Klaus Eisele

Fine-tuning DNA/albumin polyelectrolyte interactions to produce the efficient transfection agent cBSA-147

We present the preparation and isolation of different chemically modified BSA species with varying numbers of primary amino groups at the surface. Highly cationic albumin proteins with increased numbers of amino groups were achieved and complex formation with plasmid DNA was carefully investigated. We compare the transfection results, polyelectrolyte complexes morphologies with their impact on complex stabilities, cytotoxicities and DNA accessibility. This knowledge-driven approach led to the identification of the efficient non-viral DNA delivery agent cBSA-147, which showed high transfection efficacies and stability.

A Core–Shell Albumin Copolymer Nanotransporter for High Capacity Loading and Two-Step Release of Doxorubicin with Enhanced Anti-Leukemia Activity

The native transportation protein serum albumin represents an attractive nano-sized transporter for drug delivery applications due to its beneficial safety profile. Existing albumin-based drug delivery systems are often limited by their low drug loading capacity as well as noticeable drug leakage into the blood circulation. Therefore, a unique albumin-derived core-shell doxorubicin (DOX) delivery system based on the protein denaturing-backfolding strategy was developed. 28 DOX molecules were covalently conjugated to the albumin polypeptide backbone via an acid sensitive hydrazone linker. Polycationic and pegylated human serum albumin formed two non-toxic and enzymatically degradable protection shells around the encapsulated DOX molecules. This core-shell delivery system possesses notable advantages, including a high drug loading capacity critical for low administration doses, a two-step drug release mechanism based on pH and the presence of proteases, an attractive biocompatibility and narrow size distribution inherited from the albumin backbone, as well as fast cellular uptake and masking of epitopes due to a high degree of pegylation. The IC50 of these nanoscopic onion-type micelles was found in the low nanomolar range for Hela cells as well as leukemia cell lines. In vivo data indicate its attractive potential as anti-leukemia treatment suggesting its promising profile as nanomedicine drug delivery system.

A quantum dot photoswitch for DNA detection, gene transfection, and live-cell imaging.

Quantum dots (QDs) coated with an albumin-derived copolymer shell exhibit significant photoresponsiveness to DNA loading and have great potential for investigating gene delivery processes. The QDs reported herein are positively charged, have attractive optical properties, and are noncytotoxic and notably stable in live cells. Their complex formation with plasmid DNA leads to proportionally decreased photoluminescence and efficient gene transfection is observed. Therefore, they are suitable for live-cell bioimaging and mechanistic studies of nonviral gene delivery. Fluorescence correlation spectroscopy is applied for the first time to investigate individual QDs diffusing in large endosomes inside living cells, and serves as a valuable tool to study the physical properties of QDs inside live cells. The data obtained in this study strongly support the notable stability of these QDs, even in cell endosomes.

Tailored Albumin-based Copolymers for Receptor-Mediated Delivery of Perylenediimide Guest Molecules

The synthesis of a novel and multifunctional copolymer based on a human serum albumin backbone bearing several folic acid as well as PEO groups was presented. In solution, this side-chain copolymer adopts a globular architecture and about five molecules of the water-insoluble chromophore PDI were successfully incorporated into these micelles for receptor-mediated cell uptake investigations. A significant uptake of these bioconjugates via receptor-mediated endocytosis was detected for cells expressing folic acid receptors in the cell membrane. These novel albumin-based copolymers could serve as efficient and biocompatible carrier systems facilitating the directed delivery of lipophilic drug molecules into cancer cells and they allow investigating vesicle formation and trafficking even at the single molecule level.

Cationized albumin-biocoatings for the immobilization of lipid vesicles

Tethered lipid membranes or immobilized lipid vesicles are frequently used as biomimetic systems. In this article, the authors presented a suitable method for efficient immobilization of lipid vesicles onto a broad range of surfaces, enabling analysis by quantitative methods even under rigid, mechanical conditions—bare surfaces such as hydrophilic glass surfaces as well as hydrophobic polymer slides or metal surfaces such as gold. The immobilization of vesicles was based on the electrostatic interaction of zwitterionic or negatively charged lipid vesicles with two types of cationic chemically modified bovine serum albumin (cBSA) blood plasma proteins (cBSA-113 and cBSA-147). Quantitative analysis of protein adsorption was performed as the cBSA coatings were characterized by atomic force microscopy, surface zeta potential measurement, fluorescence microscopy, and surface plasmon spectroscopy, revealing a maximal surface coverage 270–280 ng/cm2 for 0.02 mg/ml cBSA on gold. Small unilamellar vesicles as well as giant unilamellar vesicles (GUVs) were readily immobilized (∼15 min) on cBSA coated surfaces. GUVs with 5–10 mol% negatively charged 1,2,-dipalmitoyl-sn-glycero-3-phosphoglycerol remained stable in liquid for at least 5 weeks.

Host–guest interactions in polycationic human serum albumin bioconjugates

Polycationic human serum albumin, cHSA, as well as cHSA conjugates with multiple polyethylene(oxide) chains of two different lengths were synthesized, and the uptake and release of spin-labeled fatty acid (FA) ligands were quantitatively analyzed by continuous wave (CW) electron paramagnetic resonance (EPR) spectroscopy and nanoscale distance measurements with double electron–electron resonance (DEER) spectroscopy. It is found that the seven FA binding pockets of native HSA are less well accessible by FAs in cHSA and its PEO-conjugates. A large number (at least 25) of FAs can diffusely and electrostatically be bound to the surface of all highly cationic serum albumin variants. These bound FAs show a remarkable resilience against release from both cHSA and PEO conjugates. Conjugation of PEO chains to cHSA had only minute effects on all EPR data, indicating that PEO grafts can be used without reduction of effectiveness in the ligand binding.

Bioactive Unnatural Somatostatin Analogues through Bioorthogonal Iodo‐ and Ethynyl‐Disulfide Intercalators

Iodo- and ethynyl-containing bisalkylating bioconjugation agents 5 and 8 were achieved and allow the introduction of reactive unnatural substituents into proteins and peptides whilst the bioactive 3D structure is retained. Derivatives of the peptide hormone somatostatin bearing a single iodo or ethynyl group were prepared through intercalation into the disulfide bridge. For the first time, the exact reaction mechanism of the intercalation was elucidated by applying 2D NMR experiments and it was shown that, during the reaction, somatostatin diastereomers were formed. Site-directed modification of the ethynyl-modified peptide with a coumarin chromophore was achieved through a [1,3] dipolar Huisgen cycloaddition reaction; this suggests that such a derivative could serve as an attractive platform to prepare artificial somatostatin compound libraries. The biological activity and specificity of a representative modified somatostatin derivative was demonstrated and efficient receptor-mediated cell uptake occurred in a dose-dependent manner into receptor positive cells only. The iodo and ethynyl bioconjugation reagents presented herein could be applied for introducing such substituents into alternative peptides and proteins and, in principle, could facilitate the efficient design of a broad variety of artificial protein and peptide analogues with previously unknown bioactivities.

cBSA-147 for the preparation of bacterial biofilms in a microchannel reactor

Whole cells are attractive biocatalysts, particularly if the reaction requires cofactors or involves multiple transformations. Immobilization of the catalyst is often a prerequisite for continuous processes. The highly cationic chemically modified plasma protein bovine serum albumin (cBSA-147) has been applied for the electrostatically mediated immobilization of the planktonic bacterium E. coli BL21 star (DE3), and the resulting biofilms were superior to those formed on poly-L-lysine coated surfaces. The biocatalyst was immobilized in a capillary column (inside diameter of 530 μm and L=30 m) and evaluated in the enantioselective reduction of ethyl acetoacetate to R-(−)ethyl hydroxybutyrate. In continuous operation in the microreactor format, the productivity of the cells was about 30% higher than that determined in a bench-scale fermentation system. This increase is attributed to the improved mass transfer over short geometrical dimensions. The similarity in the results indicates that studies on a biofilm-coated microreactor can be used for the accelerated collection of data for process optimization.

PEGylated Cationic Serum Albumin for Boosting Retroviral Gene Transfer.

Retroviral vectors are common tools for introducing genes into the genome of a cell. However, low transduction rates are a major limitation in retroviral gene transfer, especially in clinical applications. We generated cationic human serum albumin (cHSA) protected by a shell of poly(ethylene glycol) (PEG); this significantly enhanced retroviral gene transduction with potentially attractive pharmacokinetics and low immunogenicity. By screening a panel of chemically optimized HSA compounds, we identified a very potent enhancer that boosted the transduction rates of viral vectors. Confocal microscopy revealed a drastically increased number of viral particles attached to the surfaces of target cells. In accordance with the positive net charge of cationic and PEGylated HSA, this suggests a mechanism of action in which the repulsion of the negatively charged cellular and viral vector membranes is neutralized, thereby promoting attachment and ultimately transduction. Importantly, the transduction‐enhancing PEGylated HSA derivative evaded recognition by HSA‐specific antibodies and macrophage activation. Our findings hold great promise for facilitating improved retroviral gene transfer.