Solid Lipid Nanoparticles For Hydrophilic Biotech Drugs: Optimization And Cell Viability Studies (caco-2 & Hepg-2 Cell Lines).

Insulin was used as model protein to developed innovative Solid Lipid Nanoparticles (SLNs) for the delivery of hydrophilic biotech drugs, with potential use in medicinal chemistry. SLNs were prepared by double emulsion with the purpose of promoting stability and enhancing the protein bioavailability. Softisan(®)100 was selected as solid lipid matrix. The surfactants (Tween(®)80, Span(®)80 and Lipoid(®)S75) and insulin were chosen applying a 2(2) factorial design with triplicate of central point, evaluating the influence of dependents variables as polydispersity index (PI), mean particle size (z-AVE), zeta potential (ZP) and encapsulation efficiency (EE) by factorial design using the ANOVA test. Therefore, thermodynamic stability, polymorphism and matrix crystallinity were checked by Differential Scanning Calorimetry (DSC) and Wide Angle X-ray Diffraction (WAXD), whereas the effect of toxicity of SLNs was check in HepG2 and Caco-2 cells. Results showed a mean particle size (z-AVE) width between 294.6 nm and 627.0 nm, a PI in the range of 0.425-0.750, ZP about -3 mV, and the EE between 38.39% and 81.20%. After tempering the bulk lipid (mimicking the end process of production), the lipid showed amorphous characteristics, with a melting point of ca. 30 °C. The toxicity of SLNs was evaluated in two distinct cell lines (HEPG-2 and Caco-2), showing to be dependent on the concentration of particles in HEPG-2 cells, while no toxicity in was reported in Caco-2 cells. SLNs were stable for 24 h in in vitro human serum albumin (HSA) solution. The resulting SLNs fabricated by double emulsion may provide a promising approach for administration of protein therapeutics and antigens.

Inhibitory Effects Of A Cured Antibacterial Bonding System On Viability And Metabolic Activity Of Oral Bacteria.

To evaluate the antimicrobial efficacy of Clearfil SE Protect (CP) and Clearfil SE Bond (CB) after curing and rinsed against five individual oral microorganisms as well as a mixture of bacterial culture prepared from the selected test organisms. Bacterial suspensions were prepared from single species of Streptococcus mutans, Streptococcus sobrinus, Streptococcus gordonii, Actinomyces viscosus and Lactobacillus lactis, as well as mixed bacterial suspensions from these organisms. Dentin bonding system discs (6 mm×2 mm) were prepared, cured, washed and placed on the bacterial suspension of single species or multispecies bacteria for 15, 30 and 60 min. MTT, Live/Dead bacterial viability (antibacterial effect), and XTT (metabolic activity) assays were used to test the two dentin system's antibacterial effect. All assays were done in triplicates and each experiment repeated at least three times. Data were submitted to ANOVA and Scheffe's f-test (5%). Greater than 40% bacteria killing was seen within 15 min, and the killing progressed with increasing time of incubation with CP discs. However, a longer (60 min) period of incubation was required by CP to achieve similar antimicrobial effect against mixed bacterial suspension. CB had no significant effect on the viability or metabolic activity of the test microorganisms when compared to the control bacterial culture. CP was significantly effective in reducing the viability and metabolic activity of the test organisms. The results demonstrated the antimicrobial efficacy of CP both on single and multispecies bacterial culture. CP may be beneficial in reducing bacterial infections in cavity preparations in clinical dentistry.

Exercise Increases Pancreatic β-cell Viability In A Model Of Type 1 Diabetes Through Il-6 Signaling.

Type 1 diabetes (T1D) is provoked by an autoimmune assault against pancreatic β cells. Exercise training enhances β-cell mass in T1D. Here, we investigated how exercise signals β cells in T1D condition. For this, we used several approaches. Wild-type and IL-6 knockout (KO) C57BL/6 mice were exercised. Afterward, islets from control and trained mice were exposed to inflammatory cytokines (IL-1β plus IFN-γ). Islets from control mice and β-cell lines (INS-1E and MIN6) were incubated with serum from control or trained mice or medium obtained from 5-aminoimidazole-4 carboxamide1-β-d-ribofuranoside (AICAR)-treated C2C12 skeletal muscle cells. Subsequently, islets and β cells were exposed to IL-1β plus IFN-γ. Proteins were assessed by immunoblotting, apoptosis was determined by DNA-binding dye propidium iodide fluorescence, and NO(•) was estimated by nitrite. Exercise reduced 25, 75, and 50% of the IL-1β plus IFN-γ-induced iNOS, nitrite, and cleaved caspase-3 content, respectively, in pancreatic islets. Serum from trained mice and medium from AICAR-treated C2C12 cells reduced β-cell death, induced by IL-1β plus IFN-γ treatment, in 15 and 38%, respectively. This effect was lost in samples treated with IL-6 inhibitor or with serum from exercised IL-6 KO mice. In conclusion, muscle contraction signals β-cell survival in T1D through IL-6.-Paula, F. M. M., Leite, N. C., Vanzela, E. C., Kurauti, M. A., Freitas-Dias, R., Carneiro, E. M., Boschero, A. C., and Zoppi, C. C. Exercise increases pancreatic β-cell viability in a model of type 1 diabetes through IL-6 signaling.

Scaphoid Fracture Nonunion: Correlation Of Radiographic Imaging, Proximal Fragment Histologic Viability Evaluation, And Estimation Of Viability At Surgery: Diagnosis Of Scaphoid Pseudarthrosis.

The purpose of this study was to correlate the pre-operative imaging, vascularity of the proximal pole, and histology of the proximal pole bone of established scaphoid fracture non-union. This was a prospective non-controlled experimental study. Patients were evaluated pre-operatively for necrosis of the proximal scaphoid fragment by radiography, computed tomography (CT) and magnetic resonance imaging (MRI). Vascular status of the proximal scaphoid was determined intra-operatively, demonstrating the presence or absence of puncate bone bleeding. Samples were harvested from the proximal scaphoid fragment and sent for pathological examination. We determined the association between the imaging and intra-operative examination and histological findings. We evaluated 19 male patients diagnosed with scaphoid nonunion. CT evaluation showed no correlation to scaphoid proximal fragment necrosis. MRI showed marked low signal intensity on T1-weighted images that confirmed the histological diagnosis of necrosis in the proximal scaphoid fragment in all patients. Intra-operative assessment showed that 90% of bones had absence of intra-operative puncate bone bleeding, which was confirmed necrosis by microscopic examination. In scaphoid nonunion MRI images with marked low signal intensity on T1-weighted images and the absence of intra-operative puncate bone bleeding are strong indicatives of osteonecrosis of the proximal fragment.

An Efficient Protocol For The Generation Of Monocyte Derived Dendritic Cells Using Serum-free Media For Clinical Applications In Post Remission Aml Patients.

Protocols for the generation of dendritic cells (DCs) using serum as a supplementation of culture media leads to reactions due to animal proteins and disease transmissions. Several types of serum-free media (SFM), based on good manufacture practices (GMP), have recently been used and seem to be a viable option. The aim of this study was to evaluate the results of the differentiation, maturation, and function of DCs from Acute Myeloid Leukemia patients (AML), generated in SFM and medium supplemented with autologous serum (AS). DCs were analyzed by phenotype characteristics, viability, and functionality. The results showed the possibility of generating viable DCs in all the conditions tested. In patients, the X-VIVO 15 medium was more efficient than the other media tested in the generation of DCs producing IL-12p70 (p=0.05). Moreover, the presence of AS led to a significant increase of IL-10 by DCs as compared with CellGro (p=0.05) and X-Vivo15 (p=0.05) media, both in patients and donors. We concluded that SFM was efficient in the production of DCs for immunotherapy in AML patients. However, the use of AS appears to interfere with the functional capacity of the generated DCs.

Silk Fibroin And Sodium Alginate Blend: Miscibility And Physical Characteristics.

Films of silk fibroin (SF) and sodium alginate (SA) blends were prepared by solution casting technique. The miscibility of SF and SA in those blends was evaluated and scanning electron microscopy (SEM) revealed that SF/SA 25/75 wt.% blends underwent microscopic phase separation, resulting in globular structures composed mainly of SF. X-ray diffraction indicated the amorphous nature of these blends, even after a treatment with ethanol that turned them insoluble in water. Thermal analyses of blends showed the peaks of degradation of pristine SF and SA shifted to intermediate temperatures. Water vapor permeability, swelling capacity and tensile strength of SF films could be enhanced by blending with SA. Cell viability remained between 90 and 100%, as indicated by in vitro cytotoxicity test. The SF/SA blend with self-assembled SF globules can be used to modulate structural and mechanical properties of the final material and may be used in designing high performance wound dressing.

Cytogenetic And Morphologic Approaches Of Hybrids From Experimental Crosses Between Triatoma Lenti Sherlock & Serafim, 1967 And T. Sherlocki Papa Et Al., 2002 (hemiptera: Reduviidae).

The reproductive capacity between Triatoma lenti and Triatoma sherlocki was observed in order to verify the fertility and viability of the offspring. Cytogenetic, morphological and morphometric approaches were used to analyze the differences that were inherited. Experimental crosses were performed in both directions. The fertility rate of the eggs in crosses involving T. sherlocki females was 65% and 90% in F1 and F2 offspring, respectively. In reciprocal crosses, it was 7% and 25% in F1 and F2 offspring, respectively. The cytogenetic analyses of the male meiotic process of the hybrids were performed using lacto-acetic orcein, C-banding and Feulgen techniques. The male F1 offspring presented normal chromosome behavior, a finding that was similar to those reported in parental species. However, cytogenetic analysis of F2 offspring showed errors in chromosome pairing. This post-zygotic isolation, which prevents hybrids in nature, may represent the collapse of the hybrid. This phenomenon is due to a genetic dysregulation that occurs in the chromosomes of F1. The results were similar in the hybrids from both crosses. Morphological features, such as color and size of connexive and the presence of red-orange rings on the femora, were similar to T. sherlocki, while wins size was similar to T. lenti in F1 offspring. The eggshells showed characteristics that were similar to species of origin, whereas the median process of the pygophore resulted in intermediate characteristics in the F1 and a segregating pattern in F2 offspring. Geometric morphometric techniques used on the wings showed that both F1 and F2 offspring were similar to T. lenti. These studies on the reproductive capacity between T. lenti and T. sherlocki confirm that both species are evolutionarily closed; hence, they are included in the brasiliensis subcomplex. The extremely reduced fertility observed in the F2 hybrids confirmed the specific status of the species that were analyzed.

Graphene And Carbon Nanotube Nanocomposite For Gene Transfection.

Graphene and carbon nanotube nanocomposite (GCN) was synthesised and applied in gene transfection of pIRES plasmid conjugated with green fluorescent protein (GFP) in NIH-3T3 and NG97 cell lines. The tips of the multi-walled carbon nanotubes (MWCNTs) were exfoliated by oxygen plasma etching, which is also known to attach oxygen content groups on the MWCNT surfaces, changing their hydrophobicity. The nanocomposite was characterised by high resolution scanning electron microscopy; energy-dispersive X-ray, Fourier transform infrared and Raman spectroscopies, as well as zeta potential and particle size analyses using dynamic light scattering. BET adsorption isotherms showed the GCN to have an effective surface area of 38.5m(2)/g. The GCN and pIRES plasmid conjugated with the GFP gene, forming π-stacking when dispersed in water by magnetic stirring, resulting in a helical wrap. The measured zeta potential confirmed that the plasmid was connected to the nanocomposite. The NIH-3T3 and NG97 cell lines could phagocytize this wrap. The gene transfection was characterised by fluorescent protein produced in the cells and pictured by fluorescent microscopy. Before application, we studied GCN cell viability in NIH-3T3 and NG97 line cells using both MTT and Neutral Red uptake assays. Our results suggest that GCN has moderate stability behaviour as colloid solution and has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity and good transfection efficiency.

Carbon Nanoparticles For Gene Transfection In Eukaryotic Cell Lines.

For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed π-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests.

Antioxidant And Antiproliferative Activities Of Methanolic Extract From A Neglected Agricultural Product: Corn Cobs.

Neglected agricultural products (NAPs) are defined as discarded material in agricultural production. Corn cobs are a major waste of agriculture maize. Here, a methanolic extract from corn cobs (MEC) was obtained. MEC contains phenolic compounds, protein, carbohydrates (1.4:0.001:0.001). We evaluated the in vitro and in vivo antioxidant potential of MEC. Furthermore, its antiproliferative property against tumor cells was assessed through MTT assays and proteins related to apoptosis in tumor cells were examined by western blot. MEC showed no hydroxyl radical scavenger capacity, but it showed antioxidant activity in Total Antioxidant Capacity and DPPH scavenger ability assays. MEC showed higher Reducing Power than ascorbic acid and exhibited high Superoxide Scavenging activity. In tumor cell culture, MEC increased catalase, metallothionein and superoxide dismutase expression in accordance with the antioxidant tests. In vivo antioxidant test, MEC restored SOD and CAT, decreased malondialdehyde activities and showed high Trolox Equivalent Antioxidant Capacity in animals treated with CCl4. Furthermore, MEC decreased HeLa cells viability by apoptosis due an increase of Bax/Bcl-2 ratio, caspase 3 active. Protein kinase C expression increased was also detected in treated tumor cells. Thus, our findings pointed out the biotechnological potential of corn cobs as a source of molecules with pharmacological activity.

Changes In Chromatin Structure In Nih 3t3 Cells Induced By Valproic Acid And Trichostatin A.

Valproic acid (VPA) and trichostatin A (TSA) are known histone deacetylase inhibitors (HDACIs) with epigenetic activity that affect chromatin supra-organization, nuclear architecture, and cellular proliferation, particularly in tumor cells. In this study, chromatin remodeling with effects extending to heterochromatic areas was investigated by image analysis in non-transformed NIH 3T3 cells treated for different periods with different doses of VPA and TSA under conditions that indicated no loss of cell viability. Image analysis revealed chromatin decondensation that affected not only euchromatin but also heterochromatin, concomitant with a decreased activity of histone deacetylases and a general increase in histone H3 acetylation. Heterochromatin protein 1-α (HP1-α), identified immunocytochemically, was depleted from the pericentromeric heterochromatin following exposure to both HDACIs. Drastic changes affecting cell proliferation and micronucleation but not alteration in CCND2 expression and in ratios of Bcl-2/Bax expression and cell death occurred following a 48-h exposure of the NIH 3T3 cells particularly in response to higher doses of VPA. Our results demonstrated that even low doses of VPA (0.05 mM) and TSA (10 ng/ml) treatments for 1 h can affect chromatin structure, including that of the heterochromatin areas, in non-transformed cells. HP1-α depletion, probably related to histone demethylation at H3K9me3, in addition to the effect of VPA and TSA on histone H3 acetylation, is induced on NIH 3T3 cells. Despite these facts, alterations in cell proliferation and micronucleation, possibly depending on mitotic spindle defects, require a longer exposure to higher doses of VPA and TSA.

In Vitro And In Vivo Anti-angiogenic Effects Of Hydroxyurea.

Hydroxyurea (HU), or hydroxycarbamide, is used for the treatment of some myeloproliferative and neoplastic diseases, and is currently the only drug approved by the FDA for use in sickle cell disease (SCD). Despite the relative success of HU therapy for SCD, a genetic disorder of the hemoglobin β chain that results in red-cell sickling, hemolysis, vascular inflammation and recurrent vasoocclusion, the exact mechanisms by which HU actuates remain unclear. We hypothesized that HU may modulate endothelial angiogenic processes, with important consequences for vascular inflammation. The effects of HU (50-200 μM; 17-24 h) on endothelial cell functions associated with key steps of angiogenesis were evaluated using human umbilical vein endothelial cell (HUVEC) cultures. Expression profiles of the HIF1A gene and the miRNAs 221 and 222, involved in endothelial function, were also determined in HUVECs following HU administration and the direct in vivo antiangiogenic effects of HU were assessed using a mouse Matrigel-plug neovascularization assay. Following incubation with HU, HUVECs exhibited high cell viability, but displayed a significant 75% inhibition in the rate of capillary-like-structure formation, and significant decreases in proliferative and invasive capacities. Furthermore, HU significantly decreased HIF1A expression, and induced the expression of miRNA 221, while downregulating miRNA 222. In vivo, HU reduced vascular endothelial growth factor (VEGF)-induced vascular development in Matrigel implants over 7 days. Findings indicate that HU is able to inhibit vessel assembly, a crucial angiogenic process, both in vitro and in vivo, and suggest that some of HU's therapeutic effects may occur through novel vascular mechanisms.

The Effect Of Celastrol, A Triterpene With Antitumorigenic Activity, On Conformational And Functional Aspects Of The Human 90kda Heat Shock Protein Hsp90α, A Chaperone Implicated In The Stabilization Of The Tumor Phenotype.

Hsp90 is a molecular chaperone essential for cell viability in eukaryotes that is associated with the maturation of proteins involved in important cell functions and implicated in the stabilization of the tumor phenotype of various cancers, making this chaperone a notably interesting therapeutic target. Celastrol is a plant-derived pentacyclic triterpenoid compound with potent antioxidant, anti-inflammatory and anticancer activities; however, celastrol's action mode is still elusive. In this work, we investigated the effect of celastrol on the conformational and functional aspects of Hsp90α. Interestingly, celastrol appeared to target Hsp90α directly as the compound induced the oligomerization of the chaperone via the C-terminal domain as demonstrated by experiments using a deletion mutant. The nature of the oligomers was investigated by biophysical tools demonstrating that a two-fold excess of celastrol induced the formation of a decameric Hsp90α bound throughout the C-terminal domain. When bound, celastrol destabilized the C-terminal domain. Surprisingly, standard chaperone functional investigations demonstrated that neither the in vitro chaperone activity of protecting against aggregation nor the ability to bind a TPR co-chaperone, which binds to the C-terminus of Hsp90α, were affected by celastrol. Celastrol interferes with specific biological functions of Hsp90α. Our results suggest a model in which celastrol binds directly to the C-terminal domain of Hsp90α causing oligomerization. However, the ability to protect against protein aggregation (supported by our results) and to bind to TPR co-chaperones are not affected by celastrol. Therefore celastrol may act primarily by inducing specific oligomerization that affects some, but not all, of the functions of Hsp90α. To the best of our knowledge, this study is the first work to use multiple probes to investigate the effect that celastrol has on the stability and oligomerization of Hsp90α and on the binding of this chaperone to Tom70. This work provides a novel mechanism by which celastrol binds Hsp90α.

Chitosan/tripolyphosphate Nanoparticles Loaded With Paraquat Herbicide: An Environmentally Safer Alternative For Weed Control.

Paraquat is a fast acting nonselective contact herbicide that is extensively used worldwide. However, the aqueous solubility and soil sorption of this compound can cause problems of toxicity in nontarget organisms. This work investigates the preparation and characterization of nanoparticles composed of chitosan and sodium tripolyphosphate (TPP) to produce an efficient herbicidal formulation that was less toxic and could be used for safer control of weeds in agriculture. The toxicities of the formulations were evaluated using cell culture viability assays and the Allium cepa chromosome aberration test. The herbicidal activity was investigated in cultivations of maize (Zea mays) and mustard (Brassica sp.), and soil sorption of the nanoencapsulated herbicide was measured. The efficiency association of paraquat with the nanoparticles was 62.6 ± 0.7%. Encapsulation of the herbicide resulted in changes in its diffusion and release as well as its sorption by soil. Cytotoxicity and genotoxicity assays showed that the nanoencapsulated herbicide was less toxic than the pure compound, indicating its potential to control weeds while at the same time reducing environmental impacts. Measurements of herbicidal activity showed that the effectiveness of paraquat was preserved after encapsulation. It was concluded that the encapsulation of paraquat in nanoparticles can provide a useful means of reducing adverse impacts on human health and the environment, and that the formulation therefore has potential for use in agriculture.

Developmental Rate Of Immatures Of Two Fly Species Of Forensic Importance: Sarcophaga (liopygia) Ruficornis And Microcerella Halli (diptera: Sarcophagidae).

Since insect species are poikilothermic organisms, they generally exhibit different growth patterns depending on the temperature at which they develop. This factor is important in forensic entomology, especially for estimating postmortem interval (PMI) when it is based on the developmental time of the insects reared in decomposing bodies. This study aimed to estimate the rates of development, viability, and survival of immatures of Sarcophaga (Liopygia) ruficornis (Fabricius 1794) and Microcerella halli (Engel 1931) (Diptera: Sarcophagidae) reared in different temperatures: 10, 15, 20, 25, 30, and 35 ± 1 °C. Bovine raw ground meat was offered as food for all experimental groups, each consisting of four replicates, in the proportion of 2 g/larva. To measure the evolution of growth, ten specimens of each group were randomly chosen and weighed every 12 h, from initial feeding larva to pupae, and then discarded. Considering the records of weight gain, survival rates, and stability of growth rates, the range of optimum temperature for the development of S. (L.) ruficornis is between 20 and 35 °C, and that of M. halli is between 20 and 25 °C. For both species, the longest times of development were in the lowest temperatures. The survival rate at extreme temperatures (10 and 35 °C) was lower in both species. Biological data such as the ones obtained in this study are of great importance to achieve a more accurate estimate of the PMI.