Red Blood Cell Membrane Research Articles

Synthesis of 2-Phenoxyacetic Acid Analogs for Evaluation of the Anti-inflammatory Potential and Subsequent <i>In Silico</i> Analysis

Several 2-phenoxyacetic acid derivatives and their isosteres were synthesized in moderate to high yield (57–96%) for searching newer and safer alternatives to these existing anti-inflammatory agents. The compounds were characterized by 1H-NMR, 13C-NMR and HRMS. The synthesized compounds have been subjected to in vitro anti-inflammatory activity using egg albumin denaturation and human red blood cell membrane stabilization methods. The compounds were also evaluated for their anti-inflammatory activity in the mice model. The synthesized compounds were subsequently subjected to docking analysis. The in vitro pharmacokinetic properties were analyzed by applying the pkCSM software and drug-likeness was checked by using Lipinski’s rule. Some synthesized esters and amides, like SM61, SM81, SM82, SM83, SM91 and SM101, were significantly more powerful as anti-inflammatory agents. The efficacies were correlated with their orientations in the binding pocket of the cyclooxygenase enzyme. The encouraging in vitro and in vivo activities, the optimum pharmacokinetic profile, and good drug-likeness indicated that these scaffolds could be considered for future discovery of newer nonsteroidal anti-inflammatory drugs (NSAIDs).

Enzyme-armed nanocleaner provides superior detoxification against organophosphorus compounds via a dual-action mechanism

By inhibiting acetylcholinesterase (AChE) activity, organophosphate compounds (OPs) can quickly cause severe injury to the nervous system and death, making it extremely difficult to rescue victims after OP exposure. However, it is quite challenging to construct scavengers that neutralize and eliminate these harmful chemical agents promptly in the blood circulation system. Herein, we report an enzyme-armed biomimetic nanoparticle that enables a ‘targeted binding and catalytic degradation’ action mechanism designed for highly efficient in vivo detoxification (denoted as ‘Nanocleaner’). Specifically, the resulting Nanocleaner is fabricated with polymeric cores camouflaged with a modified red blood cell membrane (RBC membrane) that is inserted with the organophosphorus hydrolase (OPH) enzyme. In such a subtle construct, Nanocleaner inherits abundant acetylcholinesterase (AChE) on the surface of the RBC membrane, which can specifically lure and neutralize OPs through biological binding. The OPH enzyme on the membrane surface breaks down toxicants catalytically. The in vitro protective effects of Nanocleaner against methyl paraoxon (MPO)-induced inhibition of AChE activity were validated using both preincubation and competitive regimens. Furthermore, we selected the PC12 neuroendocrine cell line as an experimental model and confirmed the cytoprotective effects of Nanocleaner against MPO. In mice challenged with a lethal dose of MPO, Nanocleaner significantly reduces clinical signs of intoxication, rescues AChE activity and promotes the survival rate of mice challenged with lethal MPO. Overall, these results suggest considerable promise of enzyme-armed Nanocleaner for the highly efficient removal of OPs for clinical treatment.

Antimicrobial and Anti-Inflammatory Potential of Euphorbia paralias (L.): a bioprospecting study with phytoconstituents analysis.

The phytochemicals in the aerial parts of Euphorbia paralias (also known as Sea Spurge) and their anti-inflammatory and antimicrobial activities were investigated. The methanolic extract was characterized using GC-MS and HPLC techniques. The anti-inflammatory feature was estimated through a Human Red Blood Cell (HRBC) membrane stabilization technique, while the antimicrobial feature was evaluated by the disc diffusion agar technique, minimum bactericidal concentration, and minimum inhibitory concentration (MIC) via micro-broth dilution method. The GC/MS results demonstrated the existence of various phytochemicals, such as n-hexadecenoic acid, cis-11-eicosenoic acid, and methyl stearate, recognized for their anti-inflammatory and antibacterial features. The similarity of the phytochemical composition with other Euphorbia species emphasizes the genus-wide similarity. The anti-inflammatory activity exhibited a noteworthy inhibitory effect comparable to the reference drug indomethacin. The extract's antimicrobial potential was tested against a range of microorganisms, demonstrating significant action against Gram-positive bacteria and Candida albicans. The quantification of total phenolics and flavonoids further supported the therapeutic potential of the extract. The methanolic extract from E. paralias emerges as a successful natural source of important active constituents with potential applications as anti-inflammatory and antimicrobial agents. This research provides a first step to valorize Euphorbia paralias insights as a source of worthwhile phytochemicals that have potential applications in the pharmaceutical industry.

Design, Synthesis, and Anti-Prostate Cancer Potential of 2-(4-Nitrobenzyl) Malonates In Vitro and DAL Acute Oral Toxicity Assessment In Vivo.

New 4-nitrobenzyl derivatives were designed and synthesised by nucleophilic substitution reactions of 4-nitrobenzyl bromide with malonic acid and its derivatives. The synthesised molecules were characterised using mass analysis and spectroscopic techniques and tested for their antioxidant properties using various methods, such as nitric oxide, DPPH, and hydrogen peroxide radical scavenging methods. The anti-inflammatory activities of the molecules were assessed using RBC membrane stabilisation and albumin denaturation methods. We evaluated the compounds' potential anti-prostate cancer activity using the DU145 cell line. The MTT assay determined the cell viability, indicating good anti-proliferative activity. The molecule 3 c exhibited the highest potency, with a CTC50 of 11.83 μg/mL. Molecular dynamics simulations were performed to study the stability of the ligand within the protein after docking and the resulting protein-ligand complex. The in vivo analysis of molecule 3 c in the DAL xenograft model demonstrated promising results. The increase in life span, reduction in tumor volume, and comparable effects to standard drugs are encouraging features that suggest that molecule 3 c may possess significant potential as an anti-cancer agent. The research also implies that these molecules might be potential lead compounds for developing new prostate cancer drugs.

β-Citronellol: a potential anti-inflammatory and gastro-protective agent-mechanistic insights into its modulatory effects on COX-II, 5-LOX, eNOS, and ICAM-1 pathways through in vitro, in vivo, in silico, and network pharmacology studies

BackgroundThe current study aimed to evaluate the anti-inflammatory, anti-oxidant, and pronounced gastro-protective activities of β- Citronellol using in vitro, in vivo assays and in silico approaches.MethodsIn vitro assays, denaturation of bovine serum albumin, egg protein, and human Red Blood Cells (RBCs) membrane stabilization were performed, using Piroxicam as standard. For in vivo assessment, Histamine (0.1 ml from 1% w/v) and Formaldehyde (0.1 ml from 2% v/v) were used to mediate inflammation. In silico molecular docking and network pharmacology were employed to probe the possible target genes mediating gastroprotective effect of β-Citronellol at 25, 50, and 100 mg/kg, using indomethacin-induced (25 mg/kg i.p) gastric ulcer in rats. Moreover, Gastric tissues were evaluated for morphological, histopathological, and bio-chemical analysis of PGE2, COX-I, COX-II, 5-LOX, eNOS, ICAM-1, oxygen-free radical scavengers (SOD, CAT), and oxidative stress marker (MDA).Resultsβ-Citronellol prevented denaturation of proteins and RBCs membrane stabilization with maximum effect observed at 6,400 µg/mL. Citronellol decreased rat’s paw edema. Network pharmacology and docking studies revealed gastro-protective potential of Citronellol possibly mediated through arachidonic acid pathways by targeting COX-I, COX-II, PGE2, and 5-LOX. Citronellol reduced the ulcer indices, and histopathological changes. Further, β-Citronellol (50 and 100 mg/kg) increased gastric PGE2, COX-1, and eNOS; while suppressing COX-2, 5-LOX and ICAM-1. Citronellol markedly enhanced the oxidative balance in isolated rat stomach tissues.ConclusionsThe anti-inflammatory, anti-oxidant, and gastro-protective effects of β-Citronellol against indomethacin-induced gastric ulcer model in rats through mediating COX-I, COX-II, PGE2, 5-LOX, eNOS, and ICAM-1 inflammatory markers.Graphical abstract

Antioxidant, membrane stabilizing and thrombolytic potentials of the leaves of erythrina fusca lour

Erythrina fusca (Family: Fabaceae) is famous for many traditional healing. In the present study, the leaves of E. fusca were collected, dried, powdered and then extracted with methanol. It was further partitioned into n-hexane, dichloromethane and aqueous soluble fractions. All the extracts were subjected to antioxidant, membrane stabilizing and thrombolytic assays. In case of free radical scavenging assay, the aqueous soluble fraction showed highest scavenging activity. In ferric reducing anti-oxidant power assay, the dicholoromethane extract showed highest reduction of ferric ion in a dose dependent manner. In total antioxidant assay, the methanol extract and dichloromethane extract showed stronger capacity as antioxidant. During the determination of total phenolic and flavonoid contents, the methanol extract and dichloromethane soluble fraction showed higher contents of phenolics and flavonoids, respectively. The erythrocyte membrane stabilizing assay revealed the capacities of methanol extract and its aqueous fraction to stabilize the RBC membrane in both hypotonic solution- and heat- induced hemolytic conditions. However, the clot lysis ability of the E. fusca leaf extracts was not so prominent. Bangladesh J. Bot. 53(3): 439-445, 2024 (September)

Multi-step synthesis, kinetics and in silico explorations of indole-Phenyl-1,2,4-triazole Bi-heterocyclic hybrids unified with 3-substituted benzamides as elastase inhibitors

In the present research work, a library of unique indole-phenyltriazole hybrids comprising 3-substituted-benzamide moiety was synthesized through convergent multi-step strategy. The structural confirmation of all synthesized molecules was corroborated by IR, 1H NMR, 13C MMR, EI-MS and CHN analysis data. The results of in vitro inhibitory potential of novel benzamides against elastase enzyme revealed that all molecules were potent inhibitors and 10d was most active compound among them having IC50 value of (0.197 ± 0.027 µM), relative to the standard (13.421 ± 0.016 µM). The Kinetics mechanism analyzed by Lineweaver-Burk plots which exposed that 10d inhibited this enzyme competitively by forming an enzyme-inhibitor complex. The inhibition constant Ki determined from Dixon plot for compound 10d was 0.85 µM. Moreover, cytotoxicity of these compounds was also profiled by hemolytic activity and it was observed that almost all these benzamides compounds displayed low cytotoxicity. These molecules also exhibited mild cytotoxicity toward red blood cell membrane. In addition, the in silico computational explorations fully supported the in vitro enzyme inhibitory results. The binding energy ranges from -7.1 to -9.1 kcal/mol, which showed that compound possessed good interaction tendencies to the pancreatic elastase target protein. So, it was anticipated from the experimental results and computational investigations of the current research that these derivatives might lead to further research gateways for obtaining better and safe nontoxic medicinal scaffolds for dealing with the elastase related ailments such as lungs diseases, pruritic skin disease and liver infection.

Synergistic Therapy of Melanoma by Co-Delivery of Dacarbazine and Ferroptosis-Inducing Ursolic Acid Using Biomimetic Nanoparticles.

Melanoma is one of the most aggressive types of cancer and is prone to metastasis, making current clinical treatment quite difficult. The usage of the first-line medication dacarbazine (DTIC) for melanoma is limited due to harsh side effects, limited water solubility, and a short half-life. To tackle these disadvantages, polylactic acid-hydroxyacetic acid copolymer nanoparticles (NPs) loaded with dacarbazine and ursolic acid (NPs) were fabricated, which were further encapsulated with a red blood cell membrane (RNPs). MTT, apoptosis assay, wound healing assay, colony formation assay, and immunohistochemistry were used to assess the antitumor effect of NPs and RNPs. Ferroptosis evaluation was implemented using GSH detection and the malondialdehyde assay. We found that RNPs exhibited stability and biosafety in vitro and in vivo and achieved superior anticancer ability against xenograft tumors compared with single agents and NPs, which indicated the synergistic and biomimetic efficacy. Furthermore, ferroptotic activity was observed in RNPs-treated tumor cells, and ferroptosis inhibition could partially rescue melanoma cells from RNPs-induced cell death. Collectively, this study evaluated the potential of RNPs as a novel biomimetic nanomedicine for synergistic melanoma therapy by eliciting ferroptosis in tumor cells with both anticancer activity and biosafety.

Comparative study of stability and activity of wild-type and mutant human carbonic anhydrase II enzymes using molecular dynamics and docking simulations

The human carbonic anhydrase II (HCA II) enzyme is a cytosolic protein that catalyzes the reversible hydration of carbon dioxide (CO2). Considering the critical role of the HCA II and the effects of some mutations on the activity and stability of the enzyme in humans, several computational methods are used to study the structure and dynamics of the wild-type and the mutant enzymes with three ligands, CO2, 4-nitrophenyl acetate and acetazolamide. Our results of MD simulation of a wild-type enzyme with 4-nitrophenyl acetate show that it created essential effects on the fluctuation of this enzyme and made it more unstable and less compact than the same enzyme without ligand. In the MD of the mutant enzyme with 4-nitrophenyl acetate ligand, no significant difference is observed between with and without ligand. The affinity of the wild-type enzyme to the 4-nitrophenyl acetate is notably higher than the mutant enzyme with the same ligand. Furthermore, results showed that wild-type and mutant enzymes with CO2 are more favorable in stability and flexibility than the same enzymes without ligand. The MD results of wild-type with acetazolamide indicate instability compare without ligand, but in MD of mutant enzyme with acetazolamide show that it more stable and compact than the same enzyme without ligand. Finally, Comparing protein trajectories to assess the impact of ligands on the stability and activity of HCA II enzymes can have medical applications and can in the engineering and design of new variants of carbonic anhydrase enzyme.

Biofilm-camouflaged Prussian blue synergistic mitochondrial mass enhancement for Alzheimer's disease based on Cu2+ chelation and photothermal therapy

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases characterized by cognitive and memory impairment. Metal ion imbalance and Mitochondrial dysfunction, leading to abnormal aggregation of β-amyloid protein (Aβ), are key factors in the pathogenesis of AD. Therefore, we designed a composite nanometer system of red blood cell (RBC) membranes-encapsulated Prussian blue nanoparticles (PB/RBC). Prussian blue nanoparticles (PBNPs) can chelate Cu2+ and reduce reactive oxygen species (ROS). The RBC membranes are a kind of natural long-lasting circulating carrier. At the same time, through NIR irradiation, the excellent photothermal ability of PBNPs can also temporarily open the blood-brain barrier (BBB), enhance the transmission efficiency of PB/RBC across the BBB, and depolymerize the formed Aβ deposits, thereby achieving the optimal therapeutic effect. In vitro and in vivo studies demonstrated that PB/RBC could inhibit Cu2+-induced Aβ monomers aggregation, eliminate the deposition of Aβ plaques, improve the quality of mitochondria, restore the phagocytic function of microglia, alleviate neuroinflammation in APP/PS1 mice, and repair memory damage. In conclusion, our biofilm-camouflaged nano-delivery system provides significant neuroprotection by inhibiting Cu2+-induced Aβ monomers aggregation, photothermally depolymerizing Aβ fibrils and reducing the level of ROS, thus effectively ameliorating and treating AD.

Biomimetic Iron-Based Nanoparticles Remodel Immunosuppressive Tumor Microenvironment for Metabolic Immunotherapy.

Immunotherapy has led to a paradigm shift in reinvigorating treatment of cancer. Nevertheless, tumor associated macrophages (TAMs) experience functional polarization on account of the generation of suppressive metabolites, contributing to impaired antitumor immune responses. Hence, metabolic reprogramming of tumor microenvironment (TME) can synergistically improve the efficacy of anti-tumor immunotherapy. Herein, we engineered an iron-based nanoplatform termed ERFe3O4 NPs. This platform features hollow Fe3O4 nanoparticles loaded with the natural product emodin, the outer layer is coated with red blood cell membrane (mRBCs) inserted with DSPE-PEG2000-galactose. This effectively modulates lactate production, thereby reversing the tumor immune suppressive microenvironment (TIME). The ERFe3O4 NPs actively targeted TAMs on account of their ability to bind to M2-like TAMs with high expression of galectin (Mgl). ERFe3O4 NPs achieved efficient ability to reverse TIME via the production of reducing lactate and prompting enrichment iron of high concentrations. Furthermore, ERFe3O4 NPs resulted in heightened expression of CD16/32 and enhanced TNF-α release, indicating promotion of M1 TAMs polarization. In vitro and in vivo experiments revealed that ERFe3O4 NPs induced significant apoptosis of tumor cells and antitumor immune response. This study combines Traditional Chinese Medicine (TCM) with nanomaterials to synergistically reprogram TAMs and reverse TIME, opening up new ideas for improving anti-tumor immunotherapy.

Optimization of Polyphenol Extraction Conditions from Rhizomes of Curcuma zedoaria with Antioxidant, Anti-Inflammatory and Anti-Diabetic Activities In Vitro

The recovery of polyphenol compounds is seen as an arduous task because polyphenol compounds are available as free aglycones, as sugar or ester conjugates, or as polymers with several monomeric components. The response surface method (RSM) as a tool to optimize the factors that affect extraction efficacy as well as to obtain maximum recovery of the compounds of interest. This study optimizes ultrasound-assisted polyphenol extraction from Curcuma zedoaria rhizomes using response surface methodology (RSM). Three variables were considered: Ethanol concentration (70 - 90 %, v/v), temperature (60 - 80 °C), and ultrasound time (15 - 25 min). Data were analyzed by ANOVA, yielding an R2 of 0.9993, a significant interaction effect (p < 0.0001), and an insignificant lack-of-fit test (p = 0.6684). Optimal conditions for maximum polyphenol content (TPC = 31.05 ± 0.53 mg GAE/g powder) were 80.02 % ethanol, 68 °C, 20.47 min ultrasound time, and 1/10 (w/v) raw material/solvent ratio. Experimental values matched RSM predictions, confirming successful extraction optimization from Curcuma zedoaria rhizomes. The polyphenol-rich optimum extract from Curcuma zedoaria rhizomes has been studied for its antioxidant, anti-inflammatory and anti-inflammatory properties in vitro. The results were found, the optimum extract of Curcuma zedoaria rhizomes could perform effective neutralization activities of free radicals performed in DPPH test (IC50 = 5.89 ± 0.23 µg/mL), NO· (IC50 = 5.89 ± 0.23 µg/mL) and ABTS·+ (IC50 = 8.28 ± 0.12 µg/mL). Besides, this optimum extract had the ability to protect red blood cell membranes and inhibit protein denaturation due to heat with IC50 times values ​​such as 29.06 ± 0.35 and 30.24 ± 0.32 µg/mL. In addition, it also significantly inhibited α-amylase, α-glucosidase enzyme activities with IC50 values ​​of 5.89 ± 0.23 and 9.62 ± 0.11 μg/mL, respectively. This investigation showed that the polyphenol-rich optimum extract from Curcuma zedoaria rhizomes was a promising antioxidant, anti-inflammatory and anti-diabetic agent. HIGHLIGHTS Using ultrasound and the Box-Behnken design to extract polyphenols from Curcuma zedoaria rhizomes can speed up and improve extraction efficiency. The goal of this study is to maximize the extraction parameters for polyphenols from the rhizomes of Curcuma zedoaria. There is strong agreement between the experimental data and the model that predicts the polyphenol content in the Box-Behnken design. In vitro biological activities such as anti-inflammatory, antidiabetic and antioxidant properties are significantly correlated with polyphenol concentration. The findings of our investigation show a noteworthy enhancement in the bioavailability of polyphenol compounds through increased extraction efficiency and bioactivity. GRAPHICAL ABSTRACT

Epitope prediction of malarial MSP-1 protein of plasmodium sp. using in silico method

Due to its pivotal function in parasite adherence to the RBC membrane during erythrocyte invasion, Msp1 represents a potential vaccine candidate. Three or four fragments of MSP1 are produced during the proteolytic maturation process, and these fragments stay together as a complex on the cell surface. Msp1 fragments have been investigated as potential vaccine candidate, and malaria antibody immunoassays have made use of the fragmented polypeptide, which exhibits a considerable degree of intra-species conservation. Because of its polymorphism, Msp1 is also useful in strain differentiation and carries a crucial genetic marker. To comprehend genetic variation among species and find epitopes for the development of a disease vaccine, Msp1 needs to be investigated. The preliminary investigation on the evolutionary history using the Maximum Likelihood method and the General Time Reversible model (GTR) models were used. The phylogenetic relationship of the MSP1 gene was inferred and it represents the level of similarity between the MSP1 gene of Plasmodium species from various locations in South-East Asia. The top 30 MHC class II epitopic regions were predicted using Bepipred server with epitope mean score above 0.05.

Biomimetic Gd-Metal-Organic Framework Radiosensitizer for Near-Infrared Fluorescence Imaging-Guided Radiotherapy toward Nasopharyngeal Carcinoma.

Radiotherapy (RT) is recognized as a primary treatment modality for Nasopharyngeal carcinoma (NPC). However, enhancing RT's targeting accuracy and selectivity remains a significant challenge. In this study, we present an innovative radiosensitizer, Gd-metal-organic framework (MOF)-based nanocarrier coated with indocyanine green (ICG) and red blood cell membrane (RBCM), designed to bypass immune clearance and achieve prolonged circulation within the bloodstream. This design significantly enhances tumor localization and systemic circulation, as evidenced by in vivo analyses. The strategic accumulation of the Gd-MOF-ICG nanocarrier at the tumor site facilitates precise tumor localization and sensitization to RT, leveraging the RBCM camouflage to enhance the tumor uptake potential. Our comprehensive study introduces a potent approach for optimizing RT in NPC treatment through this advanced theranostic nanoplatform, which combines material science with biomedical engineering to augment the effectiveness of RT and underscores the significance of precision in cancer therapy. This strategy offers a promising avenue for clinical application and further research in targeted cancer treatments.

Bioactivity evaluations from stem bark extract fractions of Myrica esculenta Buch. ‐Ham.ex D. Don

AbstractThe chemical constituents of Myrica esculenta (a plant distributed across Nepal, India, China, and Pakistan) are known for their potent antioxidant properties and reported pharmacological effects. The study used methanol extract to assess antioxidant activity (DPPH assay), anthelmintic effects (standard; albendazole and experimental organism; Eisenia fetida), anti‐inflammatory properties (human red blood cell membrane stabilization), and cytotoxicity (brine shrimp lethality bioassay). The study revealed 39.06% yield with methanol and 2.01% with hexane. Phenolic and flavonoid content were measured at 96.43 mg GAE/g and 14.54 mg QE/g, respectively. Methanolic extract exhibited IC50 of 60.89 μg/mL and LC5o of 160.5298 μg/mL. It also demonstrated anti‐inflammatory activity, notably achieving a 37.8% membrane stabilizing action at 500 μg/mL. Anthelmintic potential was observed at different concentrations (50 mg/mL, 75 mg/mL, and 100 mg/mL), leading to paralysis and death of worms with corresponding times reported. Brine Shrimp Lethality Bioassay revealed concentration‐dependent effects. Methanolic extract of M. esculenta displayed robust antioxidant and anthelmintic activities. The extract was identified as bioactive, holding promise as a source for plant‐derived antitumor compounds. However, further investigation is essential to validate these findings.

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.

Enhanced delivery of CRISPR/Cas9 system based on biomimetic nanoparticles for hepatitis B virus therapy

The persistent presence of covalently closed circular DNA (cccDNA) in hepatocyte nuclei poses a significant obstacle to achieving a comprehensive cure for hepatitis B virus (HBV). Current applications of CRISPR/Cas9 for targeting and eliminating cccDNA have been confined to in vitro studies due to challenges in stable cccDNA expression in animal models and the limited non-immunogenicity of delivery systems. This study addresses these limitations by introducing a novel non-viral gene delivery system utilizing Gemini Surfactant (GS). The developed system creates stable and targeted CRISPR/Cas9 nanodrugs with a negatively charged surface through modification with red blood cell membranes (RBCM) or hepatocyte membranes (HCM), resulting in GS-pDNA@Cas9-CMs complexes. These GS-pDNA complexes demonstrated complete formation at a 4:1 w/w ratio. The in vitro transfection efficiency of GS-pDNA-HCM reached 54.61%, showing homotypic targeting and excellent safety. Additionally, the study identified the most effective single-guide RNA (sgRNA) from six sequences delivered by GS-pDNA@Cas9-HCM. Using GS-pDNA@Cas9-HCM, a significant reduction of 96.47% in in vitro HBV cccDNA and a 52.34% reduction in in vivo HBV cccDNA were observed, along with a notable decrease in other HBV-related markers. The investigation of GS complex uptake by AML-12 cells under varied time and temperature conditions revealed clathrin-mediated endocytosis (CME) for GS-pDNA and caveolin-mediated endocytosis (CVME) for GS-pDNA-HCM and GS-pDNA-RBCM. In summary, this research presents biomimetic gene-editing nanovectors based on GS (GS-pDNA@Cas9-CMs) and explores their precise and targeted clearance of cccDNA using CRISPR/Cas9, demonstrating good biocompatibility both in vitro and in vivo. This innovative approach provides a promising therapeutic strategy for advancing the cure of HBV.

Convergent synthesis, kinetics and computational attributions of indole-N-phenyltriazole hybrids bearing N-(aryl)butanamides as alkaline phosphatase inhibitors

In the research delineate herein, an innovative sequence of new series of multi-functional target molecules (9a-i) having indole-N-phenyltriazole bi-heterocyclic hybrids unified with N-arylated butanamides was synthesized as alkaline phosphatase inhibitor. The structural validation of all the formulated compounds was accomplished through IR, EI-MS, 1H NMR, 13C NMR and CHN analysis data. The in vitro enzyme inhibitory investigation revealed the efficacy of these bi-heterocyclic derivatives, 9a–i, as potent inhibitors of alkaline phosphatase relative to the standard used. The compound 9h was found to be the most active compound (IC50 = 0.062 ± 0.017 μM), and its inhibitory activity is about 10 times higher than potassium dihydrogen phosphate (KH2PO4) (IC50 = 5.251 ± 0.468 μM). The kinetics mechanism was attributed by evaluating the Lineweaver–Burk plots, which revealed that compound 9h inhibited the alkaline phosphatase non-competitively to form an enzyme–inhibitor complex. The inhibition constant Ki determined from Dixon plots for this compound was 0.045 μM. The computational study was in full agreement with the experimental records and these ligands exhibited good interactions and binding energy values. These molecules also demonstrated mild cytotoxicity toward red blood cell membranes when analyzed through hemolysis. So, based on the presented results, these molecules, being the promising inhibitors of alkaline phosphatase, might be deliberated as suitable medicinal scaffolds to render normal calcification of bones and teeth.

Influence of inhibiting methemoglobin formation on erythrocyte antioxidant defense

We aimed to study the influence of preventing methemoglobin (metHb) formation, in the roles of peroxiredoxin 2 (Prx2), glutathione peroxidase (GPx) and catalase (CAT) on the erythrocyte antioxidant defense system. We performed in vitro assays using healthy erythrocytes, with and without inhibition of autoxidation of Hb (saturation with carbon monoxide), followed by H2O2-induced oxidative stress. We assessed the enzyme activities and amounts of CAT, GPx and Prx2 in the red blood cell (RBC) cytosol and membrane and several biomarkers of oxidative stress, such as the reduced and oxidized glutathione levels, thiobarbituric acid reactive substances (TBARS) levels, membrane bound hemoglobin and total antioxidant status. When autoxidation of Hb was inhibited, no significant changes were found for GPx and CAT; Prx2 was observed only in the monomeric form in the cytosol and none bound to the membrane. Blocking the function of Hb as a pseudo-peroxidase does not seem to have an impact on the function of the RBC peroxidases.

Single-shot spatial light interference microscopy for dynamic monitoring of membrane fluctuations.

Single-shot spatial light interference microscopy (SS-SLIM) with a pair of non-polarizing beam splitters is proposed for substantially enhancing the speed and efficiency of conventional SLIM systems. Traditional methods are limited by the need for multiple-frame serial modulation and acquisition by spatial light modulators and detectors. Our approach integrates non-polarizing beam splitters to simultaneously capture four phase-shifted intensity images, increasing the imaging speed by at least fourfold while maintaining high quality. This capability is crucial for effectively monitoring the dynamic fluctuations of red blood cell membranes. Furthermore, the potential applications of the SS-SLIM system in biomedical research are demonstrated, particularly in scenarios requiring high temporal resolution and label-free imaging.