Search Results (158)

Background/objectives: Foodborne diseases remain a significant global public health concern, requiring innovative and effective antimicrobial strategies to control food pathogens. Encapsulation of natural antimicrobials have attracted increasing interest. In this study, liposomes encapsulating pink pepper essential oil (PPEO), nisin, or their combination were developed, aiming to potentiate antimicrobial performance against foodborne pathogens. Methods: Phosphatidylcholine liposomes were prepared by the thin-film method and characterized by DLS and FTIR. The antimicrobial activity of nisin, PPEO, and liposomes was investigated by the agar diffusion method against foodborne pathogens like Staphylococcus aureus, Listeria monocytogenes, and Salmonella Typhimurium. Results: The liposomes exhibited nanometric size ranging from 91 to 107 nm, low polydispersity, and zeta potential between −3.73 and −7.39 mV, indicating well-defined vesicles with negative surface charges. Encapsulation enhanced antimicrobial efficacy, with nisin–PPEO liposomes stored for 21 days under refrigeration showing a sustained inhibition of L. monocytogenes, outperforming liposomes containing nisin alone. The combined antimicrobials also inhibited Gram-positive bacteria in milk agar, used as a simulated food system. Additionally, the antioxidant activity of PPEO was preserved upon encapsulation, especially under refrigeration, reinforcing the protective role of the liposomes. Conclusions: The co-encapsulation approach strengthened the stability and bioactivity of natural antimicrobials, highlighting liposomal delivery as a promising strategy to control foodborne bacteria. Full article

Pulmonary drug delivery represents a promising approach in the treatment of respiratory diseases, allowing for passive targeting and enhanced drug efficacy. Background/Objectives: The aim of the present study was to develop inhalable dry powders from lyophilized sildenafil citrate (SC)-loaded liposomes made from phosphatidylcholine and either cholesterol (CH) or resveratrol (RSV). Methods: Liposomes were prepared via a pH gradient method to increase drug entrapment efficiency and drug loading, and then the liposomes were lyophilized using different proportions of ethanol, mannitol, and lactose as excipients. The resulting dry cakes were converted into powders and evaluated for aerodynamic performance using a custom-designed air-blowing device. Notably, this is the first time that resveratrol has been used as a substitute for cholesterol in SC-loaded liposomes. Results: Our results demonstrate that RSV is a suitable liposome bilayer component and improves drug loading. Our findings prove that lyophilized cakes containing liposomes produce a dry powder that is suitable for aerosolization with potential application to pulmonary delivery of sildenafil citrate. The results suggest that RSV represents a potential alternative to traditional cholesterol-based liposomal formulations. Conclusions: This work presents a novel strategy for the pulmonary delivery of sildenafil, using biocompatible and FDA-approved mannitol and lactose for this administration route. Full article

Achieving a high nutritional value of food often involves fortifying microorganisms (such as bacteria and yeast) used in baking and dairy industry with essential elements. The aim of this study was to investigate the effect of a pulsed electric field (PEF) on the penetration and accumulation of Ca²⁺ and Mg²⁺ ions into model membranes of the food-grade yeast Saccharomyces cerevisiae. Simplified model membranes (monolayers and liposomes) were constructed using the phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC). The Langmuir monolayer technique, dynamic light scattering (DLS) and microelectrophoresis were employed to characterize the physicochemical properties of the model membranes investigated. The results showed significant molecular-level differences in the interactions of the selected cations with lipid monolayers and bilayers in liposome structures. Both cations deeply penetrated the membrane’s hydrophilic region, yet two competing effects were evident: expansion induced by hydrated Mg²⁺ and condensation driven by Ca²⁺ bridging. Furthermore, the application of PEF increased the concentration of ions absorbed by the liposomes. Specifically, optimized PEF parameters resulted in cation accumulation within the model membranes, ranging from 6 to 13%. This finding correlates well with the increased Ca²⁺ and Mg²⁺ uptake observed in real yeast cells, providing a deeper understanding of the cell membrane-environment interface and the underlying processes. Full article

Background: Lutein and zeaxanthin (LZ) are macular xanthophyll carotenoids with antioxidant and blue-light filtering properties, but their oral bioavailability is limited. Lipid-based delivery systems may enhance absorption. Methods: We compared four single-dose LZ delivery systems in male Sprague–Dawley rats: (G1) LZ in medium-chain triglyceride (MCT) oil; (G2) LZ in MCT + phosphatidylcholine (PC); (G3) LZ in MCT + phosphatidylserine (PS); (G4) LZ in liposomal powder. Following an overnight fast, each group (n = 6) received an oral gavage of the assigned formulation. Serial blood samples were collected up to 24 h post-dose. Plasma lutein + zeaxanthin concentrations were quantified by a validated LC–MS/MS method. Non-compartmental pharmacokinetic (PK) parameters were computed (Phoenix WinNonlin^®), and one-way ANOVA was used to make inter-group comparisons on ln-transformed metrics with Dunnett’s post hoc tests. Results: The PS-complexed formulation (G3) yielded the highest LZ exposure (mean C_max 69.63 ± 0.78 ng/mL; AUC_0-t 620.23 ± 16.41 ng·h/mL), significantly exceeding the MCT oil control (G1: 52.54 ± 0.70 ng/mL; 494.51 ± 13.70 ng·h/mL; p < 0.001). The PC-enriched oil (G2) and liposomal powder (G4) also produced higher C_max and AUC than G1 (p < 0.01). No differences in elimination half-life (t1/2 ≈ eight h) were observed between formulations. Conclusions: Phospholipids, especially PS, substantially improved the systemic availability of lutein and zeaxanthin compared with MCT oil alone. PS-based lipid complexes appear particularly effective, supporting their use in ocular-health formulations to maximise xanthophyll bioavailability. Full article

The study focused on essential oils (EOs) of plant origin, which are of great interest to scientists in the context of medical applications due to their biological properties, such as antimicrobial, anti-inflammatory, antioxidant, and anticancer effects. The objective of the study was to determine chemical profiles and biological activities of the essential oils extracted from five mixtures (M1 [Thymus vulgaris, Ammi visnaga, Syzygium aromaticum, Citrus sinensis]; M2 [Thymus vulgaris, Ammi visnaga, Cinnamomum verum, Citrus sinensis]; M3 [Mentha pulegium, Lavandula angustifolia, Zingiber officinale, Citrus sinensis]; M4 [Mentha pulegium, Lavandula angustifolia, Cinnamomum verum, Citrus sinensis]; M5 [Ammi visnaga, Lavandula angustifolia, Zingiber officinale, Syzygium aromaticum]). Each mixture was derived from a blend of four selected plants used in traditional medicine in Mostaganem, Algeria. When selecting the best composition, the interactions between plant components were considered in terms of potential therapeutic benefits. The chemical compositions of the EO mixtures were analyzed using GC-MS. The acute toxicity of the EO mixtures was evaluated in vivo following oral administration. The sensitivity of the microorganisms to the EO mixtures was determined using the agar diffusion method. Virucidal testing was performed using the quantitative suspension method to determine virucidal activity, as described in the European standard for disinfectants used in the medical field. The antioxidant activity of the EO mixtures was evaluated using a model membrane system based on liposomes derived from soybean phosphatidylcholine. Chemopreventive activity was assessed in vitro using cell culture. The main compounds identified were carvacrol and thymol in M1; geranial, cinnamylaldehyde, and carvacrol in M2; pulegone and limonene in M3; geranial and cinnamylaldehyde and limonene in M4; and eugenol and caryophyllene in M5. The selection of the “best” blend depended on the biological activity deemed most critical for the specific application. Specifically, M3, M4, and M5 exhibited the strongest anti-HSV-1, anti-HAdV-5, and anticancer activity, respectively. In contrast, M1, a potent antioxidant, demonstrated the strongest antibacterial and anticancer activity. These results indicate that M1, M3, M4, and M5 EOs have promising applications in the pharmaceutical industry and medical research. Full article

Background/Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) poses a significant global health threat due to its increasing resistance to conventional antibiotics. Antimicrobial peptides (AMPs) derived from natural sources represent a promising alternative. Fragments of spider membrane-active toxins can serve as AMPs with anti-MRSA activity. Methods: To demonstrate this, amino acid sequences of approximately 2000 linear spider venom peptides were fragmented into 9–22-residue-long moieties (75,235 in total) and pre-trained neural networks were used to predict their anti-MRSA activity. As many as 15 peptides with high predicted activity were synthesized, and three AMPs with high anti-MRSA and low hemolytic activities were selected. One of these peptides was studied using high-resolution ¹H-, ¹³C-, and ¹⁵N-NMR spectroscopy in an aqueous solution and lyso-palmitoylphosphatidylglycerol (LPPG) micelles. Wide-line ³¹P-NMR was applied to multilamellar phospholipid liposomes composed of phosphatidylcholine (PC) or phosphatidylglycerol (PG). Results: Low hemolytic activity is explained by non-specific interaction with PC whereas high antibacterial activity arises from specific interaction with PG accompanied with the formation of a tight complex between the N-terminal tripeptide fragment and PG headgroup. The structure of a such complex, stabilized by an ionic interaction between the N-terminal NH₃⁺ group and the lipid phosphate, was determined based on peptide–LPPG NOEs. The most favorable ratio between anti-MRSA and hemolytic activities, i.e., selectivity of the peptides, is attained when the tripeptide consists exclusively of phenylalanine and tryptophan residues. Confocal microscopy confirmed that the most selective peptide deteriorates the plasma membrane of S. aureus. Conclusions: This approach may enable the production of highly selective AMPs against Stapylococci, including MRSA. Full article

L-asparaginase (L-ASNase) is a vital enzymatic drug widely used for treating acute lymphoblastic leukemia (ALL) and certain lymphomas. However, its clinical application is often limited by a short plasma half-life, pronounced immunogenicity, and systemic toxicities. To address these challenges, we recently developed conjugates of L-ASNase with cationic polymers, enhancing its cytostatic activity by increasing enzyme binding with cancer cells. The present study focuses on the development of liposomal formulations of E. coli L-asparaginase (EcA) and its conjugates with cationic polymers: the natural oligoamine spermine (spm) and a synthetic polyethylenimine–polyethyleneglycol (PEI-PEG) copolymer. This approach aims to improve enzyme encapsulation efficiency and stability within liposomes. Various formulations—including EcA conjugates with polycations incorporated into 100 nm and 400 nm phosphatidylcholine/cardiolipin (PC/CL, 80/20) anionic liposomes—were synthesized as a delivery system of high enzyme load. Fourier Transform Infrared (FTIR) spectroscopy confirmed successful enzyme association with liposomal carriers by identifying characteristic changes in the vibrational bands corresponding to both protein and lipid components. In vitro release studies demonstrated that encapsulating EcA formulations in liposomes more than doubled their half-release time (T_1/2), depending on the formulation. Cytotoxicity assays against Raji lymphoma cells revealed that liposomal formulations, particularly 100 nm EcA-spm liposomes, exhibited markedly superior anti-proliferative activity, reducing cell viability to 4.5%, compared to 35% for free EcA. Confocal Laser Scanning Microscopy (CLSM) provided clear visual and quantitative evidence that enhanced cellular internalization of the enzyme correlates directly with its cytostatic efficacy. Notably, formulations showing higher intracellular uptake produced greater cytotoxic effects, emphasizing that hydrolysis of asparagine inside cancer cells, rather than extracellularly, is critical for therapeutic success. Among all tested formulations, the EcA-spermine liposomal conjugate demonstrated the highest fluorescence intensity within cells providing enhanced cytotoxicity. These results strongly indicate that encapsulating cationically modified L-ASNase in liposomes is a highly promising strategy to improve targeted cellular delivery and prolonged enzymatic activity. This strategy holds significant potential for developing more effective and safer antileukemic therapies. Full article

Background/Objectives: The rapid emergence of antibiotic-resistant oral pathogens has rendered many conventional therapies increasingly ineffective. Antimicrobial peptides (AMPs) have emerged as a promising therapeutic alternative due to their unique mechanisms of action and low propensity for inducing resistance. The delivery of novel therapeutic AMPs against oral cavity bacterial infections requires effective pharmaceutical dosage formulations. This study investigated the potential of two liposomal formulations for the association and delivery of the antimicrobial peptide (AMP) LL17-32 against the dental bacterial pathogen Porphyromonas gingivalis. Methods: Liposomes composed of either negatively charged soya lecithin (SL) or neutrally charged dioleoyl-phosphatidylcholine (DOPC) phospholipids were formulated and characterized based on their hydrodynamic size distribution, ζ-potential, morphology, membrane fluidity, peptide association efficiency, stability and release of peptide in vitro under physiological conditions. The characterization of their biological activity included efficiency of bacterial killing, bacterial adherence, and mammalian cell cytotoxicity using human gingival keratinocyte (TIGK) cells. Results: Both liposomal formulations exhibited spherical morphology with hydrodynamic diameters smaller than ~170 nm and demonstrated good colloidal stability. LL17-32 showed high association efficiency with both liposomal membranes, with no detectable LL17-32 in vitro release. In biological assays, peptide-loaded DOPC liposomes exhibited dose-dependent bactericidal activity against P. gingivalis, whereas SL liposomes significantly attenuated the bactericidal effect of LL17-32. Both formulations displayed reduced cytotoxicity toward human gingival keratinocyte (TIGK) cells versus free peptide. Conclusions: These findings suggest that DOPC liposomes represent a promising delivery system for LL17-32 by adhering to P. gingivalis and exhibiting minimal cytotoxicity to mammalian cells. This study emphasises the critical role of lipid charge in designing AMP delivery systems for antibacterial applications, while it additionally demonstrates the utility of flow cytometry as a quantitative tool to assess liposome–bacteria association. Full article

The recruitment of peripheral membrane proteins is tightly regulated by membrane lipid composition and local electrostatic microenvironments. Our experimental observations revealed that Vp54, a viral matrix protein, exhibited preferential binding to lipid bilayers enriched in anionic lipids such as phosphatidylglycerol (PG) and phosphatidylserine (PS), compared to neutral phosphatidylcholine/phosphatidylethanolamine liposomes, and this occurred in a curvature-dependent manner. To elucidate the molecular basis of this selective interaction, we performed a series of computational analyses including helical wheel projection, electrostatic potential calculations, electric field lines simulations, and electrostatic force analysis. Our results showed that the membrane-proximal region of Vp54 adopted an amphipathic α-helical structure with a positively charged interface. In membranes containing PG or PS, electrostatic potentials at the interface were significantly more negative, enhancing attraction with Vp54. Field line and force analyses further confirmed that both the presence and spatial clustering of anionic lipids intensify membrane–Vp54 electrostatic interactions. These computational findings align with experimental binding data, jointly demonstrating that membrane lipid composition and organization critically modulate Vp54 recruitment. Together, our findings highlight the importance of electrostatic complementarity and membrane heterogeneity in peripheral protein targeting and provide a framework applicable to broader classes of membrane-binding proteins. Full article

Background/Objectives: Intranasal delivery is a promising approach for targeting the central nervous system (CNS); however, most of the drugs show poor permeability through the nasal mucosa. Nanocarriers such as liposomes can improve nasal drug absorption; however, the surface charge of liposomes has a key role in the nasal mucosal uptake process. Therefore, the present study aimed to formulate and compare the intranasal applicability of oppositely charged liposomes loaded with donepezil hydrochloride (DPZ) as CNS-active model compound using two different charge inducers, the negatively charged dicethyl phosphate (DCP) and the positively charged stearylamine (SA). Methods: Liposomes were prepared with a fixed phosphatidylcholine (PC)/cholesterol (CH) 7:2 molar ratio, while the effect of DCP and SA was studied in a 0.5:2 molar ratio. The most important properties for intranasal administration were studied, e.g., colloidal parameters, drug release and permeability behavior, and mucoadhesion. Results: It has been revealed that the reduction in liposome vesicle size is directly proportional to the amount of DCP, while it is inversely proportional to the amount of SA. This was also supported by the drug release studies—the lower vesicle size resulted in faster drug release. Both charge inducers increased the drug encapsulation efficiency (~60–80%) through tighter packing or increased spacing of the lipid bilayer structure. DCP also improved the in vitro nasal permeability compared to the initial DPZ solution. The positively charged SA showed more remarkable mucoadhesive properties than DCP. Conclusions: We can conclude that both charge inducers can be useful for improving nasal absorption of liposomal carriers, DCP in higher (PC:CH:DCP 7:2:2), while SA in lower concentrations (PC:CH:SA 7:2:0.5). Full article

Introduction: Antimicrobial resistance has become a global concern, and few new antimicrobials are currently being developed. Fluopsin C has proven broad-spectrum activity, being a promising candidate for new antimicrobial development. To optimize antimicrobial activity, this research aimed at fluopsin C (Flp) encapsulation in liposomes to achieve controlled release and reduce cytotoxicity. Methods: Liposomal formulations were prepared by extruding formulations based on soy phosphatidylcholine (SPC) or poly (ethylene glycol)-distearoylphosphatidylethanolamine (DSPE-PEG) plus cholesterol, and were characterized by their size, polydispersity index, zeta potential, encapsulation efficiency, shelf-life stability, in vitro release profile, cytotoxicity, and antimicrobial activity against Klebsiella pneumoniae in vitro and in vivo. Results: The results indicated that the DSPE-PEG DMSO+Flp formulation presented superior physicochemical stability and unaltered antimicrobial activity. In vitro, CC₅₀ decreased by 54%. No lethal dose was obtained in mice within the concentration range tested. The most effective doses in vivo were 2 × 2 mg/kg for free fluopsin C and 1 × 2 mg/kg for DSPE-PEG DMSO+Flp, resulting in a 40% reduction in mortality from bacteremia. Only discrete inflammatory infiltration was detected in the liver, while kidney necrosis ranged from discrete to moderate. Encapsulation of fluopsin C in liposomes showed promising features supporting to use against infections by MDR K. pneumoniae. Full article

Condensed tannins from grape seed residues show high antioxidant activity but low oral bioavailability because of their high degree of polymerization and covalent interactions with proteins. This study aimed to improve their bioaccessibility through depolymerization and encapsulation. Depolymerization was carried out using microwave-assisted SN1 reactions with gallic acid as a nucleophile under food-grade conditions, mainly producing epicatechin monomers with 99.8% polymer degradation efficiency. Importantly, the inhibition of ABTS●+ and DPPH● radicals remained unaffected (p > 0.05), indicating that depolymerization preserved the antioxidants’ redox function, maintaining about 90% of their inhibition activity. The products were encapsulated in phosphatidylcholine liposomes, which had nanometric sizes and high encapsulation efficiency (83.11%), and remained stable for up to 60 days. In vitro release of nanoliposomal epicatechin in a D1 simulant was less than 10% after 48 h, fitting a Weibull model (β = 0.07), suggesting sub-diffusive transport and demonstrating high bioactive retention capacity in aqueous systems. During in vitro digestion, bioaccessibility of gallic acid and epicatechin reached 95.61 ± 0.58% and 98.56 ± 0.81%, respectively, with a 2333% increase in the bioaccessible mass of flavan-3-ols in native liposomal condensed tannins, which otherwise showed no detectable bioaccessibility. These findings highlight the potential of polyphenols from agro-industrial waste with enhanced bioaccessibility for applications in nutraceuticals and functional foods. Full article

Background/Objectives: Recent evidence challenges the classical chemiosmotic theory, suggesting that proton movement along membrane surfaces—not bulk-phase gradients—drives bioenergetic processes. Proton accumulation on membranes like the myelin sheath and endoplasmic reticulum (ER) may represent a universal mechanism for cellular energy storage. This study investigates whether phospholipids from these membranes, combined with anionic bee venom proteins, enhance proton absorption, potentially elucidating a novel bioenergetic pathway. Methods: Five phospholipids (phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, phosphatidylcholine) from rat liver were isolated to model myelin/ER membranes. Anionic proteins (pI 5.65–5.80) were purified from bee venom via cation exchange chromatography. Liposomes (with/without proteins) were prepared, and proton absorption was quantified by pH changes in suspensions versus pure water. Statistical significance was assessed via ANOVA and t-tests. Results: All phospholipid liposomes examined in this study absorbed protons under the tested conditions, with phosphatidylethanolamine showing the highest capacity (pH increase: 7.00 → 7.18). Liposomes enriched with anionic proteins exhibited significantly greater proton absorption (e.g., phosphatidylserine + proteins: pH 8.15 vs. 7.15 alone; p < 2.43 × 10⁻⁶). Sphingomyelin-protein liposomes absorbed the most protons, suggesting that protein–phospholipid interactions modulate surface proton affinity. Conclusions: Anionic bee venom proteins amplify proton absorption by phospholipid membranes, supporting the hypothesis that lipid–protein complexes act as “proton capacitors”. This mechanism may underpin extramitochondrial energy storage in myelin and ER. Pharmacologically, targeting these interactions could mitigate bioenergetic deficits in aging or disease. Further research should define the structural basis of proton capture by membrane-anchored proteins. Full article

Liposomes are lipid bilayer vesicles that are highly biocompatible, able to interact with the cell membrane, and able to release their cargo easily. The improvement of the physicochemical properties of liposomes, such as surface charge, lipid composition, and functionalization, makes these vesicles eligible delivery nanosystems for the gene therapy of many pathological conditions. In the present study, pre-formulation analysis was conducted to develop liposomes that facilitate the delivery of nucleic acids to neuronal cells, with the aim of future delivery of a CRISPR/Cas9 system designed to silence genes responsible for autosomal dominant neurodegenerative disorders. To this aim, different nucleic acid cargo models, including λ phage DNA, plasmid DNA, and mRNA encoding GFP, were considered. Liposomes with varying lipid compositions were produced using the ethanol injection method and analyzed for their dimensional stability and ability to interact with DNA. The selected formulations were tested in vitro using a neuroblastoma cell line (SH-SY5Y) to evaluate their potential toxicity and the ability to transfect cells with a DNA encoding the green fluorescent protein (pCMV-GFP). Among all formulations, the one containing phosphatidylcholine, phosphatidylethanolamine, pegylated 1,2-distearoyl-sn-glycero-3-phosphethanolamine, cholesterol, and dioctadecyl-dimethyl ammonium chloride (in the molar ratio 1:2:4:2:2) demonstrated the highest efficiency in mRNA delivery. Although this study was designed with the goal of ultimately enabling the delivery of a CRISPR/Cas9 system for treating autosomal dominant neurodegenerative disorders such as polyglutamine spinocerebellar ataxias (SCAs), CRISPR/Cas9 components were not delivered in the present work, and their application remains the objective of future investigations. Full article

Background/Objectives: Cancer is one of the deadliest diseases worldwide. Despite the existing treatments, the adverse side effects and the increasing drug resistance to the current therapies lead to a reduced quality of life for patients and poor prognosis. The pyrimido[5,4-d]pyrimidine compound (PP) was identified as a promising new anticancer drug due to its potent activity against colorectal and triple-negative breast cancers; however it showed poor aqueous solubility and safety profile. This study aimed the synthesis of compound PP, its encapsulation in liposomal formulations based on phosphatidylcholines (PC), the characterization of liposomal formulations and its biological evaluation. Methods: A new synthesis method for PP was developed. The compound was incorporated into different liposomal formulations. The hydrodynamic size, polydispersity, and zeta potential of loaded and non-loaded formulations were measured by DLS. The cytotoxic effects of compound PP, placebo nanoformulations, and PP-loaded nanoformulations were assessed in colorectal (HCT 116) and triple-negative breast cancer (MDA-MB-231) cell lines, as well as in non-tumor BJ-5ta cells. Results: The PP compound was efficiently synthesized. The PP-loaded liposomal formulations exhibit sizes below 150 nm, low polydispersity, and long-time stability upon storage at 4 °C. The antitumor compound was encapsulated with excellent efficiency, and sustained release profiles were obtained. The PP compound showed high activity against HCT 116 (IC₅₀ = 2.04 ± 0.45 µM) and MDA-MB-231 (IC₅₀ = 5.24 ± 0.24 µM) cell lines. DPPC-containing formulations were effective against cancer cells, but showed toxicity comparable to free PP in BJ-5ta normal cells. Conversely, PP-EggPC-Chol-L formulation displayed strong anticancer activity with residual toxicity to normal cells. Conclusions: The PP-loaded liposomal formulation, composed of 70% PC from egg yolk (EggPC) and 30% cholesterol (Chol), designated as PP-EggPC-Chol-L, was the most promising formulation, showing effective anticancer activity in both cancer cell lines and a significant improvement in the safety profile which is of utmost importance to progress to the next phase of drug development. Full article