František Rypáček

Microdomain structure in polylactide-block-poly(ethylene oxide) copolymer films

Structured surface is an important property of polymer biomaterials for tissue engineering, for its capacity to expose domains with different surface energy and functional groups. For this purpose, amphiphilic A-B-A block copolymers with polylactide (PLA) as A blocks and poly(ethylene oxide) (PEO 3, Mn = 3090; PEO6, Mn = 6110) as B block were synthesized by ring-opening polymerization of either L-lactide (L-LA) or DL-lactide (DL-LA), using poly(ethylene glycol)s as macroinitiators and tin(II) octanoate (Sn(Oct)2) as a catalyst. Differential scanning calorimetry (DSC) and electron microscopy were used to study the phase separation of the hydrophobic (PLA) and hydrophilic (PEO) segments in films made of the copolymers and their blends with high-molecular-weight PLA homopolymers. Hydrophilic (PEO) and hydrophobic (PLA) domains were formed at the polymer film surface due to the separation of phases. The phase separation was affected by the copolymer composition and the stereoregularity of PLA blocks in the copolymers.

Poly(ethylene oxide) Layers Grafted to Dopamine-melanin Anchoring Layer: Stability and Resistance to Protein Adsorption

In this study, we propose substrate-independent modification for creating a protein-repellent surface based on dopamine-melanin anchoring layer used for subsequent binding of poly(ethylene oxide) (PEO) from melt. We verified that the dopamine-melanin layer can be formed on literally any substrate and could serve as the anchoring layer for subsequent grafting of PEO chains. Grafting of PEO from melt in a temperature range 70-110 °C produces densely packed PEO layers showing exceptionally low protein adsorption when exposed to the whole blood serum or plasma. The PEO layers prepared from melt at 110 °C retained the protein repellent properties for as long as 10 days after their exposure to physiological-like conditions. The PEO-dopamine-melanin modification represents a simple and universal surface modification method for the preparation of protein repellent surfaces that could serve as a nonfouling background in various applications, such as optical biosensors and tissue engineering.

Nonfouling poly(ethylene oxide) layers end-tethered to polydopamine

Nonfouling surfaces capable of reducing protein adsorption are highly desirable in a wide range of applications. Coating of surfaces with poly(ethylene oxide) (PEO), a water-soluble, nontoxic, and nonimmunogenic polymer, is most frequently used to reduce nonspecific protein adsorption. Here we show how to prepare dense PEO brushes on virtually any substrate by tethering PEO to polydopamine (PDA)-modified surfaces. The chain lengths of hetero-bifunctional PEOs were varied in the range of 45-500 oxyethylene units (M(n) = 2000-20,000). End-tethering of PEO chains was performed through amine and thiol headgroups from reactive polymer melts to minimize excluded volume effects. Surface plasmon resonance (SPR) was applied to investigate the adsorption of model protein solutions and complex biologic medium (human blood plasma) to the densely packed PEO brushes. The level of protein adsorption of human serum albumin and fibrinogen solutions was below the detection limit of the SPR measurements for all PEO chains end-tethered to PDA, thus exceeding the protein resistance of PEO layers tethered directly on gold. It was found that the surface resistance to adsorption of lysozyme and human blood plasma increased with increasing length and brush character of the PEO chains end-tethered to PDA with a similar or better resistance in comparison to PEO layers on gold. Furthermore, the chain density, thickness, swelling, and conformation of PEO layers were determined using spectroscopic ellipsometry (SE), dynamic water contact angle (DCA) measurements, infrared reflection-absorption spectroscopy (IRRAS), and vibrational sum-frequency-generation (VSFG) spectroscopy, the latter in air and water.

Biomimetic non-fouling surfaces: extending the concepts

In this study, we propose a substrate-independent biomimetic modification route for the creation of antifouling polymer brushes. This modification route consists of the formation/deposition of a biomimetic polydopamine anchor layer followed by a well-controlled surface-initiated atom transfer radical polymerization of antifouling polymer brushes initiated by 2-bromo-2-methylpropanoyl groups covalently attached to the hydroxyl and amine groups present in the anchor layer. In this way, we synthesized polymer brushes of methoxy- and hydroxy-capped oligoethylene glycol methacrylate, 2-hydroxyethyl methacrylate and carboxybetaine acrylamide. Spectroscopic ellipsometry (SE) indicated well-controlled polymerization kinetics of the brushes, thus the thickness of the ultra-thin films could be precisely tuned at a nanometer scale. The covalent structure and organization of the brushes grown from the polydopamine anchor layer were accessed by infrared reflection-adsorption spectroscopy (IRRAS) while the change in hydrophilicity caused by the presence of the brush was determined by dynamic water contact angle measurements. Surface plasmon resonance as well as ex situ IRRAS and SE measurements were applied to investigate the adsorption of model protein solutions and undiluted human blood plasma to the brushes. The biomimetic brushes completely suppressed the fouling from single protein solutions and reduced the fouling from plasma to less than 3% from the fouling measured on bare gold surfaces. The proposed modification procedure is non-destructive and does not require any chemical pre-activation or the presence of reactive groups on the substrate surface. Contrary to other antifouling modifications the coating can be performed on various classes of substrates and preserves its properties even in undiluted blood plasma. This work offers a promising technology for the facile fabrication of different surface-based biotechnological and biomedical devices able to perform tailor-made functions while resisting the fouling from the complex biological media where they operate.

Click & seed approach to the biomimetic modification of material surfaces.

A simple, versatile, protein-repulsive, substrate-independent biomimetic surface modification is presented that is based on the creation of a PEO brush on a polydopamine anchoring layer and its capacity for selective follow-up modifications with various ligands using a copper-catalyzed alkyne-azide cycloaddition reaction. The desired surface concentration of peptide biomimetic ligands can be controlled by adjusting the peptide concentration in the reaction mixture, then measuring the activity of (125)I-radiolabeled peptides that are immobilized on the substrates. The performance of the prepared substrates is tested in cell cultures with MEF cells and a human ECC line.

Solute Absorption from the Airways of the Isolated Rat Lung. IV. Mechanisms of Absorption of Fluorophore-Labeled Poly-α,β-[N(2-Hydroxyethyl)-DL-Aspartamide]

The pulmonary absorption kinetics of a single molecular weight distribution (MWD) of fluorophore-labeled poly-α,β-[N(2-hydroxyethyl)-DL-aspartamide] (F-PHEA), a hydrophilic and biocompatible synthetic polypeptide, were studied in the isolated, perfused rat lung (iprl) as functions of administered polymer concentration, dose, vehicle, and presence and absence of fluorophore. The MWD was characterized before and after absorption by measurement of weight- and number-averaged molecular weights (Mwand Mn, respectively) using high-performance gel-permeation chromatography. Values for Mw and Mn were 8.6 and 5.3 kD before, and 6.7 and 4.7 kD after, absorption into the perfusate; there was no significant metabolism and the MWD of the absorbed polymer was independent of both dose and sampling time over a 3-hr period. F-PHEA failed to show any evidence of aggregation in solution or changes in dose distribution within the airways as functions of increasing polymer concentration and dose. A concentration ranging study indicated the presence of a saturable, carrier-mediated transport process for F-PHEA with a maximum absorption rate, Vmax, of approximately 180 µg or 0.027 µmol/hr. Coadministration of fluorophore-free PHEA was capable of depressing the absorption of F-PHEA. The transport process for F-PHEA appeared to have a molecular weight limit of about 7 kD for this hydrophilic polymer.

Synthetic poly(amino acid) hydrogels with incorporated cell-adhesion peptides for tissue engineering

Preparation of soft poly(amino acid) hydrogels containing biomimetic cell‐adhesive peptides was investigated. Covalently crosslinked gels were formed by radical co‐polymerization of methacryloylated macromonomer poly[N5‐(2‐hydroxyethyl)‐L‐glutamine‐stat‐L‐alanine‐stat‐methacryloyllysine] with 2‐hydroxyethyl methacrylate (HEMA) as minor co‐monomer. Hydrogels carrying biomimetic peptides were prepared by using methacryloylated peptides, such as methacryloyl–GGGRGDSG–OH and methacryloyl–GGGYIGSR–OH, as additional monomers in the polymerization mixture. Mechanical stability and swelling in water of the hydrogels obtained for different solid:water and polypeptide:HEMA ratios were evaluated. The microporosity of gels (5–20 µm), dependent on the polyHEMA phase separation in water, was followed by low‐vacuum SEM. The effect of biomimetic modification of hydrogels with RGDS and YIGSR peptides on the seeding efficiency of porcine mesenchymal stem cells (MSCs) was studied in vitro. While unmodified hydrogels showed very low cell adhesion, due to their highly hydrophilic nature, the incorporation of adhesive peptides significantly improved the adhesion and viability of seeded cells. Copyright

AFM imaging and analysis of local mechanical properties for detection of surface pattern of functional groups

In this work we evaluate the applicability of different atomic force microscopy (AFM) modes, such as Phase Shift Imaging, Atomic Force Acoustic Microscopy (AFAM) and Force Spectroscopy, for mapping of the distribution pattern of low-molecular-weight biomimetic groups on polymer biomaterial surfaces. Patterns with either random or clustered spatial distribution of bioactive peptide group derived from fibronectin were prepared by surface deposition of functional block copolymer nano-colloids and grafted with RGDS peptide containing the sequence of amino acids arginine-glycine-aspartic acid-serine (conventionally labeled as RGDS) and carrying biotin as a tag. The biotin-tagged peptides were labeled with 40nm streptavidin-modified Au nanospheres. The peptide molecules were localized through the detection of bound Au nanospheres by AFM, and thus, the surface distribution of peptides was revealed. AFM techniques capable of monitoring local mechanical properties of the surface were proved to be the most efficient for identification of Au nano-markers. The efficiency was successfully demonstrated on two different patterns, i.e. random and clustered distribution of RGDS peptides on structured surface of the polymer biomaterial.

Self-assembled anchor layers/polysaccharide coatings on titanium surfaces: a study of functionalization and stability

Summary Composite materials based on a titanium support and a thin, alginate hydrogel could be used in bone tissue engineering as a scaffold material that provides biologically active molecules. The main objective of this contribution is to characterize the activation and the functionalization of titanium surfaces by the covalent immobilization of anchoring layers of self-assembled bisphosphonate neridronate monolayers and polymer films of 3-aminopropyltriethoxysilane and biomimetic poly(dopamine). These were further used to bind a bio-functional alginate coating. The success of the titanium surface activation, anchoring layer formation and alginate immobilization, as well as the stability upon immersion under physiological-like conditions, are demonstrated by different surface sensitive techniques such as spectroscopic ellipsometry, infrared reflection–absorption spectroscopy and X-ray photoelectron spectroscopy. The changes in morphology and the established continuity of the layers are examined by scanning electron microscopy, surface profilometry and atomic force microscopy. The changes in hydrophilicity after each modification step are further examined by contact angle goniometry.

Solute Absorption from the Airways of the Isolated Rat Lung. V. Charge Effects on the Absorption of Copolymers of N(2-hydroxyethyl)-DL-Aspartamide with DL-Aspartic Acid or Dimethylaminopropyl-DL-Aspartamide

AbstractPurpose. To determine the effects of ionized substituents upon the pulmonary absorption of 6-8 kDa synthetic, hydrophilic polypeptides. Methods. Fluorophore-labeled poly (hydroxyethylaspartamide), F-PHEA (neutral at pH 7.4) and its copolymer derivatives poly (hydroxyethylaspartamide-co-dimethylaminopropylaspartamide), F-P(HEA-DMAPA) (positive at pH 7.4) and poly (hydroxyethylaspartamide-co-aspartic acid), F-P(HEA-AA) (negative at pH 7.4) were synthesized and administered in different concentrations to the airways of the isolated rat lung preparation. The time and molecular weight dependencies of polypeptide absorption into perfusate were determined at intervals by gel permeation chromatography. Results. For all polypeptides, molecular weights in perfusate were about 1 kDa less than those which were administered, due to preferential absorption of smaller molecules. The absorption, up to 70% of the administered dose over 3 h, of the anionic F-P(HEA-AA), was significantly faster than that of the neutral F-PHEA or the polycationic F-P(HEA-DMAPA). The latter derivative produced greatest edema in the lung. Absorption showed both active [dose-dependent kinetics] and passive [diffusive] components for all three polymers. Conclusions. Pulmonary absorption of similarly sized macromolecular PHEA derivatives, either neutral, positively or negatively charged, occured via carrier-mediated and diffusive mechanisms. The highest rate of absorption was observed with the polyanionic derivative.