Transcriptional study of genes involved in the passage from teliospore to hyphae stage in the fungus Thecaphora frezii, the causal agent of peanut smut.

Peanuts (Arachis hypogaea L.) are among the most important leguminous crops in Argentina. During the growing season, they are frequently attacked by fungal diseases, including Thecaphora frezii. The spores of T. frezii are structures that confer resistance to this phytopathogen. The transition from teliospore to hypha is a characteristic process of some fungi, which is essential for completing their life cycle. Using the transcriptomes of teliospores and hyphae of T. frezii, we aimed to identify genes that were differentially expressed during this transition, and we found 134 up-regulated and 66 down-regulated genes, which would participate in different cellular processes such as: (a) cell cycle and DNA processing; (b) cell fate; (c) rescue, defense and cellular virulence; (d) detoxification by CYP450; (e) energy; (f) nutrient interaction and nutritional adaptation; (g) metabolism; (g) proteins with binding functions or cofactor requirements; (h) stress, cell differentiation and biogenesis of cell components; and (i) transport, cell communication and transcription. The identification of genes in T. frezii and their expression levels during different stages of differentiation could contribute to our understanding of the biological mechanisms in this fungus.

Eudragit Films as Carriers of Lipoic Acid for Transcorneal Permeability.

Diabetes mellitus (DM) is a highly prevalent disease affecting almost 10% of the world population; it is characterized by acute and chronic conditions. Diabetic patients have twenty-five times higher risk of going blind and developing cataracts early than the general population. Alpha-lipoic acid (LA) is a highly valuable natural antioxidant for the prevention and treatment of ophthalmic complications, such as diabetic keratopathy and retinopathy. However, its applicability is limited due to its low solubility in water; therefore, suitable systems are required for its formulation. In this work we developed an erodible insert based on Eudragit E100 (E PO) and Lipoic Acid (LA) for the delivery of this compound for the preventive treatment of ocular diseases especially in diabetic patients. Film evaluation was carried out by mechanical and thermal properties, mucoadhesivity, drug release, dynamic light scattering and corneal permeability as the concentration of LA increased. It was shown that upon LA release, it forms nanoparticles in combination with E PO that favor corneal permeation and LA retention in the cornea. These E PO-LA films also resulted non-irritable hence they are promising for their application in the treatment of ocular diseases.

Changes of lipids composition in different ontogenetic stages of Thecaphora frezii: expression of key enzymes for lipid biosynthetic pathways.

AIMS: It is known that Thecaphora frezii produces peanut smut that generates numerous economic losses. For this reason, it is a priority to search for control strategies. In this sense, we investigated the lipid profile of this pathogen, as possible antifungal targets, regarding polar lipid composition, fatty acid profile, and transcriptional regulation of genes involved in each stage of the development. METHOD AND RESULTS: Lipids from T. frezii teliospores, basidiospores, and hyphae were analyzed by HPLC/CAD and CG/FID. We found differences in the unsaturation levels as well as in the long-chain fatty acids along the stages. Phosphatidylcholine was the main component in the three development stages, followed by cardiolipins. Phosphatidylinositol, phosphatidylethanolamine, and lyso-phosphatidylethanolamine were found in similar amounts in all stages. Although ergosterol was not detected, we found two unsaponifiable lipids. In addition, we found transcripts that encode 28 enzymes involved in the biosynthesis of three lipids by RNA-Seq. CONCLUSIONS: Thecaphora frezii shows changes in the composition of membrane lipids in different ontogenetic stages as well as in the expression of transcripts for enzymes involved in lipid biosynthesis.

Expression and localization of tubulin isotypes and its mRNAs during Thecaphora frezii developments.

Thecaphora frezii is a phytopathogenic fungus that infects Arachys hypogaea L. and produces peanut smut. It has three ontological stages teliospores, basidiospores, and hyphae. Microtubules are cellular structures that participate in various important cellular processes. In this work, we analyzed the presence and location of α-tubulin isotypes and enzymes that participate in tyrosination-detyrosination in the three stages of T. frezii. Although both tyrosinated and detyrosinated tubulin seem to be associated with a membrane fraction component that gives it a similar behavior to integral proteins, in the soluble cytosolic fraction, only detyrosinated tubulin was detected, not tyrosinated tubulin. The presence of α-tubulin was not detected using the monoclonal antibody DM1A as neither acetylated tubulin. The RNA-Seq analysis showed the presence of α, ß, and γ-tubulins and the genes that codes for tyrosine-tubulin ligase and cytosolic carboxypeptidase 1, enzymes that are involved in post-translational modification processes. These sequences showed a high percentage of identity and homology with Ustilago maydis, Thecaphora thlaspeos, and Anthracocystis flocculosa. This is the first report for tubulins subpopulations and the cellular distribution in T. frezii, which together with the data obtained by RNA-Seq contribute to the knowledge of the pathogen, which will allow the development of control strategies.

Nebulization with alkaline hipertonic ibuprofen induces a rapid increase in platelets circulating in COVID-19 patients but not in healthy subjects.

We analyze changes in circulating platelets in COVID-19 positive patients who received conventional treatment Dexamethasone and Enoxaparin (Dexa-Enoxa) compared to patients treated with conventional therapy plus nebulization with alkaline hypertonic ibuprofenate (AHI). Results show that after 24 h of nebulization with AHI, circulating platelets shows an increase about 40% at 24 h and reach 65% at 96 h. In patients with platelets content below 200,000 by microliter the increase was 49% and 79% at 24 and 96 h respectively. In patients with platelets above 200,000 by microliter the increase was 24% and 31% at 24 and 96 h, respectively. The increase of platelets via AHI was similar in both, men and women.To evaluate whether this action of AHI was related to platelets from COVID-19 positive patients or also for healthy people, two controls were included: one of them with 10 healthy volunteers and another one with COVID-19 positive patients hospitalized and treated only with Dexa-Enoxa. Results show that, in healthy volunteers, the number of circulating platelets remains unchanged even after 7 days of treatment with AHI. In COVID-19 positive patients treated only with Dexa-Enoxa for 4 days, platelets increased only 16%.

Biocompatible Hydrogel for Intra-Articular Implantation Comprising Cationic and Anionic Polymers of Natural Origin: In Vivo Evaluation in a Rabbit Model.

We describe the functional capability of a cross-linked hydrogel composed of sulfated glycosaminoglycans and a cationic cellulose by conducting trials on experimental animal models using intra-articular implants to treat an articular disease called osteoarthritis. Forty-eight mature New Zealand white rabbits were divided into three experimental groups: A, B, and C. Group A and B underwent unilateral anterior cruciate ligament transection (ACLT) of the right knee. Subsequently, both knees of group A were treated with the injectable formulation under study. Meanwhile, group B was treated with sterile PBS (placebo). The animals of group C were surgically operated in both knees: Commercial hyaluronic acid (HA) was implanted in the left knee, and the formulation under study was implanted in the right knee. After implantation, all specimens underwent several evaluations at 3, 6, and 12 months postoperatively. At 6 months, no significant differences were detected between the right and left knees of the different groups. However, significant differences were observed between both knees at 12 months in group C, with less cartilage damage in the right knees implanted with our hydrogel. Therefore, in vivo studies have demonstrated hydrogel safety, superior permanence, and less cartilage damage for long-term follow up 12 months after implantation for the formulation under study compared with commercial HA.

Reversal of SARS-CoV2-Induced Hypoxia by Nebulized Sodium Ibuprofenate in a Compassionate Use Program.

INTRODUCTION: Sodium ibuprofenate in hypertonic saline (NaIHS) administered directly to the lungs by nebulization and inhalation has antibacterial and anti-inflammatory effects, with the potential to deliver these benefits to hypoxic patients. We describe a compassionate use program that offered this therapy to hospitalized COVID-19 patients. METHODS: NaIHS (50 mg ibuprofen, tid) was provided in addition to standard of care (SOC) to hospitalized COVID-19 patients until oxygen saturation levels of > 94% were achieved on ambient air. Patients wore a containment hood to diminish aerosolization. Outcome data from participating patients treated at multiple hospitals in Argentina between April 4 and October 31, 2020, are summarized. Results were compared with a retrospective contemporaneous control (CC) group of hospitalized COVID-19 patients with SOC alone during the same time frame from a subset of participating hospitals from Córdoba and Buenos Aires. RESULTS: The evolution of 383 patients treated with SOC + NaIHS [56 on mechanical ventilation (MV) at baseline] and 195 CC (21 on MV at baseline) are summarized. At baseline, NaIHS-treated patients had basal oxygen saturation of 90.7 ± 0.2% (74.3% were on supplemental oxygen at baseline) and a basal respiratory rate of 22.7 ± 0.3 breath/min. In the CC group, basal oxygen saturation was 92.6 ± 0.4% (52.1% were on oxygen supplementation at baseline) and respiratory rate was 19.3 ± 0.3 breath/min. Despite greater pulmonary compromise at baseline in the NaIHS-treated group, the length of treatment (LOT) was 9.1 ± 0.2 gs with an average length of stay (ALOS) of 11.5 ± 0.3 days, in comparison with an ALOS of 13.3 ± 0.9 days in the CC group. In patients on MV who received NaIHS, the ALOS was lower than in the CC group. In both NaIHS-treated groups, a rapid reversal of deterioration in oxygenation and NEWS2 scores was observed acutely after initiation of NaIHS therapy. No serious adverse events were considered related to ibuprofen therapy. Mortality was lower in both NaIHS groups compared with CC groups. CONCLUSIONS: Treatment of COVID-19 pneumonitis with inhalational nebulized NaIHS was associated with rapid improvement in hypoxia and vital signs, with no serious adverse events attributed to therapy. Nebulized NaIHS s worthy of further study in randomized, placebo-controlled trials (ClinicalTrials.gov: NCT04382768).

Self-assembled micelles of the (lipo) glycopeptides, teicoplanin, as taxane nanocarriers.

The use of nanoparticles is one of the strategies currently studied to minimize the toxicity and lack of tissue specificity of many cancer drugs used in chemotherapy. In this research the physicochemical and biological behavior of a novel self-assembled nanostructure of the antibiotic Teicoplanin (Teico) was characterized as a nanocarrier system for solubilizing highly hydrophobic drugs like Paclitaxel (Ptx) in aqueous media. The Teico micelles were loaded with Ptx in DMSO or PEG-400. The interaction between the loaded micelles and Albumin human serum albumin (HSA) was then studied by size exclusion chromatography. Transmission electron microscopy, dynamic light scattering and high-resolution liquid chromatography were also used to characterize the physicochemical and structural properties of the micelles to form the Teico/Ptx and Teico/Ptx/HSA micelles. Cellular uptake of Ptx was evaluated by fluorescent microscopy. Thein vitrocytotoxicity of the complexes was studied on Hep-2 tumor cells, by a Crystal Violet assay. Teico cosolvent-free micelles can solubilize up to 20 mg.ml-1of Ptx dissolved in PEG, increasing four times the solubility of Ptx in water compared to Abraxane, and 20 000 times the intrinsic solubility of Ptx in water. In addition, Teico/Ptx micelles binds spontaneously HSA through hydrophobic interaction. Teico and Teico/HSA micelles as a Ptx transporter does not affect its release or biological activity. Therefore, Teico/Ptx or Teico/Ptx/HSA complexes appear as new alternatives for transporting larger amounts of hydrophobic drugs that offer advantages, turning it an interesting option for further study.

Maternal intake of alpha-lipoic acid prevents development of symptoms associated with a fructose-rich diet in the male offspring in Wistar rats.

The hypothesis was that maternal intake of the antioxidant alpha-lipoid acid (ALA), during the developmental period of the hypothalamic orexigenic neurons, causes a permanent beneficial effect in offspring metabolism. Pregnant Wistar rats were fed with standard diet (food) + ALA (0.4% wt/wt) from day 14 of gestation to day 20 of lactation (n = 4) or food (n = 4). At 3 months of age, male offspring born from ALA-fed rats or controls (CT) were randomly assigned to be fed with food + 10% fructose solution in drinking water (F) or food + tap water (C), resulting in four groups: ALAF, ALAC, CTF, and CTC (n = 5/group). Food intake and body weight (BW) were measured twice a week for 31 days. Metabolites' levels in blood, mRNA expressions of Npy, Agrp (hypothalamus), Fasn, Srebf1, Ppard, and Pparg (liver), and the antioxidant capacity of the liver were determined. Results significance was set at p < 0.05. Average BW gain, daily BW gain, and intraabdominal fat tissue at necropsy were higher in CTF group followed by CTC, ALAF, and ALAC groups. There were no differences between groups in Kcal intake per day. mRNA expressions of hypothalamic and hepatic genes and plasmatic levels of glucose and triglycerides were higher in CTF group followed by ALAF, CTC, and ALAC groups. Fructose intake affected the oxidative capacity of the liver, but this effect was not observed in the ALAF group. In conclusion, maternal ALA intake protected the adult offspring to develop metabolic symptoms associated with high fructose in the drinking water.

Testosterone-loaded GM1 micelles targeted to the intracellular androgen receptor for the specific induction of genomic androgen signaling.

Androgens play a central role in homeostatic and pathological processes of the prostate gland. At the cellular level, testosterone activates both the genomic signaling pathway, through the intracellular androgen receptor (AR), and membrane-initiated androgen signaling (MIAS), by plasma membrane receptors. We have previously shown that the activation of MIAS induces uncontrolled proliferation and fails to stimulate the beneficial immunomodulatory effects of testosterone in prostatic cells, becoming necessary to investigate if genomic signaling mediates homeostatic effects of testosterone. However, the lack of specific modulators for genomic androgen signaling has delayed the understanding of this mechanism. In this article, we demonstrate that monosialoganglioside (GM1) micelles are capable of delivering testosterone into the cytoplasm to specifically activate genomic signaling. Stimulation with testosterone-loaded GM1 micelles led to the activation of androgen response element (ARE)-regulated genes in vitro as well as to the recovery of normal prostate size and histology after castration in mice. In addition, these micelles avoided MIAS, as demonstrated by the absence of rapid signaling pathway activation and the inability to induce uncontrolled cell proliferation. In conclusion, our results validate a novel tool for the specific activation of genomic androgen signaling and demonstrate the importance of selective pathway activation in androgen-mediated proliferation.

Ibuprofen, a traditional drug that may impact the course of COVID-19 new effective formulation in nebulizable solution.

The traditional formulation of ibuprofen is poorly soluble in water, so the administered dose must be 10 times higher than the dose required for a therapeutic effect. The development of a hydrosoluble form of ibuprofen can be a strategy to reach a high concentration in the lungs by using modern inhalation devices. Therefore, the development of an inhalable formulation with high bioavailability in the lungs was the leitmotiv of our investigation. The hypertonic ibuprofen solution to be nebulized (NIH) presents great relevant characteristics: bactericidal, virucidal, mucolytic and has a known anti-inflammatory property. Bactericidal and virucidal effects are related to the physico-chemical properties of Na-ibuprofenate as an amphipathic molecule. It has the capability to insert into the bilayer membranes destabilizing the structure, altering its biological properties and avoiding the duplication or infection. Our preliminary results indicate that the presence of this high ionic strength solution reduces 10 times the amount of ibuprofen necessary to kill bacteria, but also the time to kill 1x106 bacteria, from 4 h (in its absence) to only three minutes (in its presence). That was observed using Pseudomona aeruginosa, methicillin-resistant Staphylococcus aureus and Burkholderia cepacia. Also, "in vitro'' ibuprofen demonstrated virucidal activity against the so-called enveloped virus, a family that includes coronavirus strain (2019-nCoV). We observed too, the markedly reduced local inflammation in the airways after administering NIH lays on its ability to inhibit the enzyme cyclooxygenase and to markedly diminish reactive oxygen species (ROS). Other investigators also showed the importance of actin in the rapid spread of virus infection. Furthermore, reorganization of the actin filaments is a key step in lung inflammation induced by systemic inflammatory responses caused by SARS-CoV-2. These findings suggest that the interaction between actin proteins and S1 is involved in the 2019-nCoV infection and pathogenesis. Consequently, the possibility of interfering in this interaction could represent a valid hypothesis for the development of promising therapeutic and prevention strategies. In conclusion, we consider that treating people with COVID-19 with NIH may be beneficial and an opportunity to contribute for the current global health emergency.

Monosialoganglioside GM1 reduces toxicity of Ptx and increase anti-metastasic effect in a murine mammary cancer model.

Having demonstrated the ability of monosialoganglioside GM1 micelles as oncology drug transporter, this work focuses on evaluating its application in an in vivo system, studying the toxicity and antitumoral effect of GM1-Ptx micellar formulation. The maximum tolerated dose (MTD) obtained after intravenous administration of GM1-Ptx in mice was 55 mg/kg and the 50% lethal dose (LD50) was 70 mg/kg. This value is higher than those described for the commercial formulations TAXOL and ABRAXANE, with LD50 of 30 and 45 mg/kg respectively. The antitumor activity, mortality and incidence of metastasis were studied on a murine model of mammary gland cancer. The GM1-Ptx formulation was administered i.v. at different doses for 9 weeks using empty GM1 micelles and saline as treatment controls. Once the treatments were completed, biochemical markers were quantified and histological tissue tests were performed. The most promising results were obtained with the treatment at a dose of 15 mg/kg/twice a week, condition in which a longer survival and significant reduction in the incidence of animals with metastasis, since only one 25% of the mice showed presence of pulmonary micro metastases.

Loss of the tRNALysCUU encoding gene, Chr-11 tRNA-Lys-CUU, is not associated with Type 2 diabetes mellitus.

AIM: To investigate the presence/absence of the Chr-11 tRNA-Lys-CUU gene as a marker for genetic predisposition to Type 2 diabetes mellitus (T2DM). METHODS: We enrolled 122 patients diagnosed with T2DM and 77 non-diabetic individuals. We evaluated clinical and biochemical parameters (body mass index, hypertension, cholesterol levels, glycosylated hemoglobin, triglycerides, etc.), and performed a genotypic profiling of Chr-11 tRNA-Lys-CUU by polymerase chain reaction analyses. RESULTS: Approximately one third of the population lacked Chr-11 tRNA-Lys-CUU. We did not observe a statistically significant association between the presence/absence of Chr-11 tRNA-Lys-CUU and T2DM. CONCLUSION: The genotypic distribution of Chr-11 tRNA-Lys-CUU in our population was consistent to that reported by others. This gene failed as a marker for T2DM predisposition.

High Concentrations of Sodium Chloride Improve Microbicidal Activity of Ibuprofen against Common Cystic Fibrosis Pathogens.

Ibuprofen (IBU-H), a widely used anti-inflammatory, also shows a marked antimicrobial effect against several bacterial species, including those involved in cystic fibrosis such as Pseudomona aeruginosa, methicillin resistant Staphylococcus aureus and Burkholderia cepacia complex. Additionally, our results show significant synergy between water soluble Na-ibuprofen (IBU-Na) and ionic strength. Salt concentrations above 0.5 M modify the zeta potential promoting the action of Na-IBU; thus, with 1 M sodium chloride, IBU-Na is ten times more efficient than in the absence of ionic strength, and the minimum effective contact time is reduced from hours to minutes. In short time periods, where neither IBU-Na nor controls with 1 M NaCl show activity, the combination of both leads to a reduction in the bacterial load. We also analyzed whether the changes caused by salt on the bacterial membrane also promoted the activity of other microbicide compounds used in cystic fibrosis like gentamicin, tobramycin and phosphomycin. The results show that the presence of ionic strength only enhanced the bactericidal activity of the amphipathic molecule of IBU-Na. In this respect, the effect of saline concentration was also reflected in the surface properties of IBU-Na, where, in addition to the clear differences observed between 145 mM and 1 M, singular behaviors were also found, different in each condition. The combination of anti-inflammatory activity and this improved bactericidal effect of Na-IBU in hypertonic solution provides a new alternative for the treatment of respiratory infections of fibrotic patients based on known and widely used compounds.

Silver Nanoparticles with High Loading Capacity of Amphotericin B: Characterization, Bactericidal and Antifungal Effects.

The purpose of this study was to evaluate the most appropriate conditions to generate silver nanoparticles (AgNPs) loaded with a potent antimycotic drug like amphotericin B (AmB), characterize the physicochemical properties, and to evaluate the cytotoxic effect and biological activity of these new nanostructures as a potential nanocarrier for hydrophobic drugs. It was determined that the optimal molar ratio between Ag and AmB is 1/1 given the uniformity of size around 170 nm of the nanoparticles generated as well as their strongly negative ζ potential of -35 mV, a condition that favors repulsions between AgNPs and inhibiting their aggregation. In this condition, only 0.8 mg.mL-1 of Ag is needed to solubilize 5 mg.mL-1 of AmB, a concentration currently used in commercial formulations. It is important to emphasize that the loading capacity (w/w) of this nanostructure is much higher than that of micellar and liposomal formulations. These AgNP-AmB nanoparticles retain both the bactericidal effect of silver and the cytotoxic and antifungal effect of AmB. However, it was shown that these nanoparticles are spontaneously associated with plasma lipoproteins (LDL and HDL), inhibiting their cytotoxic effects on red blood cells and on at least two cell lines, Vero and H1299 and slightly reducing its bactericidal effect on P. aeruginosa. In contrast, the antifungal effect of the formulation is maintained and is even higher than that when the nanoparticle is not associated with lipoproteins, indicating that this association is of the reversible type. The characterization of these nanoparticles is discussed as a potential new model formulation able to improve the antifungal therapeutic efficiency of AmB.

Maternal Triclosan consumption alters the appetite regulatory network on Wistar rat offspring and predispose to metabolic syndrome in the adulthood.

The objectives of this study were to evaluate the effects of maternal oral exposure to the antibacterial Triclosan (TCS) during gestation and lactation on the metabolic status of the adult offspring and on the expression of main genes controlling the appetite regulatory network. Pregnant rats were fed ad-libitum with ground food + TCS (1 mg/kg) from day 14 of gestation to day 20 of lactation (n=3) or ground food (n=3). After litter reduction, 12 males and 12 females born from the TCS exposed rats (TCS, n=24) or not (Control, n=24) were used to evaluate monthly body weight, food intake, plasma levels of cholesterol, glucose and triglycerides, and the hypothalamic mRNA expression of agouti-related protein (Agrp), neuropeptide Y (Npy) and propiomelanocortin (Pomc). Body weight for rats in the TCS group was 12.5% heavier for males at 4 months (p<0.001) and 19% heavier for females at 8 months (p=0.01). Food intake was significantly higher for rats in the TCS group at 5 months of age (p<0.01). Cholesterol and glucose levels were significantly higher for rats in the TCS group at 8 months (p<0.05). mRNA expression of Npy and Agrp were significantly increased in hypothalami of rats in the TCS group at 2 months for males or 8 months for females (p<0.05). In conclusion, low doses of oral TCS consumption by the pregnant and lactating dam increase the hypothalamic expression of the orexigenic neuropeptides Npy and Agrp in the offspring and alter their metabolic status during adulthood, resembling development of the metabolic syndrome.

Sialoganglioside Micelles for Enhanced Paclitaxel Solubility: In Vitro Characterization.

Efficiency of mono-sialogangliosides to load Paclitaxel (Ptx) has recently been found to depend on the structure of the polysaccharide chain. In this study, we demonstrated that incorporation of only one more sialic acid into the ganglioside molecule, independently of its position, causes a 4-fold increase in Ptx-loading capacity, the maximum being at a 5:1 molar ratio (di-sialoganglioside/Paclitaxel, GD/Ptx). These complexes are stable in solution for at least 3 months, and over 90% of Ptx remains loaded in the micelles after extreme stress conditions such as high-speed centrifugation, lyophilization, or freeze-thaw cycles. Ganglioside micelles protect 50% of the initially loaded Ptx from alkaline hydrolysis after 24 h at pH 10. Dynamic light scattering studies revealed that GD micelles increase their size from 9 to 12 nm when loaded with Ptx. Transmission electron microscopy shows a homogeneous population of spherical micelles either with or without Ptx. In vitro biological activity was similar to that of the free drug. These results provide further options of self-assembled nanostructures of di- and tri-sialogangliosides with a higher loading capacity.

Thermodynamic and Kinetic Aspects Involved in the Development of Nanocarriers and Drug Delivery Systems Based on Cationic Biopolymers.

During the last years we have seen an increasing number of reports describing new properties and potential applications of cationic polymers and derived nanostructures. This review gives a summary of their applications in drug delivery, the preparation methods for nano and microstructures and will attempt to give a glimpse on how their structure, chemical composition and properties may be affected or modulated as to make them suitable for an intended application as drug delivery nanocarriers. The compositional complexity with the existence of several reacting groups makes cationic nanostructures critically sensitive to the contribution of thermodynamic and kinetic parameters in the determination of the type and stability of a particular structure and its ability to respond to changes in environmental conditions in the right time frame. Curiously, and contrarily to what could be expected, despite the fact that cationic polymers can form strong electrostatic interactions the contribution of the entropic component has been often found to be very important for their association with negatively charged supramolecular structures. Some general considerations indicate that when considering a complex multimolecular system like a nanocarrier containing an active ingredient it is frequently possible to find conditions under which enthalpic and entropic contributions are compensated leading to stable structures with a marginal thermodynamic stability (free energy change close to zero) which make them able to respond relatively fast to changes in the environmental conditions and therefore suitable for the design of smart drug delivery systems. Like with other nanocarriers, it should always be kept in mind that the properties of cationic nanocarriers will depend not only on their chemical composition but also on the properties of the structures formed by them.

Interfacial stabilization of the antitumoral drug Paclitaxel in monolayers of GM1 and GD1a gangliosides.

Molecular interactions between the anti-cancer agent Paclitaxel (Ptx), and two gangliosides with different sialic acid content, GM1 and GD1a, were investigated using the Langmuir film balance technique. Ptx showed interfacial activity reducing the air/water surface tension by 18 mN·m(-1). However, the drug was able to insert into preformed ganglioside monolayers at much higher surface pressures, indicating a preferential interaction of Ptx with GM1 and GD1a. Compression isotherms of binary mixtures of Ptx and GM1 or GD1a also indicated non-ideal mixed monolayers in which the drug became stabilized at the interface in the presence of gangliosides. Ptx reached much higher surface pressure values in the mixed monolayers than those sustained in pure Ptx, although partial desorption of the drug from the interface into the subphase was also observed at high Ptx contents. The mean molecular area of the mixtures showed condensation, mainly in the case of GD1a, whereas Ptx induced a decrease in the compressibility of monolayers when mixed with either GM1 or GD1a. Additionally, Brewster angle microscopy analysis indicated that higher amounts of Ptx are present at the mixed ganglioside/Ptx interface when compared to pure drug monolayers. Finally, GD1a micelles increased in size in the presence of Ptx, whereas GM1 micelles kept their diameter, according to dynamic light scattering measurements, which could be explained by the different properties of ganglioside monolayers. The results obtained on ganglioside-Ptx interactions allowed interpreting the different Ptx loading capacity of GM1 and GD1a, enabling them to act as potential drug carriers.

Biochemical characterization of the interactions between doxorubicin and lipidic GM1 micelles with or without paclitaxel loading.

Doxorubicin (Dox) is an anthracycline anticancer drug with high water solubility, whose use is limited primarily due to significant side effects. In this study it is shown that Dox interacts with monosialoglycosphingolipid (GM1) ganglioside micelles primarily through hydrophobic interactions independent of pH and ionic strength. In addition, Dox can be incorporated even into GM1 micelles already containing highly hydrophobic paclitaxel (Ptx). However, it was not possible to incorporate Ptx into Dox-containing GM1 micelles, suggesting that Dox could be occupying a more external position in the micelles. This result is in agreement with a higher hydrolysis of Dox than of Ptx when micelles were incubated at alkaline pH. The loading of Dox into GM1 micelles was observed over a broad range of temperature (4°C-55°C). Furthermore, Dox-loaded micelles were stable in aqueous solutions exhibiting no aggregation or precipitation for up to 2 months when kept at 4°C-25°C and even after freeze-thawing cycles. Upon exposure to blood components, Dox-containing micelles were observed to interact with human serum albumin. However, the amount of human serum albumin that ended up being associated to the micelles was inversely related to the amount of Dox, suggesting that both could share their binding sites. In vitro studies on Hep2 cells showed that the cellular uptake and cytotoxic activity of Dox and Ptx from the micellar complexes were similar to those of the free form of these drugs, even when the micelle was covered with albumin. These results support the idea of the existence of different nano-domains in a single micelle and the fact that this micellar model could be used as a platform for loading and delivering hydrophobic and hydrophilic active pharmaceutical ingredients.