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.

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.

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.

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.

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.

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.

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.

Biochemical characterization of GM1 micelles-Amphotericin B interaction.

In this work a thorough characterization of the GM1 micelle-Amphotericin B (AmB) interaction was performed. The micelle formation as well as the drug loading occurs spontaneously, although influenced by the physicochemical conditions, pH and temperature. The chromatographic profile of GM1-AmB complexes at different molar ratios shows the existence of two populations. The differential absorbance of GM1, monomeric and aggregate AmB, allowed us to discriminate the presence of all of them in both fractions. Thus, we noted that at higher proportion of AmB in the complex, increases the larger population which is composed mainly of aggregated AmB. The physical behavior of these micelles shows that both GM1- AmB complexes were stable in solution for at least 30 days. However upon freeze-thawing or lyophilization-solubilization cycles, only the smallest population, enriched in monomeric AmB, showed a complete solubilization. In vitro, GM1-AmB micelles were significantly less toxic on cultured cells than other commercial micellar formulations as Fungizone, but had a similar behavior to liposomal formulations as Ambisome. Regarding the antifungal activity of the new formulation, it was very similar to that of other formulations. The characterization of these GM1-AmB complexes is discussed as a potential new formulation able to improve the antifungal therapeutic efficiency of AmB.

Self-assembled micelles of monosialogangliosides as nanodelivery vehicles for taxanes.

We demonstrate herein that taxanes (paclitaxel (Ptx) and docetaxel (Dtx)) can be spontaneously loaded into ganglioside nanomicelles. The efficiency of gangliosides to solubilize taxanes was highly dependent on their self-aggregating structure. Thus, GM3 that forms unilamellar vesicles was less efficient to solubilize taxanes than gangliosides that form micelles (i.e. GM1 and GM2). Sialic acid cyclization of GM1 by acid treatment led to an important reduction in its capacity to solubilize taxanes, as also did the replacement of the fatty acid of ceramide by a dicholoracetyl group. Water solubility of paclitaxel (Ptx) is less than 1 µg mL⁻¹ and increased up to 6.3mg.mL⁻¹ upon its association with GM1 micelles. The incorporation of Ptx in GM1 reached an optimum at GM1/Ptx 20/1 molar ratio when performed at room temperature. An increase in the solubilization capacity of GM1 micelles was observed upon dehydration of their polar head group by pre-treatment at 55 °C. Loading of Ptx into the micelle induced a structural reorganization that led to an important protection of Ptx reducing its hydrolysis at alkaline pH. Diffusion of either GM1 or Ptx was restricted upon mixed-micelle formation indicating that they are kinetically more stable than pure ganglioside micelles. X-ray powder diffraction of lyophilized GM1 micelles with Ptx showed a change in their internal structure from a crystalline state to completely amorphous. Taxane-ganglioside mixed micelles were stable in solution for at least 4months and also upon freeze-thawing or lyophilization-solubilization cycles. Upon mixing with human blood constituents, GM1/Ptx micelles did not induce hemolysis or platelet aggregation and were spontaneously covered with human serum albumin (HSA), which could aid in the delivery of micellar content to tumors. In vitro antimitotic activity of GM1/Ptx mixed micelles was qualitatively equivalent to that of free drug in DMSO solution.

Eudragit E100 surface activity and lipid interactions.

Eudragit E100 (E100) is a cationic methacrylate polymer that interacts with viral and cell membranes. We studied the effect of pH, ionic strength and the presence of lipid monolayers on the surface activity of the polymer. E100 forms stable monolayers at the air-water interface, either by spreading or when added into the subphase. This behavior is highly influenced by the pH and saline concentration of the subphase. At pH 5 or higher, the adsorption of the polymer to the air-water interface begins immediately after its injection into the subphase, while at pH below 5 E100 remains in the subphase with a particularly slow adsorption to the interface. In addition, low ionic strength (10 mM) in the subphase results in a fast adsorption of the polymer to the interface, even at pH under 5. On the other hand, in the presence of non-ionic (cholesterol) or anionic (monosialoganglioside) lipid monolayers, E100 shows a fast adsorption to the interface, [comma] reaching surface pressures of 25 and 36 mN m(-1), respectively. However, E100 barely interacts with monolayers of a zwitterionic lipid (hydrogenated soy lecithin) with a cut-off pressure of 11 mN m(-1). The interaction of E100 with GM1 micelles in the subphase reduces its surface activity. Altogether these results show that E100 can effectively penetrate into model membranes and that its amphipathic character is largely dependent on the chemical composition of the aqueous environment and the lipid composition of the membrane.