Cytotoxic activity of Crotalus molossus molossus snake venom-loaded in chitosan nanoparticles against T-47D breast carcinoma cells.

Nanomedicine has led to the development of new biocompatible and biodegradable materials able to improve the pharmaceutical effect of bioactive components, broadening the options of treatment for several diseases, including cancer. Additionally, some snake venom toxins have been reported to present cytotoxic activity in different tumor cell lines, making them an auspicious option to be used as cancer drugs. The present study aims to evaluate the cytotoxic activity of the northern black-tailed rattlesnake (Crotalus molossus molossus) venom-loaded chitosan nanoparticles (Cs-Venom NPs) against the T-47D breast carcinoma cell line. To do so, we first identified the significant proteins composing the venom; afterward, hemocompatibility and cytotoxic activity against tumoral cells were evaluated. The venom was then loaded into chitosan nanoparticles through the ionotropic gelation process, obtaining particles of 415.9±21.67 nm and ζ-potential of +28.3±1.17 mV. The Cs-Venom complex delivered the venom into the breast carcinoma cells, inhibiting their viability and inducing morphological changes in the T-47D cells. These features indicate that these nanoparticles are suitable for the potential use of C. m. molossus venom toxins entrapped within polymer nanoparticles for the future development and research of cancer drugs.

Nanoproteomic Approach for Isolation and Identification of Potential Biomarkers in Human Urine from Adults with Normal Weight, Overweight and Obesity.

In this work, previously synthesized and characterized core-shell silica nanoparticles (FCSNP) functionalized with immobilized molecular bait, Cibacron blue, and a porous polymeric bis-acrylamide shell were incubated with pooled urine samples from adult women or men with normal weight, overweight or obesity for the isolation of potential biomarkers. A total of 30 individuals (15 woman and 15 men) were included. FCSNP allowed the capture of a variety of low molecular weight (LMW) proteins as evidenced by mass spectrometry (MS) and the exclusion of high molecular weight (HMW) proteins (>34 kDa) as demonstrated by SDS-PAGE and 2D SDS-PAGE. A total of 36 proteins were successfully identified by MS and homology database searching against the Homo sapiens subset of the Swiss-Prot database. Identified proteins were grouped into different clusters according to their abundance patterns. Four proteins were found only in women and five only in men, whereas 27 proteins were in urine from both genders with different abundance patterns. Based on these results, this new approach represents an alternative tool for isolation and identification of urinary biomarkers.

Insights into the Structural Features, Conformational Stability and Functional Activity of the Olneya tesota PF2 Lectin.

BACKGROUND: The O. tesota lectin PF2 is a tetrameric protein with subunits of 33 kDa that recognizes only complex carbohydrates, resistant to proteolytic enzymes and has insecticidal activity against Phaseolus beans pest. OBJECTIVE: To explore PF2 lectin features at different protein structural levels and to evaluate the effect of temperature and pH on its functionality and conformational stability. METHODS: PF2 lectin was purified by affinity chromatography. Its primary structure was resolved by mass spectrometry and analyzed by bioinformatic tools, including its tertiary structure homology modeling. The effect of temperature and pH on its conformational traits and stability was addressed by dynamic light scattering, circular dichroism, and intrinsic fluorescence. The hemagglutinating activity was evaluated using a suspension of peripheral blood erythrocytes. RESULTS: The proposed PF2 folding comprises a high content of beta sheets. At pH 7 and 25°C, the hydrodynamic diameter (Dh) was found to be 12.3 nm which corresponds to the oligomeric native state of PF2 lectin. Dh increased under the other evaluated pH and temperature conditions, suggesting protein aggregation. At basic pH, PF2 exhibited low conformational stability. The native PF2 (pH 7) retained its full hemagglutinating activity up to 45°C and exhibited one transition state with a melting temperature of 76.8°C. CONCLUSION: PF2 showed distinctive characteristics found in legume lectins. The pH influences the functionality and conformational stability of the protein. PF2 lectin displayed a relatively narrow thermostability to the loss of secondary structure and hemagglutinating activity.

Gene Sequences of Potential Targets of Insecticidal PF2 Lectin Identified from the Larval De Novo Transcriptome of the Mexican Bean Weevil (Zabrotes Subfasciatus; Boheman 1833).

The available genomic and proteomic information of non-model organisms is often underrepresented in public databases hindering their study at molecular, cellular, and physiological levels. Information on Zabrotes subfasciatus (Mexican bean weevil) is poorly represented in databases, yet it is a major pest of common beans. We report the transcriptome of Z. subfasciatus larvae; transcripts were sequenced using an Illumina RNA-Seq technology and assembled de novo identifying 29,029 unigenes with an average size of 1168 bp and an N50 value of 2196 bp. About 15,124 unigenes (52%) were functionally annotated and categorized. Further analysis revealed 30 unigene sequences encoding putative targets of the insecticidal PF2 lectin. The complete deduced amino acid sequences of eight selected proteins potentially related to insecticidal mechanism of Palo Fierro 2 (PF2) were used for predicting probable N-glycosylation sites and analyzing phylogenetic relationships with insect sequences. This work provides a dramatic increase in the genetic resources available for Coleopterans and set the basis for developing future studies on biological aspects and potential control strategies for Z. subfasciatus.

Bifunctional nickel-iminodiacetic acid-core-shell silica nanoparticles for the exclusion of high molecular weight proteins and purification of His-tagged recombinant proteins.

Herein, silica nanoparticles were synthesized and chemically modified with iminodiacetic acid (IDA) and Ni2+ ions surrounded by a bis-acrylamide polymeric shell to obtain a new core-shell immobilized metal affinity chromatography (IMAC) based material. These Ni2+-IDA-core-shell silica nanoparticles (Ni2+-IDA-CSS-NP) represent a new alternative for purification of His-tagged proteins and exclusion of high molecular weight (HMW) proteins at the same time. Nanoparticles presented a final size of 479.6 ± 6.9 nm determined by dynamic light scattering (DLS) and a surface charge of -37.2 ± 0.5 mV. Successful incorporation of the different compounds at every phase of synthesis was evidenced by ATR-FTIR analysis. Ni2+-IDA-CSS-NP were used for isolation of His-tagged spo0F (6His-spo0F) from E. coli lysate. Ni2+-IDA-CSS-NP presented a capacity of 4.16 ± 0.45 µg mg-1. Purification of 6His-spo0F with high selectivity and the effective exclusion of HMW proteins were evidenced by SDS-PAGE and validated through mass spectrometry analysis.

Molecular recognition of glyconanoparticles by RCA and E. coli K88 - designing transports for targeted therapy.

The targeted drug delivery has been studied as one of the main methods in medicine to ensure successful treatments of diseases. Pharmaceutical sciences are using micro or nano carriers to obtain a controlled delivery of drugs, able to selectively interact with pathogens, cells or tissues. In this work, we modified bovine serum albumin (BSA) with lactose, obtaining a neoglycan (BSA-Lac). Subsequently, we synthesized glyconanoparticles (NPBSA-Lac) with the premise that it would be recognized by microbial galactose specific lectins. NPBSA-Lac were tested for bio-recognition with adhesins of E. coli K88 and Ricinus communis agglutinin I (RCA). Glycation of BSA with lactose was analyzed by electrophoresis, infrared spectroscopy and fluorescence. Approximately 41 lactoses per BSA molecule were estimated. Nanoparticles were obtained using water in oil emulsion method and spheroid morphology with a range size of 300-500 nm was observed. Specific recognition of NPBSA-Lac by RCA and E. coli K88 was displayed by aggregation of nanoparticles analyzed by dynamic light scattering and atomic force microscopy. The results indicate that the lactosylated nanovectors could be targeted at the E. coli K88 adhesin and potentially could be used as a transporter for an antibacterial drug.

Novel Synthesis of Core-Shell Silica Nanoparticles for the Capture of Low Molecular Weight Proteins and Peptides.

Silica nanoparticles were functionalized with immobilized molecular bait, Cibacron Blue, and a porous polymeric bis-acrylamide shell. These nanoparticles represent a new alternative to capture low molecular weight (LMW) proteins/peptides, that might be potential biomarkers. Functionalized core-shell silica nanoparticles (FCSNP) presented a size distribution of 243.9 ± 11.6 nm and an estimated surface charge of -38.1 ± 0.9 mV. The successful attachment of compounds at every stage of synthesis was evidenced by ATR-FTIR. The capture of model peptides was determined by mass spectrometry, indicating that only the peptide with a long sequence of hydrophobic amino acids (alpha zein 34-mer) interacted with the molecular bait. FCSNP excluded the high molecular weight protein (HMW), BSA, and captured LMW proteins (myoglobin and aprotinin), as evidenced by SDS-PAGE. Functionalization of nanoparticles with Cibacron Blue was crucial to capture these molecules. FCSNP were stable after twelve months of storage and maintained a capacity of 3.1-3.4 µg/mg.

Binding of PF2 lectin from Olneya tesota to gut proteins of Zabrotes subfasciatus larvae associated with the insecticidal mechanism.

Zabrotes subfasciatus (Boheman) is the main pest of common beans ( Phaselous vulgaris ). Wild legume seeds from Olneya tesota contain a lectin, PF2, that shows insecticidal activities against this insect. The binding of PF2 to midgut glycoproteins of 20-day-old larvae was evaluated using PF2 affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the proteins retained on the gel revealed several putative glycoproteins, ranging in mass from 17 to 97 kDa. Subsequent protein digestion and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) provided amino acid fragments that identified an α-tubulin, cytochrome c oxidase subunit I, an odorant receptor, and a lysozyme from available insect sequence databases. The potential of these proteins to serve as part of the mechanisms involved in the insecticidal activity of PF2 to Z. subfasciatus is discussed.

Conjugates of bovine serum albumin with chitin oligosaccharides prepared through the Maillard reaction.

Chitin neoglycoconjugates (BSA-CO) were obtained by the conjugation of bovine serum albumin (BSA) with chitin oligosaccharides (CO) through the Maillard reaction (nonenzymatic glycation). CO produced by acid hydrolysis of chitin were fractionated using an ultrafiltration membrane system (1-3 kDa cutoff). The Maillard reaction was carried out by heating a freeze-dried mixture containing BSA and CO at 60 °C (under 43% relative humidity for 6 and 12 h). BSA-CO were characterized by available amino groups content, intrinsic tryptophan emission spectra, gel electrophoresis, and mass spectrometry. Biological assays included interaction with wheat germ agglutinin (WGA) and with bacterial adhesins of Escherichia coli K88+ and Salmonella choleraesuis. Glycation of BSA was revealed by reduction of available amino groups and fluorescence intensity and also retarded migration through SDS-PAGE. Conjugation of BSA with chitin oligomers appeared to be time dependent and was confirmed by mass spectrometry, by which molecular mass increase for monomers and dimers was observed. Monomers were estimated to contain either one or two glycation sites (at 6 and 12 h of treatment, respectively), with one or two tetrasaccharide units attached. Consequently, dimers showed two or four glycation sites. BSA-CO presented biological recognition by WGA and E. coli K88+ and S. cholerasuis adhesins. The strategy used in this work represents a simple method to obtain glycoconjugates to study applications involving protein-carbohydrate recognition.

Insecticidal action of PF2 lectin from Olneya tesota (Palo Fierro) against Zabrotes subfasciatus larvae and midgut glycoconjugate binding.

Zabrotes subfasciatus (Boheman) is the main pest of common beans (Phaselous vulgaris). Some wild legume seeds may contain lectins with insecticidal activities against this insect. The larval developments of Z. subfasciatus on seeds of Olneya tesota (a desert wild legume) and on artificial seeds containing purified PF2 lectin were evaluated. PF2 susceptibility to proteolysis was assessed by incubation with midgut extract at different times. PF2 binding to midgut glycoconjugates was assessed by histochemistry. A reduced level of oviposition and a lack of emergence of adult beetles were observed in O. tesota seeds (compared to common beans), and in artificial seeds containing PF2 at 0.5 and 1%. PF2 was resistant to larval midgut proteolysis for 24 h, while PHA-E (lectin control) was fully digested after 4 h. Histochemistry analysis of midguts incubated with PF2 showed recognition for microvillae and possibly with peritrophic gel. On the other hand, PHA-E exhibited no interaction with larval midgut glycoproteins. Proteolysis resistance and glycan recognition could in part explain why PF2 is toxic to Z. subfasciatus while PHA is not.