor assays, predominantly for the generation-three (G3) dendrimer. An eyedrop formulation containing G3 cationic carbosilane dendrimers (5 µM) and ACZ (0.07%) (289.4 mOsm; 5.6 pH; 41.7 mN/m) induced a rapid (onset time 1 h) and extended (up to 7 h) hypotensive effect, and led to a significant increment in the efficacy determined by AUC0(8h) and maximal intraocular pressure reduction. This work takes advantage of the high-affinity interaction between cationic carbosilane dendrimers and ocular transmembrane mucins, as well as the tensioactive behavior observed for these polymers. Our results indicate that low amounts of cationic carbosilane dendrimers are well tolerated and able to improve the hypotensive effect of an acetazolamide solution. Our results suggest that carbosilane dendrimers can be used in a safe range of concentrations to enhance the bioavailability of drugs topically administered in the eye.

Mesoporous Silica Nanoparticles Decorated with Carbosilane Dendrons as New Non-viral Oligonucleotide Delivery Carriers.

A novel nanosystem based on mesoporous silica nanoparticles covered with carbosilane dendrons grafted on the external surface of the nanoparticles is reported. This system is able to transport single-stranded oligonucleotide into cells, avoiding an electrostatic repulsion between the cell membrane and the negatively charged nucleic acids thanks to the cationic charge provided by the dendron coating under physiological conditions. Moreover, the presence of the highly ordered pore network inside the silica matrix would make possible to allocate other therapeutic agents within the mesopores with the aim of achieving a double delivery. First, carbosilane dendrons of second and third generation possessing ammonium or tertiary amine groups as peripheral functional groups were prepared. Hence, different strategies were tested in order to obtain their suitable grafting on the outer surface of the nanoparticles. As nucleic acid model, a single-stranded DNA oligonucleotide tagged with a fluorescent Cy3 moiety was used to evaluate the DNA adsorption capacity. The hybrid material functionalised with the third generation of a neutral dendron showed excellent DNA binding properties. Finally, the cytotoxicity as well as the capability to deliver DNA into cells, was tested in vitro by using a human osteoblast-like cell line, achieving good levels of internalisation of the vector DNA/carbosilane dendron-functionalised material without affecting the cellular viability.

Synthesis of new anionic carbosilane dendrimers via thiol-ene chemistry and their antiviral behaviour.

A synthetic strategy has been developed for the preparation of anionic carbosilane dendrimers bearing sulfonate or carboxylate groups at their periphery by means of thiol-ene chemistry. It offers significant advantages, such as milder reaction conditions, shorter reaction times and more facile purification methods, when compared with other synthetic protocols used previously, e.g. hydrosilylation followed by a Michael-type addition or azide-alkyne coupling reactions. Molecular dynamics simulations of the second generation anionic dendrimers addressing shape and size effects of the terminal groups and conformational variability indicated that the core eccentricity and flexibility might need to be taken into account for toxicity and interaction with viral and/or cellular receptors, respectively. The biocompatibility of anionic carbosilane dendrimers has been explored showing differences between silicon-cored and polyphenoxo-cored dendrimers. In addition, silicon-cored dendrimers achieved 85-90% of HIV inhibition without inducing inflammation or vaginal irritation in mice, which makes them likely candidates for readily available, good and safe topical vaginal microbicides against HIV.

Enhanced HPLC Method for Boar Taint Quantification.

Boar taint is an unpleasant odour found in the carcasses of entire male pigs, resulting from androstenone and skatole accumulation during pubertal development, and impacting pork quality. This study proposes the validation of an adapted chromatographic method for quantifying skatole and androstenone in the pigs' liquid fat using fluorescence detection. A good chromatographic separation was achieved, with skatole (SKA) and androstenone (AND) elution at 4.4 and 9.9 min., respectively. An external calibration method was applied, with calibration curves correlation coefficient of 0.9999 for both analytes. Detection limit values were 1.53 and 16.02 ng/g for SKA and AND, respectively. SKA recovery was 99.72±2.34 % (2.34 % RSD) and 102.84±1.62 % (1.57 % RSD) for AND. Results showed good precision values (repeatability <2.46 % RSD for SKA, <6.85 % RSD for AND; intermediate precision <2.87 % RSD for SKA, <6.98 % RSD for AND). The method's robustness was tested and the values were within the reference ranges. The validation results proved that the adaptation of an existing method resulted in good assessments of robustness, reliability and accuracy.