Facile Photochemical Modification of Silk Protein-Based Biomaterials.

Silk protein-based materials show promise for application as biomaterials for tissue engineering. The simple and rapid photochemical modification of silk protein-based materials composed of either Bombyx mori silkworm silk or engineered spider silk proteins (eADF4(C16)) is reported. Radicals formed on the silk-based materials initiate the polymerization of monomers (acrylic acid, methacrylic acid, or allylamine) which functionalize the surface of the silk materials with poly(acrylic acid) (PAA), poly(methacrylic acid) (PMAA), or poly(allylamine) (PAAm). To demonstrate potential applications of this type of modification, the polymer-modified silks are mineralized. The PAA- and PMAA-functionalized silks are mineralized with calcium carbonate, whereas the PAAm-functionalized silks are mineralized with silica, both of which provide a coating on the materials that may be useful for bone tissue engineering, which will be the subject of future investigations.

Polyacrylamide "revisited": UCST-type reversible thermoresponsive properties in aqueous alcoholic solutions.

Combining experiments and all-atom molecular dynamics simulations, we study the conformational behavior of polyacrylamide (PAM) in aqueous alcohol mixtures over a wide range of temperatures. This study shows that even when the microscopic interaction is dictated by hydrogen bonding, unlike its counterparts that present a lower critical solution temperature (LCST), PAM shows a counterintuitive tunable upper critical solution temperature (UCST)-type phase transition in water/alcohol mixtures that was not reported before. The phase transition temperature was found to be tunable between 4 and 60 °C by the type and concentration of alcohol in the mixture as well as by the solution concentration and molecular weight of the polymer. In addition, molecular dynamics simulations confirmed a UCST-like behaviour of the PAM in aqueous alcoholic solutions. Additionally, it was observed that the PAM is more swollen in pure alcohol solutions than in 80% alcoholic solutions due to θ-like behaviour. Additionally, in the globular state, the size of the aggregates was found to increase with increasing solvent hydrophobicity and polymer concentration of the solutions. Above its phase transition temperature, PAM might be present as individual polymer chains in the coil state (≤10 nm). As PAM is a widespread polymer in many biomedical applications (gel electrophoresis, etc.), this finding could be of high relevance for many more practical applications in high performance pharmaceuticals and/or sensors.

Hybrid Silicon-Based Organic/Inorganic Block Copolymers with Sol-Gel Active Moieties: Synthetic Advances, Self-Assembly and Applications in Biomedicine and Materials Science.

Hybrid silicon-based organic/inorganic (multi)block copolymers are promising polymeric precursors to create robust nano-objects and nanomaterials due to their sol-gel active moieties via self-assembly in solution or in bulk. Such nano-objects and nanomaterials have great potential in biomedicine as nanocarriers or scaffolds for bone regeneration as well as in materials science as Pickering emulsifiers, photonic crystals or coatings/films with antibiofouling, antibacterial or water- and oil-repellent properties. Thus, this Review outlines recent synthetic efforts in the preparation of these hybrid inorganic/organic block copolymers, gives an overview of their self-assembled structures and finally presents recent examples of their use in the biomedical field and material science.

A Dendritic Amphiphile for Efficient Control of Biomimetic Calcium Phosphate Mineralization.

The phase behavior of a dendritic amphiphile containing a Newkome-type dendron as the hydrophilic moiety and a cholesterol unit as the hydrophobic segment is investigated at the air-liquid interface. The amphiphile forms stable monomolecular films at the air-liquid interface on different subphases. Furthermore, the mineralization of calcium phosphate beneath the monolayer at different calcium and phosphate concentrations versus mineralization time shows that at low calcium and phosphate concentrations needles form, whereas flakes and spheres dominate at higher concentrations. Energy-dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, and electron diffraction confirm the formation of calcium phosphate. High-resolution transmission electron microscopy and electron diffraction confirm the predominant formation of octacalcium phosphate and hydroxyapatite. The data also indicate that the final products form via a complex multistep reaction, including an association step, where nano-needles aggregate into larger flake-like objects.

Dielectric analysis of the upper critical solution temperature behaviour of a poly(acrylamide-co-acrylonitrile) copolymer system in water.

A copolymer consisting of acrylamide (AAm) and acrylonitrile (AN) in aqueous solution was investigated using broadband dielectric spectroscopy at frequencies between 10-1 Hz and 106 Hz in the temperature range from 2 °C to 60 °C. This system shows an UCST phase behavior. The phase transition and aggregation behavior is monitored by both the temperature and frequency dependence of the complex conductivity σ*(f, T), where the AN fraction and the concentration of the solution were varied. Additionally, the dielectric data are compared with the results obtained from dynamic light scattering measurements. The temperature dependence of the DC conductivity (σDC) of the copolymer solution is monitored and the phase transition temperature (PTT) of the poly(AAm-co-AN) copolymer is deduced from a change in the T-dependence of the DC conductivity. The change in σDC can be explained by decreased effective charge carrier mobility and a reduction of the effective charge number density at temperatures below the phase transition temperature of the poly(AAm-co-AN) solution. A pronounced interfacial polarization effect on the frequency dependence of the real part of the conductivity (σ') is observed at temperatures below the phase transition temperature. The charge carriers are blocked at the formed aggregates giving rise to this interfacial polarization. The dependence of the interfacial polarization on the acrylonitrile fraction in the copolymer and the concentration of the solution is studied in detail and conclusions concerning the internal structures of the copolymer aggregates are drawn.

Vesicles from Amphiphilic Dumbbells and Janus Dendrimers: Bioinspired Self-Assembled Structures for Biomedical Applications.

The current review focuses on vesicles obtained from the self-assembly of two types of dendritic macromolecules, namely amphiphilic Janus dendrimers (forming dendrimersomes) and amphiphilic dumbbells. In the first part, we will present some synthetic strategies and the various building blocks that can be used to obtain dendritic-based macromolecules, thereby showing their structural versatility. We put our focus on amphiphilic Janus dendrimers and amphiphilic dumbbells that form vesicles in water but we also encompass vesicles formed thereof in organic solvents. The second part of this review deals with the production methods of these vesicles at the nanoscale but also at the microscale. Furthermore, the influence of various parameters (intrinsic to the amphiphilic JD and extrinsic-from the environment) on the type of vesicle formed will be discussed. In the third part, we will review the numerous biomedical applications of these vesicles of nano- or micron-size.

Phase transition and aggregation behaviour of an UCST-type copolymer poly(acrylamide-co-acrylonitrile) in water: effect of acrylonitrile content, concentration in solution, copolymer chain length and presence of electrolyte.

An UCST-type copolymer of acrylamide (AAm) and acrylonitrile (AN) (poly(AAm-co-AN)) was prepared by reversible addition fragmentation chain transfer (RAFT) polymerization and its temperature-induced phase transition and aggregation behaviour studied by turbidimetry, static and dynamic light scattering, small angle neutron scattering (SANS) and cryo-transmission electron microscopy (cryo-TEM) measurements. The phase transition temperature was found to increase with increasing AN content in the copolymer, concentration of the solutions and copolymer chain length. A significant effect was observed onto the phase transition temperature by addition of different electrolytes into the copolymer solution. The copolymer chains were aggregated below the phase transition temperature and disaggregated above it. The size of the aggregates increases with increasing AN contents and concentration of the copolymer solutions below the phase transition temperature. The copolymer chains were expanded and weekly associated in solution above the phase transition temperature. A model is proposed to explain such association-aggregation behaviour of poly(AAm-co-AN) copolymers depending on AN contents and concentration of the copolymer solutions as a function of temperature.

Tuning the Surface of Nanoparticles: Impact of Poly(2-ethyl-2-oxazoline) on Protein Adsorption in Serum and Cellular Uptake.

Due to the adsorption of biomolecules, the control of the biodistribution of nanoparticles is still one of the major challenges of nanomedicine. Poly(2-ethyl-2-oxazoline) (PEtOx) for surface modification of nanoparticles is applied and both protein adsorption and cellular uptake of PEtOxylated nanoparticles versus nanoparticles coated with poly(ethylene glycol) (PEG) and non-coated positively and negatively charged nanoparticles are compared. Therefore, fluorescent poly(organosiloxane) nanoparticles of 15 nm radius are synthesized, which are used as a scaffold for surface modification in a grafting onto approach. With multi-angle dynamic light scattering, asymmetrical flow field-flow fractionation, gel electrophoresis, and liquid chromatography-mass spectrometry, it is demonstrated that protein adsorption on PEtOxylated nanoparticles is extremely low, similar as on PEGylated nanoparticles. Moreover, quantitative microscopy reveals that PEtOxylation significantly reduces the non-specific cellular uptake, particularly by macrophage-like cells. Collectively, studies demonstrate that PEtOx is a very effective alternative to PEG for stealth modification of the surface of nanoparticles.

Temperature-Triggered Protein Adsorption on Polymer-Coated Nanoparticles in Serum.

The protein corona, which forms on the nanoparticle's surface in most biological media, determines the nanoparticle's physicochemical characteristics. The formation of the protein corona has a significant impact on the biodistribution and clearance of nanoparticles in vivo. Therefore, the ability to influence the formation of the protein corona is essential to most biomedical applications, including drug delivery and imaging. In this study, we investigate the protein adsorption on nanoparticles with a hydrodynamic radius of 30 nm and a coating of thermoresponsive poly(2-isopropyl-2-oxazoline) in serum. Using multiangle dynamic light scattering (DLS) we demonstrate that heating of the nanoparticles above their phase separation temperature induces the formation of agglomerates, with a hydrodynamic radius of 1 µm. In serum, noticeably stronger agglomeration occurs at lower temperatures compared to serum-free conditions. Cryogenic transmission electron microscopy (cryo-TEM) revealed a high packing density of agglomerates when serum was not present. In contrast, in the presence of serum, agglomerated nanoparticles were loosely packed, indicating that proteins are intercalated between them. Moreover, an increase in protein content is observed upon heating, confirming that protein adsorption is induced by the alteration of the surface during phase separation. After cooling and switching the surface back, most of the agglomerates were dissolved and the main fraction returned to the original size of approximately 30 nm as shown by asymmetrical flow-field flow fractionation (AF-FFF) and DLS. Furthermore, the amounts of adsorbed proteins are similar before and after heating the nanoparticles to above their phase-separation temperature. Overall, our results demonstrate that the thermoresponsivity of the polymer coating enables turning the corona formation on nanoparticles on and off in situ. As the local heating of body areas can be easily done in vivo, the thermoresponsive coating could potentially be used to induce the agglomeration of nanoparticles and proteins and the accumulation of nanoparticles in a targeted body region.

Self-assembly of amphiphilic Janus dendrimers into uniform onion-like dendrimersomes with predictable size and number of bilayers.

A constitutional isomeric library synthesized by a modular approach has been used to discover six amphiphilic Janus dendrimer primary structures, which self-assemble into uniform onion-like vesicles with predictable dimensions and number of internal bilayers. These vesicles, denoted onion-like dendrimersomes, are assembled by simple injection of a solution of Janus dendrimer in a water-miscible solvent into water or buffer. These dendrimersomes provide mimics of double-bilayer and multibilayer biological membranes with dimensions and number of bilayers predicted by the Janus compound concentration in water. The simple injection method of preparation is accessible without any special equipment, generating uniform vesicles, and thus provides a promising tool for fundamental studies as well as technological applications in nanomedicine and other fields.

"Single-single" amphiphilic janus dendrimers self-assemble into uniform dendrimersomes with predictable size.

An accelerated modular synthesis of six libraries containing 29 amphiphilic Janus dendrimers, employed to discover and predict functions via primary structures, is reported. These dendrimers were constructed from a single hydrophobic and a single hydrophilic dendron, interconnected with l-Ala to form two constitutional isomeric libraries, with Gly to produce one library, and with l-propanediol ester to generate two additional constitutional isomeric libraries. They are denoted "single-single" amphiphilic Janus dendrimers. Assemblies obtained by injection of their ethanol solution into water were analyzed by dynamic light scattering and cryogenic transmission electron microscopy. A diversity of complex structures including soft and hard dendrimersomes, cubosomes, solid lamellae, and rod-like micelles were obtained in water. It was discovered that the "single-single" amphiphilic Janus dendrimers containing three triethylene glycol groups in the hydrophilic dendron favored the formation of dendrimersomes. Assemblies in bulk analyzed by differential scanning calorimetry and powder X-ray diffraction revealed that the amphiphilic Janus dendrimers with melting point or glass transition below room temperature self-assemble into soft dendrimersomes in water, while those with higher temperature transitions produce hard assemblies. In the range of concentrations where their size distribution is narrow, the diameter of the dendrimersomes is predictable by the d-spacing of their assemblies in bulk. These results suggested the synthesis of Library 6 containing two simpler constitutional isomeric benzyl ester based amphiphilic Janus dendrimers that self-assemble in water into soft dendrimersomes and multidendrimersome dendrimersomes with predictable dimensions.

Modular synthesis of amphiphilic Janus glycodendrimers and their self-assembly into glycodendrimersomes and other complex architectures with bioactivity to biomedically relevant lectins.

The modular synthesis of 7 libraries containing 51 self-assembling amphiphilic Janus dendrimers with the monosaccharides D-mannose and D-galactose and the disaccharide D-lactose in their hydrophilic part is reported. These unprecedented sugar-containing dendrimers are named amphiphilic Janus glycodendrimers. Their self-assembly by simple injection of THF or ethanol solution into water or buffer and by hydration was analyzed by a combination of methods including dynamic light scattering, confocal microscopy, cryogenic transmission electron microscopy, Fourier transform analysis, and micropipet-aspiration experiments to assess mechanical properties. These libraries revealed a diversity of hard and soft assemblies, including unilamellar spherical, polygonal, and tubular vesicles denoted glycodendrimersomes, aggregates of Janus glycodendrimers and rodlike micelles named glycodendrimer aggregates and glycodendrimermicelles, cubosomes denoted glycodendrimercubosomes, and solid lamellae. These assemblies are stable over time in water and in buffer, exhibit narrow molecular-weight distribution, and display dimensions that are programmable by the concentration of the solution from which they are injected. This study elaborated the molecular principles leading to single-type soft glycodendrimersomes assembled from amphiphilic Janus glycodendrimers. The multivalency of glycodendrimersomes with different sizes and their ligand bioactivity were demonstrated by selective agglutination with a diversity of sugar-binding protein receptors such as the plant lectins concanavalin A and the highly toxic mistletoe Viscum album L. agglutinin, the bacterial lectin PA-IL from Pseudomonas aeruginosa, and, of special biomedical relevance, human adhesion/growth-regulatory galectin-3 and galectin-4. These results demonstrated the candidacy of glycodendrimersomes as new mimics of biological membranes with programmable glycan ligand presentations, as supramolecular lectin blockers, vaccines, and targeted delivery devices.

Predicting the size and properties of dendrimersomes from the lamellar structure of their amphiphilic Janus dendrimers.

Dendrimersomes are stable, monodisperse unilamellar vesicles self-assembled in water from amphiphilic Janus dendrimers. Their size, stability, and membrane structure are determined by the chemical structure of Janus dendrimer and the method of self-assembly. Comparative analysis of the periodic arrays in bulk and dendrimersomes assembled by ethanol injection in water of 11 libraries containing 108 Janus dendrimers is reported. Analysis in bulk and in water was performed by differential scanning calorimetry, X-ray diffraction, dynamic light scattering, and cryo-TEM. An inverse proportionality between size, stability, mechanical properties of dendrimersomes, and thickness of their membrane was discovered. This dependence was explained by the tendency of alkyl chains forming the hydrophobic part of the dendrimersome to produce the same local packing density regardless of the branching pattern from the hydrophobic part of the dendrimer. For the same hydrophobic alkyl chain length, the largest, toughest, and most stable dendrimersomes are those with the thinnest membrane that results from the interdigitation of the alkyl groups of the Janus dendrimer. A simplified spherical-shell model of the dendrimersome was used to demonstrate the direct correlation between the concentration of Janus dendrimer in water, c, and the size of self-assembled dendrimersome. This concentration-size dependence demonstrates that the mass of the vesicle membrane is proportional with c. A methodology to predict the size of the dendrimersome based on this correlation was developed. This methodology explains the inverse proportionality between the size of dendrimersome and its membrane thickness, and provides a good agreement between the experimental and predicted size of dendrimersome.

Dendronized iron oxide nanoparticles for multimodal imaging.

The synthesis of small-size dendrons and their grafting at the surface of iron oxide nanoparticles were achieved with the double objective to obtain a good colloidal stability with a mean hydrodynamic diameter smaller than 100 nm and to ensure the possibility of tuning the organic coating characteristics including morphology, functionalities, physico-chemical properties, grafting of fluorescent or targeting molecules. Magnetic resonance and fluorescence imaging are then demonstrated to be simultaneously possible using such versatile superparamagnetic iron oxide nanocrystals covered by a dendritic shell displaying either carboxylate or ammonium groups at their periphery which could be further labelled with a fluorescent dye. The grafting conditions of these functionalized dendrons at the surface of SPIO NPs synthesized by co-precipitation have been optimized as a function of the nature of the peripheral functional group. The colloidal stability has been investigated in water and osmolar media, and in vitro and in vivo MRI and optical imaging measurements have been performed showing encouraging biodistribution.

In vitro neurotoxicity of magnetic resonance imaging (MRI) contrast agents: influence of the molecular structure and paramagnetic ion.

Interest in contrast agent's (CA) neurotoxicity has greatly increased due to the growing need of new compounds dedicated to brain imaging. Magnetic resonance imaging (MRI) CA have been evaluated by means of different toxicological assays with cultured rat primary neurons (evaluation of neurite specific parameters via immunostaining of the cells and LDH leakage). To determine the potential neurotoxicity of a precise paramagnetic ion in a defined structure (architecture and molecular weight), novel hydrosoluble dendritic Manganese (II) and Gadolinium (III) complexes derived from diethylenetriamine pentaacetic acid (DTPA) have been studied and compared to a linear homologue (same molecular weight) and commercially available low molecular weight MRI CA like Mn-DPDP (Teslascan, GE Healthcare) and Gd-DTPA (Magnevist, Schering). The range of CA concentrations studied was 0.1-10mM, suitable for MRI examinations. This set of experiments allows a toxicity ranking of these reagents as a function of molecular structure and nature of the paramagnetic ion. We could determine that the architecture (linear vs. dendritic) does not play an important role in the in vitro neurotoxicity, whereas the structure of the chelating cage is of greater importance.

Poly(2-oxazoline)s as Smart Bioinspired Polymers.

Poly(2-alkyl-2-oxazoline)s can be regarded as pseudo-peptides or bioinspired polymers, which are available through living/controlled cationic polymerization and polymer ("click") modification procedures. Materials and solution properties may be adjusted via the nature of the side chain (hydrophilic-hydrophobic, chiral, bio-functional, etc.), opening the way to stimulus-responsive materials and complex colloidal structures in aqueous environments. Herein, we give an overview over the macromolecular engineering of polyoxazolines, including the synthesis of biohybrids, and the "smart"/bioinspired aggregation behavior in solution.

Development of a dendritic manganese-enhanced magnetic resonance imaging (MEMRI) contrast agent: synthesis, toxicity (in vitro) and relaxivity (in vitro, in vivo) studies.

A new dendritic manganese(II) chelate 1 has been evaluated by in vivo (relaxivity) and in vitro (toxicity and relaxivity) experiments as a manganese enhanced magnetic resonance imaging (MEMRI) contrast agent. Also, a comparison with its corresponding gadolinium(III) homologue 2 and the commercially available MEMRI agent MnDPDP (Teslascan, Amersham Health) was achieved in order to determine respectively the real influence of the paramagnetic ion in terms of toxicity and relaxivity for this precise treelike structure and the potential of 1 to be a favorable candidate for brain-targeting MRI. Complexes 1 and 2 displayed high hydrosolubility (0.1 M) and revealed no in vitro neuronal toxicity at concentrations as high as 1 mM. Considering manganese(II) complex 1, the in vivo nontoxicity at 20 mM (100% rats survival) is very likely due to a slow diffusion of the compound, meaning a controlled release of the paramagnetic ions. Finally, T(1) relaxivity of 4.2 mM(-1).s(-1) for 2 and T(2) relaxivity of 17.4 mM(-1).s(-1) for 1 at 4.7 T were measured and are higher than that of the commercial MRI contrast agents GdDTPA and MnDPDP, respectively.