RBC Lysis Research Articles

HER-2 Receptor and αvβ3 Integrin Dual-Ligand Surface-Functionalized Liposome for Metastatic Breast Cancer Therapy.

Human epidermal growth factor receptor-2 (HER2)-positive breast cancer metastasis remains the primary cause of mortality among women globally. Targeted therapies have revolutionized treatment efficacy, with Trastuzumab (Trast), a monoclonal antibody, targeting HER2-positive advanced breast cancer. The tumor-homing peptide iRGD enhances the intratumoral accumulation and penetration of therapeutic agents. Liposomes serve as versatile nanocarriers for both hydrophilic and hydrophobic drugs. Gefitinib (GFB) is a potential anticancer drug against HER2-positive breast cancer, while Lycorine hydrochloride (LCH) is a natural compound with anticancer and anti-inflammatory properties. This study developed TPGS-COOH-coated liposomes co-loaded with GFB and LCH, prepared by the solvent injection method, and surface-functionalized with Trast and iRGD. The dual surface-decorated liposomes (DSDLs) were characterized for their particle size (PS), polydispersity index (PDI), zeta potential (ZP), surface chemistry, surface morphology, and their crystallinity during in-vitro drug release, drug encapsulation, and in-vitro cell line studies on SK-BR-3 and MDA-MB-231 breast cancer cells. The half-maximum inhibitory concentration (IC-50) values of single decorated liposomes (SDLs), iRGD-LP, and Trast-LP, as well as DSDLs (iRGD-Trast-LP) on SK-BR-3 cells, were 6.10 ± 0.42, 4.98 ± 0.36, and 4.34 ± 0.32 μg/mL, respectively. Moreover, the IC-50 values of SDLs and DSDLs on MDA-MB-231 cells were 15.12 ± 0.68, 13.09 ± 0.59, and 11.08 ± 0.48 μg/mL, respectively. Cellular uptake studies using confocal laser scanning microscopy (CLSM) showed that iRGD and Trast functionalization significantly enhanced cellular uptake in both cell lines. The wound-healing assay demonstrated a significant reduction in SDL and DSDL-treated MDA-MB-231 cell migration compared to the control. Additionally, the blood compatibility study showed minimal hemolysis (less than 5% RBC lysis), indicating good biocompatibility and biosafety. Overall, these findings suggest that TPGS-COOH-coated, GFB and LCH co-loaded, dual-ligand (iRGD and Trast) functionalized, multifunctional liposomes could be a promising therapeutic strategy for treating HER2-positive metastatic breast cancer.

The Immunological Profile of Adipose Mesenchymal Stromal/Stem Cells after Cell Expansion and Inflammatory Priming.

AT-MSCs display great immunoregulatory features, making them potential candidates for cell-based therapy. This study aimed to evaluate the "RBC lysis buffer" isolation protocol and immunological profiling of the so-obtained AT-MSCs. We established an immune-comparative screening of AT-MSCs throughout in vitro cell expansion (PM, P1, P2, P3, P4) and inflammatory priming regarding the expression of 28 cell-surface markers, 6 cytokines/chemokines, and 10 TLR patterns. AT-MSCs were highly expandable and sensitive to microenvironment challenges, hereby showing plasticity in distinct expression profiles. Both cell expansion and inflammation differentially modulated the expression profile of CD34, HLA-DR, CD40, CD62L, CD200 and CD155, CD252, CD54, CD58, CD106, CD274 and CD112. Inflammation resulted in a significant increase in the expression of the cytokines IL-6, IL-8, IL-1β, IL-1Ra, CCL5, and TNFα. Depending on the culture conditions, the expression of the TLR pattern was distinctively altered with TLR1-4, TLR7, and TLR10 being increased, whereas TLR6 was downregulated. Protein network and functional enrichment analysis showed that several trophic and immune responses are likely linked to these immunological changes. AT-MSCs may sense and actively respond to tissue challenges by modulating distinct and specific pathways to create an appropriate immuno-reparative environment. These mechanisms need to be further characterized to identify and assess a molecular target that can enhance or impede the therapeutic ability of AT-MSCs, which therefore will help improve the quality, safety, and efficacy of the therapeutic strategy.

Chloride Gradient Is Involved in Ammonium Influx in Human Erythrocytes.

The ammonia/ammonium (NH3/NH4+, AM) concentration in human erythrocytes (RBCs) is significantly higher than in plasma. Two main possible mechanisms for AM transport, including simple and facilitated diffusion, are described; however, the driving force for AM transport is not yet fully characterized. Since the erythroid ammonium channel RhAG forms a structural unit with anion exchanger 1 (eAE1) within the ankyrin core complex, we hypothesized the involvement of eAE1 in AM transport. To evaluate the functional interaction between eAE1 and RhAG, we used a unique feature of RBCs to swell and lyse in isotonic NH4+ buffer. The kinetics of cell swelling and lysis were analyzed by flow cytometry and an original laser diffraction method, adapted for accurate volume sensing. The eAE1 role was revealed according to (i) the changes in cell swelling and lysis kinetics, and (ii) changes in intracellular pH, triggered by eAE1 inhibition or the modulation of eAE1 main ligand concentrations (Cl- and HCO3-). Additionally, the AM import kinetics was analyzed enzymatically and colorimetrically. In NH4+ buffer, RBCs concentration-dependently swelled and lysed when [NH4+] exceeded 100 mM. Cell swelling and hemolysis were tightly regulated by chloride concentration. The complete substitution of chloride with glutamate prevented NH4+-induced cell swelling and hemolysis, and the restoration of [Cl-] dose-dependently amplified the rates of RBC swelling and lysis and the percentage of hemolyzed cells. Similarly, eAE1 inhibition impeded cell swelling and completely prevented hemolysis. Accordingly, eAE1 inhibition, or a lack of chloride anions in the buffer, significantly decreased NH4+ import. Our data indicate that the eAE1-mediated chloride gradient is required for AM transport. Taken together, our data reveal a new player in AM transport in RBCs.

Pre- and post-radical surgery comparative quantification of circulating tumor cells and circulating tumor-associated cells.

e15056 Background: Classical Circulating Tumor Cells (CTCs) are defined as EPCAM+, PanCK+, CD45-, whereas, Circulating Tumor Associated Cells (CTACs) are defined as EPCAM+, PanCK+, CD45+/-. CTACs comprise a heterogenous mix of tumor-associated cells including classical CTCs, cancer stem cells, circulating micro-emboli, tumor cell fused macrophages and other tumor-educated cells including junction endothelial and stromal cells. These cells play either a causative or synergistic role in the metastatic spread of cancer. Although it has been speculated that CTCs and CTACs would be rapidly cleared once tumor burden diminishes, the effect of radical surgery in cases of solid organ cancers and the change in the quantum of these cells after surgery has not been fully explored. Methods: We obtained 15 ml peripheral blood from 20 patients diagnosed with solid organ cancers comprising head and neck (11/20, 55%), breast (4/20, 20%), and colorectal (5/20, 25%) before and immediately after (within 24 hrs.) radical surgery. Peripheral Blood Mononucleated Cells (PBMCs) were isolated after RBC lysis and CTCs and CTACs were identified and enumerated from the same sample using differential apoptosis based negative enrichment followed by fluorescent imaging. Results: Pre-surgery, CTCs ranged from 12 to 0 cells per ml and 0 to 2 per ml post-surgery. CTACs ranged from 17 to 11057 cells per ml and, post-surgery, from 4 to 2041 cells per ml of blood. After surgery, CTCs were eliminated in 19/20 (95%) specimens, and in 1/20 (5%) specimens, there was a slight increase in the number of CTCs post-surgery. In 16/20 (80%) patients, the CTACs reduced by > 80%; in 2/20 (10%) patients, the CTACs reduced by > 50%-<80% and, in 2/20 (10%) patients, CTACs increased by > 5%. Conclusions: The enumeration of CTCs appears to correlate with the tumor burden pre- and post-surgery. On the other hand, although there appears to be a significant reduction in CTACs, residual cells of this phenotype appear to persist. The clinical correlation of this divergent trajectory of CTCs and CTACs pre- and post-surgery warrants further long-term studies.

Ayurvedic herbal formulations Haridra Khanda and Manjisthadi Kwath (brihat) in the management of allergic rhinitis: A pharmacological study

This study aims to pharmacologically validate Haridra Khanda (HK) and Manjishthadi Kwatham (brihat) (MMK) in allergy management using invivo and invitro studies to rationalize the prescription of these two ayurvedic polyherbal drug formulations, which are currently used in Indian government hospitals. Experimental animals received HK and MMK orally from day 0 to day 14 and histamine (1 mg/kg b.w/i.v) and 1 % evans blue (EB) (0.1 mL) via tail vein on day 14. The compound 48/80 (intracutaneous) challenged mice model followed the same technique. The former mimicked acute anaphylaxis and the latter mast cell degranulation. For both models, EB dye leakage was quantified spectrophotometrically to determine vascular permeability. Plasma histamine was measured in Compound 48/80-induced animals using LC-ESI-MS/MS. The guineapig received HK and MMK p.o. and 0.6 % histamine sprayed in a histamine chamber to simulate allergic rhinitis. Blood eosinophil count and sneeze rate were measured in histamine-challenged guineapigs. Goat R.B.C. membrane stability assay (mammalian cell membrane toxicity) and intracellular histamine-induced cytosolic Ca2+ release assay in Chinese hamster ovary (CHO) cells were performed in vitro. For both histamine and Compound 48/80 challenged animals, HK (22.81 % and 14.58 %) and MMK (19.71 % and 22.40 %) significantly reduced EB dye leakage (p < 0.05). Both formulations, HK and MMK considerably (p < 0.05) decreased plasma histamine (29.62 % and 25.37 % respectively) in mice and eosinophilic count (11.56 % and 9.94 % respectively) and sneeze rate (42.58 % and 29.03 % respectively) in guinea pigs. In membrane stability experiment, HK and MMK reduced RBC lysis. Both HK and MMK raw/dialysate blocked CHO cell cytosolic Ca2+ release. HK and MMK activities mimic mast cell stabilization with possible H1 receptor inactivation seen by decreased Ca2+ efflux and thus indicate potential for allergic rhinitis management. The combination of activities is usually related with curative and prophylactic therapy and might lead future clinical trials and therapies.

Dual Drug Delivery for Augmenting Bacterial Wound Complications via Tailored Ultradeformable Carriers.

Addressing the complex challenge of healing of bacterially infected wounds, this study explores the potential of lipid nanomaterials, particularly advanced ultradeformable particles (UDPs), to actively influence the wound microenvironment. The research introduces a novel therapeutic approach utilizing silver sulfadiazine (SSD) coupled with vitamin E (VE) delivered through UDPs (ethosomes/transferosomes/transethosomes). Comparative physicochemical characterization of these nanosized drug carriers reveals the superior stability of transethosomes, boasting a zeta potential of -36.5 mV. This method demonstrates reduced side effects compared to conventional therapies, with almost 90% SSD and 72% VE release achieved in wound pH in a sustained manner. Cytotoxicity assessment shows 60% cell viability even at the highest concentration (175 μg/mL), while hemolysis test demonstrates RBC lysis below 5% at a concentration of 250 μg/mL. Vitamin E-SSD-loaded transethosomes (VSTEs) significantly enhance cellular migration and proliferation, achieving 95% closure within 24 h, underscoring their promising efficacy. The synergistic method effectively reduces bacterial burden, evidenced by an 80% reduction in Escherichia coli and Staphylococcus aureus within the wound microenvironment. This approach offers a promising strategy to address complications associated with skin injuries.

Drug repurposing: insights into the antimicrobial effects of AKBA against MRSA

Staphylococcus aureus is a major threat in infectious diseases due to its varied infection types and increased resistance. S. aureus could form persister cells under certain condition and could also attach on medical apparatus to form biofilms, which exhibited extremely high resistance to antibiotics. 3-Acetyl-11-keto-beta-boswellic acid (AKBA) is a well-studied anti-tumor and antioxidant drug. This study is aimed to determine the antimicrobial effects of AKBA against S. aureus and its persister cells and biofilms. The in vitro antimicrobial susceptibility of AKBA was assessed by micro-dilution assay, disc diffusion assay and time-killing assay. Drug combination between AKBA and conventional antibiotics was detected by checkerboard assay. And the antibiofilm effects of AKBA against S. aureus were explored by crystal violet staining combined with SYTO/PI probes staining. Next, RBC lysis activity and CCK-8 kit were used to determine the cytotoxicity of AKBA. In addition, murine subcutaneous abscess model was used to assess the antimicrobial effects of AKBA in vivo. Our results revealed that AKBA was found to show effective antimicrobial activity against methicillin-resistant S. aureus (MRSA) with the minimal inhibitory concentration of 4–8 µg/mL with undetectable cytotoxicity. And no resistant mutation was induced by AKBA after 20 days of consecutive passage. Further, we found that AKBA could be synergy with gentamycin or amikacin against S. aureus and its clinical isolates. By crystal violet and SYTO9/PI staining, AKBA exhibited strong biofilm inhibitory and eradication effects at the concentration of 1 ~ 4 µg/mL. In addition, the effective antimicrobial effect was verified in vivo in a mouse model. And no detectable in vivo toxicity was found. These results indicated that AKBA has great potential to development as an alternative treatment for the refractory S. aureus infections.

Preparation and characterization of a jelly fig (Ficus awkeotsang Makino) polysaccharide-based bioactive 3D scaffold for improved vascularization and skin tissue engineering applications

Jelly fig polysaccharides (JFP) were extracted from Ficus awkeotsang Makino achenes. The yield of JFP was approximately 10–15 %. FT-IR spectrum of the extracted JFP confirmed that it was made of low methoxyl pectin (LMP). 3D scaffolds of JFP (JFP scaffold) were fabricated using ionic crosslinking of 2 % (w/v) JFP solution with Ca2+ ions and freeze-drying. The JFP scaffold showed 73.46 ± 1.97 % porosity and a 12-fold swelling capacity. The porous morphology was also observed in SEM micrographs. JFP scaffolds were completely degraded in 14 days when incubated in 1 mg/mL lysozyme solution, compared to the 50 % degradation observed in PBS alone. The antioxidant activity of the JFP and JFP scaffold was approximately 40 %. The hemolytic assay of the JFP scaffold showed <5 % (3.0 ± 0.4) RBC lysis. The cytocompatibility of the JFP scaffold was evaluated using L929 mouse fibroblasts and human dermal fibroblasts (HDF). The in vitro studies using L929 cells showed that the JFP scaffold is cytocompatible. HDF cells cultured in the presence of JFP scaffolds show a higher fold cell viability, proliferation, and migration. Collagen expression and deposition were also studied, and no significant changes occurred with JFP scaffold treatment. In vivo CAM assay showed an increase in the number and thickness of blood vessels by 1.185-fold and 1.19-fold, respectively. These results confirm the angiogenic property of the JFP scaffold. These biocompatible and bioactive properties of the JFP scaffold could be beneficial for tissue engineering and regenerative medicine applications.

Isolation methods determine human neutrophil responses after stimulation.

Studying neutrophils is challenging due to their limited lifespan, inability to proliferate, and resistance to genetic manipulation. Neutrophils can sense various cues, making them susceptible to activation by blood collection techniques, storage conditions, RBC lysis, and the isolation procedure itself. Here we assessed the impact of the five most used methods for neutrophil isolation on neutrophil yield, purity, activation status and responsiveness. We monitored surface markers, reactive oxygen species production, and DNA release as a surrogate for neutrophil extracellular trap (NET) formation. Our results show that neutrophils isolated by negative immunomagnetic selection and density gradient methods, without RBC lysis, resembled untouched neutrophils in whole blood. They were also less activated and more responsive to milder stimuli in functional assays compared to neutrophils obtained using density gradients requiring RBC lysis. Our study highlights the importance of selecting the appropriate method for studying neutrophils, and underscores the need for standardizing isolation protocols to facilitate neutrophil subset characterization and inter-study comparisons.

A Novel Strategy for Square-Wave Micromixers: A Survey of RBC Lysis for Further Biological Analysis

A Novel Strategy for Square-Wave Micromixers: A Survey of RBC Lysis for Further Biological Analysis

Characterization of Secondary Metabolites Identified in Lentinula edodes and Determination of Their Biological Activities by in‐silico Antioxidant Screening

AbstractMushrooms, like other fungi, are unique in the living world They have been assigned to the kingdom of Myceteae. Lentinula edodes(shiitake), is second‐largest and most often consumed edible mushroom in the world. Shiitake mushroom possess large capability to be used as food as it contains carbohydrates, proteins, vitamins, minerals, fibers, fatty acids, amino acids, etc. In current study powdered form mushroom used to form semisolid extract in selected fractions (Butanol, ethyl acetate, hexane, chloroform and aqueous). The aim of the current study was to explore phytoconstituents which have biological activities in selected fractions (n. hexane chloroform, and ethyl acetate) of methanolic extract of Lentinula edodes.GC‐MS, and HPLC analysis presented the secondary metabolites in all the selected fractions under taken. Chloroform fraction had the highest total phenolic, and total flavonoid which exhibited the maximum in vitro antioxidant ABTS potential (74.78±0.83) potential at 350 μg/mL while ethyl acetate has moderate and n‐hexane shows least concentration. Hemolysis essay showed less than 10 % lysis of RBC, s which indicates that fractions under study are active, and safe to use as herbal medicine. Biomolecules detected through HPLC and GC‐MS in fractions under study showed significant inhibition in paw inflammation (1.18±0.06) and yeast induced pyrexia (36.8±0.68) in dose dependent manner. Furthermore, In‐silico study showed that 4H‐Furo[3,2‐b] pyrrole‐5‐carboxylic acid have a strong interaction to NADPH oxidase, Myeloperoxidase as compared to standard Melatonin which shows best antioxidant compound which was confirmed in chloroform fraction.The outcomes of this research indicated that L. edodes significantly reduced inflammation and have ability to scavenge free radicals, prevent protein denaturation, also indicate the possible use in food and nutraceutical applications as an antioxidant, antipyretic, anti‐inflammatory, and antidiabetic agent.

P06.04.B NEW APPROACHES FOR EXPANDING GLIOBLASTOMA INFILTRATING LYMPHOCYTES

Abstract BACKGROUND Adoptive cell therapy using tumor infiltrating lymphocytes (TILs) has shown promise in melanoma and Non-Small Cell Lung Cancer. TILs culture and expansion from GBM remains challenging due to paucity of infiltrated T-cells that are also exhausted. This hinders creation of potent TILs. We have developed a new efficient method to manufacture potent TILs from GBM samples. METHODS We have developed efficient TILs manufacturing methods using both solid and aspirated liquid samples collected during vacuum assisted excision containing tumor fragments that may stem predominantly from the tumor margins and provide a source for TILs. We followed the traditional two steps: initial expansion (preREP) where T-cells are expanded with tumor cells to selectively expand tumor antigen-reactive TILs, followed by a large-scale expansion (REP) after further activation. For both steps we optimized media and reagents. RESULTS TILs cultures from solid tumors were established from enzymatically digested tissue and by fragment plating. Successful TILs yield from liquid samples required enzymatic digestion, debris removal and RBC lysis prior to culture initiation. PreREP TILs yields ranged from a few million to 2 billion viable cells for solid and liquid samples. REP of TILs from liquid samples expanded similarly from solid samples. REP of TILs reached up to 556-fold expansion (mean: 346, range: 95-556) providing promising numbers of activated TILs. The potency of the TILs was demonstrated with tumor specific Interferon-gamma production when cultured with matched tumor digest or neurospheres. CONCLUSIONS We successfully expanded TILs that are rich in cytotoxic T-cells, demonstrated their tumor reactivity and further expanded the preREP TILs to reach therapeutic doses. We have characterized the TILs by scRNA and TCRseq and analyze paired TCR reads using scRNA sequencing. Overrepresented TCR alpha and beta chains will be linked by a P2A site and will be cloned into a lentiviral vector to establish a TCR library which will be transduced into novel Jurkat Triple Parameter Reporter Cell Lines under the control of different T cell activation programs. TCR Jurkat Libraries will be cocultured with original tumor material and sorted to identify tumor reactive TCRs; enabling us to define the percentage of TCRs that are enriched by TIL isolation that are specifically reactive to tumor vs bystander expanded TCR cells. Ultimately, we aim to bring this therapy to patients in a clinical trial.

Comparison of Protective Effects of Phenolic Acids on Protein Glycation of BSA Supported by In Vitro and Docking Studies.

Several diabetic complications are associated with forming advanced glycation end products (AGEs). Different chemical and natural compounds are able to prevent the development of these products. In this study, glycosylation was induced as a model by incubating bovine serum albumin (BSA) with glucose. Consequently, BSA was treated with glucose and different concentrations (1.25, 2.5, and 5 μM) of syringic acid, gallic acid, ellagic acid, ferulic acid, paracoumaric acid, and caffeic acid for 4 and 6 weeks. Biochemical experiments comprise measurements of fluorescent AGEs, protein carbonyl contents, total thiol, hemolysis tests, and also malondialdehyde (MDA) levels in RBC. These demonstrated the antiglycating mechanism of these phenolic acids. Most of the phenolic acids used in this study reduced MDA levels and protected thiol residues in protein structures. They also inhibited the formation of fluorescent AGEs and RBC lysis, except gallic acid. Moreover, ferulic acid, paracoumaric acid, and caffeic acid proteins significantly prevent carbonylation. Molecular docking and simulation studies showed that ellagic, caffeic, gallic, and syringic acids could interact with lysine and arginine residues in the active site of BSA and stabilize its structure to inhibit the formation of AGEs. Our results suggest that phenolic acid could be used as a potential phytochemical against protein glycation and related diabetic complications.

Calcium Phosphate Treatment Enhances Osteogenic Differentiation of Porcine Adipose-Derived Stem Cells on Fibrin Scaffolds

This study was designed to develop a fibrin scaffold for optimum in vitro osteogenic differentiation of porcine ASCs. Fibrin scaffolds physically and chemically modified to be stiffer or have higher concentrations of calcium and phosphate ions were hypothesized to enhance osteogenic differentiation compared to control fibrin scaffolds. Treatments during coagulation resulted in six scaffold types for comparison: whole blood controls, red blood cell lysis buffer, calcium chloride, calcium hydrogen phosphate, vacuum, and mechanical compression. Based on the results, fibrin supplemented with granules of calcium hydrogen phosphate (CaHPO4) was determined to be the most suitable formulation of those examined. Using calcium phosphate resulted in a fibrin scaffold that coagulated faster (p = 0.022), had a rougher surface, higher stiffness, and desirable properties for practical use during surgical operations and scaffolds used in bone tissue engineering. Further, osteogenic differentiation was enhanced on scaffolds treated with calcium phosphate. In addition, fibrin scaffolds treated with RBC lysis buffer were also stiffer than untreated controls. These results are partly explained by ASC attachment and fibrin polymerization during coagulation.

MASP3 Deficiency in Mice Reduces but Does Not Abrogate Alternative Pathway Complement Activity Due to Intrinsic Profactor D Activity.

Complement factor D (FD) is a rate-limiting enzyme of the alternative pathway (AP). Recent studies have suggested that it is synthesized as an inactive precursor and that its conversion to enzymatically active FD is catalyzed by mannan-binding lectin-associated serine protease 3 (MASP3). However, whether MASP3 is essential for AP complement activity remains uncertain. It has been shown that Masp1/3 gene knockout did not prevent AP complement overactivation in a factor H-knockout mouse, and a human patient lacking MASP3 still retained AP complement activity. In this study, we have assessed AP complement activity in a Masp3-knockout mouse generated by CRISPR/Cas9 editing of the Masp1/3 gene. We confirmed specific Masp3 gene inactivation by showing intact MASP1 protein expression and absence of mature FD in the mutant mice. Using several assays, including LPS- and zymosan-induced C3b deposition and rabbit RBC lysis tests, we detected plasma concentration-dependent AP complement activity in Masp3 gene-inactivated mice. Thus, although not measurable in 5% plasma, significant AP complement activity was detected in 20-50% plasma of Masp3 gene-inactivated mice. Furthermore, whereas FD gene deletion provided more than 90% protection of CD55/Crry-deficient RBCs from AP complement-mediated extravascular hemolysis, Masp3 gene deletion only provided 30% protection in the same study. We also found pro-FD to possess intrinsic catalytic activity, albeit at a much lower level than mature FD. Our data suggest that MASP3 deficiency reduces but does not abrogate AP complement activity and that this is explained by intrinsic pro-FD activity, which can be physiologically relevant invivo.

Husk-like Zinc Oxide Nanoparticles Induce Apoptosis through ROS Generation in Epidermoid Carcinoma Cells: Effect of Incubation Period on Sol-Gel Synthesis and Anti-Cancerous Properties

This study effectively reports the influence of experimental incubation period on the sol-gel production of husk-like zinc oxide nanoparticles (ZNPs) and their anti-cancerous abilities. The surface morphology of ZNPs was studied with the help of SEM. With the use of TEM, the diameter range of the ZNPs was estimated to be ~86 and ~231 nm for ZNPA and ZNPB, prepared by incubating zinc oxide for 2 and 10 weeks, respectively. The X-ray diffraction (XRD) investigation showed that ZNPs had a pure wurtzite crystal structure. On prolonging the experimental incubation, a relative drop in aspect ratio was observed, displaying a distinct blue-shift in the UV-visible spectrum. Furthermore, RBC lysis assay results concluded that ZNPA and ZNPB both demonstrated innoxious nature. As indicated by MTT assay, reactive oxygen species (ROS) release, and chromatin condensation investigations against the human epidermoid carcinoma (HEC) A431 cells, ZNPB demonstrated viable relevance to chemotherapy. Compared to ZNPB, ZNPA had a slightly lower IC50 against A431 cells due to its small size. This study conclusively describes a simple, affordable method to produce ZNP nano-formulations that display significant cytotoxicity against the skin cancer cell line A431, suggesting that ZNPs may be useful in the treatment of cancer.

Anticancer effect of zinc oxide nanoparticles prepared by varying entry time of ion carriers against A431 skin cancer cells in vitro.

Although, zinc oxide nanoparticles (ZRTs) as an anti-cancer agent have been the subject of numerous studies, none of the reports has investigated the impact of the reaction entry time of ion-carriers on the preparation of ZRTs. Therefore, we synthesized variants of ZRTs by extending the entry time of NaOH (that acts as a carrier of hydroxyl ions) in the reaction mixture. The anti-proliferative action, morphological changes, reactive oxygen species (ROS) production, and nuclear apoptosis of ZRTs on human A431 skin carcinoma cells were observed. The samples revealed crystallinity and purity by X-ray diffraction (XRD). Scanning electron microscopy (SEM) images of ZRT-1 (5min ion carrier entry) and ZRT-2 (10min ion carrier entry) revealed microtubule like morphology. On prolonging the entry time for ion carrier (NaOH) introduction in the reaction mixture, a relative ascent in the aspect ratio was seen. The typical ZnO band with a slight shift in the absorption maxima was evident with UV-visible spectroscopy. Both ZRT-1 and ZRT-2 exhibited non-toxic behavior as evident by RBC lysis assay. Additionally, ZRT-2 showed better anti-cancer potential against A431 cells as seen by MTT assay, ROS generation and chromatin condensation analyses. At 25μM of ZRT-2, 5.56% cells were viable in MTT test, ROS production was enhanced to 166.71%, while 33.0% of apoptotic cells were observed. The IC50 for ZRT-2 was slightly lower (6μM) than that for ZRT-1 (8μM) against A431 cells. In conclusion, this paper presents a modest, economical procedure to generate ZRT nano-structures exhibiting strong cytotoxicity against the A431 cell line, indicating that ZRTs may have application in combating cancer.

Establishment of a Patient-Derived Organoid Culture Platform for Mantle Cell Lymphoma Drug Sensitivity and CAR T Cell Anti-Tumor Activity Assay

Background: With BTK inhibitors and brexucabtagene autoleucel CAR-T cell therapy approved by FDA to treat B-cell malignancies, survival of patients with mantle cell lymphoma (MCL) has been greatly improved. However, frequent therapeutic relapses in patients remains as an unmet medical challenge. Preclinical tumor mouse models such as patient-derived xenografts (PDXs) are often used for drug sensitivity and treatment prediction. However, these conventional cancer models are time-consuming and expensive. In recent years, development and application of patient-derived organoid (PDO) models have led to many discoveries in various types of solid tumors. PDO models have demonstrated many advantages over PDX models. For example, PDOs can be established using biopsies as small as needle biopsies yet with much higher success rates. However, a PDO model has not been reported yet in MCL or in a few other hematologic malignancies. Therefore, in this study, with successful establishment of MCL PDO models, we optimized our protocols and applied this model for drug sensitivity screens and rapid evaluation of immunotherapies. Methods: Patient samples were lysed with RBC lysis buffer and washed with PBS, then cell pellets were resuspended in culture medium containing cytokine cocktails. The cell suspension was aliquoted into V-bottom 96-well plates and spun. The cell aggregates within 50% Matrigel were gently transferred into multiple-well plates as needed. After solidification of the cell-Matrigel mixture, pre-warmed culture medium was added to cover them. The PDOs in 24 or 96-well plates were treated with drug or co-cultured with CAR T cells for 3-5 days. The cell viability and T cell killing activity were determined using CellTiter-Glo 3D assay kits. Results: We successfully established and optimized an organoid culture procedure and culture medium for diverse types of biopsy from patients with MCL. The success rate of MCL organoid development from diverse MCL biopsies was greater than 90%, much higher than for PDX. H&E staining of both tumors and their derived organoids revealed similar histological features (Figure 1A). To examine the drug sensitivity of primary MCL cells, organoid cultures derived from the patient apheresis biopsies that were either BTK inhibitor-sensitive (naive) or multiple drug resistant (to acalabrutinib, rituximab, and venetoclax) were treated with the BTK inhibitors ibrutinib or pirtobrutinib or else doxorubicin (as a control) for 48 h. Cell viability assays indicated that MCL cells in organoid cultures maintained the responses of the original tumor cells to drugs used in clinical treatment. We also demonstrated that the MCL organoids had the same major cell lineage populations as the original biopsy. These results support the notion that the PDO in vitro assay has great potential to evaluate and predict patient responses. In addition, we applied this PDO platform to evaluate CAR T cell anti-tumor activity by co-culturing the primary MCL cells with CD19-targeting CAR T cells in organoids for 3 days. To visualize and quantitate tumor killing potential, the MCL cells were labelled with the green dye 5-chloromethyl fluorescein diacetate. The cocultures were examined by imaging and FACS analysis. Compared to the same batch of peripheral blood monocytes used to generating CAR T cells, the CD19-CAR T cells very effectively eradicated CD19+ MCL cells from the co-cultures (Figure 1B), indicating that the PDO platform is a promising in vitro assay for rapid examining CAR T cell activity. Importantly, we validated drug responses in parallel in PDO and PDX models derived from the same tumors. Our preliminary results displayed high consistency between these assay systems, suggesting that PDO-based assay provides an encouraging platform for predicting clinical treatment response. Conclusion: We have successfully established and optimized an MCL PDO platform that can be used to perform reliable and fast assays for drug sensitivity and CAR T cell anti-tumor activity in vitro. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Efficacy of Trevesia palmata (Roxb. ex Lindl.) Vis. Extract on MG 63 cell lines and arthritis-induced animal models

Ethnopharmacological relevanceDespite widespread use of herbal remedies for treating arthritis and osteosarcoma, many plants are still not pharmacologically evaluated for their efficacy. Contrary to many non-steroidal, immunosuppressants, antibiotics, and antineoplastic drugs that have adverse effects, phytotherapeutic compounds have promising benefits with fewer complications. In this study the unexplored Northeastern India indigenous plant Trevesia palmata (Roxb. ex Lindl.) Vis. used in traditional medicine to cure bone fractures is chosen for studying anti-proliferative and anti-rheumatic properties. Aim of the studyThis study designed to explore the polyphenolic composition, antioxidant, anti-inflammatory and anti-arthritic potential of T. palmata leaf extracts. Further, the cellular activity was studied using MG 63 osteoblast cell lines and pharmacologically evaluated using Complete Freund's Adjuvant (CFA) induced arthritic rat model. Materials and methodsIn vitro free radical scavenging activity, anti-inflammatory and anti-arthritic activities of extracts were analyzed using standardized methods. The polyphenolic profiling and apoptosis inducing ability of T. palmata ethyl acetate (TPEA) extract on MG 63 osteoblast cell lines were analyzed. The in vivo pharmacological studies were carried out with low dose 250 mg/kg and high dose of 500 mg/kg of T. palmata. The biochemical and haematological parameters and in vivo antioxidant activity were evaluated for the control and treated groups. Radiological and histological study were done to understand the impact and penetration of inflammatory arthritis from tissues to joint bones. ResultsTPEA showed highest free radical scavenging activity (DPPH - 4.72 IC50, ABTS - 242.33 ± 6.81 mM TE/g extract), anti-inflammatory (40.04% inhibition of RBC lysis) and anti-arthritic activity (32.4% inhibition of protein denaturation) with the presence of gallic acid, catechin, caffeic acid, rutin, quercetin and naringenin. The TPEA extract inhibited cell proliferation of MG 63 osteoblast cells and induced apoptosis by arresting cell cycle at different phases. After acute toxicity studies the doses 250 mg/kg and 500 mg/kg were fixed and showed better results in CFA-induced arthritic animals. Thus, the extract phytoconstituents may have immense potential against chronic inflammation, joint ailments, bone cancer and arthritis which serves as a phytomedicine contrary to synthetic medications. ConclusionsThe potential treatment of polyphenolic compounds in the T. palmata extract on osteosarcoma and arthritis was demonstrated from this study. Thus, cellular inflammatory infiltrates are significantly reduced in bone and joint tissues as well.

In Vitro and In Vivo Bactericidal and Antibiofilm Efficacy of Alpha Mangostin Against Staphylococcus aureus Persister Cells.

The formation of persister cells is associated with recalcitrance and infections. In this study, we examined the antimicrobial property of alpha mangostin, a natural xanthone molecule, against methicillin-resistant Staphylococcus aureus (MRSA) persisters and biofilm. The MIC of alpha mangostin against MRSA persisters was 2 µg/ml, and activity was mediated by causing membrane permeabilization within 30 min of exposure. The membrane activity of alpha mangostin was further studied by fast-killing kinetics of MRSA persiste r cells and found that the compound exhibited 99.99% bactericidal activity within 30 min. Furthermore, alpha mangostin disrupted established MRSA biofilms and inhibited bacterial attachment as biofilm formation. Alpha mangostin down-regulated genes associated with the formation of persister cells and biofilms, such as norA, norB, dnaK, groE, and mepR, ranging from 2 to 4-folds. Alpha mangostin at 16 μg/ml was non-toxic (> 95% cell survival) to liver-derived HepG2 and lung-derived A549 cells, similarly. Still, alpha mangostin exhibited 50% cell lysis of human RBC at 16 μg/ml. Interestingly, alpha mangostin was effective in vivo at increasing the survival up to 75% (p<0.0001) of Galleria mellonella larvae infected with MRSA persister for 120 h. In conclusion, we report that alpha mangostin is active against MRSA persisters and biofilms, and these data further our understanding of the antistaphylococcal activity and toxicity of this natural compound.