Exploring the interplay between the TGF-ßpathway and SLN-mediated transfection: implications for gene delivery efficiency in prostate cancer and non-cancer cells.

Solid lipid nanoparticles (SLN) are widely recognized for their biocompatibility, scalability, and long-term stability, making them versatile formulations for drug and gene delivery. Cellular interactions, governed by complex endocytic and signaling pathways, are pivotal for successfully applying SLN as a therapeutic agent. This study aims to enhance our understanding of the intricate interplay between SLN and cells by investigating the influence of specific endocytic and cell signaling pathways, with a focus on the impact of the TGF-ßpathway on SLN-mediated cell transfection in both cancerous and non-cancerous prostate cells. Here, we systematically explored the intricate mechanisms governing the interactions between solid lipid nanoparticles and cells. By pharmacologically manipulating endocytic and signaling pathways, we analyzed alterations in SLNplex internalization, intracellular traffic, and cell transfection dynamics. Our findings highlight the significant role of macropinocytosis in the internalization and transfection processes of SLNplex in both cancer and non-cancer prostate cells. Moreover, we demonstrated that the TGF-ßpathway is an important factor influencing endosomal release, potentially impacting gene expression and modulating cell transfection efficiency. This study provides novel insights into the dynamic mechanisms governing the interaction between cells and SLN, emphasizing the pivotal role of TGF-ßsignaling in SLN-mediated transfection, affecting internalization, intracellular transport, and release of the genetic cargo. These findings provide valuable insight for the optimization of SLN-based therapeutic strategies in prostate-related applications.

Cationic solid lipid nanoparticles (SLN) complexed with plasmid DNA enhance prostate cancer cells (PC-3) migration.

Nanotechnology applications in biomedicine have increased in recent decades, primarily as therapeutic agents, drugs, and gene delivery systems. Among the nanoparticles used in medicine, we highlight cationic solid lipid nanoparticles (SLN). Given their nontoxic properties, much research has focused on the beneficial effects of SLN for drug or gene delivery system. However, little attention has been paid to the adverse impacts of SLN on the cellular environment, particularly their influence on intracellular signaling pathways. In this work, we investigate the effects triggered by cationic SLN on human prostate non-tumor cells (PNT1A) and tumor cells (PC-3). Our results demonstrate that cationic SLN enhances the migration of PC-3 prostate cancer cells but not PNT1A non-tumor prostate cells, an unexpected and unprecedented development. Furthermore, we observed that the enhanced cell migration velocity is a concentration-dependent and nanoparticle-dependent effect, and not related to any individual nanoparticle component. Moreover, cationic SLN increased vimentin expression (p < 0.05) but SLN did not affect Smad2 nuclear translocation. Meanwhile, EMT-related (epithelial-to-mesenchymal transition) proteins, such as ZEB1, underwent nuclear translocation when treated with cationic SLN, thereby affecting PC-3 cell motility through ZEB1 and vimentin modulation. From a therapeutic perspective, cationic SLN could potentially worsen a patient's condition if these results were reproduced in vivo. Understanding the in vitro molecular mechanisms triggered by nanomaterials and their implications for cell function is crucial for defining their safe and effective use.

Biogenic silver nanoparticles' antibacterial activity and cytotoxicity on human hepatocarcinoma cells (Huh-7).

Exploring diverse synthetic pathways for nanomaterial synthesis has emerged as a promising direction. For example, silver nanoparticles (AgNPs) are synthesized using different approaches yielding nanomaterials with distinct morphological, physical and biological properties. Hence, the present study reports the biogenic synthesis of silver nanoparticles using the aqueous secretome of the fungus Fusarium oxysporum f. sp. cubense (AgNP@Fo) and orange peel extract (AgNP@OR). The physical and morphological properties of synthesized nanoparticles were similar, with AgNP@Fo measuring 56.43 ± 19.18 nm and AgNP@OR measuring 39.97 ± 19.72 nm in size. The zeta potentials for the nanoparticles were low, -26.8 ± 7.55 and -26.2 ± 2.87 mV for AgNP@Fo and AgNP@OR, respectively, demonstrating a similar negative charge. The spherical morphologies of both nanoparticles were evidenced by Scanning Transmission Electron Microscopy (STEM) and Atomic Force Microscopy (AFM). However, despite their similar physical and morphological properties, AgNPs demonstrated different bioactivities. We evaluated and compared the antimicrobial efficacy of these nanoparticles against a range of bacteria, such as Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, and Escherichia coli. The AgNP@Fo showed Minimum Inhibitory Concentration (MIC) values ranging from 0.84 to 1.68 µg mL-1 and were around ten times more potent compared to AgNP@OR. The anticancer activities of both nanoparticles were investigated using human hepatocarcinoma cells (Huh-7), where AgNP@Fo exhibited around 20 times higher cytotoxicity than AgNP@OR with an IC50 value of 0.545 µmol L-1. Anticancer effects were demonstrated by the MTT, confirmed by the calcein-AM assay and fluorescence imaging. This study establishes solid groundwork for future exploration of molecular interactions of nanoparticles synthesized through distinct biosynthetic routes, particularly within bacterial and cancerous cell environments.

A multiparametric and orthogonal approach indicates low toxicity for zein nanoparticles in a repellent formulation.

The incidence of viruses such as Zika, Dengue, and Chikungunya affects human health worldwide, and insect repellents are recommended for individual protection. Formulations incorporating nanotechnology should be carefully assessed for toxicity, particularly regarding the security levels established for human health and the environment. This study evaluates the cytotoxicity of a repellent formulation containing zein nanoparticles (NP) loading geraniol (Ger) and icaridin (Ica) in three cell lines: NIH/3T3, HaCaT, and SIRC. To address formulation hazards, IC50 values were determined by MTT and Calcein-AM assays. In both NIH/3T3 and HaCaT, the IC50 values for NP + Ger + Ica formulation were around 0.2%. For risk assessment, cell viability was also determined after a single exposure and repeated exposure to the formulation. No evidence of cytotoxicity was observed for NP + Ger + Ica formulation-treated cells. The risk assessment for eye damage revealed cytotoxicity in SIRC cells when exposed to a 5% concentration, which may be attributed to ocular geraniol toxicity, because zein nanoparticles alone did not exhibit any signs of toxicity. Cell internalization indicated low uptake in NIH/3T3 and HaCaT cells. Phenotypic profiling resulted in similar phenotypes for untreated cells and cells exposed to NP + Ger + Ica formulation. The toxicological profile outlined by the multiparametric and orthogonal approach suggests that the NP + Ger + Ica formulation poses no significant risk to the topical application under the tested conditions. Adopting an orthogonal approach brings robustness to our findings.

Interpreting Image-based Profiles using Similarity Clustering and Single-Cell Visualization.

Image-based profiling quantitatively assesses the effects of perturbations on cells by capturing a breadth of changes via microscopy. Here, we provide two complementary protocols to help explore and interpret data from image-based profiling experiments. In the first protocol, we examine the similarity among perturbed cell samples using data from compounds that cluster by their mechanisms of action. The protocol includes steps to examine feature-driving differences between samples and to visualize correlations between features and treatments to create interpretable heatmaps using the open-source web tool Morpheus. In the second protocol, we show how to interactively explore images together with the numerical data, and we provide scripts to create visualizations of representative single cells and image sites to understand how changes in features are reflected in the images. Together, these two tutorials help researchers interpret image-based data to speed up research. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Exploratory analysis of profile similarities and driving features Basic Protocol 2: Image and single-cell visualization following profile interpretation.

Nanotechnology as a tool to overcome macromolecules delivery issues.

Nanocarriers can deliver drugs to specific organs or cells, potentially bridging the gap between a drug's function and its interaction with biological systems such as human physiology. The untapped potential of nanotechnology stems from its ability to manipulate materials, allowing control over physical and chemical properties and overcoming drug-related problems, e.g., poor solubility or poor bioavailability. For example, most protein drugs are administered parenterally, each with challenges and peculiarities. Some problems faced by bioengineered macromolecule drugs leading to poor bioavailability are short biological half-life, large size and high molecular weight, low permeability through biological membranes, and structural instability. Nanotechnology emerges as a promising strategy to overcome these problems. Nevertheless, the delivery system should be carefully chosen considering loading efficiency, physicochemical properties, production conditions, toxicity, and regulations. Moving from the bench to the bedside is still one of the major bottlenecks in nanomedicine, and toxicological issues are the greatest challenges to overcome. This review provides an overview of biotech drug delivery approaches, associated nanotechnology novelty, toxicological issues, and regulations.

Mechanisms of solid lipid nanoparticles-triggered signaling pathways in eukaryotic cells.

Solid lipid nanoparticles (SLN) are used in various fields such as pharmaceutical, cosmetic, and biomedical research and show promising results in delivering biomolecules. SLN formulations are made with solid lipids (at room and body temperature) stabilized with surfactants and co-surfactants that may guarantee specific properties. Typically, these compounds have high stability, allow large-scale production, and are biodegradable. Since most of these SLNs are formulated with biodegradable materials, they are assumed to have low toxicity or are nontoxic. Therefore, this assumption introduced experimental bias, making SLN toxicity an often overlooked area; moreover, few studies have focused on this topic. Here, we critically review the literature, focusing on blank controls (i.e., SLN formulations without cargo) and their ability to trigger signaling pathways, cellular outcomes, and cytotoxicity. We found that SLN can trigger or disturb many cell signaling pathways; thus, we emphasize the importance of testing the biocompatibility and cytotoxicity of empty SLN. Overall, more attention should be paid to the possible cytotoxic effects of SLN, which is still an open topic, showing that this topic needs further investigation. Therefore, a detailed understanding of SLN toxicity, particularly for biomedical applications, can significantly impact the transfer of SLN formulations from the laboratory bench to the bedside.

Improved efficacy of meglumine antimoniate incorporated in anionic liposomes against Leishmania infantum infecting canine macrophages.

OBJECTIVES: Leishmaniasis is a zoonotic disease and several drugs have been used in the treatment, including meglumine antimoniate (AME). The chemotherapy reaches clinical cure but does not eliminate parasites, contributing to drug resistance. To improve AME efficacy we incorporated it in anionic liposomes. The antiparasitic activity and intracellular localization were investigated in canine macrophages infected with Leishmania infantum. METHODS: Liposomes (L-AME) is composed of egg phosphatidylcholine, cholesterol, palmitoyl oleoyl phosphatidyl serine and α-tocopherol (4 : 3 : 0.4 : 0.07 mol%) plus AME. L-AME size, polydispersity, zeta potential and morphology were analysed as well as antileishmanial activity and intracellular localization in DH82 macrophages. KEY FINDINGS: Liposomes (360 nm) zeta potential range from -40 to -65 mV, had 23% encapsulation efficiency and were stable for 180 days at 4°C. Free AME was cytotoxic towards L. infantum infected macrophages (ID50 = 0.012 M) while L-AME did not reduce cell viability. L-AME colocalized with parasites inside macrophages in a time-dependent manner, and reduced the percentage of infected cells and the number of intracellular parasites, decreasing the infection index (75-80%) twice as compared with AME treatment. CONCLUSIONS: Liposomal AME is a promising delivery system for treating visceral leishmaniasis, improving meglumine efficacy against L. infantum and minimizing its cytotoxicity towards canine macrophages.

Dact1 is expressed during chicken and mouse skeletal myogenesis and modulated in human muscle diseases.

Vertebrate skeletal muscle development and repair relies on the precise control of Wnt signaling. Dact1 (Dapper/Frodo) is an important modulator of Wnt signaling, interacting with key components of the various Wnt transduction pathways. Here, we characterized Dact1 mRNA and protein expression in chicken and mouse fetal muscles in vivo and during the differentiation of chick primary and mouse C2C12 myoblasts in vitro. We also performed in silico analysis to investigate Dact1 gene expression in human myopathies, and evaluated the Dact1 protein structure to seek an explanation for the accumulation of Dact1 protein aggregates in the nuclei of myogenic cells. Our results show for the first time that in both chicken and mouse, Dact1 is expressed during myogenesis, with a strong upregulation as cells engage in terminal differentiation, cell cycle withdrawal and cell fusion. In humans, Dact1 expression was found to be altered in specific muscle pathologies, including muscular dystrophies. Our bioinformatic analyses of Dact1 proteins revealed long intrinsically disordered regions, which may underpin the ability of Dact1 to interact with its many partners in the various Wnt pathways. In addition, we found that Dact1 has strong propensity for liquid-liquid phase separation, a feature that explains its ability to form nuclear aggregates and points to a possible role as a molecular 'on'-'off' switch. Taken together, our data suggest Dact1 as a candidate, multi-faceted regulator of amniote myogenesis with a possible pathophysiological role in human muscular diseases.

Mechanistic insights into the intracellular release of doxorubicin from pH-sensitive liposomes.

pH-sensitive liposomes are interesting carriers for drug-delivery, undertaking rapid bilayer destabilization in response to pH changes, allied to tumor accumulation, a desirable behavior in the treatment of cancer cells. Previously, we have shown that pH-sensitive liposomes accumulate in tumor tissues of mice, in which an acidic environment accelerates drug delivery. Ultimately, these formulations can be internalized by tumor cells and take the endosome-lysosomal route. However, the mechanism of doxorubicin release and intracellular traffic of pH-sensitive liposomes remains unclear. To investigate the molecular mechanisms underlying the intracellular release of doxorubicin from pH-sensitive liposomes, we followed HeLa cells viability, internalization, intracellular trafficking, and doxorubicin's intracellular delivery mechanisms from pH-sensitive (SpHL-DOX) and non-pH-sensitive (nSpHL-DOX) formulations. We found that SpHL-DOX has faster internalization kinetics and intracellular release of doxorubicin, followed by strong nuclear accumulation compared to nSpHL-DOX. The increased nuclear accumulation led to the activation of cleaved caspase-3, which efficiently induced apoptosis. Remarkably, we found that chloroquine and E64d enhanced the cytotoxicity of SpHL-DOX. This knowledge is paramount to improve the efficiency of pH-sensitive liposomes or to be used as a rational strategy for developing new formulations to be applied in vivo.

Leucine-Rich Diet Modulates the Metabolomic and Proteomic Profile of Skeletal Muscle during Cancer Cachexia.

Background: Cancer-cachexia induces a variety of metabolic disorders, including skeletal muscle imbalance. Alternative therapy, as nutritional supplementation with leucine, shows a modulatory effect over tumour damage in vivo and in vitro. Method: Adult rats distributed into Control (C), Walker tumour-bearing (W), control fed a leucine-rich diet (L), and tumour-bearing fed a leucine-rich diet (WL) groups had the gastrocnemius muscle metabolomic and proteomic assays performed in parallel to in vitro assays. Results: W group presented an affected muscle metabolomic and proteomic profile mainly related to energy generation and carbohydrates catabolic processes, but leucine-supplemented group (WL) recovered the energy production. In vitro assay showed that cell proliferation, mitochondria number and oxygen consumption were higher under leucine effect than the tumour influence. Muscle proteomics results showed that the main affected cell component was mitochondria, leading to an impacted energy generation, including impairment in proteins of the tricarboxylic cycle and carbohydrates catabolic processes, which were modulated and improved by leucine treatment. Conclusion: In summary, we showed a beneficial effect of leucine upon mitochondria, providing information about the muscle glycolytic pathways used by this amino acid, where it can be associated with the preservation of morphometric parameters and consequent protection against the effects of cachexia.

Biogenic silver nanoparticles: In vitro and in vivo antitumor activity in bladder cancer.

Bladder cancer is the fifth most common disease in the United States, and the treatment and alternatives for patients have not changed in the last decades. Silver nanoparticles (AgNP) have been used in the treatment of various cancer, mainly because of the antineoplastic activity; however, their use and the molecular mechanisms towards bladder cancer still unexplored. Therefore, this work aims to evaluate the in vitro and in vivo antitumoral mechanisms of biogenic silver nanoparticles synthesized from Fusarium sp. First, AgNP showed cytotoxicity in a dose- and time-response relationship and detailed analysis demonstrated the induction of cell death via apoptosis, also inhibiting cell migration and proliferation in bladder carcinoma cell line 5637. Next, it was evaluated the antitumoral activity of AgNP against non-muscle invasive bladder cancer (NMIBC). Bladder cancer was chemically induced with N-methyl-N-nitrosourea (MNU) on C57BL/6JUnib female mice and treated by intravesical route with AgNP concentrations of 0.5, 0.2, and 0.05 mg/mL. Finally, treatment with AgNP (0.05 mg/mL) led to 57.13% of tumor regression, with 14.28% of the animals showing normal urothelium, and 42.85% showing flat hyperplasia, considered to be a benign lesion. Overall, these findings demonstrated that AgNP might be a cost-effective alternative and promising candidate for the treatment of bladder cancer.

PyScratch: An ease of use tool for analysis of scratch assays.

BACKGROUND AND OBJECTIVE: Image acquisition has greatly benefited from the automation of microscopes and has been followed by an increasing amount and complexity of data acquired. Here, we present the PyScratch, a new tool for processing spatial and temporal information from scratch assays. PyScratch is an open-source software implemented in Python that analyses the migration area in an automated fashion. METHODS: The software was developed in Python. Wound healing assays were used to validate its performance. The images were acquired using Cytation 5™ during 60 h. Data were analyzed using One-Way ANOVA. RESULTS: PyScratch performed a robust analysis of confluent cells, showing that high plating density affects cell migration. Additionally, PyScratch was approximately six times faster than a semi-automated analysis. CONCLUSIONS: PyScratch offers a user-friendly interface allowing researches with little or no programming skills to perform quantitative analysis of in vitro scratch assays.

Bacterial anti-microbial peptides and nano-sized drug delivery systems: The state of the art toward improved bacteriocins.

Antimicrobial peptides (AMP) are molecules consisting of 12-100 amino acids synthesized by certain microbes and released extracellularly to inhibit the growth of other microbes. Among the AMP molecules, bacteriocins are produced by both gram-positive and gram-negative bacterial species and are used to kill or inhibit other prokaryotes in the environment. Due to their broad-spectrum antimicrobial activity, some bacteriocins have the potential of becoming the next generation of antibiotics for use in the crisis of multi antibiotic-resistant bacteria. Recently, bacteriocins have even been used to treat cancer. However, bacteriocins present a few drawbacks, such as sensitivity to proteases, immunogenicity issues, and the development of bacteriocin resistance by pathogenic bacteria. In this regard, nanoscale drug delivery systems (Nano-DDS) have led to the expectation that they will eventually improve the treatment of many diseases by addressing these limitations and improving bacteriocin pharmacokinetics and pharmacodynamics. Thus, combining bacteriocins with nano-DDS may be useful in overcoming these drawbacks and thereby reveal the full potential of bacteriocins. In this review article, we highlight the importance of tailoring nano-DDS to address bacteriocin limitations, the successes and failures of this technology thus far, the challenges that this technology still has to overcome before reaching the market, and future perspectives. Therefore, the purpose of this review is to highlight, categorize, compare and contrast the different nano-DDS described in the literature so far, and compare their effectiveness in order to improve the next generation of bacteriocin nano-sized drug delivery systems (Nano-DDS).

Protective effect of N-acetylcysteine on the toxicity of silver nanoparticles: Bioavailability and toxicokinetics in Enchytraeus crypticus.

We previously demonstrated that N-acetylcysteine (NAC) could reduce the toxicity of silver (Ag) materials (nanoparticles (NPs) and Ag nitrate) to the soil invertebrate Enchytraeus crypticus (Oligochaeta). It remains however, unclear whether the antitoxic mechanism of NAC was caused by NAC-Ag binding in the soil or inside the organisms. This study aimed at determining the bioavailability of Ag in the soil in a 21-day toxicity test as well as the Ag uptake and elimination kinetics in E. crypticus exposed to AgNPs in LUFA 2.2 standard soil amended with low (100 mg/kg dry soil) and high (600 mg/kg dry soil) NAC concentrations. The addition of NAC to the soil alleviated the toxicity of AgNPs by decreasing the internal Ag concentration of E. crypticus in a dose-dependent manner. Indeed, NAC reduced the binding of Ag to the soil, which probably was due to the formation of soluble but biologically unavailable Ag-cysteine complexes. The reduced Ag uptake in the enchytraeids was explained from an increased elimination at high NAC levels. These findings reinforce the view that metal complexing-compounds like NAC play a key role in the modulation of AgNP toxicity and bioavailability in terrestrial environments. Further, it may inform on the potential of NAC as a remediation solution for Ag or other metal-contaminated soils.

Evaluation of cytotoxicity of nanolipid carriers with structured Buriti oil in the Caco-2 and HepG2 cell lines.

Buriti oil is rich in monounsaturated fatty acids, carotenoids and tocopherols and it is used for the treatment of various diseases. One strategy to restructure the triglycerides is enzymatic interesterification and nanocarriers have been employed to improve the solubility, bioavailability and stability of active compounds. This work aims to investigate the in vitro cytotoxicity of this structured oil in nanoemulsions and nanostructured lipid carriers to expand the applicability of the crude oil. None of the samples had a cytotoxic effect on Caco-2 and HepG2 cell lines at the concentrations tested. Structured lipids acted protecting against oxidative stress and lipid peroxidation. Additionally, no consumption of glutathione has been observed in both cells, and the compounds present in buriti oil are possibly acting as antioxidants. Thus, nanoparticles prepared with interesterified buriti oil had low cytotoxicity and high oxidative stability, with great potential for future applications.

The in vivo toxicological profile of cationic solid lipid nanoparticles.

Cationic solid lipid nanoparticles (cSLNs) are considered as one of the most effective lipid nanocarriers for delivery of low water-solubility compounds and genetic materials. As the excipients used in the cSLN production are generally regarded as safe (GRAS), the formulations are granted as non-toxic. However, the toxicological profile of new SLN-based formulations should always be performed to confirm that the delivery systems themselves may not impose risks to the human health. Therefore, in this study, we delineate the toxicological profile of the cSLN formulation at 24 and 72 h after single intravenous injection to male Wistar rats. Hematological, biochemical, and histopathological evaluations of the spleen, lungs, liver, and kidneys indicated short-lived alterations including neutrophilia. We found increases in the population of macrophages in the lungs, liver, and spleen and also migration of circulating neutrophils into inflamed tissue and a decrease in blood urea nitrogen. We also observed the presence of cSLNs within the brain parenchyma without any sign of damage to the blood-brain barrier. These side effects appeared to be mild and transitory (< 72 h). These findings reinforce the importance of investigating the toxicity of SLN-based formulations before the incorporation of drugs/genetic material to the formulation and its translation to the clinic.

Butyrate Protects Mice from Clostridium difficile-Induced Colitis through an HIF-1-Dependent Mechanism.

Antibiotic-induced dysbiosis is a key factor predisposing intestinal infection by Clostridium difficile. Here, we show that interventions that restore butyrate intestinal levels mitigate clinical and pathological features of C. difficile-induced colitis. Butyrate has no effect on C. difficile colonization or toxin production. However, it attenuates intestinal inflammation and improves intestinal barrier function in infected mice, as shown by reduced intestinal epithelial permeability and bacterial translocation, effects associated with the increased expression of components of intestinal epithelial cell tight junctions. Activation of the transcription factor HIF-1 in intestinal epithelial cells exerts a protective effect in C. difficile-induced colitis, and it is required for butyrate effects. We conclude that butyrate protects intestinal epithelial cells from damage caused by C. difficile toxins via the stabilization of HIF-1, mitigating local inflammatory response and systemic consequences of the infection.

A Mechanistic View of Interactions of a Nanoherbicide with Target Organism.

Atrazine is one of the most used herbicides and has been associated with persistent surface and groundwater contamination, and novel formulations derived from nanotechnology can be a potential solution. We used poly(ε-caprolactone) nanoencapsulation of atrazine (NC+ATZ) to develop a highly effective herbicidal formulation. Detailed structural study of interaction between the formulation and Brassica juncea plants was carried out with evaluation of the foliar uptake of nanoatrazine and structural alterations induced in the leaves. Following postemergent treatment, NC+ATZ adhered to the leaf and penetrated mesophyll tissue mainly through the hydathode regions. NC+ATZ was transported directly through the vascular tissue of the leaves and into the cells where it degraded the chloroplasts resulting in herbicidal activity. Nanocarrier systems, such as the one used in this study, have great potential for agricultural applications in terms of maintenance of herbicidal activity at low concentrations and a substantial increase in the herbicidal efficacy.