María Rosa Aguilar

Macroporous Scaffolds Based on Chitosan and Bioactive Molecules

Chitosan-based macroporous scaffolds for tissue engineering applications are developed by cryogelation in aqueous media. The cryogels obtained are modified using a new RGD-containing peptide developed in this laboratory. A RGD-containing peptide is chemically attached to the surface of the cryogels to improve cell adhesion to the 3D-structure chitosan-based scaffolds. The synthesis, physico-chemical, and biological evaluations of the system are described, and the optimization of the formulations is carried out by varying the reaction parameters. Fibroblasts and endothelial cells are used in cell cultures to determine cell behavior and the cytocompatibility of the macroporous cryogels. Cell spreading and actin cytoskeleton organization process are assessed by confocal microscopy. Cells colonize the porous structure of the chitosan-based cryogel and are observed to be growing inside the pores.

Using environmental niche models to test the 'everything is everywhere' hypothesis for Badhamia

It is often discussed whether the biogeography of free-living protists is better explained by the ‘everything is everywhere’(EiE) hypothesis, which postulates that only ecology drives their distribution, or by the alternative hypothesis of ‘moderate endemicity’ in which geographic barriers can limit their dispersal. To formally test this, it would be necessary not only to find organisms restricted to a geographical area but also to check for their presence in any other place with a similar ecology. We propose the use of environmental niche models to generate and test null EiE distributions. Here we have analysed the distribution of 18S rDNA variants (ribotypes) of the myxomycete Badhamia melanospora (belonging to the protozoan phylum Amoebozoa) using 125 specimens from 91 localities. Two geographically structured groups of ribotypes congruent with slight morphological differences in the spores can be distinguished. One group comprises all populations from Argentina and Chile, and the other is formed by populations from North America together with human-introduced populations from other parts of the world. Environmental climatic niche models constructed separately for the two groups have significant differences, but show several overlapping areas. However, only specimens from one group were found in an intensively surveyed area in South America where both niche models overlap. It can be concluded that everything is not everywhere for B. melanospora. This taxon constitutes a complex formed by at least two cryptic species that probably diverged allopatrically in North and South America.

Quantitative analysis of protein adsorption via atomic force microscopy and surface plasmon resonance.

Surface properties have a significant influence on the performance of biomedical devices. The influence of surface chemistry on the amount and distribution of adsorbed proteins has been evaluated by a combination of atomic force microscopy (AFM) and surface plasmon resonance (SPR). Adsorption of albumin, fibrinogen, and fibronectin was analyzed under static and dynamic conditions, employing self-assembled monolayers (SAMs) as model surfaces. AFM was performed in tapping mode with antibody-modified tips. Phase-contrast images showed protein distribution on SAMs and phase-shift entity provided information on protein conformation. SPR analysis revealed substrate-specific dynamics in each system investigated. When multi-protein solutions and diluted human plasma interacted with SAMs, SPR data suggested that surface chemistry governs the equilibrium composition of the protein layer.

Chitosan-gelatin biopolymers as carrier substrata for limbal epithelial stem cells

The aim of this work was to evaluate semi-synthetic biopolymers based on chitosan (CH) and gelatin (G) as potential in vitro carrier substrata for human limbal epithelial cells (hLECs). To that end, human corneal epithelial cells (HCE) were cultured onto different CH–G membranes. None of the polymers were cytotoxic and cell proliferation was higher when CH was functionalized with G. Expression levels of corneal epithelial markers (K3, K12, E-caherin, desmoplakin, and zonula occludens (ZO)-1) were better maintained in HCE cells grown on CH–G 20:80 membranes than other proportions. Consequently, CH–G 20:80 was chosen for the subsequent expansion of hLECs. Cells derived from limbal explants were successfully expanded on CH–G 20:80 membranes using a culture medium lacking components of non-human animal origin. The expression levels found for corneal (K3 and K12) and limbal epithelial stem cells (K15) specific markers were similar to or higher than those found in limbal cells grown onto the control substratum. Our results demonstrate that CH–G 20:80 membranes are suitable for the expansion and maintenance of stem cells derived from the limbal niche. These results strongly support the use of polymers as alternative substrata for the transplantation of cultivated limbal cells onto the ocular surface.

Bioactive bilayered dressing for compromised epidermal tissue regeneration with sequential activity of complementary agents

The article deals with the design, preparation, and evaluation of a new bilayered dressing for application in the healing of compromised wounds. The system is based on the sequential release of two complementary bioactive components to enhance the activation of the regeneration of dermal tissue. The internal layer is a highly hydrophilic and biodegradable film of gelatin and hyaluronic acid (HG), crosslinked with the natural compound genipin, which reacts with the amine groups of gelatin. This film is loaded with the proangiogenic, anti-inflammatory, and antibacterial peptide, proadrenomedullin N-terminal 20 peptide (PAMP), that is released slowly in the wound site. The external layer, more stable and less hydrophilic, is constituted by a biodegradable polyurethane derived from poly(caprolactone) and pluronic L61. This layer is loaded with resorbable nanoparticles of bemiparin (a fractionated low molecular weight heparin), which promotes the activation of growth factors, FGF and VEGF, and provides a good biomechanical stability and controlled permeability of the bilayered dressing. Experiments carried out in mice demonstrate the excellent angiogenic effect of the HG film in the dermal tissue. Application of the bilayered dressing in the wound healing rabbit ear model shows an improved cicatrization of the wound in both ischemic and non-ischemic defects, favoring epithelialization and reducing noticeably the contraction and the inflammation.

Encapsulation of low molecular weight heparin (bemiparin) into polymeric nanoparticles obtained from cationic block copolymers: properties and cell activity

Bemiparin (fractionated low molecular weight heparin)-loaded nanoparticles were prepared by two consecutive w/o emulsions and an inversion to an o/w emulsion for various polymer systems as controlled release formulations. New synthetic block copolymers, poly(methyl methacrylate-b-trimethyl aminoethyl methacrylate) (PMMA-b-PMAETMA), with controlled microstructure and molecular weight, were prepared by RAFT (Reversible Addition-Fragmentation chain-Transfer) polymerization creating a set of polymers with different amounts of cationic charges. For comparison, a non-biodegradable positively charged polymer, Eudragit® RS PO, and a biodegradable polymer poly(lactic-co-glycolic acid), PLGA, were used. The microstructural arrangement of MMA and MAETMA sequences in PMMA-b-PMAETMA results in self-assembled core-shell nanoparticles in water with a positively charged surface, which interacts with bemiparin. The formulations were evaluated in terms of particle size, zeta potential and morphology by scanning electron microscopy (SEM). The entrapment of bemiparin molecules was confirmed by a negatively increased zeta potential value and the detection of a sulfur signal by energy dispersive X-ray spectroscopy (EDAX). High encapsulation efficiency was reached with all the polymeric matrices, ranging from 89 to 98%. Systems prepared with synthetic block copolymers PMMA-b-PMAETMA and PLGA showed higher in vitro bemiparin release than Eudragit® RS PO systems. For each formulation, bemiparin released from nanoparticles preserved its biological activity as shown by the BaF32 cell proliferation assay in the presence of fibroblast growth factor (FGF2).

Antimitogenic Polymer Drugs Based on AMPS: Monomer Distribution-Bioactivity Relationship of Water-Soluble Macromolecules

A number of polysulfonated molecules have demonstrated their interaction with fibroblast growth factor (FGF), hampering their binding to its receptors (low affinity heparan sulfate proteoglycans (HSPG) and high affinity tyrosine kinase FGF receptors) and inhibiting the intracellular signaling and mitogenic response in cultured endothelial cells. The aim of this work was the synthesis and characterization of new copolymers based on 2-acrylamido-2-methylpropane sulfonic acid (AMPS) with antiproliferative activity for antitumoral applications. N-Vinylpyrrolidone (VP) or butyl acrylate (BA) was copolymerized with the sulfonated monomer to obtain macromolecules with different hydrophilic/hydrophobic balance and distribution of the sulfonated groups within the macromolecules. In vitro cell culture proliferative assays showed that monomer distribution affected the inhibition of the proliferative action of FGF. Reactivity ratios of the systems were determined following the free radical copolymerization by in situ (1)H NMR, and the correlation of the monomer sequence distribution with the bioactivity is discussed.

Anticancer and antiangiogenic activity of surfactant-free nanoparticles based on self-assembled polymeric derivatives of vitamin E: Structure-activity relationship

α-Tocopheryl succinate (α-TOS) is a well-known mitochondrially targeted anticancer compound, however, it is highly hydrophobic and toxic. In order to improve its activity and reduce its toxicity, new surfactant-free biologically active nanoparticles (NP) were synthesized. A methacrylic derivative of α-TOS (MTOS) was prepared and incorporated in amphiphilic pseudoblock copolymers when copolymerized with N-vinylpyrrolidone (VP) by free radical polymerization (poly(VP-co-MTOS)). The selected poly(VP-co-MTOS) copolymers formed surfactant-free NP by nanoprecipitation with sizes between 96 and 220 nm and narrow size distribution, and the in vitro biological activity was tested. In order to understand the structure-activity relationship three other methacrylic monomers were synthesized and characterized: MVE did not have the succinate group, SPHY did not have the chromanol ring, and MPHY did not have both the succinate group and the chromanol ring. The corresponding families of copolymers (poly(VP-co-MVE), poly(VP-co-SPHY), and poly(VP-co-MPHY)) were synthesized and characterized, and their biological activity was compared to poly(VP-co-MTOS). Both poly(VP-co-MTOS) and poly(VP-co-MVE) presented triple action: reduced cell viability of cancer cells with little or no harm to normal cells (anticancer), reduced viability of proliferating endothelial cells with little or no harm to quiescent endothelial cells (antiangiogenic), and efficiently encapsulated hydrophobic molecules (nanocarrier). The anticancer and antiangiogenic activity of the synthesized copolymers is demonstrated as the active compound (vitamin E or α-tocopheryl succinate) do not need to be cleaved to trigger the biological action targeting ubiquinone binding sites of complex II. Poly(VP-co-SPHY) and poly(VP-co-MPHY) also formed surfactant-free NP that were also endocyted by the assayed cells; however, these NP did not selectively reduce cell viability of cancer cells. Therefore, the chromanol ring of the vitamin E analogues has an important role in the biological activity of the copolymers. Moreover, when succinate moiety is substituted and vitamin E is directly linked to the macromolecular chain through an ester bond, the biological activity is maintained.

1 – Introduction to smart polymers and their applications

: The scientific community tries to mimic nature in the way that living organisms adopt their behavior as a function of environmental conditions to improve survival. In this sense, smart polymers offer materials that respond to numerous stimuli (temperature, pH, electric and magnetic fields, light intensity, biological molecules, etc.), and scientists must devise the best way to apply them in all research areas. This chapter comprehensively summarizes the content of this book which tries to provide a wide overview of smart polymers and the most interesting applications developed recently.

Smart heparin-based bioconjugates synthesized by a combination of ATRP and click chemistry

This article describes the synthesis of novel well defined polysaccharide–polymer bioconjugates. Alkyne-containing bioconjugates were prepared using Cu(I) catalyzed azide–alkyne [3 + 2] dipolar cycloaddition, i.e. ‘click’ chemistry between an alkyne functionalized low molecular weight heparin (LMWH) and α-azide functionalized stimuli-responsive copolymers synthesized by atom transfer radical polymerization using Activators Generated by Electron Transfer (AGET). The alkyne functionality was incorporated into the heparin by conducting a high yield amidation of the polysaccharide using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride. The structure and composition of the novel bioconjugates were confirmed by FTIR, NMR and thermogravimetric analysis (TGA).