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As a growing number of nanotherapeutics enters the market, improved analytical techniques for measuring the drug release are required. Biorelevant release tests have become a standard in the prediction of in vivo pharmacokinetics but also in quality control of novel dosage forms. In the present study, two methods for testing the drug release from nanocarriers, namely the filtration technique and the dispersion releaser technology, have been investigated. Initially, the in vitro release rates were determined using two different biorelevant media. Additionally, the effect of each method on a simulated in vivo pharmacokinetic profile was studied using advanced PBPK modelling. The two methods resulted in slightly different release profiles. Applying the filtration method, an early plateau of 91.0 ± 5.3% was reached at the first sampling time point. In comparison, the release rate steadily increased to a maximum of 100.9 ± 4.1% when the dispersion releaser technology was used. Sensitivity analysis revealed how these differences translated into the PBPK-based simulation. A change in the total dissolution rate of 10% resulted in cmax values of +1.6% and -11.0%, respectively, when using input data obtained with the dispersion releaser. Data obtained by filtration translated into cmax values of ±1.8%.

Enzymatically-induced degradation of bovine serum albumin (BSA) by serine proteases (trypsin and α-chymotrypsin) in various concentrations was monitored by means of Fourier transform infrared (FT-IR) and ultraviolet circular dichroism (UV-CD) spectroscopy. In this study, the applicability of both spectroscopies to monitor the proteolysis process in real time has been proven, by tracking the spectral changes together with secondary structure analysis of BSA as proteolysis proceeds. On the basis of the FTIR spectra and the changes in the amide I band region, we suggest the progression of proteolysis process via conversion of α-helices (1654 cm(-1)) into unordered structures and an increase in the concentration of free carboxylates (absorption of 1593 and 1402 cm(-1)). For the first time, the correlation between the degree of hydrolysis and the concentration of carboxylic groups measured by FTIR spectroscopy was revealed as well. The far UV-CD spectra together with their secondary structure analysis suggest that the α-helical content decreases concomitant with an increase in the unordered structure. Both spectroscopic techniques also demonstrate that there are similar but less spectral changes of BSA for the trypsin attack than for α-chymotrypsin although the substrate/enzyme ratio is taken the same.

The aim of the present investigation was to develop a reliable method which can be applied to the measurement of in vitro drug release from nanocarriers. Since the limited membrane transport is one major obstacle to the assessment of drug release with dialysis techniques, the determination of this parameter was our objective. Therefore, a novel drug release automatic monitoring system (DREAMS) was designed to conduct continuous measurements during the dialysis process. Moreover, a mathematical model was used for evaluation of the experimental data. This combination of mathematical and analytical tools enabled the quantification of the total amount of free drug in the system. Eudragit(®) RS 100 nanoparticles loaded with the model compound 5,10,15,20-tetrakis(m-hydroxypheny)chlorin (mTHPC) were investigated and the drug release was continuously monitored by using a fluorescence spectrometer that is part of the setup. Free drug and drug-loaded nanoparticles were tested to discriminate between the two formulations. In addition, two types of membranes composed of different materials were evaluated and the kinetics of membrane transport was determined. The data obtained from the apparatus were further treated by a mathematical model, which yielded distinguishable release profiles between samples of different compositions. The method offers a promising option for release testing of nanoparticles.

Time-resolved studies were performed for tryptic proteolysis of β-casein in media containing 10–40 % (v/v) ethanol at 37 °C and pH 7.9. The peptide bond demasking, the process which implies the removal of steric obstacles shielding polypeptide sites against enzymatic attack, was quantitatively evaluated with fluorescence spectroscopy by monitoring the exposure of Trp residues to the aqueous polar medium. This process obeys a first-order kinetic law that allowed us the determination of the rate constants of demasking k d . The fraction of initially masked bonds m, being able to convert during proteolysis to the demasked ones, and the fraction of unhydrolysable bonds n were calculated within the framework of a two-step model with consecutive demasking and hydrolysis steps. Parameters m and n were shown to decrease and increase, respectively, with the addition of ethanol.

Immunosurveillance of tumor cells depends on NKp30, a major activating receptor of human natural killer (NK) cells. The human BCL2-associated athanogene 6 (BAG-6, also known as BAT3; 1126 amino acids) is a cellular ligand of NKp30. To date, little is known about the molecular details of this receptor ligand system. Within the current study, we have located the binding site of NKp30 to a sequence stretch of 250 amino acids in the C-terminal region of BAG-6 (BAG-6686–936). BAG-6686–936 forms a noncovalent dimer of 57–59 kDa, which is sufficient for high affinity interaction with NKp30 (KD < 100 nm). As our most important finding, BAG-6686–936 inhibits NKp30-dependent signaling, interferon-γ release, and degranulation of NK cells in the presence of malignantly transformed target cells. Based on these data, we show for the first time that BAG-6686–936 comprises a subdomain of BAG-6, which is sufficient for receptor docking and inhibition of NKp30-dependent NK cell cytotoxicity as part of a tumor immune escape mechanism. These molecular insights provide an access point to restore tumor immunosurveillance by NK cells and to increase the efficacy of cellular therapies.

Time-resolved studies were performed for tryptic proteolysis of β-casein. The accumulation and decay of the proteolysis products was shown to proceed in the form of nanoscale aggregates that exhibited considerable scattering cross sections for visible light. Monitoring the time course of degradation of these aggregates by static light scattering allowed the determination of the rate constants of aggregate degradation. Additionally, another set of rate constants was determined with fluorescence spectroscopy by monitoring the exposure of Trp residues to the aqueous polar medium. These constants were interpreted as the rate constants of peptide bond demasking, the process that implies the removal of steric obstacles shielding polypeptide sites against enzymatic attack. Both methods gave comparable values for the rate constants. It was concluded that for the trypsinolysis of β-casein, demasking proceeds in parallel with the degradation of aggregates that arise at the initial stage of proteolysis.

As a rapidly growing class of therapeutics, biopharmaceuticals have conquered the global market. Despite the great potential from a therapeutic perspective, such formulations often require frequent injections due to their short half-life. Aiming to establish a parenteral dosage form with prolonged release properties, a biodegradable implant was developed, based on a combination of nanoencapsulation of protein-heparin complexes, creation of a slow release matrix by freeze-drying, and compression using hyaluronan and methylcellulose. In order to investigate this novel delivery system, formulations containing IFN-v-1a and trypsinogen as model proteins were developed. No degradation of the proteins was observed at any stage of the formulation processing. The potential of the delivery system was evaluated in vivo and in vitro after fluorescence-labeling of the biopharmaceuticals. An optimized agarose gel was utilized as in vitro release medium to simulate the subcutaneous environment in a biorelevant manner. In addition, the formulations were administered to female SJL mice and release was innovatively tracked by fluorescence imaging, setting up an in vitro-in vivo correlation. A prolonged time of residence of approximately 12 days was observed for the selected formulation design.

Demasking of peptide bonds during proteolysis of β-casein and β-lactoglobulin by trypsin was monitored by the measurement of the overall spectral shift of intrinsic protein fluorescence. A clear shift of the apparent emission maxima from approximately 340–345 nm to 355–360 nm during proteolysis was observed, with a time course, which follows protein degradation and structural opening. In contrast to procedures using extrinsic fluorescence labels, this label-free procedure does not bear the risk of structural alterations. It is easy to perform, fast, and has a relatively high accuracy of determination. Proteolysis was modelled as simple two-step process with consecutive demasking and hydrolysis stages. It was shown that the fluorescence shift can be attributed to the demasking stage. Formally, kinetics of the peptide bond demasking obeys a first-order kinetic law. Both the theoretical simulations and experiment are in accordance giving the similar dependences of the hydrolysis degree on the degree of peptide bond demasking.

Nanoparticles consisting of human serum albumin (HSA) play an emerging role in the development of new drug delivery systems. Many of these protein-based colloidal carriers are prepared by the well-known desolvation technique, which has shown to be a robust and reproducible method for the laboratory-scale production of HSA nanoparticles. The aim of the present study was to upscale the ethanolic desolvation process utilizing the paddle stirring systems Nanopaddle I and II in combination with a HPLC pump in order to find the optimal conditions for the controlled desolvation of up to 2000 mg of the protein. For characterization of the HSA nanoparticles particle size, zeta potential as a function of the pH, polydispersity index and particle content were investigated. The particle content was determined by microgravimetry and by a turbidimetry to allow optimized in-process control for the novel desolvation technique. Furthermore the sedimentation coefficient was measured by analytical ultracentrifugation (AUC) to gain deeper insight into the size distribution of the nanoparticles. The formed nanocarriers were freeze dryed to achieve a solid preparation for long-term storage and further processing. Particles ranging in size between 251.2 ± 27.0 and 234.1 ± 1.5 nm and with a polydispersity index below 0.2 were achieved.

Fourier transform infrared (FT-IR)- and UV-circular dichroism (UV-CD) spectroscopy have been used to study real-time proteolytic digestion of β-lactoglobulin (β-LG) and β-casein (β-CN) by trypsin at various substrate/enzyme ratios in D2O-buffer at 37 °C. Both techniques confirm that protein substrate looses its secondary structure upon conversion to the peptide fragments. This perturbation alters the backbone of the protein chain resulting in conformational changes and degrading of the intact protein. Precisely, the most significant spectral changes which arise from digestion take place in the amide I and amide II regions. The FT-IR spectra for the degraded β-LG show a decrease around 1634 cm−1, suggesting a decrease of β-sheet structure in the course of hydrolysis. Similarly, the intensity around the 1654 cm−1 band decreases for β-CN digested by trypsin, indicating a reduction in the α-helical part. On the other hand, the intensity around ∼1594 cm−1 and ∼1406 cm−1 increases upon enzymatic breakdown of both substrates, suggesting an increase in the antisymmetric and symmetric stretching modes of free carboxylates, respectively, as released digestion products. Observation of further H/D exchange in the course of digestion manifests the structural opening of the buried groups and accessibility to the core of the substrate. On the basis of the UV-CD spectra recorded for β-LG and β-CN digested by trypsin, the unordered structure increases concomitant with a decrease in the remaining structure, thus, revealing breakdown of the intact protein into smaller fragments. This model study in a closed reaction system may serve as a basis for the much more complex digestion processes in an open reaction system such as the stomach.

Purpose To develop a liver-mimicking MRI gel phantom for use in the development of temperature mapping and coagulation progress visualization tools needed for the thermal tumor ablation methods, including laser-induced interstitial thermotherapy (LITT) and radiofrequency ablation (RFA). Methods A base solution with an acrylamide concentration of 30 vol. % was prepared. Different components were added to the solution; among them are bovine hemoglobin and MR signal-enhancing contrast agents (Magnevist as T1 and Lumirem as T2 contrast agent) for adjustment of the optical absorption and MR relaxation times, respectively. The absorption was measured in samples with various hemoglobin concentrations (0%-7.5%) at different temperatures (25-80 degrees C) using the near-infrared spectroscopy, measuring the transmitted radiation through the sample. The relaxation times were measured in samples with various concentrations of T1 (0.025%-0.325%) and T2 (0.4%-1.6%) contrast agents at different temperatures (25-75 degrees C), through the MRI technique, acquiring images with specific sequences. The concentrations of the hemoglobin and contrast agents of the gel were adjusted so that its absorption coefficient and relaxation times are equivalent to those of liver. To this end, the absorption and relaxation times of the gel samples were compared to reference values, measured in an ex vivo porcine liver at different temperatures through the same methods used for the gel. For validation of the constructed phantom, the absorption and relaxation times were measured in samples containing the determined amounts of the hemoglobin and contrast agents and compared with the corresponding liver values. To qualitatively test the heat resistance of the phantom, it was heated with the LITT method up to approximately 120 degrees C and then was cut to find out if it has been melted. Results In contrast to liver, where the absorption change with temperature showed a sigmoidal form with a jump at T approximately equal 45 degrees C, the absorption of the gel varied slightly over the whole temperature range. However, the gel absorption presented a linear increase from approximately 1.8 to approximately 2.2 mm(-1) with the rising hemoglobin concentration. The gel relaxation times showed a linear decrease with the rising concentrations of the respective contrast agents. Conversely, with the rising temperature, both T1 and T2 increased linearly and showed almost the same trends as in liver. The concentrations of hemoglobin and T1 and T2 contrast agents were determined as 3.92 +/- 0.42 vol. %, 0.098 +/- 0.023 vol. %, and 2.980 +/- 0.067 vol. %, respectively. The measured ex vivo liver T1 value increased from approximately 300 to approximately 530 ms and T2 value from approximately 45 to approximately 52 ms over the temperature range. The phantom validation experiments resulted in absorption coefficients of 2.0-2.1 mm(-1) with variations of 1.5%-2.95% compared to liver below 50 degrees C, T1 of 246.6-597.2 ms and T2 of 40.8-67.1 ms over the temperature range of 25-75 degrees C. Using the Bland-Altman analysis, a difference mean of -6.1/1.9 ms was obtained for T1/T2 between the relaxation times of the phantom and liver. After heating the phantom with LITT, no evidence of melting was observed. Conclusions The constructed phantom is heat-resistant and MR-compatible and can be used as an alternative to liver tissue in the MR-guided thermal ablation experiments with laser to develop clinical tools for real-time monitoring and controlling the thermal ablation progress in liver.

RUNX1/ETO, the fusion protein resulting from the chromosomal translocation t(8;21), is one of the most frequent translocation products in acute myeloid leukemia. Several in vitro and in vivo studies have shown that the homo-tetramerization domain of ETO, the nervy homology region 2 (NHR2), is essential for RUNX1/ETO oncogenic activity. We analyzed the energetic contribution of individual amino acids within the NHR2 to RUNX1/ETO dimer-tetramer transition and found a clustered area of 5 distinct amino acids with strong contribution to the stability of tetramers. Substitution of these amino acids abolishes tetramer formation without affecting dimer formation. Similar to RUNX1/ETO monomers, dimers failed to bind efficiently to DNA and to alter expression of RUNX1-dependent genes. RUNX1/ETO dimers do not block myeloid differentiation, are unable to enhance the self-renewal capacity of hematopoietic progenitors, and fail to induce leukemia in a murine transplantation model. Our data reveal the existence of an essential structural motif (hot spot) at the NHR2 dimer-tetramer interface, suitable for a molecular intervention in t(8;21) leukemias.

Hydrophobic drugs, loperamide and paclitaxel, were loaded in poly(butyl cyanoacrylate) nanoparticles by polymerization of n-butyl-2-cyanoacrylate in aqueous-organic media in the presence of a drug. The particles were stabilized by dextran 70,000 and poloxamer 188 or by 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] sodium salt. It was shown that in the presence of dichloromethane, methanol or ethanol the encapsulation efficiency of loperamide in the nanoparticles reached 80%. Loading of paclitaxel was efficient only in the presence of the lipid. The organic solvents did not significantly influence the nanoparticle morphology or their physicochemical parameters. Thus produced poly(butyl cyanoacrylate) nanoparticles enabled delivery of loperamide across the blood-brain barrier, which was evidenced by the drug analgesic effect evaluated by the tail-flick test.

We present here a study on proteorhodopsin (PR) 2D crystals with analytical ultracentrifugation, circular dichroism and Fourier transform infrared (FTIR) spectroscopy. The aim of our experiments was to test the activity of 2D crystal sample preparations and to gain further insight in PR structure, stability and function with these techniques. Our results demonstrate higher stability compared to detergent-solubilized or reconstituted samples. For different pH values, low pH 2D crystals tend to form bigger aggregates and are less stable than at basic pH. The pH 9 sample shows a sharp phase transition during heat denaturation and there is also evidence for protein-protein interaction due to the close proximity of the proteins in the 2D crystals. In the FTIR measurements at cryogenic temperatures (77 K), we characterized the first step in the PR photocycle. At pH 9, the K intermediate could be observed and the samples showed no orientation effects. At pH 5, we could trap the K/L intermediate, characterized by its negative IR signal at 1741 cm(-1). In rapid-scan FTIR experiments, we could also identify the M intermediate of the photocycle at basic pH. We conclude that the PR 2D crystals exhibit a fully functional photocycle and are therefore well suited for further studies on the proton transport mechanism of PR.

Es wurde eine neue, direkte und einfache Methode zur Bestimmung des Heparinspiegels im Blut entwickelt, die auf der Komplexbildung von Heparin mit Protamin beruht. Heparin bildet mit Protamin im Uberschuss nanoskalige Partikel der Grose von ca. 100–200 nm, die mit Lichtstreuverfahren nachgewiesen werden konnen. Die Lichtstreuung ist proportional zur Partikelkonzentration und damit zum vorliegenden Heparinspiegel. Diese Nachweismethode ist sowohl fur fraktionierte, niedermolekulare Heparine als auch fur unfraktionierte Heparine anwendbar und benotigt nur eine geringe Menge ( 500 I.E./kg KG) eingesetzt werden. Da die Methode spezifisch auf die Heparinmolekule anspricht, konnen durch diesen Test die Nachteile verschiedener Methoden zur Bestimmung der Blutgerinnungsfahigkeit (ACT, Hepcon, Heptest, usw.) vermieden werden.

Nanoparticles prepared by self-assembly from oligonucleotides (ONs), protamine free base, and human serum albumin (“ternary proticles”) are spheres of diameters around 200 nm. Substitution of the protamine free base by protamine sulfate leads to proticles of only around 40 nm in diameter with otherwise unchanged properties. The availability of drug delivery systems of very similar composition but grossly different size may be advantageous when dealing with cells which show size-dependent particle uptake. These nanoparticles are promising candidates for ON delivery to cells because of the following reasons: (1) They are stable for several hours in solutions of up to physiological ionic strength; (2) they are efficiently taken up by cells; (3) after cellular uptake, they easily release the ONs even when these are present as phosphorothioates.

Micelles prepared from amphiphilic block copolymers in which a poly(styrene) segment is connected to a poly(ethylene oxide) block via a bis-(2,2':6',2"-terpyridine-ruthenium) complex have been intensely studied. In most cases, the micelle populations were found to be strongly heterogeneous in size because of massive micelle/ micelle aggregation. In the study reported in this article we tried to improve the homogeneity of the micelle population. The variant preparation procedure developed, which is described here, was used to prepare two "protomer"-type micelles: PS20-[Ru]PEO70 and PS20-[Ru]-PEO375. The dropwise addition of water to a solution of the compounds in dimethylformamide was replaced by the controlled addition of water by a syringe pump. The resulting micelles were characterized by sedimentation velocity and sedimentation equilibrium analyses in an analytical ultracentrifuge and by transmission electron microscopy of negatively stained samples. Sedimentation analysis showed virtually unimodal size distributions, in contrast to the findings on micelles prepared previously. PS20-[Ru]PEO70 micelles were found to have an average molar mass of 318,000 g/mol (corresponding to 53 protomers per micelle, which is distinctly less than after micelle preparation by the standard method) and an average hydrodynamic diameter (d(h)) of 18 nm. For PS20-[Ru]-PEO375 micelles, the corresponding values were M = 603,000 g/mol (31 protomers per micelle) and dh = 34 nm. The latter particles were found to be identical to the "equilibrium" micelles prepared in pure water. Both micelle types had a very narrow molar mass distribution but a much broader distribution of s values and thus of hydrodynamic diameters. This indicates a conformational heterogeneity that is stable on the time scale of sedimentation velocity analysis. The findings from electron microscopy were in disagreement with those from the sedimentation analysis both in average micelle diameter and in the width of the distributions, apparently because of imperfections in the staining procedure. The preparation procedure described also may be useful in micelle formation from other types of protomers. (C) 2004 Wiley Periodicals, Inc.

Nanoparticles prepared by desolvation and subsequent crosslinking of human serum albumin (HSA) represent promising carriers for drug delivery. Particle size is a crucial parameter, in particular for the in vivo behaviour of nanoparticles after intravenous injection. The objective of the present study is the development of a desolvation procedure for the preparation of HSA-based nanoparticles under the aspect of a controllable particle size between 100 and 300 nm in combination with a narrow size distribution. A pump-controlled preparation method was established which enabled particle preparation under defined conditions. Several factors of the preparation process, such as the rate of addition of the desolvating agent, the pH value and the ionic composition of the HSA solution, the protein concentration, and the conditions of particle purification were evaluated. The pH value of the HSA solution prior to the desolvation procedure was identified as the major factor determining particle size. Varying this parameter, (mean) particle diameters could be adjusted between 150 and 280 nm, higher pH values leading to smaller nanoparticles. Washing the particles by differential centrifugation led to significantly narrower size distributions. The reproducibility of the particle size and particle size distribution under the proposed preparation conditions was demonstrated by sedimentation velocity analysis in the analytical ultracentrifuge and the cellular uptake of those nanoparticles was studied by confocal microscope imaging and FACS analysis. The stability of the resulting nanoparticles was evaluated by pH and buffer titration experiments. Only pH values distinctly outside the isoelectric pH range of HSA and low salt concentrations were able to prevent nanoparticle agglomeration.

Industrial production of nanosized drug delivery devices is still an obstacle to the commercialization of nanomedicines. This study encompasses the development of nanoparticles for peroral application in photodynamic therapy, optimization according to the selected product specifications, and the translation into a continuous flow process. Polymeric nanoparticles were prepared by nanoprecipitation of Eudragit® RS 100 in presence and in absence of glycofurol. The photosensitizer temoporfin has been encapsulated into these carrier devices. Process parameters were optimized by means of a Design of Experiments approach and nanoparticles with optimal characteristics were manufactured by using microreactor technology. The efficacy was determined by means of cell culture models in A-253 cells. Physicochemical properties of nanoparticles achieved by nanoprecipitation from ethanolic solutions were superior to those obtained from a method based upon glycofurol. Nanoencapsulation of temoporfin into the matrix significantly reduced toxicity of this compound, while the efficacy was maintained. The release profiles assured a sustained release at the site of action. Finally, the transfer to continuous flow technology was achieved. By adjusting all process parameters, a potent formulation for application in the GI tract was obtained. The essential steps of process development and scale-up were part of this formulation development.

Estimation of optical properties of biologic tissue is crucial for theoretical modeling of laser treatments in medicine. Tissue highly absorbs and scatters the light between 650 nm and 1300 nm, where the laser provides therapeutic effects. Among other properties, the characteristic of biological tissues to scatter the light traveling trough, is described by the anisotropy coefficient (g). The relationship between g and the distribution of the scattered light at different angles is described by Henyey-Greenstein phase function. The measurement of angular distribution of scattered light is performed by the goniometric technique. This paper describes the estimation of g and attenuation coefficient, μt, of swine liver at 850 nm, performed by an ad hoc designed goniometric-based system, where a spectrometer measures intensities of scattered light at fixed angles (0°, 30°, 45°, 60, 120°, 135° and 150°). Both one-term and two-term Henyey-Greenstein phase function have been employed to estimate anisotropy coefficient for forward (gfs) and backward scattering (gbs). Measurements are performed on samples of two thicknesses (60 um and 30 urn) to investigate the influence of this factor on g, and repeated 6 times for each thickness. The estimated values of gfs were 0.947 and 0.951 for thickness of 60 μm and 30 μm, respectively; the estimations of gfs were -0.498 and -0.270 for thickness of 60 μm and 30 μm, respectively. Moreover, μt of liver has been estimated (i.e., 90±20 cm(1)), through Lambert-Beer equation. The comparison of our results with data reported in literature encourages the use of the ad hoc designed tool for performing experiments on other tissue, and at other wavelengths.

Background: Heparin is the standard drug for anticoagulation treatment and is used in many cardiac surgical interventions to prevent blood clotting. The anticoagulation status is controlled by various clotting tests. However, these tests depend on parameters like temperature, hemodilution etc. and are thus not applicable for a direct monitoring of the heparin concentration. The aim of this prospective study was to test a novel light scattering assay (LiSA) for the direct determination of heparin concentration during cardiopulmonary bypass (CPB) surgery and to compare the heparin concentrations with routinely determined activated clotting time (ACT). Methods: The patient group consisted of 50 patients undergoing coronary bypass surgery with CPB. The coagulation status was monitored by the measurement of ACT, which was performed approximately every 30 min during surgery. Parallel to each ACT measurement, the heparin concentration was measured by LiSA. Results: For 70% of the patients, ACT and heparin concentration measured by LiSA correlated reasonably over the entire time course of the intervention. For 30% of the patients, an insufficient correlation or even no correlation at all was observed. Conclusions: This study showed that LiSA enables the determination of intra-operative heparin levels. The lack of correlation between ACT and heparin concentration in a substantial group of patients shows that monitoring of heparin concentration is important. A more precise blood coagulation management, in particular, a precise administration of heparin and protamine, should be based on a combination of the measurement of heparin concentration and of ACT, but not on ACT alone.

The purpose of this study was to evaluate magnetic resonance (MR) temperature imaging of the laser-induced thermotherapy (LITT) comparing the proton resonance frequency (PRF) and T 1 thermometry methods. LITT was applied to a liver-mimicking acrylamide gel phantom. Temperature rise up to 70 °C was measured using a MR-compatible fiber-optic thermometer. MR imaging was performed by a 1.5-T scanner utilizing fast gradient echo sequences including a segmented echo planar imaging (seg-EPI) sequence for PRF and the following sequences for T 1 method: fast low-angle shot (FLASH), inversion recovery turbo flash (IRTF), saturation recovery turbo flash (SRTF), and true fast imaging (TRUFI). Temperature-induced change of the pixel values in circular regions of interest, selected on images under the temperature probe tip, was recorded. For each sequence, a calibration constant could be determined to be -0.0088 ± 0.0002 ppm °C(-1) (EPI), -1.15 ± 0.03 °C(-1) (FLASH), -1.49 ± 0.03 °C(-1) (IRTF), -1.21 ± 0.03 °C(-1) (SRTF), and -2.52 ± 0.12 °C(-1) (TRUFI). These constants were evaluated in further LITT experiments in phantom comparing the calculated temperatures with the fiber optic-measured ones; temperature precisions of 0.60 °C (EPI), 0.81 °C (FLASH), 1.85 °C (IRTF), 1.95 °C (SRTF), and 3.36 °C (TRUFI) were obtained. Furthermore, performing the Bland-Altman analysis, temperature accuracy was determined to be 0.23 °C (EPI), 0.31 °C (FLASH), 1.66 °C (IRTF), 1.19 °C (SRTF), and 3.20 °C (TRUFI). In conclusion, the seg-EPI sequence was found to be more convenient for MR temperature imaging of LITT due to its relatively high precision and accuracy. Among the T 1 method sequences, FLASH showed the highest accuracy and robustness.

To develop a liver-mimicking MRI gel phantom for use in the development of temperature mapping and coagulation progress visualization tools needed for the thermal tumor ablation methods, including laser-induced interstitial thermotherapy (LITT) and radiofrequency ablation (RFA).A base solution with an acrylamide concentration of 30 vol. % was prepared. Different components were added to the solution; among them are bovine hemoglobin and MR signal-enhancing contrast agents (Magnevist as T1 and Lumirem as T2 contrast agent) for adjustment of the optical absorption and MR relaxation times, respectively. The absorption was measured in samples with various hemoglobin concentrations (0%-7.5%) at different temperatures (25-80 degrees C) using the near-infrared spectroscopy, measuring the transmitted radiation through the sample. The relaxation times were measured in samples with various concentrations of T1 (0.025%-0.325%) and T2 (0.4%-1.6%) contrast agents at different temperatures (25-75 degrees C), through the MRI technique, acquiring images with specific sequences. The concentrations of the hemoglobin and contrast agents of the gel were adjusted so that its absorption coefficient and relaxation times are equivalent to those of liver. To this end, the absorption and relaxation times of the gel samples were compared to reference values, measured in an ex vivo porcine liver at different temperatures through the same methods used for the gel. For validation of the constructed phantom, the absorption and relaxation times were measured in samples containing the determined amounts of the hemoglobin and contrast agents and compared with the corresponding liver values. To qualitatively test the heat resistance of the phantom, it was heated with the LITT method up to approximately 120 degrees C and then was cut to find out if it has been melted.In contrast to liver, where the absorption change with temperature showed a sigmoidal form with a jump at T approximately equal 45 degrees C, the absorption of the gel varied slightly over the whole temperature range. However, the gel absorption presented a linear increase from approximately 1.8 to approximately 2.2 mm(-1) with the rising hemoglobin concentration. The gel relaxation times showed a linear decrease with the rising concentrations of the respective contrast agents. Conversely, with the rising temperature, both T1 and T2 increased linearly and showed almost the same trends as in liver. The concentrations of hemoglobin and T1 and T2 contrast agents were determined as 3.92 +/- 0.42 vol. %, 0.098 +/- 0.023 vol. %, and 2.980 +/- 0.067 vol. %, respectively. The measured ex vivo liver T1 value increased from approximately 300 to approximately 530 ms and T2 value from approximately 45 to approximately 52 ms over the temperature range. The phantom validation experiments resulted in absorption coefficients of 2.0-2.1 mm(-1) with variations of 1.5%-2.95% compared to liver below 50 degrees C, T1 of 246.6-597.2 ms and T2 of 40.8-67.1 ms over the temperature range of 25-75 degrees C. Using the Bland-Altman analysis, a difference mean of -6.1/1.9 ms was obtained for T1/T2 between the relaxation times of the phantom and liver. After heating the phantom with LITT, no evidence of melting was observed.The constructed phantom is heat-resistant and MR-compatible and can be used as an alternative to liver tissue in the MR-guided thermal ablation experiments with laser to develop clinical tools for real-time monitoring and controlling the thermal ablation progress in liver.

Heparin, a linear glycosaminoglycan, is used in different forms in anticoagulation treatment. Protamine, a highly positive charged peptide containing about 32 amino acids, acts as an antagonist for heparin to restore normal blood coagulation. The complex formation of protamine with heparin was analyzed by a combination of analytical ultracentrifugation and light scattering. Titration of heparin with protamine in blood plasma preparations results in a drastic increase of turbidity, indicating the formation of nanoscale particles. A similar increase of turbidity was observed in physiological saline solution with or without human serum albumin (HSA). Particle size analysis by analytical ultracentrifugation revealed a particle radius of approximately 30 nm for unfractionated heparin and of approximately 60 nm for low molecular weight heparin upon complexation with excess protamine, in agreement with atomic force microscopy data. In the absence of HSA, larger and more heterogeneous particles were observed. The particles obtained were found to be stable for hours. The particle formation kinetics was analyzed by light scattering at different scattering angles and was found to be complete within several minutes. The time course of particle formation suggests a condensation reaction, with sigmoidal traces for low heparin concentrations and quasi-first-order reaction for high heparin concentrations. Under all conditions, the final scattering intensity reached after several minutes was found to be proportional to the amount of heparin in the blood plasma or buffer solution, provided that excess protamine was available and no multiple scattering occurred. On the basis of a direct relation between particle concentration and the heparin concentration present before protaminization, a light scattering assay was developed which permits the quantitative analysis of the heparin concentration in blood plasma and which could complement or even replace the activated clotting time test, which is currently the most commonly used method for blood coagulation management.

Lipopolysaccharides (LPSs) from Gram-negative bacteria are strong elicitors of the human immune systems. There is strong evidence that aggregates and not monomers of LPS play a decisive role at least in the initial stages of cell activation of immune cells such as mononuclear cells. In previous reports, it was shown that the biologically most active part of enterobacterial LPS, hexa-acyl bisphosphorylated lipid A, adopts a particular supramolecular conformation, a cubic aggregate structure. However, little is known about the size and morphology of these aggregates, regarding the fact that LPS may have strong variations in the length of the saccharide chains (various rough mutant and smooth-form LPS). Thus, in the present paper, several techniques for the determination of details of the aggregate morphology such as freeze-fracture and cryo-electron microscopy, analytical ultracentrifugation, laser backscattering analysis, and small-angle X-ray scattering were applied for various endotoxin (lipid A and different LPS) preparations. The data show a variety of different morphologies not only for different endotoxins but also when comparing different applied techniques. The data are interpreted with respect to the suitability of the single techniques, in particular on the basis of available literature data.

Nanoparticles represent promising carriers for controlled drug delivery. Particle size and size distribution of the particles are important parameters for the in vivo behaviour after intravenous injection and have to be characterised precisely. In the present study, the influence of lyophilisation on the storage stability of poly(D,L lactic-co-glycolic acid) (PLGA) nanoparticles, formulated with several cryoprotective agents, was evaluated. Nanoparticles were prepared by a high pressure solvent evaporation method and freeze-dried in the presence of 1%, 2%, and 3% (m/v) sucrose, trehalose, and mannitol, respectively. Additionally, to all samples containing 3% of the excipients, L-arginine hydrochloride was added in concentrations of 2.1% or 8.4% (m/V). Dynamic light scattering (DLS), analytical ultracentrifugation and transmission electron microscopy (TEM) were used for particle characterisation before and after freeze-drying and subsequent reconstitution. In addition, glass transition temperatures were determined by differential scanning calorimetry (DSC), and the residual moisture of the lyophilisates was analysed by Karl Fischer titration. It was demonstrated that 1% sucrose or 2% trehalose were suitable to maintain particle integrity after reconstitution of lyophilised PLGA nanoparticles. The storage stability study over 3 months showed notable changes in mean particle size, size distribution, and residual moisture content, depending on the composition of the formulation.

Nanoparticles prepared from human serum albumin (HSA) are versatile carrier systems for drug delivery and can be prepared by an established desolvation process. A reproducible process with a low batch-to-batch variability is required for transfer from the lab to an industrial production. In the present study the batch-to-batch variability of the starting material HSA on the preparation of nanoparticles was investigated. HSA can build dimers and higher aggregates because of a free thiol group present in the molecule. Therefore, the quality of different HSA batches was analysed by size exclusion chromatography (SEC) and analytical ultracentrifugation (AUC). The amount of dimerised HSA detected by SEC did not affect particle preparation. Higher aggregates of the protein detected in two batches by AUC disturbed nanoparticle formation at pH values below 8.0. At pH 8.0 and above monodisperse particles between 200 and 300 nm could be prepared with all batches, with higher pH values leading to smaller particles. Besides human derived albumin a particle preparation was also feasible based on recombinant human serum albumin (rHSA). Under comparable preparation conditions monodisperse nanoparticles could be achieved and the same effects of protein aggregates on particle formation were observed. For nanoparticulate drug delivery systems the enzymatic degradation is a crucial parameter for the release of an embedded drug. For this reason, besides the particle preparation process, particle degradation in the presence of different enzymes was studied. Under acidic conditions HSA as well as rHSA nanoparticles could be digested by pepsin and cathepsin B. At neutral pH trypsin, proteinase K, and protease were suitable for particle degradation. It could be shown that the kinetics of particle degradation was dependent on the degree of particle stabilisation. Therefore, the degree of particle stabilisation will influence drug release after cellular accumulation of HSA nanoparticles.

The tetrameric state of p53, p63, and p73 has been considered one of the hallmarks of this protein family. While the DNA binding domain (DBD) is highly conserved among vertebrates and invertebrates, sequences C-terminal to the DBD are highly divergent. In particular, the oligomerization domain (OD) of the p53 forms of the model organisms Caenorhabditis elegans and Drosophila cannot be identified by sequence analysis. Here, we present the solution structures of their ODs and show that they both differ significantly from each other as well as from human p53. CEP-1 contains a composite domain of an OD and a sterile alpha motif (SAM) domain, and forms dimers instead of tetramers. The Dmp53 structure is characterized by an additional N-terminal beta-strand and a C-terminal helix. Truncation analysis in both domains reveals that the additional structural elements are necessary to stabilize the structure of the OD, suggesting a new function for the SAM domain. Furthermore, these structures show a potential path of evolution from an ancestral dimeric form over a tetrameric form, with additional stabilization elements, to the tetramerization domain of mammalian p53.

Protamine-oligonucleotide nanoparticles represent effective colloidal drug carriers for antisense phosphorothioate oligonucleotides (PTO). This study describes improvements in particle preparation and the physicochemical properties of the complexes prepared. The influence of component concentrations, length of the PTO chain and the PTO/protamine weight ratio on particle formation and size, shape and surface charge of the particles were studied in detail. Nanoparticles with diameters of 90–200 nm were obtained, using protamine free base (PFB) and phosphorothioate in water. The chemical composition of the nanoparticles was analysed. More than 90% of the PTO could be assembled in the particle matrix using a � 1 : 2 ratio (w/w) of PTO and PFB. About 53–68% of the PFB was incorporated in the particle matrix. The complexes had a zetapotential of � 19 up to þ 32 mV, depending on the PTO/PFB ratio. The kinetics of the assembly of this binary system were observed by dynamic light scattering (DLS) measurements and by sedimentation velocity analysis in the analytical ultracentrifuge (AUC). In addition, scanning electron microscopy (SEM) and atomic force microscopy (AFM) were applied to verify the results of DLS and the ultracentrifuge measurements. According to sedimentation velocity analysis, the particles were only moderately stable in water and unstable in salt solutions. However, the colloidal solution in water could be stabilized by polyethylenglycol 20 000 (PEG), which also led to an increase of stability in cell medium.

Oligodesoxynucleotides (ODNs) or the corresponding phosphorothioates (PTOs) spontaneously form spherical nanoparticles (“proticles”) with protamine in aqueous solutions. The proticles can cross cellular membranes and release the ODNs within the cells. Thus, they represent a potential drug delivery system. The major disadvantages of this system are a lack of stability in salt solutions and its inability to also release PTOs. The present study shows, using PTOs and protamine free base, that these shortcomings can be eliminated by the addition of human serum albumin (HSA) as a third component to the starting mixture. The “ternary” proticles thus obtained contain maximally a few percent of the HSA that was originally present. Nevertheless, they differ from the previously studied “binary” proticles: (1) They are stable in salt solutions for at least several hours. (2) They show a high cellular uptake into murine fibroblasts, and they readily release the PTOs after uptake. The ternary proticles therefore represent a considerable improvement over binary proticles for use as drug delivery systems.

Nanoparticles represent promising carriers for controlled drug delivery. This work focuses on the size and molecular mass characterization of polyalkylcyanoacrylate nanoparticles formed by anionic emulsion polymerization of butylcyanoacrylate in the presence of poloxamer 188 as a stabilizer. Three different methods were used to determine the size and size distribution of the particle populations: scanning electron microscopy (SEM), dynamic light scattering (DLS), and analytical ultracentrifugation (ANUC). SEM on freeze-dried and Au-shadowed samples showed a relatively narrow distribution of virtually spherical particles with a mean diameter of 167 nm. DLS yielded a monomodal distribution with hydrodynamic diameters around 199 nm (in the absence of additional stabilizer) or 184 nm (in the presence of 1% poloxamer 188). The size distribution determined by ANUC using sedimentation velocity analysis was somewhat more complex, the size of the most abundant particles being around 184 nm. Molar particle mass distributions centered around 2.3x10(9) g/mol. The advantages and disadvantages of the three sizing techniques are discussed.

In aqueous solutions, amphiphilic block copolymers in which a polystyrene (PS) segment is connected to a poly(ethylene oxide) (PEO) block via a bis(2,2':6',2"-terpyridine ruthenium) complex can form micelles. Such micelles of the protomer type PS20-[Ru]-PEO70, according to the preparation procedure representing frozen micelles, were studied by sedimentation velocity and sedimentation equilibrium analysis in an analytical ultracentrifuge and by transmission electron microscopy, with different techniques applied for the sample preparation. The particles obtained were surprisingly multifarious in size. In ultracentrifugation experiments performed at relatively low salt concentrations, the distributions of the sedimentation coefficient s(20,w) showed a pronounced peak at 9.6 S and a broad, only partly separated second peak around 14 S. The molar mass of the particles at the peak was around 430,000 g/mol, corresponding to an aggregation number of approximately 85. The average hydrodynamic diameter of the particles in the peak fraction was approximately 13 run. In electron micrographs of negatively stained samples, spheres of diameters between 10 and 25 nm were the most abundant particles, but larger ones with a wide size range were also visible. The latter particles apparently were composed of smaller ones. The data from both sedimentation analysis and electron microscopy showed that (1) the studied compound formed primary micelles of diameters around 20 nm and (2) the primary micelles had a tendency toward aggregation. (C) 2003 Wiley Periodicals, Inc.

Monotopic membrane proteins are membrane proteins that interact with only one leaflet of the lipid bilayer and do not possess transmembrane spanning segments. They are endowed with important physiological functions but until now only few of them have been studied. Here we present a detailed biochemical, enzymatic and crystallographic characterization of the monotopic membrane protein sulfide:quinone oxidoreductase. Sulfide:quinone oxidoreductase is a ubiquitous enzyme involved in sulfide detoxification, in sulfide-dependent respiration and photosynthesis, and in heavy metal tolerance. It may also play a crucial role in mammals, including humans, because sulfide acts as a neurotransmitter in these organisms. We isolated and purified sulfide:quinone oxidoreductase from the native membranes of the hyperthermophilic bacterium Aquifex aeolicus. We studied the pure and solubilized enzyme by denaturing and non-denaturing polyacrylamide electrophoresis, size-exclusion chromatography, cross-linking, analytical ultracentrifugation, visible and ultraviolet spectroscopy, mass spectrometry and electron microscopy. Additionally, we report the characterization of its enzymatic activity before and after crystallization. Finally, we discuss the crystallization of sulfide:quinone oxidoreductase in respect to its membrane topology and we propose a classification of monotopic membrane protein crystal lattices. Our data support and complement an earlier description of the three-dimensional structure of A. aeolicus sulfide:quinone oxidoreductase (M. Marcia, U. Ermler, G. Peng, H. Michel, Proc Natl Acad Sci USA, 106 (2009) 9625-9630) and may serve as a reference for further studies on monotopic membrane proteins.