Innovative tailor made dextran based membranes with excellent non-inflammatory response: In vivo assessment.

In this work, dextran based membranes with potential to be used as implantable devices in Tissue Engineering and Regenerative Medicine (TERM) were prepared by a straightforward strategy. Briefly, two polymers approved by the Food and Drug Administration, viz. dextran and poly(ε-caprolactone) (PCL) were functionalized with methacrylate moieties, and subjected to photocrosslinking. Employing different weight ratios of each polymer in the formulations allowed to obtain transparent membranes with tunable physicochemical properties and low adverse host tissue response. Independently of the material, all formulations have shown to be thermally stable up to 300 °C whilst variations in the polymer ratio resulted in membranes with different glass transition temperatures (Tg) and flexibility. The swelling capacity ranged from 50% to 200%. On the other hand, in vitro hydrolytic degradation did not show to be material-dependent and all membranes maintained their structural integrity for more than 30 days, losing only 8-12% of their initial weight. Preliminary in vitro biological tests did not show any cytotoxic effect on seeded human dental pulp stem cells (hDPSCs), suggesting that, in general, all membranes are capable of supporting cell adhesion and viability. The in vivo biocompatibility of membranes implanted subcutaneously in rats' dorsum indicate that M100/0 (100%wt dextran) and M25/75 (25 %wt dextran) formulations can be classified as "slight-irritant" and "non-irritant", respectively. From the histological analysis performed on the main tissue organs it was not possible to detect any signs of fibrosis or necrosis thereby excluding the presence of toxic degradation by-products deposited or accumulated in these tissues. In combination, these results suggest that the newly developed formulations hold great potential as engineered devices for biomedical applications, where the biological response of cells and tissues are greatly dependent on the physical and chemical cues provided by the substrate.

Stabilization of polymer lipid complexes prepared with lipids of lactic acid bacteria upon preservation and internalization into eukaryotic cells.

The physicochemical characterization of polymer liposome complexes (PLCs) prepared with lipids of lactic acid bacteria and poly(N,N-dimethylaminoethyl methacrylate) covalently bound to cholesterol (CHO-PDMAEMA) was carried out in an integrated approach, including their stability upon preservation and incorporation into eukaryotic cells. PLCs were prepared with different polymer:lipid molar ratios (0, 0.05 and 0.10). Zeta potential, particle size distribution and polydispersity index were determined. The optimal polymer:lipid ratio and the stability of both bare liposomes and PLCs were evaluated at 37 °C and at different pHs, as well as after storage at 4 °C, -80 °C and freeze-drying in the presence or absence of trehalose 250 mM. Internalization of PLCs by eukaryotic cells was assessed to give a complete picture of the system. Incorporation of CHO-PDMAEMA onto bacterial lipids (ratio 0.05 and 0.10) led to stabilization at 37 °C and pH 7. A slight decrease of pH led to their strong destabilization. Bacteria PLCs showed to be more stable than lecithin (LEC) PLCs (used for comparison) upon preservation at 4 and -80 °C. The harmful nature of the preservation processes led to a strong decrease in the stability of PLCs, bacterial formulations being more stable than LEC PLCs. The addition of trehalose to the suspension of liposomes stabilized LEC PLC and did not have effect on bacterial PLCs. In vitro studies on Raw 264.7 and Caco-2/TC7 cells demonstrated an efficient incorporation of PLCs into the cells. Preparations with higher stability were the ones that showed a better cell-uptake. The nature of the lipid composition is determinant for the stability of PLCs. Lipids from lactic acid bacteria are composed of glycolipids and phospholipids like cardiolipin and phosphatidylglycerol. The presence of negatively charged lipids strongly improves the interaction with the positively charged CHO-PDMAEMA, thus stabilizing liposomes. In addition, glycolipids and phosphatidylglycerol act as intrinsic protectants of PLCs upon preservation. This particular lipid composition of lactic acid bacteria makes them natural formulations potentially useful as drug delivery systems.

Effect of cholesterol-poly(N,N-dimethylaminoethyl methacrylate) on the properties of stimuli-responsive polymer liposome complexes.

The development of new polymer-liposome complexes (PLCs) as delivery systems is the key issue of this work. Three main areas are dealt with: polymer synthesis/characterization, liposome formulation/characterization and evaluation of the PLCs uptake by eukaryotic cells. Poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA) with low molecular weight and narrow polydispersity was synthesized by Atom Transfer Radical Polymerization (ATRP). The polymers were synthesized using two different bromide initiators (cholesteryl-2-bromoisobutyrate and ethyl 2-bromoisobutyrate) as a route to afford PDMAEMA and CHO-PDMAEMA. Both synthesized polymers (PDMAEMA and CHO-PDMAEMA) were incorporated in the preparation of lecithin liposomes (LEC) to obtain PLCs. Three polymer/lipid ratios were investigated: 5, 10 and 20%. Physicochemical characterization of PLCs was carried out by determining the zeta potential, particle size distribution, and the release of fluorescent dyes (carboxyfluorescein CF and calcein) at different temperatures and pHs. The leakage experiments showed that CHO covalently bound to PDMAEMA strongly stabilizes PLCs. The incorporation of 5% CHO-PDMAEMA to LEC (LEC_CHO-PD5) appeared to be the stablest preparation at pH 7.0 and at 37°C. LEC_CHO-PD5 destabilized upon slight changes in pH and temperature, supporting the potential use of CHO-PDMAEMA incorporated to lecithin liposomes (LEC_CHO-PDs) as stimuli-responsive systems. In vitro studies on Raw 264.7 and Caco-2/TC7 cells demonstrated an efficient incorporation of PLCs into the cells. No toxicity of the prepared PLCs was observed according to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. These results substantiate the efficiency of CHO-PDMAEMA incorporated onto LEC to assist for the release of the liposome content in mildly acidic environments, like those found in early endosomes where pH is slightly lower than the physiologic. In summary, the main achievements of this work are: (a) novel synthesis of CHO-PDMAEMA by ATRP, (b) stabilization of LEC by incorporation of CHO-PDMAEMA at neutral pH and destabilization upon slight changes of pH, (c) efficient uptake of LEC_CHO-PDs by phagocytic and non-phagocytic eukaryotic cells.

Photocrosslinkable starch-based polymers for ophthalmologic drug delivery.

This study focused on the development and characterization of a starch-based polymer with urethane linkages to be used as a controlled drug delivery system for biomedical applications. Starch was modified with 2-isocyanatoethyl methacrylate in order to obtain a polymer containing carbon-carbon double bonds in its structure. This modified starch was then used to produce films by UV irradiation using Irgacure 2959 (CIBA) as the photoinitiator. The modified polymer was characterized by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. The swelling capacity, in artificial lachrymal fluid (performed both at room temperature and physiological temperature), and water contact angles measurements were determined. The in vitro biodegradation in artificial lachrymal fluid supplemented with lysozyme was also studied. Scanning electronic microscopy (SEM) was used to characterize the morphology of the materials immediately after synthesis and after biodegradation. Timolol maleate and sodium flurbiprofen were immobilized by adsorption and their in vitro release profiles were followed spectroscopically.

Development of a new photocrosslinkable biodegradable bioadhesive.

Adhesives provide a needle-free method of wound closure and do not require local anaesthetics. Polymeric adhesives have been used for about 3 decades for joining several tissues of the organism. Also, they can accomplish other tasks, such as haemostasis and the ability to seal air leakages and have the potential to serve as delivery systems. PCL was modified with 2-isocyanatoethylmethacrylate to form a macromer that was crosslinked via UV irradiation using Irgacure 2959 by CIBA as the photoinitiating agent. The characterization of the materials was accomplished by: attenuated total reflectance-Fourier transform infrared (ATR-FTIR), swelling capacity determination, evaluation of adhesive capacity (by reaction with aminated substrates) and determination of surface energy by contact angle measurement. Thermal characterization of the adhesive was performed by dynamical mechanical thermal analysis (DMTA) and thermogravimetric analysis (TGA). The morphology of PCL networks was observed using scanning electron microscopy (SEM) both after crosslinking process and following biodegradation in human plasma. The haemocompatibility of the membranes was also evaluated by thrombosis and haemolysis tests.

Modification of the biopolymer castor oil with free isocyanate groups to be applied as bioadhesive.

Surgical adhesives have been used for several applications, including haemostasis, sealing air leakages and tissue adhesion. The aim of this work was to develop a biodegradable urethane-based bioadhesive containing free isocyanate groups. This material presents the advantage of being biodegradable, biocompatible and having the capacity of reacting with amino groups present in the biological molecules. A urethane based on castor oil (CO) was synthesized by reaction of the molecule with isophorone diisocyanate (IPD). The characterization of the material was accomplished by different techniques: ATR-FT-IR (attenuated transmittance reflection-Fourier transform infrared), swelling capacity determination, evaluation of the moisture curing kinetics, reaction with aminated substrates and determination of surface energy by contact angle measurement. The study of the urethane thermal properties was performed by DMTA (dynamical mechanical thermal analysis) and TGA (thermogravimetric analysis). The haemocompatibility of the urethane was also evaluated by thrombosis and haemolysis tests.

Synthesis and characterization of membranes obtained by graft copolymerization of 2-hydroxyethyl methacrylate and acrylic acid onto chitosan.

Chitosan based membranes to be applied on wound healing as topical drug delivery systems were developed by graft copolymerization of acrylic acid (AA) and 2-hydroxyethyl methacrylate (HEMA) onto chitosan using cerium ammonium nitrate as chemical initiator. Evidence for graft copolymerization of the vinyl monomers onto chitosan was obtained by FTIR and DMTA. Swelling degree, cytotoxicity, thrombogenicity and haemolytic activity of these membranes were evaluated. Chitosan-graft-AA-graft-HEMA showed to be the best matrix for drug delivery systems than chitosan-graft-AA because it retains good swelling properties, but the content in HEMA has improved cytocompatibility, hemocompatibility and thrombogenic character.