Improving cell adhesion: development of a biosensor for cell behaviour monitoring by surface grafting of sulfonic groups onto a thermoplastic polyurethane.

The surface properties of a material in combination with the mechanical properties are responsible for the material performance in a biological environment as well as the behaviour of the cells which contact with the material. Surface properties such as chemical, physical, biological play an important role in the biomaterials filed. In this work, the surface of a thermoplastic polyurethane film (Elastollan(®)1180A50) was tailored with sulfonic groups by grafting [2-(methacryloxyl)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide (SB) after a previous surface activation either by Argon plasma or by ultra-violet irradiation. This surface modification had the purpose of improving cell adhesion in order to develop a biosensor able to monitor cell behaviour. The surfaces were characterized by X-ray photoelectron spectroscopy, by atomic force microscopy and by contact angle measurements in order to evaluate the efficiency of the modification. Additionally, blood compatibility studies and cell adhesion tests with human bone marrow cells were performed. These methods allowed the grafting of SB and the results indicate that a higher density of grafting was achieved with previous surface plasma treatment than with UV irradiation. However, for both techniques, the presence of SB functional groups led to a decrease of hydrophobicity and roughness of the surface, together with an improvement of the materials biological performance.

An ocular insert with zero-order extended delivery: Release kinetics and mathematical models.

Ocular inserts (InEye®), were prepared based on two distinct formulations of PCL-PEG-PCL block copolymers - one with 33 % and the other with 24 % of PEG 600. Ring-open-polymerisation was used to link ε-caprolactone monomers to PEG hydroxyl end-groups. Molecular weight, PCL/PEG ratio, mass loss and swelling of different polymeric samples where determined. Based on the previously prepared block copolymers, ophthalmic inserts were assembled. These were prepared with an ellipsoidal shape by dripping melted polymer over a micro-tablet of moxifloxacin, used as drug model for this study, which therefore became entrapped in a central core coated with a polymer layer that functioned as a control-release barrier. The release kinetics of the model drug revealed a strong dependence on the PEG percentage on the polymer. Inserts' size and the amount of drug immobilized also had an important effect on the drug release profile. All release profiles followed a zero-order pattern, with 95 % of the drug being release at a constant rate. With drug releases varying from 20 to 200 days, and no initial burst, InEye® performance is unique among drug delivery systems and seems to be a very promising new formulation technology for preparing tailor-made ophthalmic inserts for prolonged and constant release of drug, which is needed for chronic diseases such as glaucoma, where compliance to treatment is essential for preventing optic-nerve lesions.

Study of Nα-benzoyl-L-argininate ethyl ester chloride, a model compound for poly(ester amide) precursors: x-ray diffraction, infrared and Raman spectroscopies, and quantum chemistry calculations.

Poly(ester amide)s (PEAs) are lacking in structural and spectroscopic information. This paper reports a structural and spectroscopic characterization of N(α)-benzoyl-L-argininate ethyl ester chloride (BAEEH(+)·Cl(-)), an important amino acid derivative and an adequate PEAs' model compound. Crystals of BAEEH(+)·Cl(-) obtained by slow evaporation in an ethanol∕water mixture were studied by different complementary techniques. X-ray analysis shows that BAEEH(+)·Cl(-) crystallizes in the chiral space group P2(1). There are two symmetry independent cations (and anions) in the unit cell. The two cations have different conformations: in one of them, the angle between the least-squares planes of the phenyl ring and the guanidyl group is 5.1(2)°, and in the other the corresponding angle is 13.3(2)°. There is an extensive network of H-bonds that assembles the ions in layers parallel to the ab plane. Experimental FT-IR and Raman spectra of BAEEH(+)·Cl(-) were recorded at room temperature in the 3750-600 cm(-1) and 3380-100 cm(-1) regions, respectively, and fully assigned. Both structural and spectroscopic analysis were supported by quantum chemistry calculations based on different models (in vacuo and solid-state DFT simulations).

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.

Impregnation of an intraocular lens for ophthalmic drug delivery.

In this work the possibility of impregnating P(MMA-EHA-EGDMA) with flurbiprofen using a clean and environmentally friendly technology, namely supercritical fluid technology was evaluated. P(MMA-EHA-EGDMA) has been proposed as a promising matrix to be used for intraocular delivery of anti-inflammatory drugs used in eye surgery and flurbiprofen is a non-steroidal anti-inflammatory agent. Fundamental studies like, the solubility of the drug in carbon dioxide, as well as the sorption degree of this polymeric matrix in the presence of carbon dioxide have been previously carried out. The aim of this research was to evaluate the effects of these two variables in the impregnation process. Different experimental conditions were tested and the results obtained suggest that the best impregnating conditions for this system are low temperatures and pressures, which at the same time correspond to a lower solubility of the drug in the supercritical fluid and a low swelling of the polymeric matrix. Experiments performed also indicate that the batch impregnation process leads to higher yields of impregnation and according to the release profiles obtained the drug can be released from the matrix up to three months, which presents great advantages for post-surgical treatments.

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.

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.

Controlled release of moxifloxacin from intraocular lenses modified by Ar plasma-assisted grafting with AMPS or SBMA: An in vitro study.

Intraocular lenses (IOLs) present an alternative for extended, local drug delivery in the prevention of post-operative acute endophthalmitis. In the present work, we modified the surface of a hydrophilic acrylic material, used for manufacturing of IOLs, through plasma-assisted grafting copolymerization of 2-acrylamido-2-methylpropane sulfonic acid (AMPS) or [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (SBMA), with the aim of achieving a controlled and effective drug release. The material was loaded with moxifloxacin (MFX), a commonly used antibiotic for endophthalmitis prevention. The characterization of the modified material showed that relevant properties, like swelling capacity, wettability, refractive index and transmittance, were not affected by the surface modification. Concerning the drug release profiles, the most promising result was obtained when AMPS grafting was done in the presence of MFX. This modification led to a higher amount of drug being released for a longer period of time, which is a requirement for the prevention of endophthalmitis. The material was found to be non-cytotoxic for rabbit corneal endothelial cells. In a second step, prototype IOLs were modified with AMPS and loaded with MFX as previously and, after sterilization and storage (30days), they were tested under dynamic conditions, in a microfluidic cell with volume and renovation rate similar to the eye aqueous humour. MFX solutions collected in this assay were tested against Staphylococcus aureus and Staphylococcus epidermidis and the released antibiotic proved to be effective against both bacteria until the 12th day of release.

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.

Structural analysis of dextran-based hydrogels obtained chemoenzymatically.

This work reports the results of structural analysis in novel dextran-acrylate (dexT70-VA) hydrogels generated chemoenzymatically. Porous structure as well as hydrogel surface and interior morphologies were evaluated by mercury intrusion porosimetry (MIP), nitrogen adsorption (NA), and scanning electron microscopy (SEM) analyses, as a function of the degree of substitution (DS), and initial water content used in the preparation of the hydrogel. MIP analysis showed that the overall networks were clearly macroporous with pore sizes ranging from 0.065 to 10 microm. As expected, the average pore size decreased as DS increased and as initial water content decreased. Moreover, the porosity values ranged from 75 up 90%, which shows that these hydrogels present an interconnected pore structure. Nitrogen adsorption analyses showed that the specific surface area of dexT70-VA hydrogels increased either by increasing the DS or by decreasing the initial water content of the hydrogel. SEM results revealed that the surface of hydrogels with lower DS presented either a porous structure or a polymeric "skin" covering the pores, whereas hydrogels with higher DS were totally porous. Furthermore, the interior morphology varied according to the DS and the initial water content of the hydrogels. Finally, the average pore size was also determined from the swelling of hydrogel using a theoretical model developed by Flory-Rehner. The comparison of the SEM and MIP results with the ones obtained by the equilibrium swelling theory of Flory-Rehner shows that this approach highly underestimates the average pore size.

Surface and hemocompatibility studies of bi-soft segment polyurethane membranes.

Cross-linked urethane/urea membranes with two soft segments were prepared by extending a poly(propylene oxide) based tri-isocyanate-terminated prepolymer (PUR) with polybutadiene diol (PBDO). The ratio of prepolymer and polybutadiene diol was varied to yield cross-linked membranes with different compositions, exhibiting different degrees of phase-separation of the PBDO segments in the bulk and of surface enrichment in PUR. In this work, surface energy and hemocompatibility aspects (hemolysis and thrombosis) of the PUR/PBDO membranes were evaluated. The results showed that the membrane surface energy increased with the PBDO content until 25% of PBDO, and decreased thereafter. The introduction of the second, more hydrophobic, soft segment (PBDO) in the PUR membranes turned hemolytic into non-hemolytic membranes and, for a blood-material contact time of 10 minutes, decreased the thrombogenicity significantly. The 10% PBDO membrane was the least thrombogenic and was also non-hemolytic. The hemolysis degree did not vary significantly with the PBDO content while, for blood-material contact times of 10 minutes, the thrombogenicity increased with an increase in PBDO content above 10%. Membrane thrombogenicity varied with the blood-material contact time. For blood contact times of 10 minutes, all membranes tested were less thrombogenic than glass.

Photocurable bioadhesive based on lactic acid.

Novel photocurable and low molecular weight oligomers based on l-lactic acid with proven interest to be used as bioadhesive were successfully manufactured. Preparation of lactic acid oligomers with methacrylic end functionalizations was carried out in the absence of catalyst or solvents by self-esterification in two reaction steps: telechelic lactic acid oligomerization with OH end groups and further functionalization with methacrylic anhydride. The final adhesive composition was achieved by the addition of a reported biocompatible photoinitiator (Irgacure® 2959). Preliminary in vitro biodegradability was investigated by hydrolytic degradation in PBS (pH=7.4) at 37 °C. The adhesion performance was evaluated using glued aminated substrates (gelatine pieces) subjected to pull-to-break test. Surface energy measured by contact angles is lower than the reported values of the skin and blood. The absence of cytoxicity was evaluated using human fibroblasts. A notable antimicrobial behaviour was observed using two bacterial models (Staphylococcus aureus and Escherichia coli). The cured material exhibited a strong thrombogenic character when placed in contact with blood, which can be predicted as a haemostatic effect for bleeding control. This novel material was subjected to an extensive characterization showing great potential for bioadhesive or other biomedical applications where biodegradable and biocompatible photocurable materials are required.

Enzyme-linked immunofiltration assay used in the screening of solid supports and immunoreagents for the development of an azinphos-methyl flow immunosensor.

Azinphos-methyl (AM), O,O-dimethyl S-[(4-oxo-1,2,3-benzotriazin-3(4H)-yl)methyl] phosphorodithioate, is a dithiophosphorous insecticide extensively used for the control of fruit culture pests. In this work the ELIFA system, initially developed and marketed to substitute conventional ELISA methods, was used for the screening of supports and immunoreagents in the development of a flow immunosensor to AM. The objective was to find the optimal antibody concentration, support quantity and enzymatic tracer concentration to develop a sensitive and reusable immunosensor. The influence of chitosan as protein stabilizing agent was also investigated. We observed that, on the basis of immunosorbent characterization, chitosan-modified silica with immobilized LIB-MFH14 monoclonal antibody (MAb) showed the best sensitivity, with a I(50) value of 6 nM AM. All of the immobilized MAbs either in alkylaminated or chitosan-modified silica showed I(50) values between 10 and 36 nM. Regarding the regeneration capability, the best desorption agent tested was 0.1 M glycine/HCl, pH 2.0, performing in most cases a 100% desorption after just one wash and maintaining the antibody activity even after 20 cycles of regeneration. The chitosan-modified silica seemed to be the best support for this purpose.

Study of the thermal stability and enzymatic activity of an immobilised enzymatic system for the bilirubin oxidation.

In this work, we have studied the immobilisation of the haemoglobin/glucose oxidase coupled enzymatic system in poly(vinyl alcohol) membranes. These are to be used as a reagent phase either in the development of an optical sensor or as an efficient bilirubin (BR) removal reactor. Poly(vinyl alcohol) (PVA) was chosen as the support for this purpose, due to its good biocompatibility, hydrophilicity and non-thrombogenic effects. A hydrogel containing the enzymatic system, consisting of PVA crosslinked with glutaraldehyde, was prepared and characterised by DSC, enzyme activity measurements and release tests. Investigating protein conformational changes as a function of temperature and the enzymatic system activity we have found that, in spite of the destabilizing effect of the glutaraldehyde in the acidic medium, the PVA insolubilisation conditions seem do not perturb either the conformation of the 'native state' nor the enzymatic system activity. Moreover, it was found that PVA/glutaraldehyde membranes offer a simple way to hold enzymatic system, with the possibility of controlling the conditions to obtain either the effective prevention of leaching of the entrapped proteins or the in situ delivery of the haemoglobin.

Study of an enzyme coupled system for the development of fibre optical bilirubin sensors.

We have established an analytical procedure for bilirubin (BR) determination by following the variation of its yellow absorption band in the presence of the biocatalytic system haemoglobin/glucose oxidase/glucose. The influence of the pH of the medium, the haemoglobin/hydrogen peroxide ratio, and the concentration of the various components in the mixture on the system activity was evaluated. The solubility of the unconjugated BR was analysed. An anionic detergent in Tris buffer, in which it presents a high critical micellar concentration, was selected for the BR/bovine serum albumin cleavage. The Michaelis-Menten constant of the system (after the optimisation of the various parameters) was determined and compared with Km values reported in the literature for similar catalytic systems. Preliminary results obtained with the haemoglobin and glucose oxidase immobilised either in poly(vinyl alcohol) or by crosslinking with glutaraldehyde, show that this system is promising as a future biosensor.

Covalent binding of urease on ammonium-selective potentiometric membranes.

As part of the development of disposable urea bioselective probes, the covalent binding of urease on ammonium-selective potentiometric membranes has been assessed. Nonactin/bis(1-butylpentyl)adipate/poly(vinylchloride) (PVC) membranes, directly applied to an internal solid contact (conductive epoxy-graphite composite), has been used as a support for covalent immobilization of urease. Two types of all-solid-state construction process have been assayed: thin layers of cellulose acetate (CA) were coated on the PVC ammonium-selective membranes (type 1) and blends of PVC and CA at various ratios were used as ammonium-selective membrane matrices (type 2). Urease was covalently attached to CA via aldehyde groups. These groups were created on the polysaccharide with sodium periodate to which the enzyme was immobilized through a spacer (hexamethylenediamine). The viability of both types of probe for the determination of ammonium ions was assessed after each step of the activation process. Results indicated that type 2 potentiometric probes are altered after the treatment with sodium periodate. Good results were obtained with type 1 probes. Their dynamic concentration range of response to urea was from 2 x 10(-5) to 0.01 M with a sensibility of 50 mV/decade.

Evaluation of poly(2-hydroxyethyl methacrylate) gels as drug delivery systems at different pH values.

Studies of dynamic and equilibrium swelling, structural characterisation and solute transport in swollen poly(2-hydroxyethyl methacrylate) gels (pHEMA) cross-linked with tripropyleneglycol diacrylate (TPGDA) were done for a wide range of TPGDA concentrations. The influence of the pH on these pHEMA properties was evaluated. In swelling studies it was found that in changing the pH from 6.5 to 12.0, a large increase in swelling occurred, from approximately 48 to 55%, for the lowest concentration of TPGDA (1 mol%), and from 40 to 80% for the highest concentration (10 mol%). Fourier transform infrared (FTIR) and differential scanning calorimetry (DSC) measurements were made after the equilibrium swelling of the gels at different pH values, to explain these results. The advantage of using these gels as controlled drug delivery systems is illustrated using salicylic acid (SA) as a model drug. The loading and the release of the SA were made at different pH values and the results obtained showed that it is possible to modulate the hydrogel performance by controlling an external factor, the pH at which the drug loading and release were performed.

Tailoring the properties of gelatin films for drug delivery applications: influence of the chemical cross-linking method.

Two types of chemically cross-linked gelatin films were prepared and characterized. The first type of films was cross-linked with 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide hydrochloride (EDC) under heterogeneous conditions and are named Gel-E. In the second type of films, gelatin was previously functionalized with methacrylamide side groups by the reaction with methacrylic anhydride and for that is named Gel-MA. The modified gelatin was subsequently cross-linked by a photoinitiated radical polymerization. These films were characterized relatively to their degree of cross-linking, buffer uptake capacity, resistance to hydrolytic and proteolytic degradation, and mechanical and thermal properties. Results show that the employed cross-linking method, together with the degree cross-linking, dictate the final properties of the films. Gel-E films have significant lower buffer uptake capacities and higher resistance to collagenase digestion when compared to Gel-MA films. Additionally, Gel-E films exhibit higher values of stress at break and lower strains at break. Moreover, the films properties could be modified by varying the extent of the chemical cross-linking, which in turn could be controlled by varying the concentration of EDC, for the first type of films (Gel-E), or by using gelatins with different degrees of functionalization, in the case of the second type of films (Gel-MA).

New drug-eluting lenses to be applied as bandages after keratoprosthesis implantation.

Corneal tissue is the most commonly transplanted tissue worldwide. This work aimed to develop a new drug-eluting contact lens that may be used as a bandage after keratoprosthesis. During this work, films were produced using poly(vinyl alcohol) (PVA) and chitosan (CS) crosslinked with glyoxal (GL). Vancomycin chlorhydrate (VA) was impregnated in these systems by soaking. Attenuated total reflectance - Fourier transform infrared spectroscopy was used to confirm crosslinking. The cytotoxic and drug release profile, hydrophilicity, thermal and biodegradation as well as swelling capacity of the samples were assessed through in vitro studies. PVA and PVA/CS films were obtained by crosslinking with GL. The films were transparent, flexible with smooth surfaces, hydrophilic and able to load and release vancomycin for more than 8h. Biodegradation in artificial lachrymal fluid (ALF) with lysozyme at 37°C showed that mass loss was higher for the samples containing CS. Also, the samples prepared with CS showed the formation of pores which were visualized by SEM. All samples revealed a biocompatible character after 24h in contact with cornea endothelial cells. As a general conclusion it was possible to determine that the 70PVA/30CS film showed to combine the necessary features to prepare vancomycin-eluting contact lenses to prevent inflammation after corneal substitution.

Synthesis of a dextran based thermo-sensitive drug delivery system by gamma irradiation.

Gamma radiation was used as the initiator/crosslinker agent for the synthesis of thermo-sensitive hydrogel networks, under the form of membranes, using dextran and N-isopropylacrylamide. The prepared membranes were loaded with Ondansetron™, a potent antiemetic drug and tested as drug delivery systems. The characterization of the materials was accomplished by: Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy, elemental analysis, lower critical solution temperature (LCST) determination, swelling behaviour evaluation, determination of surface energy by contact angle measurement and drug delivery kinetics studies. Also, the influence of irradiation time and temperature on the materials properties was evaluated.