Roxana V. Alasino

Superstimulation of ovarian follicular development in beef cattle with a single intramuscular injection of Folltropin-V.

The need to inject FSH twice daily for superstimulation of ovarian follicular development in cattle necessitates frequent attention by farm-personnel and increases the possibility of failures due to mishandling and errors in administration of treatments. A series of three experiments were designed to evaluate the feasibility of superstimulation in beef cattle with a single intramuscular (IM) injection of Folltropin-V diluted in a hyaluronan-based slow-release formulation (SRF). In Experiment 1, cows were assigned to one of three treatment groups to compare two methods of injection as compared to the twice daily IM injection protocol. Superovulatory response of cows (n=6) treated with twice daily IM injections over 4 days (Control) was greater than of cows treated with a single subcutaneous (SC) injection in SRF (n=6), while superovulatory response of cows treated with a single IM injection in SRF (n=6) was intermediate. Experiment 2 was designed to compare two concentrations of SRF (20mg/mL hyaluronan, 100% compared to 10mg/mL hyaluronan, 50%) in a single IM injection protocol. The mean number of corpora lutea (CL) were not significantly different (P≥0.05), but the numbers of total ova/embryos (P<0.05), fertilized ova (P<0.01) and transferable embryos (P<0.001) were greater in cows treated with FSH in 100% SRF (n=20) than cows treated with FSH in 50% SRF (n=20). Experiment 3 was designed to compare superovulatory response in Red Angus donor cows treated with a single IM injection of Folltropin-V diluted in 100% solution of SRF with those treated with the traditional twice-daily IM injection protocol over 4 days. Mean (±SEM) numbers of CL (13.7±1.2 compared to 13.8±1.2), total ova/embryos (12.3±1.5 compared to 13.7±2.1), fertilized ova (7.2±1.1 compared to 8.4±1.4) and transferable embryos (4.9±0.8 compared to 6.4±1.3) were not significantly different between Control (n=29) and Single injection (n=29) groups, respectively. In summary, superstimulation of beef donor cows with a single IM injection of Folltropin-V diluted in 100% solution of SRF resulted in a comparable superovulatory response to the traditional twice-daily IM administration of Folltropin-V diluted in saline over 4 days.

Vantris®, A biocompatible, synthetic, non-biodegradable, easy-to-inject bulking substance: Evaluation of local tissular reaction, localized migration and long-distance migration

Biodegradable injectable bulking agents of animal origin present a fast rate of bio-reabsorption and may cause an allergic reaction. Biodegradable elements of synthetic origin have a high rate of reabsorption after a year. Non-biodegradable agents of synthetic origin lead to the formation of a fibrotic capsule, giving stability and long-term permanence. VANTRIS is categorized into this last group; it belongs to the family of Acrylics, particles of polyacrylate polyalcohol copolymer immersed in a glycerol and physiological solution carrier. Molecular mass is very high. When injected in soft tissues, this material causes a bulkiness that remains stable through time. The carrier is a 40% glycerol solution with a pH of 6. Once injected, the carrier is eliminated by the reticular system through the kidneys, without metabolizing. Particles of this polyacrylate polyalcohol with glycerol are highly deformable by compression, and may be injected using a 23-gauge needle. The average of particles size is 320 mm. Once implanted, particles are covered by a fibrotic capsule of up to 70 microns. Particles of this new material are anionic with high superficial electronegativity, thus promoting a low cellular interaction and low fibrotic growth. The new polyacrylate polyalcohol copolymer with glycerol was tested for biocompatibility according to ISO 10993-1:2003 in vitro, showing that they are not mutagenic for the Salmonella T. strains analyzed. The extract turned out to be non-cytotoxic for cell lines in culture and non-genotoxic for mice. In in vivo studies, acrylate did not cause sensitization in mice. The macroscopic reaction of tissue irritation was not significant in subcutaneous implants and in urethras of rabbits. Seven female dogs were injected transurethrally with VANTRIS to evaluate short and long-term migration (13 weeks and 12 months respectively). No particles or signs of inflammation or necrosis are observed in any of the organs examined 13 weeks and 12 months after implantation. To conclude, this new material meets the conditions of ideal tissue bulking material.

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.

Selective Binding of Albumin to Gm1 Ganglioside Micelles Containing Paclitaxel

In our previous work we showed that taxanes (paclitaxel (Ptx) and docetaxel(Dtx)) can be spontaneously loaded into monosialoganglioside (GM1) nanomicelles, increasing their water solubility about 6,000 times, to render stable water soluble formulations that could be used as a novel strategy to deliver drugs in cancer. Here, we describe the hydrophobic interaction of Human Serum Albumin (HSA) with GM1 micelles loaded with Ptx, as a strategy that could improve tumour drug accumulation. This interaction is regulated by conditions such pH and temperature and generates ternary complexes GM1/Ptx/HSA with sizes around 19 to 24 nm and hydrodynamic radius equivalent to a globular protein of 140-180 kDa. These mixed micelles were stable in solution for at least 40 days and also upon freeze-thawing or lyophilization-solubilization cycles. The results of in vitro assays showed that the nano-structures developed are taken up by cell cultures with an antimitotic activity of Ptx on tumoral and nontumoral cell lines that was similar to that observed with the free drug in DMSO solution.

Characterization and bacterial adhesion of chitosan-perfluorinated acid films

We reported herein the study and characterization of films obtained by casting of chitosan solutions in perfluorinated acids, trifluoroacetic (TFA), perfluoropropionic (PFPA), and perfluorooctanoic (PFOA). The films were characterized by FTIR, solid state (13)C NMR, X-ray, AFM, contact angle, thermogravimetric effluent analysis by mass spectrometry, and rheology. The results showed a marked influence of chain length of the perfluorinated acids on the hydrophobic/hydrophilic ratio of the modified chitosan films which was evidenced by the different characteristics observed. The material that showed greater surface stability was chitosan-PFOA. Chitosan film with the addition of PFOA modifier became more hydrophobic, thus water vapor permeability diminished compared to chitosan films alone, this new material also depicted bacterial adhesion which, together with the features already described, proves its potential in applications for bioreactor coating.

Reversible exposure of hydrophobic residues on albumin as a novel strategy for formulation of nanodelivery vehicles for taxanes.

Background: We report herein a novel strategy for the preparation of protein-based nanode-livery vehicles for hydrophobic active pharmaceutical ingredients. Methods: The procedure consisted of three steps, ie, exposure of hydrophobic residues of a protein to a pH-induced partial unfolding: interaction between hydrophobic residues on the protein and the hydrophobic active pharmaceutical ingredient, and a final step where the structure of the protein was reversed to a native-like state by returning to neutral pH. As proof of concept, the interaction of paclitaxel with partially unfolded states of human serum albumin was evaluated as a potential method for the preparation of water-soluble complexes of the taxane with albumin. Results: We found that paclitaxel readily binds to pH-induced partially unfolded albumin, leading to the formation of optically clear water-soluble complexes. The complexes thus formed were more stable in solution when the albumin native state was at least partially restored by neutralization of the solution to a pH around 7. It was also observed that the hydrodynamic radius of human serum albumin was only slightly increased after the cycle of pH changes, remaining in a monomeric state with a size according to paclitaxel binding. Furthermore, paclitaxel binding did not affect the overall exposure of charged groups of human serum albumin, as evaluated by its interaction with an ionic exchange resin. Conclusion: The in vitro biological activity of the complexes formed was qualitatively equivalent to that of a Cremophor®-based formulation.

Characterization of physicochemical properties of perfluorodecanoic acid–polyquaternium cellulose hydrogel

We investigated the nature and stability of the interactions established between polyquaternium (PQ10) and perfluorodecanoic acid (PFDA) in terms of different variables such as composition, ionic strength, pH and temperature. The PQ10-PFDA complex formation is interpreted in view of electrostatic associations between carboxylic and quaternary amino group. The properties of the systems were characterized by rheology analysis. The adhesive properties of complex were also assessed. One of the macroscopic features of the new material formed in solution was the increase in viscosity from 6 Pas for 1% PQ10 (MW 1.7×10(6) g mol(-1)) to about 1000 Pas by the addition of enough PFDA to reach 1:0.5 ammonium:carboxylic group molar ratio. At this proportion, PQ10 and PFDA form a network structure with a maximum viscosity and storage modulus. This maximum coincides with an increased mucoadhesive work.

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.