Wet-Spun Chitosan-Sodium Caseinate Fibers for Biomedicine: From Spinning Process to Physical Properties.

We designed and characterized chitosan-caseinate fibers processed through wet spinning for biomedical applications such as drug delivery from knitted medical devices. Sodium caseinate was either incorporated directly into the chitosan dope or allowed to diffuse into the chitosan hydrogel from a coagulation bath containing sodium caseinate and sodium hydroxide (NaOH). The latter route, where caseinate was incorporated in the neutralization bath, produced fibers with better mechanical properties for textile applications than those formed by the chitosan-caseinate mixed collodion route. The latter processing method consists of enriching a pre-formed chitosan hydrogel with caseinate, preserving the structure of the semicrystalline hydrogel without drastically affecting interactions involved in the chitosan self-assembly. Thus, dried fibers, after coagulation in a NaOH/sodium caseinate aqueous bath, exhibited preserved ultimate mechanical properties. The crystallinity ratio of chitosan was not significantly impacted by the presence of caseinate. However, when caseinate was incorporated into the chitosan dope, chitosan-caseinate fibers exhibited lower ultimate mechanical properties, possibly due to a lower entanglement density in the amorphous phase of the chitosan matrix. A standpoint is to optimize the chitosan-caseinate composition ratio and processing route to find a good compromise between the preservation of fiber mechanical properties and appropriate fiber composition for potential application in drug release.

Chitosan Spray-Dried Microparticles for Controlled Delivery of Venlafaxine Hydrochloride.

Venlafaxine controlled drug delivery systems using different matrixes have been tested to reduce undesirable side effects in the treatment of depression. The legal status of chitosan (Cs) in Pharmacy has dramatically improved after its acceptance as excipient in several Pharmacopeias and, therefore, there is great interest in pharmaceutical formulations based on this polymer. In this paper, chitosan microcapsules cross-linked with sodium tripolyphosphate (TPP) for oral delivery of venlafaxine were formulated using the spray drying technique. The effect of chitosan physico-chemical properties, TPP concentration and TPP/Cs ratio on drug release was evaluated. The microcapsules were characterized in terms of size, zeta potential and morphology. The physical state of the drug was determined by X-ray diffraction (XRD) and the drug release from the microcapsules was studied in simulated gastric and intestinal fluids. The release pattern fitted well to the Peppas-Koersmeyer model with n exponents indicating anomalous transport.

Ionically-crosslinked carboxymethyl chitosan scaffolds by additive manufacturing for antimicrobial wound dressing applications.

Chitosan chemical functionalization is a powerful tool to provide novel materials for additive manufacturing strategies. The main aim of this study was the employment of computer-aided wet spinning (CAWS) for the first time to design and fabricate carboxymethyl chitosan (CMCS) scaffolds. For this purpose, the synthesis of a chitosan derivative with a high degree of O-substitution (1.07) and water soluble in a large pH range allowed the fabrication of scaffolds with a 3D interconnected porous structure. In particular, the developed scaffolds were composed of CMCS fibers with a small diameter (< 60 µm) and a hollow structure due to a fast non solvent-induced coagulation. Zn2+ ionotropic crosslinking endowed the CMCS scaffolds with stability in aqueous solutions, pH-sensitive water uptake capability, and antimicrobial activity against Escherichia coli and Staphylococcus aureus. In addition, post-printing functionalization through collagen grafting resulted in a decreased stiffness (1.6 ± 0.3 kPa) and a higher elongation at break (101 ± 9 %) of CMCS scaffolds, as well as in their improved ability to support in vitro fibroblast viability and wound healing process. The obtained results encourage therefore further investigation of the developed scaffolds as antimicrobial wound dressing hydrogels for skin regeneration.

Extraction of PLGA-microencapsulated proteins using a two-immiscible liquid phases system containing surfactants.

PURPOSE: The extraction of proteins from PLGA/PLA microspheres by a two-immiscible liquid phases system with the addition of surfactants was investigated. METHODS: First, the extraction without surfactants and the interaction between proteins (IFN-α2b and EGF) and empty microspheres (PLGA or PLA) was studied. Next, proteins stability in presence of different surfactants was evaluated by: (1) bicinchoninic acid protein assay, (2) reversed phase-high performance liquid chromatography, and (3) enzyme-linked immunosorbent assay. Then, proteins were extracted with PBS/dichloromethane including selected surfactants and characterized by the above mentioned techniques, biological activity tests, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray ionization mass spectrometry. RESULTS: Without surfactants, protein recovery was only 27-43% for IFN-α2b and 58-73% for EGF. Protein content in solutions incubated with blank microspheres decreased to 66% for IFN-α2b and 86% for EGF. It was only possible to quantify the EGF and IFN-α2b in the same manner as in PBS alone when the surfactant added was Pluronic F-68 and SDS, respectively. Addition of these surfactants allowed the complete isolation of both biomolecules from the microspheres. The extraction procedure did not affect the encapsulated proteins. CONCLUSION: Proteins can be quantitatively extracted, without changes, from PLGA/PLA microspheres using PBS/dichloromethane system that include an appropriate surfactant.

Physicochemical Properties and Cell Viability of Shrimp Chitosan Films as Affected by Film Casting Solvents. I-Potential Use as Wound Dressing.

Chitosan solubility in aqueous organic acids has been widely investigated. However, most of the previous works have been done with plasticized chitosan films and using acetic acid as the film casting solvent. In addition, the properties of these films varied among studies, since they are influenced by different factors such as the chitin source used to produce chitosan, the processing variables involved in the conversion of chitin into chitosan, chitosan properties, types of acids used to dissolve chitosan, types and amounts of plasticizers and the film preparation method. Therefore, this work aimed to prepare chitosan films by the solvent casting method, using chitosan derived from Litopenaeus vannamei shrimp shell waste, and five different organic acids (acetic, lactic, maleic, tartaric, and citric acids) without plasticizer, in order to evaluate the effect of organic acid type and chitosan source on physicochemical properties, degradation and cytotoxicity of these chitosan films. The goal was to select the best suited casting solvent to develop wound dressing from shrimp chitosan films. Shrimp chitosan films were analyzed in terms of their qualitative assessment, thickness, water vapor permeability (WVP), water vapor transmission rate (WVTR), wettability, tensile properties, degradation in phosphate buffered saline (PBS) and cytotoxicity towards human fibroblasts using the resazurin reduction method. Regardless of the acid type employed in film preparation, all films were transparent and slightly yellowish, presented homogeneous surfaces, and the thickness was compatible with the epidermis thickness. However, only the ones prepared with maleic acid presented adequate characteristics of WVP, WVTR, wettability, degradability, cytotoxicity and good tensile properties for future application as a wound dressing material. The findings of this study contributed not only to select the best suited casting solvent to develop chitosan films for wound dressing but also to normalize a solubilization protocol for chitosan, derived from Litopenaeus vannamei shrimp shell waste, which can be used in the pharmaceutical industry.

Thermoresponsive behavior of chitosan-g-N-isopropylacrylamide copolymer solutions.

Chitosan-g-N-isopropylacrylamide (NIPAm) water-soluble copolymers were synthesized and characterized by FTIR and (1)H NMR spectroscopies combined with conductometric and potentiometric titrations. Their thermoresponsive, fully reversible, behavior in aqueous solutions was characterized by means of microcalorimetry and rheology. During heating of copolymer solutions there is a well-known endothermic effect, which coincides with a marked increase in G' and a moderate decrement in G'' due to the formation of a hydrophobic network at the expense of the net amount of sol fraction. It was also found that a straight dependence between the values of G' above the LCST and the enthalpies associated with the transition reflecting that the connectivity in the gel network is governed by the net number of formed enthalpic-hydrophobic driven-junctions. Both the LCST and the enthalpy change vary with the ionic strength of copolymer solutions, but no dependence was found with the neutralization of the polyelectrolyte chain.

Chitosan Based Self-Assembled Nanoparticles in Drug Delivery.

Chitosan is a cationic polysaccharide that is usually obtained by alkaline deacetylation of chitin poly(N-acetylglucosamine). It is biocompatible, biodegradable, mucoadhesive, and non-toxic. These excellent biological properties make chitosan a good candidate for a platform in developing drug delivery systems having improved biodistribution, increased specificity and sensitivity, and reduced pharmacological toxicity. In particular, chitosan nanoparticles are found to be appropriate for non-invasive routes of drug administration: oral, nasal, pulmonary and ocular routes. These applications are facilitated by the absorption-enhancing effect of chitosan. Many procedures for obtaining chitosan nanoparticles have been proposed. Particularly, the introduction of hydrophobic moieties into chitosan molecules by grafting to generate a hydrophobic-hydrophilic balance promoting self-assembly is a current and appealing approach. The grafting agent can be a hydrophobic moiety forming micelles that can entrap lipophilic drugs or it can be the drug itself. Another suitable way to generate self-assembled chitosan nanoparticles is through the formation of polyelectrolyte complexes with polyanions. This paper reviews the main approaches for preparing chitosan nanoparticles by self-assembly through both procedures, and illustrates the state of the art of their application in drug delivery.

Biocompatibility of composites based on chitosan, apatite, and graphene oxide for tissue applications.

Novel two-dimensional films and three-dimensional (3D) scaffolds based on chitosan (CHI), apatite (Ap), and graphene oxide (GO) were developed by an in situ synthesis in which self-assembly process was conducted to direct partial reduction of GO by CHI in acidic medium. Physical-chemical characterization was carried out by optical microscopy, scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy, Raman spectroscopy, and X-ray photoelectron spectroscopy. In vitro biological studies using murine fibroblast (MC3T3) and human neuroblastoma (SH-SY5Y) cell lines were also performed. Cell growth and adherence on composites was also checked using SEM. Live and death staining by confocal microscope and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium of the samples were investigated. The results confirmed the incorporation of both Ap and GO sheets, into CHI polymeric matrix. Furthermore, it was confirmed a physical integration between inorganic Ap and organic CHI and strong chemical interaction between CHI and GO in the obtained composites. SH-SY5Y cell line showed preferential adherence on CHI/GO films surface while MC3T3 cell line displayed a good compatibility for all 3D scaffolds. This study confirms the biocompatibility of materials based on CHI, Ap, and GO for future tissues applications. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1585-1594, 2018.

Healthcare-associated infections: Appropriate empirical antibiotic treatment / Infecciones asociadas a la atención de la salud: tratamiento antibiótico empírico apropiado

Background: Health-care Associated Infections (HAI) are one of the main causes of death in critically ill patients. The aim of this paper is to establish an appropriate empirical antibiotic treatment for the main HAI in an Intensive Care Unit (ICU). Methods: A retrospective, observational, descriptive and analytical study of the culture results from January, 2014 to December, 2015. The causative microorganisms were identified, as well as sensitivity and antibiotic resistance. Results: Of the three main HAI in the ICU were Ventilator Associated Pneumonia (VAP), whose most common germs were methicillin-resistant Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa; Urinary Tract Infection Associated with Urinary Catheter (IVU-CU), Escherichia coli BLEE and Pseudomonas aeruginosa were isolated in 70%, and 56% of the bloodstream infections of the germs that caused this infection were three, the most frequent being Escherichia coli, followed by Klebsiella oxytoca and methicillin-resistant Staphylococcus aureus. Conclusions: VAP was the most frequent HAI and resistant methicillin Staphylococcus aureus was the most prevalent in this type of infection. The proposed empiric antibiotic treatment was as follows: VAP (vancomycin plus amikacin plus meropenem), IVU-CU (meropenem) and STIs (vancomycin plus cefepime).

Introducción: Las infecciones asociadas a la atención de la salud (IAAS) son una de las principales causas de muerte en pacientes en estado crítico. El objetivo de este trabajo fue identificar los gérmenes más frecuentemente asociados a las IAAS en la Unidad de Cuidados Intensivos (UCI) y determinar el tratamiento antibiótico empírico apropiado. Métodos: Estudio retrospectivo, observacional, descriptivo y analítico de los cultivos de enero de 2014 a diciembre de 2015. Se identificaron los microorganismos causantes de las IAAS, la sensibilidad y la resistencia antibiótica. Resultados: Las tres principales IAAS en la UCI fueron: la neumonía asociada a ventilador (NAV), y los gérmenes más habituales fueron Staphylococcus aureus meticilino resistente, Acinetobacter baumannii y Pseudomonas aeruginosa; la infección de vías urinarias asociada a catéter urinario (IVUCU) la Escherichia coli BLEE y Pseudomonas aeruginosa fueron aisladas en el 70% y en las infecciones del torrente sanguíneo (ITS) el 56% de los gérmenes fueron Escherichia coli, Klebsiella oxytoca y Staphylococcus aureus meticilino resistente. Conclusiones: La NAV fue la IAAS más frecuente y el Staphylococcus aureus meticilino resistente fue el más prevalente en este tipo de infección. La propuesta de tratamiento antibiótico empírico es: para NAV (vancomicina más amikacina más meropenem), IVU-CU (meropenem) y las ITS (vancomicina más cefepime).

Thermosensitive macroporous cryogels functionalized with bioactive chitosan/bemiparin nanoparticles.

Thermosensitive macroporous scaffolds of poly(N-isopropylacrylamide) (polyNIPA) loaded with chitosan/bemiparin nanoparticles are prepared by the free radical polymerization in cryogenic conditions. Chitosan/bemiparin nanoparticles of 102 ± 6.5 nm diameter are prepared by complex coacervation and loaded into polyNIPA cryogels. SEM image reveal the highly porous structure of cryogels and the integration of nanoparticles into the macroporous system. Volume phase transition temperature (VPT) and total freezing water content of cryogels are established by differential scanning calorimetry, and their porosity is determined by image-NMR. Swelling of cryogels (above and below the VPT) is highly dependent on nanoparticles concentration. In vitro release profile of bemiparin from cryogel is highly modulated by the presence of chitosan. Bemiparin released from nanoparticles preserves its biological activity, as shown by the BaF32 cell proliferation assay. Cryogels are not cytotoxic for the human fibroblast cells and present excellent properties for application on tissue engineering and controlled release of heparin.

Microencapsulation of alpha interferons in biodegradable microspheres.

The immunomodulatory, antiproliferative, and antiviral properties of interferons alpha (IFN-α) have made these cytokines attractive for numerous clinical applications. However, most of the current available IFN pharmaceutical dosage forms need to be injected frequently and may provoke adverse reactions in patients. This problem might be overcome by using biodegradable microspheres loaded with IFN. The encapsulation of IFN-α in microspheres and the current status of this technology are the main subjects reviewed here. To this end, we describe (i) the main methods and experimental parameters used to obtain IFN-loaded microspheres and (ii) characterization of these microspheres in terms of morphology, particle size, loading/encapsulation efficiency, residual water content, residual solvent content, release profile, and sterility. Also, we discuss both the characterization of the encapsulated IFN and the stabilization/protection of IFN during microencapsulation. Finally, a brief overview of preclinical and clinical studies using IFN-containing microspheres is given.

Síntesis y toxicidad de hidrogeles de poli(metacrilato de 2-hidroxietilo-co-acrilamida) obtenidos mediante irradiación con cuantos gamma / Sinthesys and toxicity of poly (2-hydroxyethyl methacrylate-co-acrylamide) hydrogels obtained by gamma photon irradiation

En el presente trabajo se reporta la obtención de hidrogeles de copolímeros de polimetacrilato de 2-hidroxietilo-co-acrilamida mediante la copolimerización y reticulación simultánea a partir de sus correspondientes monómeros y mediante irradiación con cuantos gamma. La composición del copolímero se determinó mediante análisis elemental y espectroscopia infrarroja. Adicionalmente se muestran los resultados de toxicidad de sus matrices, para ello se evaluó la acrilamida libre y se realizó el ensayo SOS en bacterias. Se comprobó que los extractos de lavados de las matrices de estos copolímeros contenían muy bajos niveles de acrilamida libre y no mostraron efecto tóxico en las células de E. coli para ninguno de los tiempos evaluados

In this paper is reported the obtaining of hydrogels from poly hydroxyethyl methacrylate-co-acryla-mide, by simultaneous crosslinking copolymerization from corresponding monomers by gamma photon irradiation. The composition of network chains in the copolymer was estimated from elemental analysis and infrared spectroscopy. Additionally, the toxicity of these materials was tested finding the free acrylamide and biological assay SOS in bacteria. The extracts of washed matrices of copolymers showed low level of free acrylamide. Moreover, they were non toxic on the E. coli cells tested at any time

Chitosan/apatite composite beads prepared by in situ generation of apatite or Si-apatite nanocrystals.

The objective of this work was to develop nanocrystalline apatite (Ap) dispersed in a chitosan (CHI) matrix as a material for applications in bone tissue engineering. CHI/Ap composites of different weight ratios (20/80, 50/50 and 80/20) and with CHI of different molecular weights were prepared by a biomimetic stepwise route. Firstly, CaHPO(4).2H(2)O (DCPD) crystals were precipitated from Ca(CH(3)COO)(2) and NaHPO(4) in the bulk CHI solution, followed by the formation of CHI/DCPD beads by coacervation. The beads were treated with Na(3)PO(4)/Na(5)P(3)O(10) solution (pH 12-13) to crosslink the CHI and to hydrolyse the DCPD to nanocrystalline Ap. This new experimental procedure ensured that complete conversion of DCPD into sodium-substituted apatite was achieved without appreciable increases in its crystallinity and particle size. In addition, composites with silicon-doped Ap were prepared by substituting Na(3)PO(4) by Na(2)SiO(3) in the crosslinking/hydrolysis step. Characterization of the resultant composites by scanning electron microscopy, X-ray powder diffraction (XRD), thermal analysis and Fourier transform infrared spectroscopy confirmed the formation, within the CHI matrix, of nanoparticles of sodium- and carbonate-substituted hydroxyapatite [Ca(10-x)Na(x)(PO(4))(6-x)(CO(3))(x)(OH)(2)] with diameters less than 20nm. Relatively good correspondence was shown between the experimentally determined inorganic content and that expected theoretically. Structural data obtained from its XRD patterns revealed a decrease in both crystal domain size and cell parameters of Ap formed in situ with increasing CHI content. It was found that the molecular weight of CHI and silicate doping both affected the nucleation and growth of apatite nanocrystallites. These effects are discussed in detail.

Cell supports of chitosan/hyaluronic acid and chondroitin sulphate systems. Morphology and biological behaviour.

Films and sponges of chitosan (CHI), chitosan/hyaluronic acid (CHI-HA) and chitosan/chondroitin sulphate (CHI-CHOS), were prepared by film deposition or lyophilization (sponges), avoiding the formation of interpolyelectrolyte complexes. The biological behaviour of the systems was analysed by studying the cell behaviour using a fibroblast cell line and standard biological MTT and Alamar Blue tests. The morphology of films, sponges and cell seeded samples was analysed by ESEM. The results obtained indicate that all the systems can be considered as good supports for cell adhesion and proliferation, but there is specific activation of the proliferative process in the presence of hyaluronic acid and chondroitin sulphate.