Multifunctional Platform for Covalent Titanium Coatings: Micro-FTIR, XPS, and NEXAFS Characterizations.

This work aims at preparing and characterizing a versatile multifunctional platform enabling the immobilization of macromolecules on a titanium surface by robust covalent grafting. Functionalized titanium is widely used in the biomedical field to improve its properties. Despite its high biocompatibility and osteointegrability, titanium implants are not very stable in the long term due to the onset of inflammation and bacterial infections. The proposed method allows the superficial insertion of three different organic linkers to be used as anchors for the attachment of biopolymers or bioactive molecules. This strategy used green solvents and is a good alternative to the proposed classic methods that employ organic solvents. The uniformly modified surfaces were characterized by micro-Fourier transform infrared spectroscopy (micro-FTIR), X-ray Photoelectron spectroscopy (XPS) and Near-Edge X-ray Absorption Fine Structure (NEXAFS). The latter made it possible to assess the orientation of the linker molecules with respect to the titanium surface. To test the efficiency of the linkers, two polymers (alginate and 2-(dimethylamino)-ethyl methacrylate (PDMAEMA)), with the potential ability to increase biocompatibility, were covalently attached to the titanium surfaces. The obtained results are a good starting point for the realization of stable polymeric coatings permanently bonded to the surface that could be used to extend the life of biomedical implants.

Influence of Nanoaggregation Routes on the Structure and Thermal Behavior of Multiple-Stimuli-Responsive Micelles from Block Copolymers of Oligo(ethylene glycol) Methacrylate and the Weak Acid [2-(Hydroxyimino)aldehyde]butyl Methacrylate.

In this work, we compare nanoaggregation driven by pH-induced micellization (PIM) and by the standard solvent displacement (SD) method on a series of pH-, light-, and thermosensitive amphiphilic block copolymers. Specifically, we investigate poly(HIABMA)-b-poly(OEGMA) and poly(HIABMA)-b-poly(DEGMA-r-OEGMA), where HIABMA = [(hydroxyimino)aldehyde]butyl methacrylate, OEGMA = oligo(ethylene glycol)methyl ether methacrylate, and DEGMA = di(ethylene glycol)methyl ether methacrylate. The weakly acidic HIA group (pKa ≈ 8) imparts stability to micelles at neutral pH, unlike most of the pH-responsive copolymers investigated in the literature. With SD, only some of our copolymers yield polymeric micelles (34-59 nm), and their thermoresponsivity is either poor or altogether absent. In contrast, PIM affords thermoresponsive, smaller micelles (down to 24 nm), regardless of the polymer composition. In some cases, cloud points are remarkably well defined and exhibit limited hysteresis. By combining turbidimetric, dyamic light scattering, and small-angle X-ray scattering measurements, we show that SD yields loose micelles with POEGMA segments partly involved in the formation of the hydrophobic core, whereas PIM yields more compact core-shell micelles with a well-defined PHIABMA core. We conclude that pH-based nanoaggregation provides advantages over block-selective solvation to obtain compact micelles exhibiting well-defined responses to external stimuli.

Polymorphic Self-Organization of Lauroyl Peptide in Response to pH and Concentration.

Amphipathic peptides are attractive building blocks for the preparation of self-assembling, bio-inspired, and stimuli responsive nanomaterials with pharmaceutical interest. The bioavailability of these materials can be improved with the insertion of d amino acid residues to avoid fast proteolysis in vivo. With this knowledge, a new lauroyl peptide consisting of a sequence of glycine, glycine, d-serine, and d-lysine was designed. In spite of its simple sequence, this lipopeptide self-assembles into spherical micelles at acid pH, when the peptide moiety adopts disordered conformations. Self-aggregates reshape toward fibers at basic pH, following the conformational transition of the peptide region from random coil to ß-sheet. Finally, hydrogels are achieved at basic pH and higher concentrations. The transition from random coil to ß-sheet conformation of the peptide headgroup obtained by increasing pH was monitored by circular dichroism and vibrational spectroscopy. A structural analysis, performed by combining dynamic light scattering, small-angle X-ray scattering, transmission electron microscopy, and molecular dynamic simulations, demonstrated that the transition allows the self-assemblies to remodel from spherical micelles to rodlike shapes, to long fibers with rectangular cross-section and a head-tail-tail-head structure. The viscoelastic behavior of the hydrogels formed at the highest pH was investigated by rheology measurements.

Block copolymers as bile salt sequestrants: intriguing structures formed in a mixture of an oppositely charged amphiphilic block copolymer and bile salt.

To study the formation and characterize the structure of mixed complexes of oppositely charged block copolymers and surfactants are of great significance for practical applications, e.g., in drug carrier formulations that are based on electrostatically assisted assembly. In this context, biocompatible block copolymers and biosurfactants (like bile salts) are particularly interesting. In this work, we report on the co-assembly in dilute aqueous solution between a cationic poly(N-isopropyl acryl amide) (PNIPAM) diblock copolymer and the oppositely charged bile salt surfactant sodium deoxycholate at ambient temperature. The cryogenic transmission electron microscopy (cryo-TEM) experiments revealed the co-existence of two types of co-assembled complexes of radically different morphology and inner structure. They are formed mainly as a result of the electrostatic attraction between the positively charged copolymer blocks and bile salt anions and highlight the potential of using linear amphiphilic block copolymers as bile salt sequestrants in the treatment of bile acid malabsorption and hypercholesterolemia. The first complex of globular morphology has a coacervate core of deoxycholate anions and charged copolymer blocks surrounded by a PNIPAM corona. The second complex has an intriguing tape-like supramolecular morphology of several micrometer in length that is striped in the direction of the long axis. A model is presented in which the stretched cationic blocks of several block copolymers interact electrostatically with the bile salt molecules that are associated to form a zipper-like structure. The tape is covered on both sides by the PNIPAM chains that stabilize the overall complex in solution. In addition to cryo-TEM, the mixed system was investigated in a range of molar charge fractions at a constant copolymer concentration by static light scattering, small angle X-ray scattering, and electrophoretic mobility measurements.

Cholinium-amino acid based ionic liquids: a new method of synthesis and physico-chemical characterization.

In the present work we report the synthesis and physico-chemical characterization in terms of the viscosity and density of a wide series of cholinium-amino acid based room temperature ionic liquids ([Ch][AA] RTILs). 18 different amino acids were used to obtain 14 room temperature ILs. Among the most common AAs, only valine did not form an RTIL but it is a liquid above 80 °C. With respect to the methods reported in the literature we propose a synthesis based on potentiometric titration which has several advantages such as shorter preparation time, stoichiometry within ±1%, very high yields (close to 100%), high reproducibility, and no use of organic solvents, thus being more environmentally friendly. We tried to prepare dianionic ILs with some AAs with two potentially ionisable groups but in all cases the salts were solids at room temperature. All the ILs were characterized by (1)H NMR to confirm the stoichiometry. Physico-chemical properties such as density, viscosity, refractive index and conductivity were measured as a function of temperature and correlated with empirical equations. The values were compared with the data already reported in the literature for some [Ch][AA] ILs. The thermal expansion coefficient αp and the molar volume Vm were also calculated from the experimental density values. Due to the high number of AAs explored and their structural heterogeneity we have been able to find some interesting correlations between the data obtained and the structural features of the AAs in terms of the alkyl chain length, hydrogen bonding ability, stacking and cyclization. Some parameters were also found to be in good agreement with those reported for other ILs. We think that these data can give an important contribution to the understanding of the structure-property relationship of ILs because they focused on the structural effect of the anions, while most data in the literature are focussed on the cations.

Self-assembling nanowires from a linear l,d-peptide conjugated to the dextran end group.

Preparation and characterization of a block-like l,d-octapeptide-dextran conjugate DEX29-(l-Val-d-Val)4 self-assembling into nanowire structures is reported. The conjugate was prepared by solid phase click-chemistry on an alkyne group N-terminus functionalized peptide with a regularly alternating enantiomeric sequence. Low molecular weight dextran (Xn = 29) with moderately low dispersity (1.30) was prepared by controlled acid hydrolysis and dialysis with selected cut-off and functionalized with an azido group on the reducing end by reductive amination. The strong hydrogen bonds and hydrophobic interactions of the (l-Val-d-Val)4 linear peptide drive the conjugate to self-assemble into long (0.1-1 µm) nanowires. To our knowledge, this is the first example of a peptide-polysaccharide conjugate that can self-assemble into a nanowire architecture.

Chitosan nanogels by template chemical cross-linking in polyion complex micelle nanoreactors.

Chitosan covalent nanogels cross-linked with genipin were prepared by template chemical cross-linking of chitosan in polyion complex micelle (PIC) nanoreactors. By using this method, we were able to prepare chitosan nanogels using only biocompatible materials without organic solvents. PIC were prepared by interaction between chitosan (X(n) = 23, 44, and 130) and block copolymer poly(ethylene oxide)-block-poly[sodium 2-(acrylamido)-2-methylpropanesulfonate] (PEO-b-PAMPS) synthesized by single-electron transfer-living radical polymerization (SET-LRP). PIC with small size (diameter about 50 nm) and low polydispersity were obtained up to 5 mg/mL. After cross-linking of chitosan with genipin, the nanoreactors were dissociated by adding NaCl. The dissociation of the nanoreactors and the formation of the nanogels were confirmed by (1)H NMR, DLS, and TEM. The size of the smallest nanogels was about 50 nm in the swollen state and 20 nm in the dry state. The amount of genipin used during reticulation was an important parameter to modulate the size of the nanogels in solution.

Dielectric properties of thermo-reversible hydrogels: the case of a dextran copolymer grafted with poly(N-isopropylacrylamide).

We investigated the dielectric properties of aqueous solutions of a grafted copolymer, consisting of a polysaccharide, Dextran, grafted with a thermo-sensitive polymer, poly(N-isopropylacrylamide), [pNIPAAM], over broad temperature and frequency ranges. The graft copolymers, prepared by atom-transfer radical polymerization [ATRP], form temperature-responsive materials that represent a class of self-assembled structures in water of great interest because of their potential use as drug delivery formulations and in diverse biotechnological applications. In these systems, in the dilute regime and below the lower critical solution temperature, relaxation modes corresponding to two different length-scales have been observed and analyzed in terms of ion fluctuation dielectric models specifically developed to describe the dielectric relaxation in highly charged polyion aqueous solutions. Regardless of whether the ions were produced by the ionization of the polymer chain, as in polyelectrolyte solutions, or not, as in the present case, they represent a probe at a microscopic level that is expected to reveal the structural characteristics of the system at different scales. We have identified a characteristic length associated with the size of the polymer coil in the dilute regime and a length due to fixed cross-links, where ions are partially localized by the local profile of the Coulombic field. These lengths are in reasonable agreement with analogous lengths derived from structural information and from the hydrodynamic radius of the polymer coils, measured by means of a dynamic light-scattering technique.

Switchable length nanotubes from a self-assembling pH and thermosensitive linear l,d-peptide-polymer conjugate.

Preparation and characterization of a pH and thermosensitive linear l,d-octapeptide-poly(dimethylamino ethyl methacrylate) ((l-Val-d-Val)4-PDMAEMA) conjugate is reported. The hydrophobic uncharged linear (l-Val-d-Val)4 octapeptide was designed to self-assemble in nanotubes by exploiting the tubular self-assembling properties of linear peptides with regularly alternating enantiomeric sequences. pH and thermosensitive PDMAEMA was obtained by atom transfer radical polymerization (ATRP). The conjugate was prepared by click-chemistry on the solid phase synthetized peptide. Because of the strong interactions between the peptide moieties, long single channel nanotubes (0.2-1.5 µm) are formed also at acidic pH with the fully charged polymer. At 25 °C and basic pH the size of the nanotubes did not change significantly. In basic conditions and temperature above the PDMAEMA lower critical solution temperature (LCST) a significant increase of the length of the nanotubes up to several micrometers is observed. The size is retained for several days after cooling back to room temperature. Sonication significantly reduces the nanotube length (0.2-0.5 µm) forming low polydisperse nanotubes. The elongation of the nanotubes is fully reversible by restoring acidic pH. This is the first example, to our knowledge, of thermosensitive peptide-polymer single channel nanotubes with length that can be varied from hundreds of nanometers to several micrometers.

Self-assembly and drug release study of linear l,d-oligopeptide-poly(ethylene glycol) conjugates.

The preparation and structural organisation of new bioinspired nanomaterials based on regular alternating enantiomeric sequence of tetra- and hexapeptides end-linked to poly(ethylene glycol) (PEG) is reported. The peptide moiety is composed of two or three repeats of l-Ala-d-Val units while the PEG has a molecular weight of 2kDa. The self-assembling properties of the two conjugates depend significantly on the length of the peptide. Nanoparticles with different sizes and morphologies are formed, the structural properties of which are compared with the previously studied l-Ala-d-Val octapeptide conjugate that self-assembles into rod-like nanoparticles. The aggregation properties were studied by NMR, circular dichroism, fluorescence spectroscopies and dynamic light scattering. The morphology and size of the nanoparticles were assessed by scanning electron microscopy and dynamic light scattering. The loading and release of a model drug were also investigated. This study demonstrates that, by changing the length of the peptide, it is possible to modulate the self-assembly and loading properties of peptide-PEG conjugates.

PEGylated ß-Sheet Breaker Peptides as Inhibitors of ß-Amyloid Fibrillization.

Three PEGylated ß-sheet breaker peptides are designed as new inhibitors of ß-amyloid fibrillization. The peptide Ac-Leu-Pro-Phe-Phe-Asp-NH2 , considered the lead compound, and hexamers in which taurine and ß-alanine substitute the acetyl group, are conjugated to poly(ethylene glycol); this conjugates self-assemble into nanoparticles. The activity of the PEGylated peptides as inhibitors of amyloid fibrillization are tested in vitro using circular dichroism spectroscopy and scanning electron microscopy. The experimental results indicate that PEGylation does not impair the ability of the ß-sheet breaker peptides to inhibit fibrillogenesis in vitro. Moreover, microscopy images of ß-amyloid incubated for 6 days with the taurine-containing peptide, suggest that this conjugate has major anti-fibrillogenesis activity and demonstrate the important role of the sulfonamide function against the amyloid aggregation.

Hyaluronic acid and alginate covalent nanogels by template cross-linking in polyion complex micelle nanoreactors.

Hyaluronic acid (HA) and alginate (AL) covalent nanogels cross-linked with l-lysine ethyl ester were prepared by template chemical cross-linking of the polysaccharide in polyion complex micelle (PIC) nanoreactors. By using this method we were able to prepare HA and AL nanogels without organic solvents. PICs were prepared by using poly(ethylene oxide)-block-poly[(3-acrylamidopropyl)-trimethylammonium chloride] (PEO-b-PAMPTMA) or poly[(N-isopropylacrylamide)-block-PAMPTMA] (PNIPAAM-b-PAMPTMA). Only PNIPAAM-b-PAMPTMA block copolymers allowed to prepare PIC with small and controlled size. Short polysaccharide chains (Xn=50 and 63 for AL and HA, respectively, where Xn is the number of monosaccharidic units present in the polysaccharide) where used to optimize PIC formation. The remarkable difference in charge density and rigidity of HA and AL did not have a significant influence on the formation of PICs. PICs with small size (diameter of about 50-80 nm) and low polydispersity were obtained up to 5mg/mL of polymer. After cross-linking with l-lysine ethyl ester, the nanoreactors were dissociated by adding NaCl. The nanogels were easily purified and isolated by dialysis. The dissociation of the nanoreactors and the formation of the nanogels were confirmed by (1)H NMR, DLS, TEM and ζ-potential measurements. The size of the smallest nanogels in solution in the swollen state was 50-70 nm in presence of salt and 80-100 nm in water.

Dielectric properties of micellar aggregates due to the self-assembly of thermoresponsive diblock copolymers.

The radiowave dielectric properties of aqueous solutions of thermosensitive copolymers, consisting of poly(2-acrylamido-2-methylpropanesulfonate) [PAMPS] and poly(N-isopropylacrylamide) [PNIPAAM] with different block lengths, have been investigated over a broad temperature and frequency range. These copolymers PAMPS(n)-b-PNIPAAM(m) form temperature responsive aggregates (micelles) that represent a class of self-assembled structures in water of great interest because of their potential use as drug delivery formulations and in diverse biotechnological applications. Copolymers formed by hydrophilic segments covalently attached to a hydrophobic segments are capable of forming a micellar structure as soon as the temperature is raised above their lower critical solution temperature. We have investigated the dielectric properties of PAMPS(n)-b-PNIPAAM(m) diblock copolymers with different lengths of the hydrophilic and hydrophobic segments during the whole aggregation process driven by the progressive increase of temperature. The process has been followed by the changes resulting in the dielectric parameters (the dielectric increment Δε and the relaxation frequency ν(0)) of the whole aqueous solution. The dielectric response of the micelles has been described within the framework of the standard electrokinetic model for charged colloidal particles, and the main characteristic parameters have been evaluated. Subsequent cross-linking of these diblock copolymers by a cationic PEO(x)-b-PAMPTMA(y) polyelectrolyte yields hybrid core-shell-corona systems, with the PNIPAAM hydrophobic blocks collapsed in the core, an interpolyelectrolyte chain complex forming the shell, and the hydrophilic PEO chains as an external corona. In this case too, the dielectric spectra can be appropriately accounted for within the same theoretical framework.

Novel thermosensitive calcium alginate microspheres: physico-chemical characterization and delivery properties.

The system described in this paper was obtained by soaking calcium alginate (CaAlg) microspheres in a water solution of poly-[(3-acrylamidopropyl)-trimethylammonium chloride-b-N-isopropylacrylamide] [poly(AMPTMA-b-NIPAAM)], a new block co-polymer recently synthesized by atom transfer radical polymerization (ATRP). The block co-polymer is characterized by a lower critical solution temperature (LCST) of 41 degrees C in aqueous 0.1 M NaCl solution, and can be anchored on the CaAlg microspheres by means of polyion interactions. Polycations (permanently positively charged blocks) and polyanions (free alginate carboxylic groups) interact, leading to microspheres with thermosensitive properties. As an effect of interaction with the microspheres the LCST of the co-polymer is lowered to 36-38 degrees C. In this temperature range a colloidal water suspension of the microspheres collapses, forming macroscopic aggregates. The new system shows, at human body temperature, an improved ability to carry and deliver both hydrophobic and hydrophilic molecules in comparison with unmodified CaAlg microspheres. The release properties of the microspheres loaded with different model drugs can be appropriately modulated by the amount of the poly(AMPTMA-b-NIPAAM). Furthermore, the microspheres show the interesting capability of retaining the activity of a loaded enzyme (horseradish peroxidase), used as a model protein. The results obtained indicate that the proposed drug delivery system may be suitable for drug depot applications.

Versatile grafting of polysaccharides in homogeneous mild conditions by using atom transfer radical polymerization.

A versatile atom transfer radical polymerization (ATRP) method for polysaccharide grafting in homogeneous mild conditions without using protecting group chemistry is presented. Water/DMF mixtures with different compositions were used as the solvent. The "grafting-from" approach was used in order to prepare suitable pullulan and dextran ATRP macroinitiators with a well controlled degree of functionalization. Methacrylate and acrylamide monomers were grafted obtaining good control over the number, molecular weight and polydispersity of the grafted chains without homopolymer formation and polysaccharide degradation. The versatility of this method allowed us to prepare comblike derivatives with a wide range of properties (amphiphilic, ionic, and thermoresponsive) by simply changing the solvent composition and the catalyst. This could make possible the synthesis of new interesting biomaterials starting from a wide range of polysaccharides.

Solid-state 13C NMR study of poly(vinyl alcohol) gels.

Poly(vinyl alcohol) (PVA) with 55% and 61% syndiotacticity, and their related dry and hydrated gels obtained by two different freeze-thawing cycles have been investigated using the solid-state 13C CP-MAS NMR technique. From a comparative analysis of the spectra, evidence was obtained that the gelation process largely disrupts the intramolecular hydrogen-bonded network of the PVA. The addition of water to the dry gels favours their swelling, destroying intra-chain hydrogen bonds between hydroxyl groups as a function of the degree of tacticity and the gelation procedure, and promotes the formation of new networks of interchain hydrogen bonds. Information on the dynamics of the polymeric domains in the kilohertz range has been obtained from the analysis of the spin relaxation times T1rho(1H) and T1rho(13C) indicating that homogeneous arrangements of the amorphous or swollen polymeric chains exist, independent of the preparation method or the tacticity of the PVA chains.

A high field NMR study of the products ensuing from konjak glucomannan C(6)-oxidation followed by enzymatic C(5)-epimerization.

Konjak glucomannan (KGM) is a water-soluble linear copolymer of (1-->4) linked beta-D-mannopyranosyl and beta-D-glucopyranosyl units. It has been selectively C6-oxidized by a 2,2,6,6-tetramethylpiperidin-1-oxy mediated reaction to obtain the corresponding uronan. Oxidized KGM has been treated with three different C-5 epimerases, AlgE4, AlgE6, and AlgE1, to obtain uronans with a various content of alpha-L-gulopyranuronate residues, namely, KGME4, KGME6, and KGME1. By use of 1D selective and 2D NMR techniques, a full assignment of the high field (600 MHz) NMR spectra of the purified native KGM and of the oxidized and epimerized derivatives has been obtained. Since in the anomeric region of the (1)H NMR spectrum of native KGM, diads sensitivity is present, the glucose-glucose, glucose-mannose, mannose-mannose, and mannose-glucose distribution has been obtained. In the (13)C spectrum of oxidized KGM, due to the presence of triad sensitivity on the C-4 resonance of glucuronic and mannuronic units, a better sequential investigation has been possible. As a result the average length of mannuronic blocks, N(M) is obtained. When AlgE4, AlgE6, and AlgE1 enzymes are used for the epimerization of oxidized KGM, the reaction products differ significantly both in the proportion and in the distribution of the mannuronic and guluronic residues. In epimerized KGM derivatives, a careful deconvolution of (1)H spectra allows the measurement of the degree of epimerization. In the case of KGME1 and KGME6, the average blocks length, N(G), of the guluronic blocks introduced in the polysaccharidic chain with the epimerization has also been calculated. Due to the shortness of mannuronic blocks in the oxidized KGM before the epimerization, N(G) in the epimerized compounds is also very low.