Vitali Vogel

Optimization of the preparation process for human serum albumin (HSA) nanoparticles

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.

Comparison of scanning electron microscopy, dynamic light scattering and analytical ultracentrifugation for the sizing of poly(butyl cyanoacrylate) nanoparticles

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.

Structural evolution of C‐terminal domains in the p53 family

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 β‐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.

Dimer-tetramer transition controls RUNX1/ETO leukemogenic activity

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.

Analysis of the Complex Formation of Heparin with Protamine by Light Scattering and Analytical Ultracentrifugation: Implications for Blood Coagulation Management

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.

A toolbox for the upscaling of ethanolic human serum albumin (HSA) desolvation.

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.

Assessing the drug release from nanoparticles: Overcoming the shortcomings of dialysis by using novel optical techniques and a mathematical model.

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.

Morphology, size distribution, and aggregate structure of lipopolysaccharide and lipid A dispersions from enterobacterial origin

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.

Physicochemical characterization of protamine-phosphorothioate nanoparticles

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 +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.

Proteolytically-induced changes of secondary structural protein conformation of bovine serum albumin monitored by Fourier transform infrared (FT-IR) and UV-circular dichroism spectroscopy.

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.