Distributional homogeneity and penetration depth assessment of antibiotic added by surface coating to pellets with mid Infrared imaging and multivariate curve resolution.

Fourier Transform Mid Infrared with Attenuated Total Reflection Imaging (FTIR-ATR imaging) and Multivariate Curve Resolution with Alternating Least Squares (MCR-ALS) were used in a multiblock fashion to study the presence, distribution and penetration depth of very low concentrations of florfenicol (FF) in a complex matrix like feed pellets for salmonids. Images from the surface, at 150 µm deep and 200 µm deep from the surface were analyzed to certify the penetration power of FF added by surface coating methodology. Besides, the unique homogeneity index was calculated in order to evaluate the distributional homogeneity of each component. The results demonstrated the reliability of MCR-ALS in studying the distributional homogeneity of FF. It was demonstrated that FF remains mostly on the surface of the pellets with almost no penetration. The rest of the components of the pellets (oil, protein and carbohydrates) were also analyzed. These three nutrients are distributed on the three layers analyzed with a relatively homogeneous location, being carbohydrates (%H = 51 ± 3) the component with the best homogeneous distribution, unlike protein (%H = 45 ± 5), and oil (%H = 40 ± 7). This is the first publication where the penetration of an antibiotic, added with surface-coating to feed pellets, was analyzed with FTIR-ATR imaging and multivariate analysis, showing the contribution these analytical tools can make to the medicated feed industry.

Evaluation of the microscopic distribution of florfenicol in feed pellets for salmon by Fourier Transform infrared imaging and multivariate analysis.

Fourier Transform infrared imaging and multivariate analysis were used to identify, at the microscopic level, the presence of florfenicol (FF), a heavily-used antibiotic in the salmon industry, supplied to fishes in feed pellets for the treatment of salmonid rickettsial septicemia (SRS). The FF distribution was evaluated using Principal Component Analysis (PCA) and Augmented Multivariate Curve Resolution with Alternating Least Squares (augmented MCR-ALS) on the spectra obtained from images with pixel sizes of 6.25 µm × 6.25 µm and 1.56 µm × 1.56 µm, in different zones of feed pellets. Since the concentration of the drug was 3.44 mg FF/g pellet, this is the first report showing the powerful ability of the used of spectroscopic techniques and multivariate analysis, especially the augmented MCR-ALS, to describe the FF distribution in both the surface and inner parts of feed pellets at low concentration, in a complex matrix and at the microscopic level. The results allow monitoring the incorporation of the drug into the feed pellets.

Factorial design optimization and characterization of poly-lactic acid (PLA) nanoparticle formation for the delivery of grape extracts.

Proanthocyanidins (PAs) extracted from grapes have several bioactive properties, giving them potential medical uses. However, PAs are unstable in the digestive tract and must be stabilized to allow oral administration, which can be accomplished by nanoencapsulation. In this study, PAs extracted from grape seed and skin were stabilized with poly-d,l-lactide (PLA) polymer by the emulsion-evaporation method. An experimental Box-Behnken design was implemented, evaluating the influence of three factors: sonication time (30-360s) for the emulsion formation, loading of grape extracts (5-20%) and concentration of stabilizing agent (polyvinyl alcohol, PVA: 1-3%). The process was optimized to achieve higher encapsulation efficiency (EE=82.7%) and a smaller size (256 nm). The nanoparticles (NPs) were physically analyzed by TEM, FT-IR, TGA and DTG to characterize the nanoencapsulation process. In vitro release studies, through stomach and intestinal simulation, showed a sustained release of PAs from PLA-NPs.

Capreomycin oleate microparticles for intramuscular administration: Preparation, in vitro release and preliminary in vivo evaluation.

Capreomycin sulfate (CS) is a second-line drug used for the treatment of multidrug-resistant tuberculosis (MDR-TB). The adverse effects profile and uncomfortable administration scheme of CS has led to the development of formulations based on liposomes and polymeric microparticles. However, as CS is a water-soluble peptide that does not encapsulate properly into hydrophobic particulate matrices, it was necessary to reduce its aqueous solubility by forming the pharmacologically active capreomycin oleate (CO) ion pair. The aim of this research was to develop a new formulation of CO for intramuscular injection, based on biodegradable microparticles that encapsulate CO in order to provide a controlled release of the drug with reduced local and systemic adverse effects. The CO-loaded microparticles prepared by spray drying or solvent emulsion-evaporation were characterized in their morphology, encapsulation efficiency, in vitro/in vivo kinetics and tissue tolerance. Through scanning electron microscopy it was confirmed that the microparticles were monodisperse and spherical, with an optimal size for intramuscular administration. The interaction between CO and the components of the microparticle matrix was confirmed on both formulations by X-ray powder diffraction and differential scanning calorimetry analyses. The encapsulation efficiencies for the spray-dried and emulsion-evaporation microparticles were 92% and 56%, respectively. The in vitro kinetics performed on both formulations demonstrated a controlled and continuous release of CO from the microparticles, which was successfully reproduced on an in vivo rodent model. The results of the histological analysis demonstrated that none of the formulations produced significant tissue damage on the site of injection. Therefore, the results suggest that injectable CO microparticles obtained by spray drying and solvent emulsion-evaporation could represent an interesting therapeutic alternative for the treatment of MDR-TB.

Development of biodegradable methylprednisolone microparticles for treatment of articular pathology using a spray-drying technique.

In this work, microparticles were prepared by spray-drying using albumin, chondroitin sulfate, and hyaluronic acid as excipients to create a controlled-release methylprednisolone system for use in inflammatory disorders such as arthritis. Scanning electron microscopy demonstrated that these microparticles were almost spherical, with development of surface wrinkling as the methylprednisolone load in the formulation was increased. The methylprednisolone load also had a direct influence on the mean diameter and zeta potential of the microparticles. Interactions between formulation excipients and the active drug were evaluated by x-ray diffraction, differential scanning calorimetry, and thermal gravimetric analysis, showing limited amounts of methylprednisolone in a crystalline state in the loaded microparticles. The encapsulation efficiency of methylprednisolone was approximately 89% in all formulations. The rate of methylprednisolone release from the microparticles depended on the initial drug load in the formulation. In vitro cytotoxic evaluation using THP-1 cells showed that none of the formulations prepared triggered an inflammatory response on release of interleukin-1ß, nor did they affect cellular viability, except for the 9.1% methylprednisolone formulation, which was the maximum test concentration used. The microparticles developed in this study have characteristics amenable to a therapeutic role in inflammatory pathology, such as arthritis.

Study on antibacterial alginate-stabilized copper nanoparticles by FT-IR and 2D-IR correlation spectroscopy.

BACKGROUND: The objective of this study was to clarify the intermolecular interaction between antibacterial copper nanoparticles (Cu NPs) and sodium alginate (NaAlg) by Fourier transform infrared spectroscopy (FT-IR) and to process the spectra applying two-dimensional infrared (2D-IR) correlation analysis. To our knowledge, the addition of NaAlg as a stabilizer of copper nanoparticles has not been previously reported. It is expected that the obtained results will provide valuable additional information on: (1) the influence of reducing agent ratio on the formation of copper nanoparticles in order to design functional nanomaterials with increased antibacterial activity, and (2) structural changes related to the incorporation of Cu NPs into the polymer matrix. METHODS: Cu NPs were prepared by microwave heating using ascorbic acid as reducing agent and NaAlg as stabilizing agent. The characterization of synthesized Cu NPs by ultraviolet visible spectroscopy, transmission electron microscopy (TEM), electron diffraction analysis, X-ray diffraction (XRD), and semiquantitative analysis of the weight percentage composition indicated that the average particle sizes of Cu NPs are about 3-10 nm, they are spherical in shape, and consist of zerovalent Cu and Cu2O. Also, crystallite size and relative particle size of stabilized Cu NPs were calculated by XRD using Scherrer's formula and FT from the X-ray diffraction data. Thermogravimetric analysis, differential thermal analysis, differential scanning calorimetry (DSC), FT-IR, second-derivative spectra, and 2D-IR correlation analysis were applied to studying the stabilization mechanism of Cu NPs by NaAlg molecules. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of stabilized Cu NPs against five bacterial strains (Staphylococccus aureus ATCC 6538P, Escherichia coli ATCC 25922 and O157: H7, and Salmonella enterica serovar Typhimurium ATCC 13311 and 14028) were evaluated with macrodilution, agar dilution plate count, and well-diffusion methods. RESULTS: On the basis of the semiquantitative analysis, there was a direct correlation between the reducing agent ratio and the percentage of zerovalent Cu. This was confirmed with the statistical analysis of population of Cu NPs from TEM micrographs. At lower reducing agent ratios, two phases coexist (Cu2O and zerovalent Cu) due to incomplete reduction of copper ions by the reducing agent; however, at higher reducing agent ratios, the Cu NPs consist mainly of zerovalent Cu. Crystallite size and relative particle size of stabilized Cu NPs showed considerable differences in results and tendencies in respect to TEM analysis. However, the relative particle size values obtained from FT of XRD data agreed well with the histograms from the TEM observations. From FT results, the relative particle size and reducing agent ratio of stabilized Cu NPs showed an inverse correlation. The incomplete reduction of copper ions at lower reducing agent ratios was also confirmed by DSC studies. FT-IR and 2D-IR correlation spectra analysis suggested the first event involved in the stabilization of Cu NPs is their electrostatic interaction with -C=O of carboxylate groups of NaAlg, followed by the interaction with the available O-C-O⁻, and finally with the -OH groups. Bacterial susceptibility to stabilized nanoparticles was found to vary depending on the bacterial strains. The lowest MIC and MBC of stabilized Cu NPs ranged between 2 mg/L and 8 mg/L for all studied strains. Disk-diffusion studies with both E. coli strains revealed greater effectiveness of the stabilized Cu NPs compared to the positive controls (cloxacillin, amoxicillin, and nitrofurantoin). S. aureus showed the highest sensitivity to stabilized Cu NPs compared to the other studied strains. CONCLUSION: Cu NPs were successfully synthesized via chemical reduction assisted with microwave heating. Average particle size, polydispersity, and phase composition of Cu NPs depended mainly on the reducing agent ratio. Likewise, thermal stability and antibacterial activity of stabilized Cu NPs were affected by their phase composition. Because of the carboxylate groups in polymer chains, the structural changes of stabilized Cu NPs are different from those of NaAlg. NaAlg acted as a size controller and stabilizing agent of Cu NPs, due to their ability to bind strongly to the metal surface. Our study on the stabilizing agent-dependent structural changes of stabilized NPs is helpful for wide application of NaAlg as an important biopolymer.

Nitric Oxide Synthase Activity and Angiogenesis Measured by Expression of CD34 in Burns Treated With Chitosan Films .

UNLABELLED:  Nitric oxide (NO) signaling appears to play a vital role in wound healing associated to improve collagen and angiogenesis. A burn wound model was used to evaluate the effects of a chitosan films on histopathological features, nitric oxide synthase (NOS) activity, and quantification of neoformed capillaries assessed with CD34. METHODS: Bilateral burns (n = 16) were made on adult Sprague-Dawley rats. The lesions on the right side of the rats were treated with chitosan films, and the lesions on the left side of the same rats were treated with gauze with NaCl 0.9% as a control. RESULTS: Histological analysis revealed accelerated burn wound healing supported by significant differences in acute inflammation, collagen, and granulation tissue formation in chitosan-treated burns. Additionally, chitosan-treated burns were associated with higher CD34 immunoreactivity antibody supported by significant differences. This analysis of NOS activity was statistically significant on treated burns in the second treatment week. NOS results are associated with the highest collagen deposition, granulation tissue formation, and new capillary formation. CONCLUSION: The use of chitosan on burns promoted re-epithelialization by means of angiogenic and NO release associated with higher cell infiltration into the wound bed during the proliferative phase.

Chitosan composite films. Biomedical applications.

Chitosan acetate films have been prepared using chitosans from shrimps (Pleuroncodes monodon) of low and high molecular weight (LMv = 68,000 g/mol and HMv = 232,000 g/mol) and deacetylation degree of 80 and 100%, respectively. The chitosan films were obtained by addition of several additives to acetic acid chitosan solutions, such as: glycerol, oleic acid and linoleic acid in different proportions. The pH of the solutions before casting ranged from 5.0 to 6.0. The composite film thickness are reported. The films have been analyzed by FTIR showing characteristic bands corresponding to the additives. The scanning electron microscopy (SEM) studies reveals the different morphology of the composite films. The films exhibit different physical properties depending upon the additives and/or mixture of them. The addition of glycerol to composite improves the elasticity of the films. The swelling in glucose and saline solutions for several films was evaluated, being higher in the glucose solution. The bactericide test against Staphylococcus aureus, Pseudomona aeruginosa and Acinetobacter baumanii in plates with either blood and or agar tripticase showed that the molecular weight influences on the bactericidal properties of the chitosan composite films and over its effect against gram positive and gram negative bacteria. Medical applications of the composite films were done in patients with burns, ulcers and injuries, the films containing glycerol showed good adhesion in comparison with those without it. The composite films tested were mainly three (1) chitosan acetate with glycerol, (2) chitosan acetate with oleic acid and (3) chitosan acetate with glycerol and oleic acid. Excellent results in the skin recovery were obtained after 7-10 days. Since the chitosan is biodegradable by the body enzymes it does not need to be removed and increases the gradual grows of the damage tissues.

Technique validation by liquid chromatography for the determination of acyclovir in plasma.

In this research project, a high-performance liquid chromatography (HPLC) method was developed for the determination of acyclovir (ACV) in plasma. The plasma samples, recharged with acyclovir and in presence of 5'-N-methylcarboxyamidoadenosine (MECA) as an internal standard, were purified using a solid-phase extraction technique with Waters Oasis HLB columns. The separation of the components from the extract was carried out in a LiChrospher 100 RP-18 column for further ultraviolet detection at a wavelength range of 250-260 nm. The mobile phase composition was 18% acetonitrile, sodium dodecylsulphate 5 mM and phosphate buffer at pH 2.6 with an analysis time of 13 min per sample. The average retention time for acyclovir was of 5.0 min and for the internal standard 11.2 min. The calibration curve was linear ranging between 0.05 and 1.80 microg/ml. The detection limit was 0.006 microg/ml with a quantification limit of 0.020 microg/ml. The ACV recuperation percentage for 250 microl of plasma was between 94.7 and 109.7% with a coefficient of variation not higher than 5.2%. This method was developed and validated for use in bioavailability and bioequivalence studies.

Preliminary pharmacokinetic study of different preparations of acyclovir with beta-cyclodextrin.

Acyclovir has absorption problems, because of its low solubility and/or its saturable absorption mechanism, that take place in the small intestine in a passive, variable, and incomplete manner. The oral bioavailability of acyclovir is thereby affected and reaches only 15-30%. The purpose of this study was to investigate the possibility of increasing the oral availability of acyclovir by forming inclusion complexes of acyclovir with beta-cyclodextrin. Acyclovir, its complex (1:1) with beta-cyclodextrin (acyclovir-beta-cyclodextrin complex), and a 50:50 mixture of acyclovir and the inclusion complex (acyclovir/complex mixture) as an aqueous suspension were administered intraintestinally to male Sprague-Dawley rats in doses equivalent to an acyclovir dose of 75 mg/kg. Sequential samples of plasma were taken by microdialysis. The samples were analyzed by high-performance liquid chromatography with ultraviolet detection. Plasma concentration versus time curves show that the complex and the mixture of acyclovir/complex have a higher bioavailability and a pharmacokinetic profile than that of the drug itself.