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In this study, we investigated galacturonic (GalAc)- and mannuronic (ManAc) acids as novel targeting ligands for receptor-mediated gene delivery. GalAc and ManAc were coupled to either polyethyleneimine (PEI) or PEIpolyethyleneglycol (PEG). Furthermore, lactobionic acid (LacAc), which comprises a GalAc-related carbohydrate ring, was coupled to each of the polymers through its open-chain gluconic acid moiety. The molar mass distributions of the polymers were characterized by analytical ultracentrifugation and size exclusion chromatography. PEI-conjugatepDNA complexes were transfected into HepG2-, HeLa-, and 16HBE14o--cells. Gene expression mediated by GalAc- and LacAc-functionalized PEI-conjugates was lower than for PEI. In contrast, gene expression mediated by ManAc-functionalized PEI-conjugates was up to three orders of magnitude higher than for the other tested PEI-conjugates, in particular for negatively charged gene vectors at low N/P ratios, independent of the cell line. Pre-incubation of cells with an excess of ManAc before transfection significantly inhibited transfection rates only for ManAc-functionalized PEI-conjugates. Coupling of methyl-α-D-mannuronic acid to PEI resulted in significantly lower transfection rates than for ManAc-PEI based complexes. Together with fluorescence microscopy images of fluorescein-labelled ManAc-functionalized dextrans and FACS analyses of cells, these results demonstrate that receptor-mediated endocytosis of ManAcPEI-conjugatepDNA complexes via ManAc-specific receptors was involved in gene transfer. In conclusion, ManAc-modification of PEI-polymers represents a novel strategy for receptor-mediated gene delivery which could be promising for in vivo application.
The radical polymerization of three monomers bearing nucleobases 1-(4-vinylbenzyl)thymine (VBT), 1-(4-vinylbenzyl)uracil (VBU) and 9-(4-vinylbenzyl)adenine (VBA) was investigated. The corresponding homopolymers could be prepared in high yields via conventional radical polymerization. However, the resulting polymers were found to be only soluble in a few polar solvents. On the other hand, copolymers of dodecyl methacrylate (DMA) with either VBT or VBA could be prepared via both free radical polymerization and atom transfer radical polymerization and could be dissolved in a large variety of organic solvents. Moreover, the formed complementary copolymers P(VBT-co-DMA) and P(VBA-co-DMA) were found to self-assemble in dilute solutions in dioxane or chloroform via base recognition, as evidenced by a significant hypochromicity effect in UV spectroscopy. Nevertheless, at higher concentrations in chloroform, both dynamic light scattering and optical microscopy indicate that P(VBT-co-DMA), P(VBA-co-DMA), or P(VBT-co-DMA)/P(VBA-co-DMA) mixtures spontaneously self-assemble into micron size spherical aggregates. 1H NMR and FTIR studies confirmed that the self-assembly process is driven in all cases via H-bond formation. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 4805-4818, 2005
The aim of this work was the synthesis of starch macroinitiators for cationic polymer grafted starches that: (i) are free of cationic homopolymer, and (ii) display a high degree of conversion of the cationic monomer. We show that this can be achieved by a free-radical polymerization reaction using the cationic monomer N-methacryloyloxyethyl-N,N-dimethyl-N-benzylammonium chloride (MADAM-BQ) and a new starch-based macroazoinitiator. For this purpose, the acid chloride of 4-tert-butylazo-4-cyanovaleric acid was synthesized and bound covalently to starch (predominantly in the C6 position) to form a nonsymmetrically substituted macroinitiator that was used to polymerize MADAM-BQ in aqueous media. Essentially no MADAM-BQ homopolymer was formed. The initiator decomposes thermally to starch radicals of high reactivity and low-molar mass radicals that do not initiate polymerization. The reason for the different reactivities of the radicals is presumably due to the nonsymmetric constitution of the starch-bound azo groups. The graft polymerization of MADAM-BQ in aqueous solution performs according to an ideal overall kinetic. The structure of the synthesized starch-graft-poly(MADAM-BQ) products is similar to that of block copolymers because of the low radical efficiency of the starch initiators in aqueous solution. Especially, starch substrates with a higher content of azo groups did not lead to graft products with shorter graft distances because the state of solution of these macroinitiators becomes worse and aggregation occurs with an increasing degree of substitution.
The new ligand 4´-(4´´´-pyridyl-N-oxide)-2,2´:6´,2´´-terpyridine (pyNoxterpy) and its homoleptic iron(II) complex have been synthesised, and structural and spectroscopic studies have been carried out. The obtained results have been compared with the reported data for the parent ligand 4´-(4´´´-pyridyl)-2,2´:6´,2´´-terpyridine (pyterpy) and its homoleptic iron(II) complex. Significant differences between the spectral and electrochemical properties of the metal complexes have been found, derived from the changes in the electronic properties of the coordinated ligands.
Solution Self-Assembly of Synthetic Copolymers bearing Complementary Nucleic Acid Funtionalities
(2005)
Superparamagnetic iron oxide particles (SPIO) of maghemite were prepared in aqueous solution and subsequently stabilized with polymers in two layer-by-layer deposition steps. The first layer around the maghemite core is formed by poly(ethylene imine) (PEI), and the second one is formed by poly(ethylene oxide)-block-poly(glutamic acid) (PEO-PGA). The hydrodynamic diameter of the particles increases stepwise from Dh = 25 nm (parent) via 35 nm (PEI) to 46 nm (PEI plus PEO-PGA) due to stabilization. This is accompanied by a switching of their -potentials from moderately positive (+28 mV) to highly positive (+50 mV) and finally slightly negative (-3 mV). By contrast, the polydispersity indexes of the particles remain constant (ca. 0.15). Mössbauer spectroscopy revealed that the iron oxide, which forms the core of the particles, is only present as Fe(III) in the form of superparamagnetic maghemite nanocrystals. The magnetic domains and the maghemite crystallites were found to be identical with a size of 12.0 ± 0.5 nm. The coated maghemite nanoparticles were tested to be stable in water and in physiological salt solution for longer than 6 months. In contrast to novel methods for magnetic nanoparticle production, where organic solvents are necessary, the procedure proposed here can dispense with organic solvents. Magnetic resonance imaging (MRI) experiments on living rats indicate that the nanoparticles are useful as an MRI contrast agent.
We synthesized a symmetric linear ABCBA pentablock copolymer consisting of poly(ethylene oxide), poly(-benzyl L-glutamate), and a poly(perfluoro ether) (fluorolink). The different blocks are highly immiscible with each other and form two-compartment micelles of mainly cylindrical shape in aqueous solution with lengths in the range of 100 to 200 nm and diameters of about 24 nm. The poly(perfluoro ether) (C blocks) forms the liquidlike center of the micelles (d = 6 nm). This is surrounded by a first shell of ca. 2 nm thickness consisting of -sheets of poly(-benzyl L-glutamate) (B blocks) and a second 7 nm shell of poly(ethylene oxide) (A blocks). The A blocks provide water solubility, and the B and C blocks form separated hydrophobic compartments. This work is a contribution to the development of multicompartment micelles devoted to mimic transport proteins such as serum albumins in long-term development.
Self-assembly of Fe(II) and the ditopic ligand 1,4-bis(2,2':6',2"-terpyridine-4'-yl)benzene results in equilibrium structures in solutions, so-called metallosupramolecular coordination polyelectrolytes (MEPEs). It is exceedingly difficult to characterize such macromolecular assemblies, because of the dynamic nature. Therefore, hardly any structural information is available for this type of material. Here, we show that from dilute solutions, where small aggregates predominate, it is possible to grow nanoscopic crystals at an interface. A near atomic resolution structure of MEPE is obtained by investigating the nanoscopic crystals with electron diffraction in combination with molecular modeling. The analysis reveals a primitive monoclinic unit cell (P21/c space group, a = 10.4 Å, b = 10.7 Å, c = 34.0 Å, α = γ = 90°, β = 95°, ρ = 1.26 g/cm³, and Z = 4). The MEPE forms linear rods, which are organized into sheets. Four sheets intersect the unit cell, while adjacent sheets are rotated by 90° with respect to each other. The pseudooctahedral coordination geometry of the Fe(II) centers is confirmed by Mössbauer spectroscopy. The combination of diffraction and molecular modeling presented here may be of general utility to address problems in structural materials science.
Oral bioavailability of cyclosporine: Solid lipid nanoparticles (SLN®) versus drug nanocrystals
(2006)
For the development of an optimized oral formulation for cyclosporine A, 2% of this drug has been formulated in solid lipid nanoparticles (SLN, mean size 157 nm) and as nanocrystals (mean size 962 nm). The encapsulation rate of SLN was found to be 96.1%. Nanocrystals are composed of 100% of drug. For the assessment of the pharmacokinetic parameters the developed formulations have been administered via oral route to three young pigs. Comparison studies with a commercial Sandimmun Neoral/Optoral® used as reference have been performed. The blood profiles observed after oral administration of the commercial microemulsion Sandimmun® revealed a fast absorption of drug leading to the observation of a plasma peak above 1000 ng/ml within the first 2 h. For drug nanocrystals most of the blood concentrations were in the range between 30 and 70 ng/ml over a period of 14 h. These values were very low, showing huge differences between the measuring time points and between the tested animals. On the contrary, administration of cyclosporine-loaded SLN led to a mean plasma profile with almost similarly low variations in comparison to the reference microemulsion, however with no initial blood peak as observed with the Sandimmun Neoral/Optoral®. Comparing the area under the curves (AUC) obtained with the tested animals it could be stated that the SLN formulation avoids side effects by lacking blood concentrations higher than 1000 ng/ml. In this study it has been proved that using SLN as a drug carrier for oral administration of cyclosporine A a low variation in bioavailability of the drug and simultaneously avoiding the plasma peak typical of the first Sandimmun® formulation can be achieved.
The self-organization of random copolymers composed of a nucleobase monomer (either 1-(4-vinylbenzyl)thymine or 9-(4-vinylbenzyl)adenine) and dodecyl methacrylate (DMA) was studied in dilute chloroform solutions. The balance between the molar fractions of the nucleobase monomer (leading to intermolecular H-bonding) and DMA (soluble moiety in chloroform) in the polymer chains was found to be the parameter that principally influences the self-organization. DMA-rich copolymers are molecularly soluble in chloroform, whereas nucleobase-rich copolymers are insoluble in this solvent. Copolymers possessing an equimolar comonomer composition self-assemble into micrometer-sized particles physically cross-linked by intermolecular H-bonds (either thymine-thymine or adenine-adenine interactions, depending on the studied copolymer). Nevertheless, when mixed together, thymine- and adenine-based colloids fuse into thermodynamically stable microspheres cross linked by adenine-thymine interactions.
We prepared crystals of di-n-alkyl esters of phosphoric acid with chain lengths of n = 10, 12, 14, 16, and 18. These were characterized by single-crystal X-ray analysis and differential scanning calorimetry (DSC). It was found that the alkyl chains are in an extended all-trans conformation and aligned close to perpendicular, forming V-shaped molecules. This is in strong contrast to the typical arrangement of the alkyl chains of phospholipids where the two alkyl chains are arranged parallel in the same direction (e.g., tuning fork configuration in bilayers). Additionally, it was found that the arrangement of the V-shaped molecules of the di-n-alkyl esters in neighboring stacks of the lamellar crystals is antiparallel for short chain lengths (n = 10 and 12) and parallel for the longer (n = 14 and 16). DSC reveals that the melting of the crystals increases systematically with increasing chain lengths from 48 to 82 C. The contribution of each methylene group to the melting enthalpy (70-133 kJ/mol) is independent of the chain length (3.9 kJ per mol CH2).
For the investigation of small sample volumes, the use of an acoustic levitator was tested as a `sample holder' for hovering droplets in a synchrotron beam. It might be advantageous to use levitated droplets instead of samples confined in solid holders, especially for the study of crystallization processes where the influence of containing walls has to be minimized. In a first experiment, the crystallization of sodium chloride in a small droplet of aqueous solution has been followed with a time resolution of 30 s. The collected diffraction peaks are compared with data in the ICSD database.
A double hydrophilic block copolymer was synthesized by controlled free radical polymerization, namely by the RAFT method, from a poly(ethylene oxide) macromonomer and a cationic acrylate. Stoichiometric complexes with sodium decanoate and with sodium perfluorodecanoate were prepared by self-assembly. Both complexes are soluble in water forming core-shell nanoparticles with hydrodynamic radii of 54 and 22 nm, respectively, and neutral potentials. The complex containing decanoate decomposed upon dilution, while the complex with perfluorodecanoate is stable even against dialysis and in salt solution. Only the nanoparticles containing perfluorodecanoate show a strong contrast in small-angle X-ray scattering (SAXS). It is produced from their cores that have an elliptical shape (short axes = 8.5 nm, long axis = 23.3 nm) and a sharp density transition to the shell. The cores exhibit an internal lamellar structure with a long period of 3.65 nm consisting of fluorocarbon rich lamellae (d1 = 3.00 nm) and lamellae enriched in ionic groups (d2 = 0.65 nm). These fluorinated nanoparticles coexist in physiological buffer with human serum albumin and fibrinogen without inducing aggregation. Moreover, the content of -helix of these serum proteins increased when in solution with the fluorinated nanoparticles.
Targeting of gene vectors to liver hepatocytes could offer the opportunity to cure various acquired and inherited diseases. Efficient gene delivery to the liver parenchyma has been obscured from efficient targeting of hepatocytes. Here we show that the thyroid hormone, triiodothyronine (T3), can be used to improve the gene transfer efficiency of nonviral gene vectors to hepatocytes in vitro and to the liver of mice in vivo. T3 conjugated to the distal ends of fluorescent labeled PEG-g-dextran resulted in T3-specific cellular endosomal uptake into the hepatocellular cell line HepG2. PEG-g-PEI graft copolymers with increasing molar PEG-ratios were synthesized, complexed with plasmid DNA, and transfected into HepG2 or HeLa cells. Gene transfer efficiency decreased as the number of PEG blocks increased. T3 conjugation to PEI and the distal ends of PEG blocks resulted in T3 specific gene transfer in HepG2 cells as evidenced by reduction of gene transfer efficiency after pre-incubation of cells with excess of T3. In vivo application of T3-PEG-g-PEI based gene vectors in mice after tail vein injection resulted in a significantly 7-fold increase of gene expression in the liver compared with PEG-g-PEI based gene vectors.
Metallosupramolecular coordination polyelectrolytes (MEPEs) based on rigid and flexible ditopic bis-terpyridine ligands and Fe(II) ions are investigated by Mössbauer spectroscopy. We demonstrate the influence of mechanical stress induced by grinding on the structure of MEPE as well as the ability of MEPE to self-repair through recrystallisation.
Solid lipid nanoparticles (SLN) were produced loaded with cyclosporine A in order to develop an improved oral formulation. In this study, the particles were characterized with regard to the structure of the lipid particle matrix, being a determining factor for mode of drug incorporation and drug release. Differential scanning calorimetry (DSC) and wide-angle X-ray scattering (WAXS) measurements were employed for the analysis of the polymorphic modifications and mode of drug incorporation. Particles were produced using Imwitor®900 as lipid matrix (the suspension consisted of 10% particles, 8% Imwitor®900, 2% cyclosporine A), 2.5% Tagat S, 0.5% sodium cholate and 87% water. DSC and WAXS were used to analyse bulk lipid, bulk drug, drug incorporated in the bulk and unloaded and drug-loaded SLN dispersions. The processing of the bulk lipid into nanoparticles was accompanied by a polymorphic transformation from the ß to the α-modification. After production, the drug-free SLN dispersions converted back to ß-modification, while the drug-loaded SLN stayed primarily in α-modification. After incorporation of cyclosporine A into SLN, the peptide lost its crystalline character. Based on WAXS data, it could be concluded that cyclosporine is molecularly dispersed in between the fatty acid chains of the liquid-crystalline α-modification fraction of the loaded SLN.
We report on the online coupling of asymmetrical flow field-flow fractionation (A4F) with small-angle X-ray scattering (SAXS) for the detection of nanoparticles. The A4F was used to fractionate superparamagnetic maghemite nanoparticles, which were prepared continuously with a micromixer. The outlet of the A4F was directly coupled to a flow capillary of a SAXSess instrument (Kratky type of camera). SAXS curves were recorded in a 1 s time interval. This was possible by using intense synchrotron radiation. The radii of gyration of the nanoparticles, as determined from Guinier plots, increased from 2 to 6 nm with increasing fractionation time of the A4F. A more detailed analysis of the scattering curves revealed that the particles were cylindrical in shape (nanorods), which we attributed to the micromixing preparation technique. The radii of the nanorods increased only slightly from 1.2 to 1.7 nm with increasing fractionation time, while the lengths increased strongly from 7.0 to 30.0 nm. The volume distribution of the nanorods was determined and described by Schultz-Zimm and log-normal distributions. Nanorod volumes increased from 45 to 263 nm³, corresponding to molar masses of 140 × 10³ to 820 × 10³ g mol-1. We propose A4F-SAXS coupling as a new method for analysis of nanoparticles of complex composition in solution. It allows precise online determination of the particles shape and size distributions. This method can be applied to mixtures of nanoparticles of arbitrary shapes and sizes (1-100 nm). Moreover, the total time needed for fractionation and online SAXS data recording is usually only 20 min.
Positively charged superparamagnetic iron oxide (SPIO) particles of maghemite were prepared in aqueous solution and subsequently stabilized with poly(ethylene oxide)-block-poly(glutamic acid) (PEOPGA) at a hydrodynamic diameter of 60 nm. Depending on the amount of PEOPGA used, this is accompanied by a switching of their zeta potentials from positive to negative charge (-33 mV). As a prerequisite for in vivo testing, the PEOPGA coated maghemite nanoparticles were evaluated to be colloidally stable in water and in physiological salt solution for longer than six months as well in various buffer systems under physiological pH and salt conditions (AFM, dynamic light scattering). We excluded toxic effects of the PEOPGA coated maghemite nanoparticles. We demonstrated by in vivo MR-imaging and 111In measurements a biodistribution of the nanoparticles into the liver comparable to carboxydextran coated superparamagnetic iron oxide nanoparticles (Resovist®) as a reference nanoscaled MRI contrast medium. This was enforced by a detailed visualization of our nanoparticles by electron microscopy of liver tissue sections. Furthermore, our results indicate that 15% of the injected PEOPGA coated maghemite nanoparticles circulate in the blood compartment for at least 60 min after i.v. application.
Hydrophobically end-modified (HM) poly(2-ethyl-2-oxazolines) (PEtOx) and poly(2-isopropyl-2-oxazolines) (PiPrOx) bearing an n-octadecyl chain on both termini or on one chain end only were prepared by cationic ring-opening polymerization of 2-ethyl-2-oxazoline and 2-isopropyl-2-oxazoline, respectively, and subsequent end-group modification. The polymers had a molar mass (Mn) ranging from 7000 to 13000 g mol-1, a size distribution Mw/Mn < 1.20, and end-group functionality > 0.97. All polymers, except the semitelechelic sample C18-PiPrOx-OH 13K (Mn = 13000 g mol-1), formed core-shell micelles in cold water with a hydrodynamic radius (RH), measured by dynamic light scattering, between 7 and 12 nm and a core of radius (Rc), determined by analysis of small-angle X-ray scattering (SAXS) data, of ~1.3 nm. Aqueous solutions of all polymers underwent a heat-induced phase transition detected by an increase in solution turbidity at a temperature (Tcp, cloud point) ranging from 32 to 62 °C, depending on polymer structure and size. Temperature-dependent light scattering (LS) measurements and fluorescence depolarization studies with the probe diphenylhexatriene (DPH) revealed that extensive intermicellar bridging takes place in solutions heated in the vicinity of Tcp leading to large assemblies (RH ≥ 1 µm). Further heating caused these assemblies to shrink into objects with RH ~ 300-700 nm, depending on the size and structure of the polymer. The formation of H-bonds between water molecules and the main-chain amide nitrogen atoms imparts distinct features to the flower/star micelles formed by telechelic/semitelechelic PiPrOx and PEtOx, compared to the micelles formed by other hydrophobically end-modified water-soluble polymers, such as poly(ethylene oxide) or poly(N-isopropylacrylamide).
The dispersed iron oxide nanoparticles of ferrofluids in aqueous solution are difficult to characterize due to their protective polymer coatings. We report on the bimodal size distribution of superparamagnetic iron oxide nanoparticles found in the MRI contrast agent Resovist, which is a representative example of commercial nanoparticle-based pharmaceutical formulations. The radii of the majority of the nanoparticles (>99%) range from 4 to 13 nm (less than 1% of the particles display radii up to 21 nm). The maxima of the size distributions are at 5.0 and 9.9 nm. The analysis was performed with in situ characterization of Resovist via online coupling of asymmetrical flow field-flow fractionation (A4F) with small-angle X-ray scattering (SAXS) using a standard copper X-ray tube as a radiation source. The outlet of the A4F was directly coupled to a flow capillary on the SAXS instrument. SAXS curves of nanoparticle fractions were recorded at 1-min time intervals. We recommend using the A4F-SAXS coupling as a routine method for analysis of dispersed nanoparticles with sizes in the range of 1-100 nm. It allows a fast and quantitative comparison of different batches without the need for sample preparation.
The spherical gold nanoparticle reference materials RM 8011, RM 8012, and RM 8013, with a nominal radius of 5, 15, and 30 nm, respectively, have been available since 2008 from NIST. These materials are recommended as standards for nanoparticle size measurements and for the study of the biological effects of nanoparticles, e.g., in pre-clinical biomedical research. We report on determination of the size distributions of these gold nanoparticles using different small-angle X-ray scattering (SAXS) instruments. Measurements with a classical Kratky type SAXS instrument are compared with a synchrotron SAXS technique. Samples were investigated in situ, positioned in capillaries and in levitated droplets. The number-weighted size distributions were determined applying model scattering functions based on (a) Gaussian, (b) log-normal, and (c) Schulz distributions. The mean radii are 4.36 ± 0.04 nm (RM 8011), 12.20 ± 0.03 nm (RM 8012), and 25.74 ± 0.27 nm (RM 8013). Low polydispersities, defined as relative width of the distributions, were detected with values of 0.067 ± 0.006 (RM 8011), 0.103 ± 0.003, (RM 8012), and 0.10 ± 0.01 (RM 8013). The results are in agreement with integral values determined from classical evaluation procedures, such as the radius of gyration (Guinier) and particle volume (Kratky). No indications of particle aggregation and particle interactions-repulsive or attractive-were found. We recommend SAXS as a standard method for a fast and precise determination of size distributions of nanoparticles.
Ionic liquids (ILs) have attracted tremendous interest in the recent past for their potential in many chemical fields. The current report explores the effects of a set of ILs based on the 1-ethyl-3-methyl-imidazolium cation and different anions on the formation of gold nanoparticles. X-Ray diffraction finds face-centered cubic gold in all cases, but transmission electron microscopy (TEM) shows that there are distinct differences in particle formation and stabilization with the ethyl sulfate (ES), trifluoromethanesulfonate (TfO) and methanesulfonate (MS) anions. With the MS anion, nanoparticles with diameters between 5 and 7 nm form, which increasingly aggregate at higher reaction temperatures. With TfO, also small 5 to 7 nm particles form, but only at low temperatures. Above ca. 160 °C, large, ill-defined and aggregated particles form. With ES, polydisperse samples form at all temperatures except 160 °C. In this case the nanoparticles appear often surrounded by an IL film, which appears to stabilize individual, ca. 15 to 20 nm particles. Dynamic light scattering and UV/Vis spectroscopy further show that in suspension the particles are, much like seen in the TEM, more strongly aggregated with higher reaction temperatures. In summary, the results suggest that there are very specific IL-gold interactions that are responsible for the formation of gold particles with an IL-specific shape, size, and aggregation behavior.
We report on a hyphenated polymer analysis method consisting of asymmetrical flow field-flow fractionation (A4F) coupled online with small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS). A mixture of six poly(styrene sulfonate)s with molar masses in the range of 6.5 × 103 to 1.0 × 106 g mol-1 was used as a model system for polyelectrolytes in aqueous solutions with a broad molar mass distribution. A complete polymer separation and analysis was performed in 60 min. Detailed information for all polymer fractions are available on i) the radii of gyration, which were determined from the SAXS data interpretation in terms of the Debye model (Gaussian chains), and ii) the diffusion coefficients (from DLS). We recommend using the A4F-SAXS-DLS coupling as a possible new reference method for the detailed analysis of complex polymer mixtures. Advantages of the use of SAXS are seen in comparison to static light scattering for polymers with radii of gyration smaller then 15 nm, for which only SAXS produces precise analytical results on the size of the polymers in solution.
Sequences and end groups of complex copolyesters were determined by fragmentation analysis by means of matrix-assisted laser desorption/ionization collision-induced dissociation tandem mass spectrometry (MALDI CID MS/MS). The complexity of the crude copolyester mixture was reduced by a chromatographic separation followed by a MALDI time-of-flight (TOF) investigation of fractions. Due to overlapping compositional and end-group information a clear assignment of end groups was very difficult. However, the fragmentation of suitable precursor ions resulted in typical fragment ion patterns and, therefore, enabled a fast and unambiguous determination of the end groups and composition of this important class of polymers.
The strategy of utilizing mechanochemical synthesis to obtain metal–organic frameworks (MOFs) with high surface areas is demonstrated for two model systems. The compounds HKUST-1 (Cu3(BTC)2, BTC = 1,3,5-benzenetricarboxylate) and MOF-14 (Cu3(BTB)2, BTB = 4,4',4''-benzenetribenzoate) were synthesized by ball milling and characterized by powder X-ray diffraction (XRD), Raman spectroscopy, scanning electron microscopy (SEM) and thermal analysis (DTA/DTG/MS). The specific surface area (SSA) of both compounds was characterized by nitrogen adsorption. To verify these results and to understand how the synthetic conditions influence the pore structure and the surface area, additional small-angle X-ray scattering (SAXS) experiments were carried out. Our investigations confirm that this synthesis approach is a promising alternative method for distinct MOFs. This facile method leads to materials with surface areas of 1713 m²/g, which is comparable to the highest given values in the literature for the respective compounds.
Pathogenic Gram-negative bacteria use a type three secretion system (TTSS) to deliver virulence factors into host cells. Although the order in which proteins incorporate into the growing TTSS is well described, the underlying assembly mechanisms are still unclear. Here we show that the TTSS needle protomer refolds spontaneously to extend the needle from the distal end. We developed a functional mutant of the needle protomer from Shigella flexneri and Salmonella typhimurium to study its assembly in vitro. We show that the protomer partially refolds from α-helix into β-strand conformation to form the TTSS needle. Reconstitution experiments show that needle growth does not require ATP. Thus, like the structurally related flagellar systems, the needle elongates by subunit polymerization at the distal end but requires protomer refolding. Our studies provide a starting point to understand the molecular assembly mechanisms and the structure of the TTSS at atomic level.
Nanosilver is increasingly used in optics, medicine and analytical chemistry. We recently reported on the synthesis and properties of novel peptide-coated chiral nanosilver [1] using a small hexapeptide based on the amino acids CKK. In a continuation of our previous work, we use the peptides to catalyse TEOS hydrolysis in order to form a dense silica layer shell around a single nanoparticle, preventing chemical etching, allowing their inclusion in other inorganics, and making them biocompatible. Because of mild reaction conditions, the peptide integrity is ensured, as the chiral information which is contained in the nanoparticle. Moreover, these novel core-shell structures remain well-dispersed and are biocompatible. The possibility of further processing (creation of metamaterials etc.) is also in the focus of our interest.
Polyelectrolytes such as PAA and its salts are widely used, but are notoriously difficult to characterize due to their polyelectrolyte properties and broad molecular mass distributions. In this paper, we report on a new PAA analysis by combining asymmetrical flow field-flow fractionation and an advanced SAXS technique using an acoustic levitator to minimize background scattering. The proof-of-principle is demonstrated with a mixture of three standard PAAs with different molecular masses. Detailed information on the PAA fractions is available on radii of gyration, polymer contour lengths, and coil conformation. Our method is expected to be applicable for a wide range of water-soluble synthetic and natural polymers and ideal for molecular masses of 5 × 103–2 × 105 g · mol-1.
Silver nanoparticles (SNP) are the subject of worldwide commercialization because of their antimicrobial effects. Yet only little data on their mode of action exist. Further, only few techniques allow for visualization and quantification of unlabeled nanoparticles inside cells. To study SNP of different sizes and coatings within human macrophages, we introduce a novel laser postionization secondary neutral mass spectrometry (Laser-SNMS) approach and prove this method superior to the widely applied confocal Raman and transmission electron microscopy. With time-of-flight secondary ion mass spectrometry (TOF-SIMS) we further demonstrate characteristic fingerprints in the lipid pattern of the cellular membrane indicative of oxidative stress and membrane fluidity changes. Increases of protein carbonyl and heme oxygenase-1 levels in treated cells confirm the presence of oxidative stress biochemically. Intriguingly, affected phagocytosis reveals as highly sensitive end point of SNP-mediated adversity in macrophages. The cellular responses monitored are hierarchically linked, but follow individual kinetics and are partially reversible.
Processing nanoparticles with A4F-SAXS for toxicological studies: Iron oxide in cell-based assays
(2011)
Nanoparticles are not typically ready-to-use for in vitro cell culture assays. Prior to their use in assays, powder samples containing nanoparticles must be dispersed, de-agglomerated, fractionated by size, and characterized with respect to size and size distribution. For this purpose we report exemplarily on polyphosphate-stabilized iron oxide nanoparticles in aqueous suspension. Fractionation and online particle size analysis was performed in a time-saving procedure lasting 50 min by combining asymmetrical flow field-flow fractionation (A4F) and small-angle X-ray scattering (SAXS). Narrowly distributed nanoparticle fractions with radii of gyration (Rg) from 7 to 21 nm were obtained from polydisperse samples. The A4F-SAXS combination is introduced for the preparation of well-characterized sample fractions originating from a highly polydisperse system as typically found in engineered nanoparticles. A4F-SAXS processed particles are ready-to-use for toxicological studies. The results of preliminary tests of the effects of fractionated iron oxide nanoparticles with a Rg of 15 nm on a human colon model cell line are reported.
Compact pnCCD-based X-ray camera with high spatial and energy resolution: a color X-ray camera
(2011)
For many applications there is a requirement for nondestructive analytical investigation of the elemental distribution in a sample. With the improvement of X-ray optics and spectroscopic X-ray imagers, full field X-ray fluorescence (FF-XRF) methods are feasible. A new device for high-resolution X-ray imaging, an energy and spatial resolving X-ray camera, is presented. The basic idea behind this so-called 'color X-ray camera' (CXC) is to combine an energy dispersive array detector for X-rays, in this case a pnCCD, with polycapillary optics. Imaging is achieved using multiframe recording of the energy and the point of impact of single photons. The camera was tested using a laboratory 30 µm microfocus X-ray tube and synchrotron radiation from BESSY II at the BAMline facility. These experiments demonstrate the suitability of the camera for X-ray fluorescence analytics. The camera simultaneously records 69696 spectra with an energy resolution of 152 eV for manganese Kα with a spatial resolution of 50 µm over an imaging area of 12.7 × 12.7 mm². It is sensitive to photons in the energy region between 3 and 40 keV, limited by a 50 µm beryllium window, and the sensitive thickness of 450 µm of the chip. Online preview of the sample is possible as the software updates the sums of the counts for certain energy channel ranges during the measurement and displays 2-D false-color maps as well as spectra of selected regions. The complete data cube of 264 × 264 spectra is saved for further qualitative and quantitative processing.
The interaction of self-assembled dendritic amphiphiles with drugs and dyes in aqueous solutions is of great significance for designing and optimizing shape-persistent delivery systems. Here we present deeper insight for two examples of low molecular weight (LMW) nonionic dendritic amphiphiles as host molecules and a series of selected aromatic guest model molecules (benzene, naphthalene, biphenyl, terphenyl, anthracene, and pyrene). Aromatic guest molecules were incorporated into the self-assemblies of dendritic nanocarriers, and the resultant complexes were studied by a combination of UV, NMR, computational simulation, and small-angle X-ray-scattering (SAXS) techniques in order to determine the loading capacity, localization, and specific interactions in dendritic amphiphiles with guest molecules. Our findings revealed that the localization of guest molecules in the hydrophobic region and the loading capacity of guest molecules are dependent on their size and the arrangement of aromatic rings instead of the loading amount. Furthermore, the shape of self-assembled host molecules was found to be ellipsoidal and highly persistent even after loading the guest molecules. To the best of our knowledge, this is the first systematic host–guest study, particularly with low molecular weight nonionic dendritic amphiphilies and aromatic guest molecules. Thus, this study opens new possibilities and ways to explore the transport behavior of aromatic drugs with such nanocarriers.
The synthesis, characterization, self-assembly, and gel formation of poly(γ-benzyl-L-glutamate) (PBLG) in a molecular weight range from ca. 7,000–100,000 g/mol and with narrow molecular weight distribution are described. The PBLG is synthesized by the nickel-mediated ring-opening polymerization and is characterized by size-exclusion chromatography coupled with multiple-angle laser light scattering, NMR, and Fourier transform infrared spectroscopy. The self-assembly and thermoreversible gel formation in the helicogenic solvent toluene is investigated by transmission electron microscopy, atomic force microscopy, small-angle X-ray scattering, and synchrotron powder X-ray diffraction. At concentrations significantly below the minimum gelation concentration, spherical aggregates are observed. At higher concentrations, gels are formed, which show a 3D network structure composed of nanofibers. The proposed self-assembly mechanism is based on a distorted hexagonal packing of PBLG helices parallel to the axis of the nanofiber. The gel network forms due to branching and rejoining of bundles of PBLG nanofibers. The network exhibits uniform domains with a length of 200±42 nm composed of densely packed PBLG helices.