Influence of Anticholinesterase Drugs on Activity and Properties of Na+,K+-ATPase in Rat Erythrocytes under Stress Caused by Intense Physical Exercise.

We studied the effect of intramuscular injection of physostigmine and neostigmine on Na+,K+-ATPase activity in erythrocytes of rats subjected to intense physical exercise. Both anticholinesterase drugs had a significant effect on the development of the stress response, which was expressed in a decrease in the manifestation of its individual components such as the concentration of ascorbic acid in the adrenal glands, stress-related erythrocyte polycythemia, and LPO indicators. Anticholinesterase drugs reverse the stress-induced decrease in Na+,K+-ATPase activity, as well as changes in its magnesium-dependent properties. There were no changes in the activity of the studied enzyme in the erythrocyte ghosts. We associate the observed differences with the correction of the functions of the cholinergic components of the hypothalamic-pituitary-adrenal axis leading to the development of a hypoergic type stress reaction.

Red Blood Cell Membrane Spontaneously Coated Nanoprodrug Based on Phosphatidylserine for Antiatherosclerosis Applications.

Atherosclerosis (AS) is characterized by the accumulation of lipids within the walls of coronary arteries, leading to arterial narrowing and hardening. It serves as the primary etiology and pathological basis for cardiovascular diseases affecting the heart and brain. However, conventional pharmacotherapy is constrained by inadequate drug delivery and pronounced toxic side effects. Moreover, the inefficacy of nanomedicine delivery systems in controlling disease progression may be attributed to nonspecific clearance by the mononuclear phagocyte system. Thus, a biomimetic platform spontaneously enveloped by red blood cell membrane is exploited for anti-atherosclerosis applications, offering favorable biocompatibility. The CLIKKPF polypeptide is introduced to develop red blood cell membrane spontaneously encapsulated nanotherapeutics only through simple coincubation. Given the functional modifications, RBC@P-LVTNPs is beneficial to facilitate the target drug delivery to the atherosclerotic lesion, responding precisely to the pathological ROS accumulation, thereby accelerating the on-demand drug release. Both in vivo and in vitro results also confirm the significant therapeutic efficacy and favorable biocompatibility of the biomimetic nanomedicine delivery system, thus providing a promising candidate for nanotherapeutics against AS.

Protective Effect of Polyphenolic Extracts from Hippophae rhamnoides L. and Reynoutria japonica Houtt. on Erythrocyte Membrane.

Sea buckthorn and Japanese knotweed are known in many traditional medicine systems to be a great source of bioactive substances. This research aims to compare the bioactivity and protective effects of the phenolic extracts of leaves from sea buckthorn and roots and leaves from the Japanese knotweed on erythrocytes. The polyphenol composition of the extract was analyzed using UPLC-PDA-ESI-MS/MS. The extracts' toxicity and impact on the erythrocytes' osmotic fragility were measured spectrophotometrically. The antioxidant activity was determined based on the inhibition of oxidation of erythrocytes and their membrane induced by 2,2'-Azobis(2-methylpropionamidine) dihydrochloride (AAPH),measured spectrophotometrically and using fluorimetry. To find the possible mechanism of the extracts' action, extract-modified cells were observed under a microscope, and the potential localization of the extract's phytochemical composition was checked using fluorescent probes. The results showed that the used extracts are not toxic to erythrocytes, increase their osmotic resistance, and successfully protect them against free radicals. Extract components localize on the outer part of the membrane, where they can scavenge the free radicals from the environment. Altogether, the presented extracts can greatly protect living organisms against free radicals and can be used to support the treatment of diseases caused by excess free radicals.

Acrolein Induces Changes in Cell Membrane and Cytosol Proteins of Erythrocytes.

High concentrations of acrolein (2-propenal) are found in polluted air and cigarette smoke, and may also be generated endogenously. Acrolein is also associated with the induction and progression of many diseases. The high reactivity of acrolein towards the thiol and amino groups of amino acids may cause damage to cell proteins. Acrolein may be responsible for the induction of oxidative stress in cells. We hypothesized that acrolein may contribute to the protein damage in erythrocytes, leading to the disruption of the structure of cell membranes. The lipid membrane fluidity, membrane cytoskeleton, and osmotic fragility were measured for erythrocytes incubated with acrolein for 24 h. The levels of thiol, amino, and carbonyl groups were determined in cell membrane and cytosol proteins. The level of non-enzymatic antioxidant potential (NEAC) and TBARS was also measured. The obtained research results showed that the exposure of erythrocytes to acrolein causes changes in the cell membrane and cytosol proteins. Acrolein stiffens the cell membrane of erythrocytes and increases their osmotic sensitivity. Moreover, it has been shown that erythrocytes treated with acrolein significantly reduce the non-enzymatic antioxidant potential of the cytosol compared to the control.

Effect of Garlic Extract on the Erythrocyte as a Simple Model Cell.

Garlic is known to have diverse effects on mammalian cells, being cytotoxic, especially to cancer cells, but also protect against oxidative stress. Mammalian erythrocyte is a simple cell devoid of intracellular organelles, protein synthesis ability, and most signaling pathways. Therefore, examination of the effects of garlic on erythrocytes allows for revealing primary events in the cellular action of garlic extract. In this study, human erythrocytes or erythrocyte membranes were exposed to garlic extract at various dilutions. Hemoglobin oxidation to methemoglobin, increased binding of hemoglobin to the membrane, and formation of Heinz bodies were observed. Garlic extract depleted acid-soluble thiols, especially glutathione, and induced a prooxidative shift in the cellular glutathione redox potential. The extract increased the osmotic fragility of erythrocytes, induced hemolysis, and inhibited hemolysis in isotonic ammonium chloride, indicative of decreased membrane permeability for Cl- and increased the membrane fluidity. Fluorescent probes indicated an increased level of reactive oxygen species and induction of lipid peroxidation, but these results should be interpreted with care since the extract alone induced oxidation of the probes (dichlorodihydrofluorescein diacetate and BODIPY C11). These results demonstrate that garlic extract induces oxidative changes in the erythrocyte, first of all, thiol and hemoglobin oxidation.

All-stage targeted red blood cell membrane-coated docetaxel nanocrystals for glioma treatment.

Challenges for glioma treatment with nanomedicines include physio-anatomical barriers (the blood-brain barrier and blood-brain tumor barrier), low drug loading capacity, and limited circulation time. Here, a red blood cell membrane-coated docetaxel drug nanocrystal (pV-RBCm-NC(DTX)), modified with pHA-VAP (pV) for all-stage targeting of glioma, was designed. The NC(DTX) core exhibited a high drug loading capacity but low in vivo stability, and the RBCm coating significantly enhanced the stability and prolonged in vivo circulation. Moreover, the Y-shaped targeting ligand pV was modified by a mild avidin-biotin interaction, which endowed RBCm-NC(DTX) with superior barrier-crossing ability and therapeutic efficacy. The integration of nanocrystal technology, cell membrane coating, and the avidin-biotin insertion method into this active targeting biomimetic formulation represents a promising drug delivery strategy for glioma.

Marine Polysaccharides Carrageenans Enhance Eryptosis and Alter Lipid Order of Cell Membranes in Erythrocytes.

Aim In the current study, hemocompatibility of three major commercially available types of carrageenans (ι, κ and λ) was investigated focusing on eryptosis. MATERIALS AND METHODS: Carrageenans of ι-, κ- and λ-types were incubated with washed erythrocytes (hematocrit 0.4%) at 0-1-5-10 g/L for either 24 h or 48 h. Incubation was followed by flow cytometry-based quantitative analysis of eryptosis parameters, including cell volume, cell membrane scrambling and reactive oxygen species (ROS) production, lipid peroxidation markers and confocal microscopy-based evaluation of intracellular Ca2+ levels, assessment of lipid order in cell membranes and the glutathione antioxidant system. Confocal microscopy was used to assess carrageenan cellular internalization using rhodamine B isothiocyanate-conjugated carrageenans. RESULTS: All three types of carrageenans were found to trigger eryptosis. Pro-eryptotic properties were type-dependent and λ-carrageenan had the strongest impact inducing phosphatidylserine membrane asymmetry, changes in cell volume, Ca2+ signaling and oxidative stress characterized by ROS overproduction, activation of lipid peroxidation and severe glutathione system depletion. Eryptosis induction by carrageenans does not require their uptake by erythrocytes. Changes in physicochemical properties of cell membrane were also type-dependent. No carrageenan-induced generation of superoxide and hydroxyl radicals was observed in cell-free milieu. CONCLUSIONS: Our findings suggest that ι-, κ- and λ-types trigger eryptosis in a type-dependent manner and indicate that carrageenans can be further investigated as potential eryptosis-regulating therapeutic agents.

Acarbose Mitigates Age-Dependent Alterations in Erythrocyte Membrane Transporters During Aging in Rats.

Acarbose (ACA), a well-studied and effective inhibitor of α-amylase and α-glucosidase, is a postprandial-acting antidiabetic medicine. The membrane of the erythrocyte is an excellent tool for analyzing different physiological and biochemical activities since it experiences a range of metabolic alterations throughout aging. It is uncertain if ACA modulates erythrocyte membrane activities in an age-dependent manner. As a result, the current study was conducted to explore the influence of ACA on age-dependent deteriorated functions of transporters/exchangers, disrupted levels of various biomarkers such as lipid hydroperoxides (LHs), protein carbonyl (PCO), sialic acid (SA), total thiol (-SH), and erythrocyte membrane osmotic fragility. In addition to a concurrent increase in Na+/H+ exchanger activity and concentration of LH, PCO, and osmotic fragility, we also detected a considerable decrease in membrane-linked activities of Ca2+-ATPase (PMCA) and Na+/K+-ATPase (NKA), as well as concentrations of SA and -SH in old-aged rats. The aging-induced impairment of the activities of membrane-bound ATPases and the changed levels of redox biomarkers were shown to be effectively restored by ACA treatment.

Appraisal of Anti-Arthritic and Anti-Inflammatory Potential of Folkloric Medicinal Plant Peganum harmala.

BACKGROUND & OBJECTIVE: Peganum harmala has been traditionally used to manage rheumatoid arthritis (RA) and other inflammatory conditions. However, its use against RA has not been scientifically evaluated. The current study was designed to assess the anti-arthritic and anti-inflammatory activities of the methanolic extract of P. harmala leaves by in vitro and in vivo methods. METHODS: The in vitro assays were carried out to determine the effect of plant extract on inhibition of egg albumin denaturation and human red blood cell membrane (HRBC) stabilization. Moreover, 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity was performed to determine the antioxidant potential. In vivo anti-arthritic activity was performed by determining the curative effect against Complete Freund's adjuvant (0.1 ml). The plant extract was administered to rats orally at 200, 400 and 600 mg/kg/day for 21 days. RESULTS: The values of IC50 of plant extract in protein denaturation, stabilization of HRBC and DPPH assays were 77.54 mg/ml, 23.90 mg/ml and 58.09 µg/ml, respectively. Moreover, the plant extract significantly attenuated the poly-arthritis and weight loss, anemia and paw edema. The plant extract restored the level of C-reactive protein, rheumatoid factor, alanine transaminase, aspartate transaminase and alkaline phosphatase in poly-arthritic rats. Moreover, the plant extract restored the immune organs' weight in treated rats. Treatment with P. harmala also significantly subdued the oxidative stress by reinstating superoxide dismutase, reduced glutathione, catalase and malondialdehyde in poly-arthritic rats. The plant extract notably restored the prostaglandin-E2 and tumor necrosis factor (TNF)-α in the serum of poly-arthritic rats. CONCLUSION: It was concluded that P. harmala extract had potential antioxidant, anti-inflammatory and antiarthritic activities, which primarily might be attributed to alkaloids, flavonoids and phenols.

Therapeutic appraisal of ephedrine against rheumatoid arthritis: A new indication.

Ephedra, natural flora has been used traditionally to treat rheumatism since decades. The scientific evidence of anti-rheumatic effect of this plant has also been reported. But the anti-rheumatic activity of major constituent of this plant (ephedrine) has not been evaluated. Based on this, the current study was aimed to assess anti-arthritic activity of ephedrine by using in vitro and in vivo approaches. Correspondingly, enzyme linked immunosorbent assay was performed for the estimation of prostaglandins E2 (PGE2) and tumor necrosis factor-α (TNF-α) in serum of formaldehyde-induced arthritic animals. The results elaborated significant reduction in albumin denaturation and remarkable progress on stabilization of red blood cells outer membrane at higher concentration during in vitro experiments. The ephedrine (40mg/kg) revealed noteworthy (p<0.001) inhibition in paw swelling in animals intoxicated with albumin as well as formaldehyde as compared to animals of control group by in vivo results. In this assay, ephedrine (20 & 40 mg/kg orally) significantly suppressed the level of these inflammatory markers (PGE2 & TNF-α). Ephedrine exhibited anti-arthritic effect by decreasing pro-inflammatory cytokines (PGE2 & TNF-α). This experimental work pharmacologically supports the use of ephedrine as anti-rheumatic drug but limited to evaluate in immunological arthritic model.

GC-MS profiling and in vitro antioxidant, cytotoxic and antimicrobial activities of Trianthema triquetra Rottl. ex Willed.

Trianthema triquetra Rottl. ex Willed is being used as herbal remedy for chronic ulcer, wound healing, diabetes, skin and inflammatory diseases in India and Pakistan. Still, no scientific data is available about the therapeutic potential and phytochemistry of the plant. The aim of the current investigation is to perform GC-MS analysis, antioxidant (total phenolic and flavonoid content, DPPH assay), antimicrobial (disc diffusion assay) and cytotoxic (XTT and RBC's cellular membrane protection assay) studies. Whole plant material was dried and extracted with methanol to get crude methanolic extract and then it was fractionated with n-hexane, ethyl-acetate, chloroform, n-butanol and water. Results showed that n-butanol fraction exhibited a significant (p<0.05) antioxidant potential measured by DPPH assay (IC50=63.35±0.13µg/mL) and also possess highest phenolic content (177±4.36mg/g GAE). Whereas, n-hexane fraction showed highest flavonoid content (14.67±1.53mg/g QE). Two major components (2, 4-Ditert-butyl-6-nitrophenol (26.79%) and Squalene (25.64%) were detected in GC-MS analysis of chloroform fraction, eluted from column chromatography. Moreover, chloroform fraction also exhibited antibacterial activity towards all the tested strains of bacteria and fungi. Significant (p<0.05) dose dependent inhibition response on cell growth against CCRF-CEM cell lines was exhibited by methanolic extract. Furthermore, hemolytic potential of methanolic extract was found to be in safe range (2.23%-6.37%). So, it can be inferred that Trianthema triquetra can be exploited as an alternative remedy for cancer, oxidative stress related disorders and in various skin diseases.

The Impact of Short-Term Shark Liver Oil Supplementation on the Fatty Acid Composition of Erythrocyte Membranes.

Fatty acid (FA) balance is strictly related to human health. The composition of fatty acids in lipid membranes seems to be influenced by diet. Shark liver oil (SLO) supplementation has been widely used recently in the prevention and treatment of human diseases. We analyzed the impact of short-term SLO supplementation on certain biochemical parameters and erythrocyte FA composition in a group of young healthy women. Our results showed that 6 weeks of SLO supplementation led to a significant decrease in C-reactive protein levels in sera and intracellular cholesterol levels in peripheral blood mononuclear cells. SLO supplementation caused a significant increase in the content of the polyunsaturated omega-3 FAs: docosahexaenoic acid, docosapentaenoic acid and α-linolenic acid. In the group of omega-6 FAs, we observed a significant elevation of arachidonic and dihomo-gamma-linoleic acid content. Due to these alterations, the omega-3 index increased significantly from 3.6% (before) to 4.2% (after supplementation). We also observed the impact of SLO supplementation on the membrane fluidity index. The ratio between saturated and unsaturated FAs decreased significantly from 13.1 to 9.9. In conclusion, our results show that even short-term SLO supplementation can improve human erythrocyte fatty acid composition and other parameters that may have health-promoting consequences.

An In Vitro Comparative Study of the Effects of Tetrabromobisphenol A and Tetrabromobisphenol S on Human Erythrocyte Membranes-Changes in ATP Level, Perturbations in Membrane Fluidity, Alterations in Conformational State and Damage to Proteins.

Brominated flame retardants (BFRs) are substances used to reduce the flammability of plastics. Among this group, tetrabormobisphenol A (TBBPA) is currently produced and used on the greatest scale, but due to the emerging reports on its potential toxicity, tetrabromobisphenol S (TBBPS)-a compound with a very similar structure-is used as an alternative. Due to the fact that the compounds in question are found in the environment and in biological samples from living organisms, including humans, and due to the insufficient toxicological knowledge about them, it is necessary to assess their impacts on living organisms and verify the validity of TBBPA replacement by TBBPS. The RBC membrane was chosen as the research model. This is a widely accepted research model for assessing the toxicity of xenobiotics, and it is the first barrier to compounds entering circulation. It was found that TBBPA and TBBPS caused increases in the fluidity of the erythrocyte membrane in their hydrophilic layer, and conformational changes to membrane proteins. They also caused thiol group elevation, an increase in lipid peroxidation (TBBPS only) and decreases in the level of ATP in cells. They also caused changes in the size and shape of RBCs. TBBPA caused changes in the erythrocyte membrane at lower concentrations compared to TBBPS at an occupational exposure level.

Combined therapy of ruthenium dendrimers and anti-cancer drugs against human leukemic cells.

Carbosilane ruthenium(ii) dendrimers have been complexed with conventional anti-cancer drugs. Due to its features, the presence of ruthenium within a dendrimer structure improves the anti-cancer properties of nanocomplexes containing 5-flurouracyl, methotrexate and doxorubicin. These dendrimers could be promising carriers of anti-cancer medicines. Ruthenium dendrimers that are positively charged can also enhance the cytotoxicity to cancer cells; moreover, they can form stable complexes with drugs. Results indicate that ruthenium dendrimers combined with doxorubicin and methotrexate significantly reduced the viability of leukaemia 1301 and HL-60 cancer cells.

Inhibition of ABCC5-mediated cGMP transport by progesterone, testosterone and their analogues.

The biodynamics and biokinetics of sex hormones are complex. In addition to the classical steroid receptors (nuclear receptors), these hormones act through several non-genomic mechanisms. Modulation of ABC-transporters by progesterone represents a non-genomic mechanism. In the present study, we employed inside out vesicles from human erythrocytes to characterize high affinity cGMP transport by ABCC5 (member 5 of the ATP-Binding Cassette subfamily C). Progesterone and testosterone inhibited the transport with respective Ki of 1.2 ± 0.3 and 2.0 ± 0.6 µmol/L. We used virtual ligand screening (VLS) to identify analogues to progesterone and testosterone. A large number of substances were screened in silico and the 19 most promising candidates were screened in vitro. Each substance was tested for a concentration of 10 µmol/L. The range of cGMP transport reduction was 21.5% to 86.2% for progesterone analogues and 8.6% to 93.8 % for testosterone analogues. Three of the most potent test compounds (TC) of each analogue class, in addition to progesterone and testosterone, were characterized for concentrations from 1 nanomol/L to 1 mmol/L. The progesterone analogues showed following Ki-values (µmol/L): TC-08: 0.61, TC-16: 0.66 and TC-15: 9.3. The Ki-values (µmol/L) for the testosterone analogues were: TC-18: 0.10, TC-07: 0.67 andTC-05: 2.0. The present study shows that VLS may be a versatile tool in the development of membrane transport modulating agents (MTMAs).

Changes in Human Erythrocyte Membrane Exposed to Aqueous and Ethanolic Extracts from Uncaria tomentosa.

Uncaria tomentosa (Willd.) DC is a woody climber species originating from South and Central America that has been used in the therapy of asthma, rheumatism, hypertension, and blood purification. Our previous study showed that U. tomentosa extracts altered human erythrocyte shape, which could be due to incorporation of the compounds contained in extracts into the erythrocyte membrane. The aim of the present study was to determine how the compounds contained in U. tomentosa extracts incorporate into the human erythrocyte membrane. The study has assessed the effect of aqueous and ethanolic extracts from leaves and bark of U. tomentosa on the osmotic resistance of the human erythrocyte, the viscosity of erythrocyte interior, and the fluidity of erythrocyte plasma membrane. Human erythrocytes were incubated with the studied extracts in the concentrations of 100, 250, and 500 µg/mL for 2, 5, and 24 h. All extracts tested caused a decrease in erythrocyte membrane fluidity and increased erythrocyte osmotic sensitivity. The ethanolic extracts from the bark and leaves increased viscosity of the erythrocytes. The largest changes in the studied parameters were observed in the cells incubated with bark ethanolic extract. We consider that the compounds from U. tomentosa extracts mainly build into the outer, hydrophilic monolayer of the erythrocyte membrane, thus protecting the erythrocytes against the adverse effects of oxidative stress.

RRx-001 Increases Erythrocyte Preferential Adhesion to the Tumor Vasculature.

Red blood cells (RBCs) serve a variety of functions beyond mere oxygen transport both in health and pathology. Notably, RRx-001, a minimally toxic pleiotropic anticancer agent with macrophage activating and vascular normalization properties currently in Phase III trials, induces modification to RBCs which could promote vascular adhesion similar to sickle cells. This study assessed whether RBCs exposed to RRx-001 adhere to the tumor microvasculature and whether this adhesion alters tumor viability. We next investigated the biomechanics of RBC adhesion in the context of local inflammatory cytokines after treatment with RRx-001 as a potential mechanism for preferential tumor aggregation. Human HEP-G2 and HT-29 tumor cells were subcutaneously implanted into nu/nu mice and were infused with RRx-001-treated and Technetium-99m (99mTc)-labeled blood. RBC adhesion was quantified in an in vitro human umbilical vein endothelial cell (HUVEC) assay under both normoxic and hypoxic conditions with administration of either lipopolysaccharide (LPS) or Tumor necrosis alpha (TNFα) to mimic the known inflammation in the tumor microenvironment. One hour following administration of 99mTc labeled RBCs treated with 10 mg/kg RRx-001, we observed an approximate 2.0-fold and 1.5-fold increase in 99mTc-labeled RBCs compared to vehicle control in HEPG2 and HT-29 tumor models, respectively. Furthermore, we observed an approximate 40% and 36% decrease in HEP-G2 and HT-29 tumor weight, respectively, following treatment with RRx-001. To quantify RBC adhesive potential, we determined τ50, or the shear stress required for 50% disassociation of RBCs from HUVECs. After administration of TNF-α under normoxia, τ50 was determined to be 4.5 dynes/cm2 (95% CI: 4.3-4.7 dynes/cm2) for RBCs treated with 10 µM RRx-001, which was significantly different (p < 0.05) from τ50 in the absence of treatment. Under hypoxic conditions, the difference of τ50 with (4.8 dynes/cm2; 95% CI: 4.6-5.1 dynes/cm2) and without (2.6 dynes/cm2; 95% CI: 2.4-2.8 dynes/cm2) 10 µM RRx-001 treatment was exacerbated (p = 0.05). In conclusion, we demonstrated that RBCs treated with RRx-001 preferentially aggregate in HEP-G2 and HT-29 tumors, likely due to interactions between RRx-001 and cysteine residues within RBCs. Furthermore, RRx-001 treated RBCs demonstrated increased adhesive potential to endothelial cells upon introduction of TNF-α and hypoxia suggesting that RRx-001 may induce preferential adhesion in the tumor but not in other tissues with endothelial dysfunction due to conditions prevalent in older cancer patients such as heart disease or diabetic vasculopathy.

Mechanism of CuSO4 cytotoxicity in goat erythrocytes after high-level in vitro exposure to isotonic media.

Copper (Cu) is a common environmental pollutant in nature. Cu-poisoning can cause liver damage and erythrocytes hemolysis. To evaluate the effect of CuSO4 poisoning on the morphological and functional characteristics of goat red blood cells. Five 10-14-month-old goats were selected for jugular vein blood sampling to obtain erythrocytes, and then the erythrocytes were processed with different concentrations (0, 10, 20, 30, 40 and 50 µmol/L) of CuSO4 for 48 h, and 40 µmol/L doses CuSO4 incubated for different time (12, 24, 36, 48 and 60 h) to process erythrocytes. We observed the changes in erythrocyte morphology through scanning electron microscopy, and detected the antioxidant function and activities of three ATPases. Additionally, biological properties were examined from the perspectives of phospholipids and membrane protein components, permeability fragility, and fluidity in erythrocytes. We found that after CuSO4 treatment, the antioxidant capacity of erythrocytes decreased, which was manifested as increased MDA content and decreased CuZn-SOD and GSH-Px activities (p < 0.05). In addition, we also found that erythrocyte fluidity decreased, osmotic fragility increased, membrane phospholipid percentage and protein composition changes abnormally, and Na+/K+-ATPase, Mg2+-ATPase and Ca2+-ATPase activities decreased (p < 0.05). From the results, it can be concluded that CuSO4 exposure causes hemolysis of goat erythrocytes through oxidative stress to the structure and function of erythrocytes, showing a dose-time effect.

Evaluation of Biological Activity of Derivatives of 1,3,4-Thiadiazole, 1,3,4-Oxadiazole and 1,2,4-Triazole.

OBJECTIVE: 1,3,4-thiadiazole (A), 1,3,4-oxadiazole (B) and 1,2,4-triazole (C) derivatives have been known for their immense pharmacotherapeutic potential. The current research article attempts to further explore and understand the probable biochemical mechanism related to antiinflammatory activity of derivatives. METHODS: The screened A, B and C derivatives were investigated for both in-vitro (Erythrocyte Membrane stabilization activity, Proteinase enzyme inhibitory activities) and in-vivo correlation using acute and chronic anti-inflammatory potential by carrageenan induced rats paw edema and cotton pellet granuloma methods, respectively. The activity was studied after interpreting acute toxicity studies results. RESULTS: In vitro studies in the case of Erythrocyte Membrane stability and Proteinase enzyme inhibitory activities exhibited by A, B, and C at 100 ppm were found to be 48.89%, 51.08% and 50.08% and 66.78%, 76.91% and 57.41%, respectively. The maximum toxic dose was found to be 2000 mg/kg. The derivatives were studied for two-dose levels viz; Lower (100 mg/kg) and higher dose (200 mg/kg). In rat paw edema, maximum decrease was obtained for A (50.05%), B (50.05%) and C (51.06%) at lower and higher dose at 68.76%, 55.61%, and 65.26%, respectively for effect up to 24 h. In the chronic model of cotton pellet granuloma viz; higher and lower doses of A, B and C exhibited 38.15%, 33.19% and 30.25 % and 19.45%, 18.55% and 17.55 %, respectively. CONCLUSION: The studied models depicted that derivatives A, B and C have the probable potential as anti-inflammatory agents. Further studies need to be undertaken to explore their potential in the different therapeutic areas.

Demonstration of intracellular trafficking, cytosolic bioavailability, and target manipulation of an antibody delivery platform.

Intracellular antibody delivery into live cells has significant implications for research and therapeutic applications. However, many delivery systems lack potency due to low uptake and/or endosomal entrapment and understanding of intracellular delivery processes is lacking. Herein, we studied the cellular uptake, intracellular trafficking and targeting of antibodies using our previously developed Hex antibody nanocarrier. We demonstrated Hex-antibodies were internalized through multiple endocytic routes into lysosomes and provide evidence of endo/lysosomal disruption and Hex-antibody release to the cytosol. Cytosolic antibodies retained their bioactivity for at least 24 h. Functional effect of Hex delivered anti-STAT3 antibodies was evidenced by inhibition of nuclear translocation of cytosolic transcription factor STAT3. This study has generated understanding of key steps in the Hex intracellular antibody delivery system and will facilitate the development of effective cytosolic antibody delivery and applications in both the therapeutic and research domains.