NanoMIPs Design for Fucose and Mannose Recognition: A Molecular Dynamics Approach.

Nanoscale molecularly imprinted polymers (nanoMIPs) are powerful molecular recognition tools with broad applications in the diagnosis, prognosis, and treatment of complex diseases. In this work, fully atomistic molecular dynamics (MD) simulations are used to assist the design of nanoMIPs with recognition capacity toward l-fucose and d-mannose as prototype disease biomarkers. MD simulations were conducted on prepolymerization mixtures containing different molar ratios of the monomers N-isopropylacrylamide (NIPAM), methacrylamide (MAM), and (4-acrylamidophenyl)(amino)methaniminium acetate (AB) and fixed molar ratios of the cross-linker ethylene glycol dimethacrylate (EGDMA) in explicit acetonitrile as the porogenic solvent. Prepolymerization mixtures containing ternary mixtures of NIPAM (50%), MAM (25%), and AB (25%) exhibit the best imprinting potential for both l-fucose and d-mannose, as they maximize (i) the stability of template-monomer plus template-cross-linker interactions, (ii) the number of functional monomers plus cross-linkers organized around the template, and (iii) the number of hydrogen bonds participating in template recognition. The studied prepolymerization mixtures exhibit an overall increased recognition capacity toward d-mannose over l-fucose, which is attributed to the higher hydrogen-bonding capacity of the former template. Our results are valuable to guide the synthesis of efficient nanoMIPs for sugar recognition and provide a computational framework extensible to any other template, monomer, or cross-linker combination, thus constituting a promising strategy for the rational design of molecularly imprinted materials.

Rational Design of Novel Glycomimetic Peptides for E-Selectin Targeting.

E-selectin is a cell-adhesion receptor with specific recognition capacity toward sialo-fucosylated Lewis carbohydrates present in leukocytes and tumor cells. E-selectin interactions mediate the progress of inflammatory processes and tumor metastasis, which aroused the interest in using this protein as a biomolecular target to design glycomimetic inhibitors for active targeting or therapeutic purposes. In this work, we report the rational discovery of two novel glycomimetic peptides targeting E-selectin based on mutations of the reference selectin-binding peptide IELLQAR. Sixteen single or double mutants at Ile1, Leu3, Leu4, and Arg7 residues were evaluated as potential candidates for E-selectin targeting using 50 ns molecular dynamics (MD) simulations. Nine peptides showing a stable association with the functional pocket were modified by adding a cysteine residue to the N-terminus to confer versatility for further chemical conjugation. Subsequent 50 ns MD simulations resulted in five cysteine-modified peptides with retained or improved E-selectin binding potential. Then, 300 ns accelerated MD (aMD) simulations were used to examine the binding properties of the best five cysteine-modified peptides. CIEELQAR and CIELFQAR exhibit the most selective association with the functional pocket of E-selectin, as revealed by potential of mean force profiles. Microscale thermophoresis experiments confirmed the E-selectin binding capacity of the selected peptides with KD values in the low micromolar range (CIEELQAR KD = 35.0 ± 1.4 µM; CIELFQAR KD = 16.4 ± 0.7 µM), which are 25-fold lower than the reported value for the native ligand sLex (KD = 878 µM). Our findings support the potential of CIEELQAR and CIELFQAR as novel E-selectin-targeting peptides with high recognition capacity and versatility for chemical conjugation, which are critical for enabling future applications in active targeting.

Mechanism-Based Rational Discovery and In Vitro Evaluation of Novel Microtubule Stabilizing Agents with Non-Taxol-Competitive Activity.

Microtubules (MT) are cytoskeletal polymers of αß-tubulin dimers that play a critical role in many cellular functions. Diverse antimitotic drugs bind to MT and disrupt their dynamics acting as MT stabilizing or destabilizing agents. The occurrence of undesired side effects and drug resistance encourages the search for novel MT binding agents with chemically diverse structures and different interaction profiles compared to known active compounds. This work reports the rational discovery of seven novel MT stabilizers using a combination of molecular modeling methods and in vitro experimental assays. Virtual screening, similarity filtering, and molecular mechanics generalized Born surface area (MM/GBSA) binding free energy refinement were employed to select seven potential candidates with high predicted affinity toward the non-taxoid site for MT stabilizers on ß-tubulin. MD simulations of 150 ns on reduced MT models suggest that candidate compounds strengthen the longitudinal interactions between tubulin dimers across protofilaments, which is a primary molecular mechanism of action for known MT stabilizers. In vitro MT polymerization assays confirmed that all candidates promote MT assembly at concentrations of >50 mM and exhibit noncompetitive MT polymerization profiles when cotreating with Taxol. Preliminary HeLa cell viability assays revealed a moderate cytotoxic effect for the compounds under study at 100 µM concentration. These results support the validity of our rational discovery strategy and the use of molecular modeling methods to pursue the search and optimization of new MT targeting agents.

In Silico Design of Novel Mutant Anti-MUC1 Aptamers for Targeted Cancer Therapy.

The transmembrane glycoprotein mucin 1 (MUC1) is an attractive tumor marker for cancer therapy and diagnosis. The nine amino acid extracellular epitope APDTRPAPG of this protein is selectively recognized by the S2.2 single-stranded DNA anti-MUC1 aptamer, which has emerged as a promising template for designing novel targeting agents for MUC1-directed therapy. In this work, 100 ns molecular dynamics (MD) simulations, MM/GBSA binding free energy calculations, and conformational analysis were employed to propose a novel prospective anti-MUC1 aptamer with increased affinity toward the MUC1 epitope resulting from the double mutation of the T11 and T12 residues with PSU and U nucleosides, respectively. The double mutant aptamer exhibits a tight interaction with the MUC1 epitope and adopts a groove conformation that structurally favors the intermolecular contact with the epitope through the intermediate T11-A18 region leaving the 3' and 5' ends free for further chemical conjugation with a nanocarrier or pharmaceutical. These results are valuable to gain understanding about the molecular features governing aptamer-epitope interactions and constitute a first key step for the design of novel aptamer-based nanocarriers for MUC1-targeted cancer therapy.

Molecular modeling study on the differential microtubule-stabilizing effect in singly- and doubly-bonded complexes with peloruside A and paclitaxel.

Microtubules (MT) are dynamic cytoskeletal components that play a crucial role in cell division. Disrupting MT dynamics by MT stabilizers is a widely employed strategy to control cell proliferation in cancer therapy. Most MT stabilizers bind to the taxol (TX) site located at the luminal interface between protofilaments, except laulimalide and peloruside A (PLA), which bind to an interfacial pocket on outer MT surface. Cryo-electron microscopy MTs reconstructions have shown differential structural effects on the MT lattice in singly- and doubly-bonded complexes with PLA, TX, and PLA/TX, as PLA is able to revert the lattice heterogeneity induced by TX association leading to more regular MT assemblies. In this work, fully-atomistic molecular dynamics simulations were employed to examine the single and double association of MT stabilizers to reduced MT models in the search for structural and energetic evidence that could be related to the differential regularization and stabilization effects exerted by PLA and TX on the MT lattice. Our results revealed that the double association of PLA/TX (a) strengthens the lateral contact between tubulin dimers compared to singly-bonded complexes, (b) favors a more parallel arrangement between tubulin dimers, and (c) induces a larger restriction in the interdimeric conformational motion increasing the probability of finding structures consistent with 13-protofilaments arrangements. These results and are valuable to increase understanding about the molecular mechanism of action of MT stabilizers, and could account for an overstabilization of MTs in doubly-bonded complexes compared to singly-bonded systems.

The role of conserved arginine in the GH70 family: a computational study of the structural features and their implications on the catalytic mechanism of GTF-SI from Streptoccocus mutans.

In this work, molecular dynamics and QM/MM calculations were employed to examine the structural and catalytic features of the retaining glucosyltransferase GTF-SI from the GH70 family, which participates in the process of caries formation. Our goal was to obtain a deeper understanding of the role of R475 in the mechanism of sucrose breakage. This residue is highly conserved in the GH70 family and so far there has been no evidence that shows what could be the role of this residue in the catalysis performed by GTF-SI. In order to understand the structural role of R475 in the native enzyme, we built full enzyme models of the wild type and the mutants R475A and R475Q. These models were addressed by means of molecular dynamics simulations, which allowed the assessment of the dynamical effect of the R475 mutation on the active site. Then, representative structures were chosen for each one of the mutant models and QM/MM calculations were carried out to unravel the catalytic role of R475. Our results show that the R475 mutation increases the flexibility of the enzyme, which triggers the entrance of water molecules in the active site. In addition, QM/MM calculations indicate that R475 is able to provide a great stabilization to the carboxylate moiety of the acid/base E515, which is an essential characteristic favoring the proton transfer process that promotes the glycosidic bond breakage of sucrose.

A QM/MM approach on the structural and stereoelectronic factors governing glycosylation by GTF-SI from Streptococcus mutans.

In this work, QM/MM calculations were employed to examine the catalytic mechanism of the retaining glucosyltransferase GTF-SI enzyme, which participates in the process of caries formation. Our goal was to characterize, with atomistic details, the mechanism of sucrose hydrolysis and the catalytic factors that modulate this reaction. Our results suggest a concerted mechanism for sucrose hydrolysis in which the first event corresponds to the glycosidic bond breakage assisted by Glu515, followed by the nucleophilic attack of Asp477, leading to the formation of the Covalent Glycosyl Enzyme (CGE) intermediate. A novel conformational itinerary of the glucosyl moiety along the reaction mechanism was identified: 2H3 → 2H3-E3 → 4C1, and the calculated energy barrier is 16.4 kcal mol-1, which is in good agreement with experimental evidence showing a major contribution coming from the glycosidic bond breakage. Our calculations also revealed that Arg475 and Asp588 play a critical role as TS-stabilizers by electrostatic and charge transfer mechanisms, respectively. This is the first report dealing with the specific features of the mechanism and catalytic residues involved in GTF-SI hydrolysis of sucrose, which is a matter of relevance in enzyme catalysis and could be valuable to aid the design of novel and specific inhibitors targeting GTF-SI.

Cytotoxicity and in vivo plasma kinetic behavior of surface-functionalized PAMAM dendrimers.

Understanding the molecular features responsible for the plasma kinetics of surface-modified polyamido amine (PAMAM) dendrimers is critical to explore novel biomedical applications for these nanomaterials. In this report, polyethylene glycol (PEG) and folic acid (FA) were employed to obtain partially-substituted PAMAM dendrimers as model biocompatible nanomaterials with different size, charge and surface functionality. Cytotoxicity assays on HEK cells at 1-500 µM concentration confirmed that PEG and FA incorporation increased the cell viability of PAMAM-based nanomaterials. Measurements of plasma kinetics in vivo revealed that PEG-PAMAM has an extended circulation time in mice blood (71.7 min) over native PAMAM (53.3 min) and FA-PAMAM (41.8 min). Molecular dynamics simulations revealed a direct relationship between circulation time and dendrimer size, thus providing valuable evidence to increase understanding about the modulation of functional properties of PAMAM-based systems through surface modification, and to guide future efforts on the rational design of novel biomedical nanomaterials.

Partially PEGylated PAMAM dendrimers as solubility enhancers of Silybin.

PEGylated PAMAM-G4 dendrimers with substitution percentages of 50% and intermediate size PEG chains (0.55 and 2.0 kDa) were synthesized and evaluated as solubility enhancers and potential supramolecular carriers for the poorly soluble drug Silybin (SIL). Aqueous solubility profiles revealed that the PEGylated system with 2.0 kDa chains induced a five-fold solubility increase for SIL and the largest drug-loading capacity within the systems under study with an average complex stoichiometry of 71:1 according to the Higuchi-Connors formulation for multiple binding sites. The supramolecular interaction between SIL and PEGylated PAMAM-G4 dendrimers was confirmed by 2D-NOESY experiments, which evidenced the simultaneous complexation of the drug in both PAMAM-G4 branches and outermost PEG chains. In vitro release studies showed that 2.0 kDa PEG chains induced a more extended release time compared with 0.5 kDa PEG chains. This result was attributed to the enhancement of PEG assistance to SIL complexation in systems with longer PEG chains, which are able to self-penetrate into dendrimer cavities and cooperate in the stabilization of SIL complexes, thus delaying the release of SIL from the supramolecular host. These results are valuable for the future design and development of novel PAMAM-based systems for SIL complexation and delivery.

Structural insight into the role of Gln293Met mutation on the Peloruside A/Laulimalide association with αß-tubulin from molecular dynamics simulations, binding free energy calculations and weak interactions analysis.

Peloruside A (PLA) and Laulimalide (LAU) are novel microtubule-stabilizing agents with promising properties against different cancer types. These ligands share a non-taxoid binding site at the outer surface of ß-tubulin and promote microtubule stabilization by bridging two adjacent αß-tubulin dimers from parallel protofilaments. Recent site-directed mutagenesis experiments confirmed the existence of a unique ß-tubulin site mutation (Gln293Met) that specifically increased the activity of PLA and caused resistance to LAU, without affecting the stability of microtubules in the absence of the ligands. In this work, fully atomistic molecular dynamics simulations were carried out to examine the PLA and LAU association with native and mutated αß-tubulin in the search for structural and energetic evidence to explain the role of Gln293Met mutation on determining the activity of these ligands. Our results revealed that Gln293Met mutation induced the loss of relevant LAU-tubulin contacts but exerted negligible changes in the interaction networks responsible for PLA-tubulin association. Binding free energy calculations (MM/GBSA and MM/PBSA), and weak interaction analysis (aNCI) predicted an increased affinity for PLA, and a weakened association for LAU after mutation, thus suggesting that Gln293Met mutation exerts its action by a modulation of drug-tubulin interactions. These results are valuable to increase understanding about PLA and LAU activity and to assist the future design of novel agents targeting the PLA/LAU binding pocket.

Mechanism of PAMAM Dendrimers Internalization in Hippocampal Neurons.

Polyamidoamine (PAMAM) dendrimers are hyperbranched macromolecules which have been described as one of the most promising drug nanocarrier systems. A key process to understand is their cellular internalization mechanism because of its direct influence on their intracellular distribution, association with organelles, entry kinetics, and cargo release. Despite that internalization mechanisms of dendrimers have been studied in different cell types, in the case of neurons they are not completely described. Considering the relevance of central nervous system (CNS) diseases and neuropharmacology, the aim of this report is to describe the molecular internalization mechanism of different PAMAM-based dendrimer systems in hippocampal neurons. Four dendrimers based on fourth generation PAMAM with different surface properties were studied: unmodified G4, with a positively charged surface; PP50, with a substitution of the 50% of amino surface groups with polyethylene glycol neutral groups; PAc, with a substitution of the 30% of amino surface groups with acrylate anionic groups; and PFO, decorated with folic acid groups in a 25% of total terminal groups. Confocal images show that both G4 and PFO are able to enter the neurons, but not PP50 and PAc. Colocalization study with specific endocytosis markers and specific endocytosis inhibitor assay demonstrate that clathrin-mediated endocytosis would be the main internalization mechanism for G4, whereas clathrin- and caveolae-mediated endocytosis would be implicated in PFO internalization. These results show the existence of different internalization mechanisms for PAMAM dendrimers in neurons and the possibility to control their internalization properties with specific chemical modifications.

Chlorin e6-Conjugated Mesoporous Titania Nanorods as Potential Nanoplatform for Photo-Chemotherapy.

Photodynamic therapy (PDT) has developed as an efficient strategy for cancer treatment. PDT involves the production of reactive oxygen species (ROS) by light irradiation after activating a photosensitizer (PS) in the presence of O2. PS-coupled nanomaterials offer additional advantages, as they can merge the effects of PDT with conventional enabling-combined photo-chemotherapeutics effects. In this work, mesoporous titania nanorods were surface-immobilized with Chlorin e6 (Ce6) conjugated through 3-(aminopropyl)-trimethoxysilane as a coupling agent. The mesoporous nanorods act as nano vehicles for doxorubicin delivery, and the Ce6 provides a visible light-responsive production of ROS to induce PDT. The nanomaterials were characterized by XRD, DRS, FTIR, TGA, N2 adsorption-desorption isotherms at 77 K, and TEM. The obtained materials were tested for their singlet oxygen and hydroxyl radical generation capacity using fluorescence assays. In vitro cell viability experiments with HeLa cells showed that the prepared materials are not cytotoxic in the dark, and that they exhibit photodynamic activity when irradiated with LED light (150 W m-2). Drug-loading experiments with doxorubicin (DOX) as a model chemotherapeutic drug showed that the nanostructures efficiently encapsulated DOX. The DOX-nanomaterial formulations show chemo-cytotoxic effects on Hela cells. Combined photo-chemotoxicity experiments show enhanced effects on HeLa cell viability, indicating that the conjugated nanorods are promising for use in combined therapy driven by LED light irradiation.

Nonlinear optical and spectroscopic properties, thermal analysis, and hemolytic capacity evaluation of quinoline-1,3-benzodioxole chalcone.

This article describes the synthesis, characterization (1H NMR, 13C NMR, FT-IR, HRMS and XRD), UV-Vis absorption and fluorescence spectra, theoretical analysis, evaluation of nonlinear optical properties (NLO), thermal analysis and determination of the hemolytic capacity of the compound (E)-N-(4-(3-(benzo[d][1,3]dioxol-5-yl)acryloyl)phenyl)quinoline-3-carboxamide (5). Radiological findings showed that compound 5 crystallized in space group Pca21. Furthermore, theoretical DFT studies performed with the B3LYP and M062X functionals showed good agreement with the experimental results and provided valuable information on the molecular and electronic structure, reactivity, polarizability, and kinematic stability of the compound. Besides, compound 5 did not show any hemolytic effect on human erythrocytes and exhibited strong NLO properties. The TG and DTA thermograms of quinoline-chalcone (5) revealed a multi-step thermal decomposition process with a total mass loss of 83.2%, including water content loss. The DTA curves exhibited endothermic peaks corresponding to decomposition steps, melting point, and thermochemical transition. Additionally, exothermic peaks in the DTA thermograms align with significant mass loss, confirming the compound's melting point and water content, as validated by X-ray diffraction analysis. These results contribute to the advancement of research on compounds with NLO properties and offer a promising avenue for the development of substances potentially applicable to optical devices in the biomedical field.

Piperonal chalcone derivative incorporating the pyrazolo[3,4-b]pyridine moiety; crystal structure, spectroscopic characterization and quantum chemical investigations.

A single crystal of a piperonal chalcone derivative was obtained, fully characterized, and crystallized by a slow evaporation technique. The synthesized compound was characterized by UV-Visible, FT-IR, HRMS, 1H NMR, and 13C NMR spectroscopic studies and X-ray crystallography, revealing that the crystal belongs to a triclinic crystal system with a P1̄ space group, Z = 2. In the present work, we focus on molecular modeling studies such as Hirshfeld surface analysis, energy framework calculations, frontier molecular orbital analysis, natural bond orbital analysis, and NLO properties of a π-conjugate system combining the chalcone and the pyrazole[3,4-b]pyridine scaffolds to describe the in-depth structural analysis thereof. Good agreement was found between the calculated results and experimental data. In addition, Hirshfeld surface analysis of the crystal structure showed that the intermolecular stabilization in the crystal packing comes mainly from H⋯H bond interactions. The chalcone crystal exhibits significant NLO properties suggesting that it could be considered a potential candidate for application in nonlinear optical devices.

Comprehensive analysis of crystal structure, spectroscopic properties, quantum chemical insights, and molecular docking studies of two pyrazolopyridine compounds: potential anticancer agents.

In this study, two pyrazolo[3,4-b]pyridine derivatives (4a and 4b) were grown using a slow evaporation solution growth technique and characterized by FT-IR, HRMS, 1H/13C NMR spectroscopy, and X-ray crystallography. The 4a and 4b structures crystallized in monoclinic and triclinic systems with space groups P21/n and P1̄, respectively. Theoretical calculations were performed at the DFT/B3LYP level for the optimized geometries. The results were in excellent agreement with the experimental data (spectroscopic and XRD). This investigation encompasses molecular modeling studies including Hirshfeld surface analysis, energy framework calculations, and frontier molecular orbital analysis. Intermolecular interactions within the crystal structures of the compounds were explored through Hirshfeld surface analysis, which revealed the notable presence of hydrogen bonding and hydrophobic interactions. This insight provides valuable information on the structural stability and potential solubility characteristics of these compounds. The research was extended to docking analysis with eight distinct kinases (BRAF, HER2, CSF1R, MEK2, PDGFRA, JAK, AKT1, and AKT2). The results of this analysis demonstrate that both 4a and 4b interact effectively with the kinase-binding sites through a combination of hydrophobic interactions and hydrogen bonding. Compound 4a had the best affinity for proteins; this is related to the fact that the compound is not rigid and has a small size, allowing it to sit well at any binding site. This study contributes to the advancement of kinase inhibitor research and offers potential avenues for the development of new therapeutic agents for cancer treatment.

Computational study on the carboligation reaction of acetohidroxyacid synthase: new approach on the role of the HEThDP- intermediate.

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate dependent enzyme that catalyses the decarboxylation of pyruvate to yield the hydroxyethyl-thiamin diphosphate (ThDP) anion/enamine intermediate (HEThDP(-)). This intermediate reacts with a second ketoacid to form acetolactate or acetohydroxybutyrate as products. Whereas the mechanism involved in the formation of HEThDP(-) from pyruvate is well understood, the role of the enzyme in controlling the carboligation reaction of HEThDP(-) has not been determined yet. In this work, molecular dynamics (MD) simulations were employed to identify the aminoacids involved in the carboligation stage. These MD studies were carried out over the catalytic subunit of yeast AHAS containing the reaction intermediate (HEThDP(-)) and a second pyruvate molecule. Our results suggest that additional acid-base ionizable groups are not required to promote the catalytic cycle, in contrast with earlier proposals. This finding leads us to postulate that the formation of acetolactate relies on the acid-base properties of the HEThDP(-) intermediate itself. PM3 semiempirical calculations were employed to obtain the energy profile of the proposed mechanism on a reduced model of the active site. These calculations confirm the role of HEThDP(-) intermediate as the ionizable group that promotes the carboligation and product formation steps of the catalytic cycle.

Colorectal cancer chemoprevention by 2 beta-cyclodextrin inclusion compounds of auraptene and 4'-geranyloxyferulic acid.

The inhibitory effects of novel prodrugs, inclusion complexes of 3-(4'-geranyloxy-3'-methoxyphenyl)-2-trans propenoic acid (GOFA) and auraptene (AUR) with beta-cyclodextrin (CD), on colon carcinogenesis were investigated using an azoxymethane (AOM)/dextran sodium sulfate (DSS) model. Male CD-1 (ICR) mice initiated with a single intraperitoneal injection of AOM (10 mg/kg body weight) were promoted by the addition of 1.5% (w/v) DSS to their drinking water for 7 days. They were then given a basal diet containing 2 dose levels (100 and 500 ppm) of GOFA/beta-CD or AUR/beta-CD for 15 weeks. At Week 18, the development of colonic adenocarcinoma was significantly inhibited by feeding with GOFA/beta-CD at dose levels of 100 ppm (63% reduction in multiplicity, p < 0.05) and 500 ppm (83% reduction in the multiplicity, p < 0.001), when compared with the AOM/DSS group (multiplicity: 3.36 +/- 3.34). In addition, feeding with 100 and 500 ppm (p < 0.01) of AUR/beta-CD suppressed the development of colonic adenocarcinomas. The dietary administration with GOFA/beta-CD and AUR/beta-CD inhibited colonic inflammation and also modulated proliferation, apoptosis and the expression of several proinflammatory cytokines, such as nuclear factor-kappaB, tumor necrosis factor-alpha, Stat3, NF-E2-related factor 2, interleukin (IL)-6 and IL-1beta, which were induced in the adenocarcinomas. Our findings indicate that GOFA/beta-CD and AUR/beta-CD, especially GOFA/beta-CD, are therefore able to inhibit colitis-related colon carcinogenesis by modulating inflammation, proliferation and the expression of proinflammatory cytokines in mice.

Bioreduction of some common carbonylic compounds mediated by yeasts.

Bioreduction of several prochiral carbonylic compounds such as acetophenone (1), ethyl acetoacetate (2) and ethyl phenylpropionate (3) to the corresponding optically active sec-alcohols 1a-3a was performed using wild-type strains of Pichia pastoris UBB 1500, Rhodotorula sp., and Saccharomyces cerevisiae. The reductions showed moderate to excellent conversion and high enantiomeric excess, in an extremely mild and environmentally benign manner in aqueous medium, using glucose as cofactor regeneration system. The obtained alcohols follow Prelog's rule, but in the reduction of 1 with P. pastoris UBB 1500 the anti-Prelog enantiopreference was observed.

Polyamidoamine-based nanovector for the efficient delivery of methotrexate to U87 glioma cells.

The purpose of this study was to design a polyamidoamine (PAMAM)-based nanovector for the efficient delivery of methotrexate to U87 glioma cells. To this end, 0-100% acetylated PAMAM dendrimers of the fourth generation were synthesized and evaluated using drug encapsulation measurements, molecular dynamics simulations, neurotoxicity assays and neuronal internalization experiments. The best system was tested as a nanovector for methotrexate delivery to U87 glioma cells. The authors found that 25% acetylated PAMAM dendrimers of the fourth-generation combine low intrinsic toxicity, large drug complexation capacity and efficient internalization into hippocampal neurons. Nanovector complexation enhances the cytotoxic response of methotrexate against U87 glioma cells compared with free drug solutions. In conclusion, 25% acetylated PAMAM dendrimers of the fourth-generation increase drug uptake by glioma cells and thereby act as efficient nanovectors for methotrexate delivery.

Chilean crab (Aegla cholchol) as a new source of chitin and chitosan with antifungal properties against Candida spp.

In the present work, the chemical composition of the Chilean freshwater crab Aegla cholchol exoskeleton was studied for the first time. α-Chitin was isolated from three main body parts (pincers, legs, carapace), and its content ranged from 9.0-10.4% (w/w). Moreover, chitosan was extracted by alkaline treatment at different temperature and time regimes. Aegla cholchol exoskeleton, chitin and chitosans were characterized by FTIR, TGA, and SEM. Chemical structure of chitin and chitosan was confirmed by NMR spectroscopy. Chitosan molecular weight determinations were carried out by GPC. The obtained chitosan samples had a degree of N-acetylation (DA) between 4 and 15% and molecular weight (Mw) in the range of 65-201 kDa. The antifungal activity of the chitosan samples and the chitooligomer were tested toward twenty isolated clinical strains of Candida yeast. Chitosan with lower DA (4%) and higher molecular weight showed the lowest minimum inhibitory concentration (MIC) values, which was evidenced by the highest antifungal activity toward Candida albicans, Candida tropicalis, Candida glabrata, and Candida parapsilosis. Results suggest that Aegla cholchol is an excellent natural source for production of bioactive materials with potential applications in the health system, to prevent infections associated with Candida strains.