Electrochemical Characterization of the Encapsulation and Release of 5-Fluorouracil in Nanocarriers Formed from Soy Lecithin Vesicles.

5-Fluorouracil (5-FU) is an antineoplastic agent known for its low bioavailability and limited cellular penetration, often resulting in adverse effects on healthy cells. Thus, finding vehicles that enhance bioavailability, enable controlled release, and mitigate adverse effects is crucial. The study focuses on encapsulating 5-FU within soy lecithin vesicles (SLVs) and assessing its impact on the carrier's properties and functionality. Results show that incorporating 5-FU does not affect SLVs' size or polydispersity, even postlyophilization. Liberation of 5-FU from SLVs requires system disruption rather than spontaneous release, with an encapsulation efficiency of approximately 43% determined using Square Wave Voltammetry. Cytotoxicity assays on colorectal cancer cells reveal SLV-based delivery's significant efficacy, surpassing free drug solution effects with 45% cell viability after 72 h vs 73% viability. The research addresses 5-FU's limited bioavailability by creating a biocompatible nanocarrier for efficient drug delivery, highlighting SLVs as promising for targeted cancer therapy due to sustained antiproliferative effects and improved cellular uptake. The study underscores the importance of tailored drug delivery systems in enhancing therapeutic outcomes and suggests SLV/5-FU formulations as a potential advancement in cancer treatment strategies.

Electrochemical and Spectroscopic Studies Performed for Indomethacin and 1-Naphthol Incorporated in 1-Butyl-3-methyl-imidazolium Bis(2-ethylhexyl) Sulfosuccinate Vesicles to Investigate Them as a Potentially pH-Sensitive Nanocarrier.

Characterization studies of 1-butyl-3-methyl-imidazolium bis(2-ethylhexyl) sulfosuccinate vesicles at different pH values have been carried out by using liquid surface tension, transmission electron microscopy, and dynamic light scattering. The results show that there are no vesicle changes in its size and negative Z potential at pH 3, 6, and 10. Furthermore, indomethacin and 1-naphthol, both pH-dependent, electroactive, and fluorescence probes, were used to further characterize the bilayer employing electrochemical and emission techniques. The partition of indomethacin and 1-naphthol between the water and bilayer pseudophases only occurs for the neutral species and does not happen for the anionic species because the highly negative Z bilayer potential prevents incorporation due to negative repulsion. For the neutral species, the partition constant values were evaluated by square wave voltammetry and emission spectroscopy. Finally, for the indomethacin incorporated into the vesicle bilayer at pH 3, the release profile was monitored over time at pH 6. It was found that a change in the pH values causes the complete release of indomethacin after 25 min, which led us to think that the vesicles presented in this work can be used as a pH-sensitive nanocarrier for neutral pH-sensitive drugs.

A simple electrochemical immunosensor for sensitive detection of transgenic soybean protein CP4-EPSPS in seeds.

Soybean is the most produced crop in Argentina, and 99 % corresponds to genetically modified soybean. One of the main produced varieties is Roundup Ready® soybean (RR), which was modified to express the enzyme CP4 5-enolpyruvylshikimate 3-phosphate synthase (CP4 EPSPS), which confers resistance to glyphosate, the main herbicide worldwide used. The possible impact of genetically modified organisms (GMO) has generated public concerns, thus increasing interest in the development of GMOs detection devices. In this work, an electrochemical immunosensor for CP4 EPSPS detection in soybean seeds was obtained, by using a gold electrode modified with an anti-CP4 EPSPS polyclonal antibody produced in our laboratory. The presented immunosensor resulted in a simple, low-cost, fast, and reproducible device. Also, labeling and/or signal amplification system was not necessary, since the sensor showed high sensibility with a low detection limit (lower at 0,038 % RR soybean, 38 ng mL-1 CP4 EPSPS).

Polyclonal antibody production anti Pc_312-324 peptide. Its potential use in electrochemical immunosensors for transgenic soybean detection.

A new polyclonal antibody that recognizes the CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4-EPSPS), which provides resistance to glyphosate in soybean (Roundup Ready®, RR soybean), was produced. New Zealand rabbits were injected with a synthetic peptide (Pc_312-324, (PEP)) present in the soybean CP4-EPSPS protein. The anti-PEP antibodies production was evaluated by electrophoresis (SDS-PAGE) and an enzyme-linked immunosorbent assay (ELISA) was developed in order to study their specificity. The ELISA showed that the polyclonal antibody was specific to PEP. In addition, the anti- PEP was immobilized onto a gold disk electrode and the antigen-antibody interaction was evaluated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Moreover, the EIS showed that the electron transfer resistance of the modified electrode increased after incubation with solutions containing CP4-EPSPS protein from RR transgenic soybean, while no changes were detected after incubation with no-RR soybean proteins. These results suggest that the CP4-EPSPS was immobilized onto the electrode, due to the specific interaction with the anti-PEP. These results show that this antigen-antibody interaction can be detected by electrochemical techniques, suggesting that the anti-PEP produced can be used in electrochemical immunosensors development to quantify transgenic soybean.

Study of lipid peroxidation and ascorbic acid protective role in large unilamellar vesicles from a new electrochemical performance.

In this contribution an electrochemical study is described for the first time of lipid peroxidation and the role of antioxidant on lipid protection using large unilamellar vesicles (LUVs). In order to simulate the cell membrane, LUVs composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were used. A vesicle-modified electrode was constructed by immobilizing DOPC LUVs onto carbon paste electrodes (CPEs). Lipid peroxidation was studied electrochemically by incubating the vesicle-modified electrodes with hydroxyl (HO) radicals generated via the Fenton reaction. Oxidative damage induced by HO was verified by using square wave voltammetry (SWV) and was indirectly measured by the increase of electrochemical peak current to [Fe(CN)6]4- which was used as the electrochemical label. Ascorbic acid (AA) was used as the antioxidant model in order to study its efficacy on free radical scavenging. The decrease of the electrochemical signal confirms the protective key role promoted by AA in the prevention of lipid peroxidation in vesicles. Through microscopy, it was possible to observe morphologic modification on vesicle structures after lipid peroxidation in the presence or absence of AA.

Determination of Benzyl-hexadecyldimethylammonium 1,4-Bis(2-ethylhexyl)sulfosuccinate Vesicle Permeability by Using Square Wave Voltammetry and an Enzymatic Reaction.

This report describes the studies performed to determine the permeability coefficient value (P) of 1-naphthyl phosphate (1-NP) through the benzyl-hexadecyldimethylammonium 1,4-bis(2-ethylhexyl)sulfosuccinate (AOT-BHD) vesicle bilayer. 1-NP was added in the external phase and must cross the bilayer of the vesicle to react with the encapsulated enzyme (alkaline phosphatase) to yield 1-naphtholate (NPh-), the product of the enzymatic hydrolysis. This product is electrochemically detected, at basic pH value, by a square wave voltammetry technique, which can be a good alternative over the spectroscopic one, to measure the vesicle solutions because scattering (due to its turbidity) does not make any influence in the electrochemical signal. The experimental data allow us to propose a mathematical model, and a value of P = (1.00 ± 0.15) × 10-9 cm s-1 was obtained. Also, a value of P = (2.0 ± 0.5) × 10-9 cm s-1 was found by using an independent technique, ultraviolet-visible spectroscopy, for comparison. It is evident that the P values obtained from both the techniques are comparable (within the experimental error of both techniques) under the same experimental conditions. This study constitutes the first report of the 1-NP permeability determination in this new vesicle. We want to highlight the importance of the introduction of a new method and the electrochemical response of the product generated through an enzymatic reaction that occurs in the inner aqueous phase of the vesicle, where the enzyme is placed.

Gold nanoparticles covalently assembled onto vesicle structures as possible biosensing platform.

In this contribution a strategy is shown to covalently immobilize gold nanoparticles (AuNPs) onto vesicle bilayers with the aim of using this nanomaterial as platform for the future design of immunosensors. A novel methodology for the self-assembly of AuNPs onto large unilamellar vesicle structures is described. The vesicles were formed with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-undecanethiol (SH). After, the AuNPs photochemically synthesized in pure glycerol were mixed and anchored onto SH-DOPC vesicles. The data provided by voltammetry, spectrometry and microscopy techniques indicated that the AuNPs were successfully covalently anchored onto the vesicle bilayer and decorated vesicles exhibit a spherical shape with a size of 190 ± 10 nm. The developed procedure is easy, rapid and reproducible to start designing a possible immunosensor by using environmentally friendly procedures.

Electrochemical and photophysical behavior of 1-naphthol in benzyl-n-hexadecyldimethylammonium 1,4-bis(2-ethylhexyl)sulfosuccinate large unilamellar vesicles.

In the present contribution, 1-naphthol is investigated in large unilamellar vesicles formed from a new catanionic surfactant, benzyl-n-hexadecyldimethylammonium 1,4-bis(2-ethylhexyl)sulfosuccinate, by electrochemical and spectroscopic techniques. The electrochemical results show that 1-naphthol experiences a partition process between the water phase and the large unilamellar vesicle bilayer phase, which is corroborated by absorption spectroscopic studies at pH = 6.40 and pH = 10.75. Interestingly, studies of 1-naphthol emission in benzyl-n-hexadecyldimethylammonium 1,4-bis(2-ethylhexyl)sulfosuccinate large unilamellar vesicles at pH = 10.75 and in sodium 1,4-bis(2-ethylhexyl)sulfosuccinate water solution show that when the 1,4-bis(2-ethylhexyl)sulfosuccinate moiety is part of the bilayer, the 1,4-bis(2-ethylhexyl)sulfosuccinate polar head interacts strongly with 1-naphthol, by favoring emission from the excited neutral species resulting in the appearance of a new band close to λ = 355 nm. It seems that the large unilamellar vesicle bilayer of the catanionic vesicle slows down the proton transfer process observed in water, where only emission from 1-naphtholate is detected.

Square Wave Voltammetry: An Alternative Technique to Determinate Piroxicam Release Profiles from Nanostructured Lipid Carriers.

A new, simple, and fast electrochemical (EC) method has been developed to determine the release profile of piroxicam, a nonsteroidal anti-inflammatory drug, loaded in a drug delivery system based on nanostructured lipid carriers (NLCs). For the first time, the samples were analyzed by using square wave voltammetry, a sensitive EC technique. The piroxicam EC responses allow us to propose a model that explains the experimental results and to subsequently determine the amount of drug loaded into the NLCs formulation as a function of time. In vitro drug release studies showed prolonged drug release (up to 5 days), releasing 60 % of the incorporated drug. The proposed method is a promising and stable alternative for the study of different drug delivery systems.

Droplet-droplet interactions investigated using a combination of electrochemical and dynamic light scattering techniques. The case of water/BHDC/benzene:n-heptane system.

In this contribution the electrochemistry of [Fe(CN)6](4-/3-) as the probe molecule was investigated in benzyl-n-hexadecyldimethylammonium chloride (BHDC) reverse micelles (RMs) varying the composition of the external solvent (benzene:n-heptane mixtures) and the surfactant concentration, at a fixed water content and probe concentration. The electrochemical and dynamic light scattering results show that in water/BHDC/benzene:n-heptane systems the aggregate sizes increase on increasing BHDC concentration. This behavior was unexpected since it is known that for water/BHDC/benzene RM systems keeping the water content constant and the surfactant concentration below 0.2 M, the droplet sizes are independent of the concentration of the surfactant. We explain the results considering that on changing the external solvent to benzene:n-heptane mixtures, RMs tend to associate in clusters and equilibrium between free RMs and droplet clusters is established. A model is presented which, using electrochemical and dynamic light scattering data, allows calculating the aggregation number of the RMs, the number of RMs that form the droplet clusters and the standard electron transfer heterogeneous rate constant.

An alternative approach to quantify partition processes in confined environments: the electrochemical behavior of PRODAN in unilamellar vesicles.

Herein, we investigate the behavior of the electroactive molecular probe 6-propionyl-2-dimethyl amino naphthalene (PRODAN) in large unilamellar vesicles (LUV) formed with the phospholipid 1,2-di-oleoyl-sn-glycero-3-phosphatidylcholine (DOPC) by using cyclic voltammetry (CV). The CV studies in pure water confirm our previous spectroscopic results that PRODAN self-aggregates due to its low water solubility. Moreover, the electrochemical results also reveal that the PRODAN aggregated species are non-electroactive within the studied electrochemical potential region. In DOPC LUV media, the redox behavior of PRODAN shows how the LUV bilayer interacts with PRODAN aggregated species to form PRODAN monomer species. Moreover, the electrochemical response of PRODAN allows us to propose a model for explaining the electrochemical experimental results and--in conjunction with our measurements--for calculating the value of the partition constant (K(p)) of PRODAN between the water and LUV bilayer pseudophases. This value coincides with that obtained through an independent technique. Moreover, our electrochemical model allows us to calculate the diffusion coefficient (D) for the DOPC LUV, which coincides with the D value obtained through dynamic light scattering (DLS). Thus, our data clearly show that electrochemical measurements could be a powerful alternative approach to investigate the behavior of nonionic electroactive molecules embed in a confined environment such as the LUV bilayer. Moreover, we believe that this approach can be used to investigate the behavior of non-optical molecular drugs embedded in bilayer media.

Sensitized photooxidation of thyroidal hormones. Evidence for heavy atom effect on singlet molecular oxygen [O2(1Deltag)]-mediated photoreactions.

Thyronine derivatives are essential indicators of thyroid gland diseases in clinical diagnosis and are currently used as standards for developing ordinary biochemical assays. Photooxidation of gland hormones of the thyronine (TN) family and structurally related compounds (TN, 3,5-diiodothyronine,3,3',5-triiodothyronine and 3,3',5,5'-tetraiodothyronine or thyroxine) was studied using rose bengal, eosin and perinaphthenone (PN) as dye sensitizers. Tyrosine (Tyr) and two iodinated derivatives (3-iodotyrosine and 3,5-diiodotyrosine) were also included in the study for comparative purposes. Irradiation of aqueous solutions of substrates containing xanthene dyes with visible light triggers a complex series of competitive interactions, which include the triplet excited state of the dye (3Xdye*) and singlet molecular oxygen [O2(1Deltag)]-mediated and superoxide ion-mediated reactions. Rate constants for interaction with the 3Xdye*, attributed to an electron transfer process, are in the order of 10(8)-10(9) M-1 s-1 depending on the dye and the particular substrate. The photosensitization using PN follows a pure Type-II (O2(1Deltag) mediated) mechanism. The presence of the phenolic group in Tyr, TN and iodinated derivatives dominates the kinetics of photooxidation of these compounds. The reactive rate constants, k(r), and the quotient between reactive and overall rate constants (k(r)/k(t) values, in the range of 0.7-0.06) behave in an opposite fashion compared with the overall rate constants and oxidation potentials. This apparent inconsistency was interpreted on the basis of an internal heavy atom effect, favoring the intersystem-crossing deactivation route within the encounter complex with the concomitant reduction of effective photooxidation.