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Flow cytometry techniques utilizing dual staining with annexin V and propidium iodide (PI) provide a robust method for quantitatively analyzing apoptosis induction. Annexin V binds phosphatidylserine exposed on the outer leaflet of the plasma membrane during early apoptosis, while PI permeates late apoptotic/necrotic cells. Simultaneous staining allows differentiation of viable, early apoptotic, and late apoptotic/necrotic populations. This approach can be enhanced by using fluorochrome-conjugated antibodies to stain specific proteins, enabling the simultaneous tracking of protein expression changes in defined cell subpopulations during apoptosis. This multiparametric approach provides key insights into signaling regulation and the mechanisms underlying the apoptotic response to cytotoxic treatments. Here we present a protocol that combines annexin V-FITC/PI staining with APC-conjugated antibody labeling in MDA-MB-231 breast cancer cells treated with doxorubicin. This protocol enables both the quantitative assessment of apoptosis induction and the tracking of decreased CD44 expression from viable to apoptotic cells. This protocol also provides guidelines for appropriate filter selection, compensation controls, gating strategies, and troubleshooting. This robust protocol holds significant potential for elucidating signaling networks involved in apoptosis and therapeutic resistance across various cellular models.
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5-Fluorouracil (5-FU) has been the primary drug used in chemotherapy for colorectal carcinoma, and localizing the drug would be effective in avoiding its side effects and improving therapeutic outcomes. One approach to achieve this is by encapsulating the drug in microbeads. Alginate microbeads, in particular, exhibit promising pH-sensitive properties, making them an attractive option for colon targeting. Thus, the main aim of this study is to formulate and characterize 5-FU-encapsulated alginate microbeads as a pH-sensitive drug delivery system for controlled release in the gastrointestinal tract. In this study, the alginate microbeads encapsulating 5-FU was manufactured using electrospray methods. This method offers the advantages of promoting the formulation of uniformly small-sized microbeads with improved performance in terms of swelling and diffusion rates. The size and shape of the 5-FU microbeads are 394.23 ± 3.077 µm and have a spherical factor of 0.026 ± 0.022, respectively, which are considered acceptable and indicative of a spherical shape. The microbeads' encapsulation efficiency was found to be 69.65 ± 0.18%, which is considered high in comparison to other literature. The attenuated total reflectance - Fourier transform infrared spectroscopy (ATR-FTIR) data confirmed the complexation of sodium alginate with calcium ions, along with the encapsulation of 5-FU in the microbeads matrix. The 5-FU microbeads displayed pH-dependent swelling, exhibiting less swelling in simulated gastric fluid (SGF) than in simulated intestinal fluid (SIF). Additionally, the release of 5-FU from the microbeads is pH-dependent, with the cumulative percentage drug release being higher in simulated intestinal fluid than in SGF. The data indicate that the 5-FU microbeads can be utilized for the delivery of 5-FU in colon-targeted therapy, potentially leading to improved tumor treatment.
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Turmeric contains curcumin, a naturally occurring compound with noted anti-inflammatory and antioxidant properties that may help fight cancer. Curcumin is readily available, nontoxic, and inexpensive. At high doses, it has minimal side effects, suggesting it is safe for human use. However, curcumin has extremely poor bioavailability and biodistribution, which further hamper its clinical applications. It is commonly administered through oral and transdermal routes in different forms, where the particle size is one of the most common barriers that decreases its absorption through biological membranes on the targeted sites and limits its clinical effectiveness. There are many studies ongoing to overcome this problem. All of this motivated us to conduct this review that discusses the fabrication of polymer-based curcumin-loaded formulation as an advanced drug delivery system and addresses different approaches to overcoming the existing barriers and improving its bioavailability and biodistribution to enhance the therapeutic effects against cancer and other diseases.
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Non-alcoholic fatty liver disease (NAFLD) is the most prevalent form of liver disease. Orthosiphon aristatus (Blume) Miq, a traditional plant in South Asia, has previously been shown to attenuate obesity and hyperglycaemic conditions. Eight weeks of feeding C57BL/6 mice with the standardized O. aristatus extract (400 mg/kg) inhibited the progression of NAFLD. Liver enzymes including alanine aminotransferase and aspartate transaminase were significantly reduced in treated mice by 74.2% ± 7.69 and 52.8% ± 7.83, respectively. Furthermore, the treated mice showed a reduction in serum levels of glucose (50% ± 5.71), insulin (70.2% ± 12.09), total cholesterol (27.5% ± 15.93), triglycerides (63.2% ± 16.5), low-density lipoprotein (62.5% ± 4.93) and atherogenic risk index relative to the negative control. Histologically, O. aristatus reversed hepatic fat accumulation and reduced NAFLD severity. Notably, our results showed the antioxidant activity of O. aristatus via increased superoxide dismutase activity and a reduction of hepatic malondialdehyde levels. In addition, the levels of serum pro-inflammatory mediators (IL-6 and TNFα) decreased, indicating anti-inflammatory activity. The aqueous, hydroethanolic and ethanolic fractions of O. aristatus extract significantly reduced intracellular fat accumulation in HepG2 cells that were treated with palmitic-oleic acid. Together, these findings suggest that antioxidant activities are the primary mechanism of action of O. aristatus underlying the anti-NAFLD effects.
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Polyethylene glycol (PEG) coated Poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) for cancer treatment are biocompatible, nonimmunogenic and accumulate in tumour sites due to the enhanced permeability and retention (EPR). Doxorubicin (DOX) is a potent but cardiotoxic anticancer agent. Hyaluronic acid (HA) occurs naturally in the extra-cellar matrix and binds to CD44 receptors which are overexpressed in cancer metastasis, proven to be characteristic of cancer stem cells and responsible for multidrug resistance. In this study, an athymic mice model of breast cancer metastasis was developed using red fluorescent protein (RFP)-labelled triple negative cancer cells. The animals were divided into four treatment groups (Control, HA-PEG-PLGA nanoparticles, PEG-PLGA nanoparticles, and Free DOX). The tumour size growth was assessed until day 25 when animals were sacrificed. Mice treated with HA-PEG-PLGA NPs inhibited tumour growth. The tumour growth at day 25 (118% ± 13.0) was significantly (p < 0.05) less than PEG-PLGA NPs (376% ± 590 and control (826% ± 970). Fluorescent microscopy revealed that HA-PEG-PLGA NPs had significantly (p < 0.05) less metastasis in liver, spleen, colon, and lungs as compared to control and to Free DOX groups. The efficacy of HA-PEG-PLGA NPs was proven in vivo. Further pharmacokinetic and toxicity studies are required for this formulation to be ready for clinical research.
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Metabolic dysfunction-associated fatty liver disease (MAFLD), proposed in 2020 is a novel term for non-alcoholic fatty liver disease (NAFLD) which was coined for the first time in 1980. It is a leading cause of the most chronic liver disease and hepatic failure all over the world, and unfortunately, with no licensed drugs for treatment yet. The progress of the disease is driven by the triggered inflammatory process, oxidative stress, and insulin resistance in many pathways, starting with simple hepatic steatosis to non-alcoholic steatohepatitis, fibrosis, cirrhosis, and liver cancer. Protein kinases (PKs), such as MAPK, ErbB, PKC, PI3K/Akt, and mTOR, govern most of the pathological pathways by acting on various downstream key points in MAFLD and regulating both hepatic gluco- lipo-neogenesis and inflammation. Therefore, modulating the function of those potential protein kinases that are effectively involved in MAFLD might be a promising therapeutic approach for tackling this disease. In the current review, we have discussed the key role of protein kinases in the pathogenesis of MAFLD and performed a protein-protein interaction (PPI) network among the main proteins of each kinase pathway with MAFLD-related proteins to predict the most likely targets of the PKs in MAFLD. Moreover, we have reported the experimental, pre-clinical, and clinical data for the most recent investigated molecules that are activating p38-MAPK and AMPK proteins and inhibiting the other PKs to improve MAFLD condition by regulating oxidation and inflammation signalling.
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Enfermedad del Hígado Graso no Alcohólico , Proteínas Quinasas , Progresión de la Enfermedad , Humanos , Inflamación , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Fosfatidilinositol 3-QuinasasRESUMEN
Poly lactic-co-glycolic acid (PLGA) nanoparticles are intensively studied nanocarriers in drug delivery because of their biodegradability and biochemical characteristics. Polyethylene glycol (PEG) coating for nanocarriers gives them long circulation time in blood and makes them invisible to the reticuloendothelial system. Breast cancer cells have greater uptake of hyaluronic acid compared to normal cells as it binds to their overexpressed CD44 receptors. Since hypoxia plays an important role in cancer metastasis; we formulated PEG-PLGA nanoparticles coated with hyaluronic acid as targeted delivery system for doxorubicin (DOX) using nanoprecipitation method, and characterized them for chemical composition, size, surface charge, shape, and encapsulation efficiency. Then we tested them in vitro on hypoxia-optimized metastatic breast cancer cells. The nanoparticles were spherical with an average size of about 106 ± 53 nm, a negative surface charge (-15 ± 3 mV), and high encapsulation efficiency (73.3 ± 4.1%). In vitro investigation with hypoxia-elevated CD44 MDA-MB-231 cells showed that hyaluronic acid-targeted nanoparticles maintained their efficacy despite hypoxia-induced drug resistance unlike free DOX and nontargeted nanoparticles. In conclusion, this study revealed a simple third generation nanoparticle formulation for targeted treatment of hypoxia-induced drug resistance in breast cancer metastatic cells. Further, optimization is needed including In vivo efficacy and nanoparticle-specific pharmacokinetic studies.
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Antibióticos Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Doxorrubicina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Nanopartículas/química , Antraciclinas/administración & dosificación , Antraciclinas/farmacología , Antibióticos Antineoplásicos/administración & dosificación , Neoplasias de la Mama/patología , Línea Celular Tumoral , Doxorrubicina/administración & dosificación , Portadores de Fármacos/química , Femenino , Humanos , Ácido Hialurónico/química , Metástasis de la Neoplasia , Tamaño de la Partícula , Polietilenglicoles/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Propiedades de SuperficieRESUMEN
Nanoprecipitation is a simple and increasingly trending method for nanoparticles preparation. The self-assembly feature of poly (ethylene glycol)-poly (lactide-co-glycolic acid) (PEG-PLGA) amphiphilic copolymer into a nanoparticle and its versatile structure makes nanoprecipitation one of the best methods for its preparation. The aim of this study is to review currently available literature for standard preparation of PEG-PLGA nanoparticles using nanoprecipitation technique in order to draw conclusive evidenceto draw conclusive evidence that can guide researchers during formulation development. To achieve this, three databases (Web of Science, Scopus and PubMed) were searched using relevant keywords and the extracted articles were reviewed based on defined inclusion and exclusion criteria. Data extraction and narrative analysis of the obtained literature was performed when appropriate, along with our laboratory observations to support those claims wherever necessary. As a result of this analysis, reports that matched our criteria conformed to the general facts about nanoprecipitation techniques such as simplicity in procedure, low surfactants requirement, narrow size distribution, and low resulting concentrations. However, these reports showed interesting advantages for using PEG-PLGA as they are frequently reported to be freeze-dried and active pharmaceutical ingredients (APIs) with low hydrophobicity were reported to successfully be encapsulated in the particles.