Confinement Assembly of Terpolymer-Based Janus Nanoparticles.

While the confinement assembly of block copolymers into functional microparticles has been extensively studied, little is known about the behavior of Janus nanoparticles (JNPs) in spherical confinement. Here, the confinement self-assembly of JNPs in drying emulsion droplets is investigated and their behavior compared to their ABC triblock terpolymer precursors. Emulsions of both materials are prepared using Shirasu Porous Glass membranes leading to narrow size distributions of the microparticles with average hydrodynamic radii in the range of Rh  = 250-500 nm (depending on the membrane pore radius, Rpore ). The internal structure of the microparticles is verified with transmission electron microscopy (TEM) on ultrathin cross sections and compared to the corresponding bulk morphologies. While the confinement assembly of terpolymers results in microparticles with ordered inner morphologies, order for JNPs diminishes when the Janus balance deviates from parity.

Controlling the Surface Functionalization of Ultrasmall Gold Nanoparticles by Sequence-Defined Macromolecules.

Ultrasmall gold nanoparticles (diameter about 2 nm) were surface-functionalized with cysteine-carrying precision macromolecules. These consisted of sequence-defined oligo(amidoamine)s (OAAs) with either two or six cysteine molecules for binding to the gold surface and either with or without a PEG chain (3400 Da). They were characterized by 1 H NMR spectroscopy, 1 H NMR diffusion-ordered spectroscopy (DOSY), small-angle X-ray scattering (SAXS), and high-resolution transmission electron microscopy. The number of precision macromolecules per nanoparticle was determined after fluorescent labeling by UV spectroscopy and also by quantitative 1 H NMR spectroscopy. Each nanoparticle carried between 40 and 100 OAA ligands, depending on the number of cysteine units per OAA. The footprint of each ligand was about 0.074 nm2 per cysteine molecule. OAAs are well suited to stabilize ultrasmall gold nanoparticles by selective surface conjugation and can be used to selectively cover their surface. The presence of the PEG chain considerably increased the hydrodynamic diameter of both dissolved macromolecules and macromolecule-conjugated gold nanoparticles.

Capsules from synthetic diblock-peptides as potential artificial oxygen carriers.

The design of an encapsulation system consisting of a synthetic peptide which is fully biodegradable into non-toxic constituents. This system should be capable of encapsulating perfluorinated hydrocarbons and should be a promising basis for oxygen carriers to be used as artificial blood replacement. A diblock-peptide is synthesised following a phosgene-free method and characterised by 1H-NMR. Subsequently, this diblock-peptide is self-assembled with perfluorodecalin (PFD) to form PFD-filled capsules as potential artificial oxygen carriers allowing for rapid oxygen uptake and release. The diblock-peptide Bu-PAsp10-PPhe10 is successfully synthesised and used to encapsulate PFD. The capsules have a spherical shape with an average diameter of 360 nm in stable aqueous dispersion. NMR measurements prove their physical capability for reversible uptake and release of oxygen. The resulting capsules are expected to be fully biodegradable and possibly could act as oxygen carriers for artificial blood replacement.

Synthesis and characterisation of aqueous haemoglobin-based microcapsules coated by genipin-cross-linked albumin.

Bovine serum albumin (BSA)-coated haemoglobin (Hb)-microcapsules prepared by co-precipitation of Hb and MnCO3 may present an alternative type of artificial blood substitute. Prepared microcapsules were analysed by Scanning electron microscopy (SEM) and Respirometry, cytotoxicity was evaluated by addition of microcapsules to murine fibroblast-derived cell line L929 (American Type Culture Collection, NCTC clone 929 of strain L). The capsules come along with a mean diameter of approximately 0.6 µm and a mean volume of 1.13 × 10-19 L, thus an average human red blood cell with a volume of 9 × 10-14 L is about 800,000 times bigger. Hb-microcapsules are fully regenerable by ascorbic acid and maintain oxygen affinity because oxygen is able to pass the BSA wall of the capsules and thereby binding to the ferrous iron of the haemoglobin entity. Therefore, these microcapsules present a suitable type of potential artificial haemoglobin-based oxygen carrier (HbOC).

Cross-linked Triblock Peptide Capsules as Potential Oxygen Carriers.

Perfluorodecalin (PFD)-filled capsules have been studied for over 15 years as artificial oxygen carriers. However, none of these capsules combines good biocompatibility, good mechanical stability and dispersion stability. Here we propose to use synthetic triblock peptides containing a central block of cysteine units as a cross-linking shell material for capsules with both good biocompatibility and stability. Together with outer aspartate units and inner phenylalanine units, the resulting amphiphilic triblock peptides can encapsulate PFD efficiently to prepare capsules with a suitable diameter, a certain mechanical strength, a large diffusion constant, fast gas exchange rates, and little cytotoxicity. Given the above advantages, these PFD-filled peptide capsules are very promising as potential artificial oxygen carriers.

Synthesis and characterization of enzymatically active micrometer protein-capsules.

This work describes a general method for the encapsulation of enzymes with albumin as wall material and the enzyme catalase as prime example. Care was taken for the preparation of biochemically active sub-micrometer particles in order to prevent oxygen toxicity induced by artificial oxygen carriers of any type. In cell culture experiments, capsules containing catalase did not exhibit any harmful activities in the absence of peroxides. In the presence of hydrogen peroxide application of low and medium dosed capsules below 0.05 vol% (final concentration 0.001 vol%) even increased the cell damaging process. However, a higher dosage of capsules (>0.05 vol%) prevented completely cellular disruption induced by 5 mM hydrogen peroxide and decreased up to 90% of cellular damage at higher peroxide concentrations. These results demonstrated that encapsulated catalase was enzymatically active and the over-all activity of prepared catalase capsules was determined to be >1900 U mL-1 vol%-1.

A supramolecular double-helix based on complementary phosphate-guanidinium pairing.

A double-helical supramolecular structure was formed by self-assembly of 1,1'-binaphthyl-based bisguanidines and bisphosphoric acids. Interestingly the homochiral (S,S) + (S,S)-pair forms a left-handed double-helix, while the heterochiral (S,S) + (R,R)-pair forms a non-helical dimer.

Characterization and Optimization of AZO Nanoparticles as Coatings for Flexible Substrates toward High IR Reflectivity.

Energy consumption by air-conditioning is expansive and leads to the emission of millions of tons of CO2 every year. A promising approach to circumvent this problem is the reflection of solar radiation: Rooms that would not heat up by irradiation will not need to be cooled down. Especially, transparent conductive metal oxides exhibit high infrared (IR) reflectivity and are commonly applied as low-emissivity coatings (low-e coatings). Indium tin oxide (ITO) coatings are the state-of-the-art application, though indium is a rare and expensive resource. This work demonstrates that aluminum-doped zinc oxide (AZO) can be a suitable alternative to ITO for IR-reflection applications. AZO synthesized here exhibits better emissivity to be used as roofing membrane coatings for buildings in comparison to commercially available ITO coatings. AZO particles forming the reflective coating are generated via solvothermal synthesis routes and obtain high conductivity and IR reflectivity without the need of any further post-thermal treatment. Different synthesis parameters were studied, and their effects on both conductive and optical properties of the AZO nanoparticles were evaluated. To this end, a series of characterization methods, especially 27Al-nuclear magnetic resonance spectroscopy (27Al-NMR) analysis, have been conducted for a deeper insight into the particles' structure to understand the differences in conductivity and optical properties. The optimized AZO nanoparticles were coated on flexible transparent textile-based roofing membranes and tested as low-e coatings. The membranes demonstrated higher thermal reflectance compared with commercial ITO materials with an emissivity value lowered by 16%.

Accelerated trypsin autolysis by affinity polymer templates.

Self-cleavage of proteins is an important natural process that is difficult to control externally. Recently a new mechanism for the accelerated autolysis of trypsin was discovered involving polyanionic template polymers; however it relies on unspecific interactions and is inactive at elevated salt loads. We have now developed affinity copolymers that bind to the surface of proteases by specific recognition of selected amino acid residues. These are highly efficient trypsin inhibitors with low nanomolar IC50 levels and operate at physiological conditions. In this manuscript we show how these affinity copolymers employ the new mechanism of polymer-assisted self-digest (PAS) and act as a template for multiple protease molecules. Their elevated local concentration leads to accelerated autolysis on the accessible surface area and shields complexed areas. The resulting extremely efficient trypsin inhibition was studied by SDS-PAGE, gel filtration, CD, CZE and ESI-MS. We also present a simple theoretical model that simulates most experimental findings and confirms them as a result of multivalency and efficient reversible templating. For the first time, mass spectrometric kinetic analysis of the released peptide fragments gives deeper insight into the underlying mechanism and reveals that polymer-bound trypsin cleaves much more rapidly with low specificity at predominantly uncomplexed surface areas.

Prevention of Decompression Sickness by Novel Artificial Oxygen Carriers.

For three decades, studies have demonstrated the therapeutic efficacy of perfluorocarbon (PFC) in reducing the onset of decompression trauma. However, none of these emulsion-based preparations are accepted for therapeutic use in the western world, mainly because of severe side effects and a long organ retention time. A new development to guarantee a stable dispersion without these disadvantages is the encapsulation of PFC in nanocapsules with an albumin shell. PURPOSE: Newly designed albumin-derived perfluorocarbon-based artificial oxygen carriers (A-AOC) are used in a rodent in vivo model as a preventive therapy for decompression sickness (DCS). METHODS: Thirty-seven rats were treated with A-AOC (n = 12), albumin nanocapsules filled with neutral oil (A-O-N, n = 12), or 5% human serum albumin solution (A-0-0, n = 13) before a simulated dive. Eleven rats, injected with A-AOC, stayed at normal pressure (A-AOC surface). Clinical, laboratory, and histological evaluations were performed. RESULTS: The occurrence of DCS depended on the treatment group. A-AOC significantly reduced DCS appearance and mortality. Furthermore, a significant improvement of survival time was found (A-AOC compared with A-0-0). Histological assessment of A-AOC-dive compared with A-0-0-dive animals revealed significantly higher accumulation of macrophages, but less blood congestion in the spleen and significantly less hepatic circulatory disturbance, vacuolization, and cell damage. Compared with nondiving controls, lactate and myoglobin showed a significant increase in the A-0-0- but not in the A-AOC-dive group. CONCLUSION: Intravenous application of A-AOC was well tolerated and effective in reducing the occurrence of DCS, and animals showed significantly higher survival rates and less symptoms compared with the albumin group (A-0-0). Analysis of histological results and fast reacting plasma parameters confirmed the preventive properties of A-AOC.

Functionality of albumin-derived perfluorocarbon-based artificial oxygen carriers in the Langendorff-heart †.

The aim of this study was to prove whether albumin-derived perfluorocarbon-based nanoparticles (capsules) can operate as a novel artificial oxygen carrier in a rat Langendorff-heart perfusion model. Hearts perfused with capsules showed increased left ventricular pressure and rate pressure product compared to hearts perfused with pure Krebs-Henseleit (KH)-buffer. The capsules prevented the myocardium from functional fail when in their absence a noxious ischemia was observed. Capsules did not change rheological properties of KH-buffer and could repeatedly reload with oxygen. This albumin-derived perfluorocarbon-based artificial oxygen carrier preserved the function of rat hearts due to the transport of oxygen in a satisfactory manner. Because of these positive results, the functionality of the applied capsules should be verified in living animals.

Albumin-derived perfluorocarbon-based artificial oxygen carriers: A physico-chemical characterization and first in vivo evaluation of biocompatibility.

Until today, artificial oxygen carriers have not been reached satisfactory quality for routine clinical treatments. To bridge this gap, we designed albumin-derived perfluorocarbon-based nanoparticles as novel artificial oxygen carriers and evaluated their physico-chemical and pharmacological performance. Our albumin-derived perfluorocarbon-based nanoparticles (capsules), composed of an albumin shell and a perfluorodecalin core, were synthesized using ultrasonics. Their subsequent analysis by physico-chemical methods such as scanning electron-, laser scanning- and dark field microscopy as well as dynamic light scattering revealed spherically-shaped, nano-sized particles, that were colloidally stable when dispersed in 5% human serum albumin solution. Furthermore, they provided a remarkable maximum oxygen capacity, determined with a respirometer, reflecting a higher oxygen transport capacity than the competitor Perftoran®. Intravenous administration to healthy rats was well tolerated. Undesirable effects on either mean arterial blood pressure, hepatic microcirculation (determined by in vivo microscopy) or any deposit of capsules in organs, except the spleen, were not observed. Some minor, dose-dependent effects on tissue damage (release of cellular enzymes, alterations of spleen's micro-architecture) were detected. As our promising albumin-derived perfluorocarbon-based nanoparticles fulfilled decisive physico-chemical demands of an artificial oxygen carrier while lacking severe side-effects after in vivo administration they should be advanced to functionally focused in vivo testing conditions.

Insertion stability of poly(ethylene glycol)-cholesteryl-based lipid anchors in liposome membranes.

Liposomes consist of a hydrophilic core surrounded by a phospholipid (PL) bilayer. In human blood, the half-life of such artificial vesicles is limited. To prolong their stability in the circulation, liposomal bilayers can be modified by inserting poly(ethylene glycol) (PEG) molecules using either PL or sterols as membrane anchors. This establishes a hydrophilic steric barrier, reducing the adsorption of serum proteins, recognition and elimination by cells of the immune system. In addition, targeting ligands (such as antibodies) are frequently coupled to the distal end of the PEG chains to direct the vesicles (then called 'immuno-liposomes') to specific cell types, such as tumor cells. To our knowledge, experiments on the stability of ligand anchoring have so far only been conducted with PL-based PEGs and not with sterol-based PEGs after insertion via the sterol-based post-insertion technique (SPIT). Therefore, our study examines the insertion stability of PEG-cholesteryl ester (Chol-PEG) molecules with PEG chains of 1000, 1500 and 2000Da molecular mass which have been inserted into the membranes of liposomes using SPIT. For this study we used different acceptor media and multiple analytical techniques, including pulsed-field-gradient nuclear magnetic resonance (PFG-NMR), free-flow electrophoresis, size exclusion chromatography and ultracentrifugation. The obtained data consistently showed that a higher molar mass of PEG chains positively correlates with higher release from the liposome membranes. Furthermore, we could detect and quantify the migration of Chol-PEG molecules from radioactively double-labeled surface-modified liposomes to negatively charged acceptor liposomes via free-flow electrophoresis. Insertion of Chol-PEG molecules into the membrane of preformed liposomes using SPIT is an essential step for the functionalization of liposomes with the aim of specific targeting. For the first time, we present a kinetic analysis of this insertion process using PFG-NMR, showing that insertion into the liposomal membranes takes place within 90s for Chol-PEG1000 molecules.

Polyalkylcyanoacrylate nanocapsules: variation of membrane permeability by chemical cross-linking.

The permeability of the polymer walls of polyalkylcyanoacrylate nanocapsules varies by different degrees of chemical cross-linking. For this reason, different amounts of bivalent alkylcyanoacrylates are added to the monovalent alkylcyanoacrylate prior to an interfacial polymerization step in order to generate capsules with various cross-linking densities. The obtained nanocapsules are characterized by observing the water molecules via pulsed field-gradient nuclear magnetic resonance using a stimulated echo sequence. The resulting echo decay plots reveal the exchange rate of the water molecules between the free and encapsulated states. The observed dwell times of water molecules in the encapsulated state are characteristic parameters for the permeability of the given capsule membranes. They show a clear dependence on the degree of cross-linking, proving the potential of this approach for a controlled variation of the capsule permeability. Also, the cross-linked nanocapsules exhibit a significantly decreased solubility in tetrahydrofuran which may lead to new applications for polyalkylcyanoacrylate nanocapsules in organic solvents.

Pulsed-field gradient NMR measurements on hydrogels from phosphocholine.

Gels from diacylphosphatidylcholine in glycerol/butylene glycol mixtures were investigated by pulsed-field gradient NMR measurements. Previous measurements had shown that the gels are formed by networks from crystalline multilamellar vesicles (MLV). The obtained self-diffusion coefficients for water and butylene glycol molecules indicate that both molecules occur in two different environments, even at temperatures above the phase transition T(m) where the system is still in a liquid crystalline state. While the larger fraction of the molecules shows a free self-diffusion process like in a homogeneous phase, the smaller fraction seems to be encapsulated in closed domains and undergoes only hindered self-diffusion. It is concluded that the hindered diffusions are due to the solvent molecules trapped between the bilayers of the multilamellar vesicles, while the free diffusion is assigned to the solvent molecules outside of the MLV. Since the fraction of the entrapped molecules does not change during phase transition, we assume that the structure of the network in the samples remains the same when gelation occurs. The gelation process is simply due to the transformation of the vesicle bilayers from the liquid crystalline to the crystalline state. The permeability of the bilayer for the solvent molecules is drastically changed by this transition. The exchange of water molecules through the bilayers slows down significantly below T(m): while the average residence time of water molecules inside the vesicles is smaller than 50 ms in the liquid crystalline state, this value increases to more than 1 s for the gel state. In the case of pure butylene glycol, no vesicles are present, and it is likely that these gels are formed from crystalline fibers.