Layer-by-layer (LbL)-coated microcarriers offer a good opportunity as transport systems for active agents into specific cells and tissues. The assembling of oppositely charged polyelectrolytes enables a modular construction of the carriers and therefore an optimized integration and application of drug molecules. Here, we report the multilayer incorporation and transport of R 1 -antitrypsin (AT) by colloidal microcarriers. AT is an anti-inflammatory agent and shows inhibitory effects toward its proinflammatory antagonist, human neutrophil elastase (HNE). The highly proteolytic enzyme HNE is released by polymorphonuclear leukocytes (PMNs) during inflammatory processes and can cause host tissue destruction and pain. The high potential of this study is based on a simultaneous intraand extracellular application of AT-functionalized LbL carriers. Carrier application in PMNs results in significant HNE inhibition within 21 h. Microcarriers phagocytosed by PMNs were time dependently decomposed inside phagolysosomes, which enables the step-by-step release of AT. Here, AT inactivates HNE before being released, which avoids a further HNE concentration increase in the extracellular space and, subsequently, reduces the risk of further tissue destruction. Additionally, AT surface-functionalized microcarriers allow the inhibition of already released HNE in the extracellular space. Finally, this study demonstrates the successful application of LbL carriers for a concurrent extra-and intracellular HNE inhibition aiming the rebalancing of protease and antiprotease concentrations and the subsequent termination of chronic inflammations.

The modular construction of Layer-by-Layer biopolymer microcarriers facilitates a highly specific design of drug delivery systems. A supported lipid bilayer (SLB) contributes to biocompatibility and protection of sensitive active agents. The addition of a lipid anchor equipped with PEG (shielding from opsonins) and biotin (attachment of exchangeable outer functional molecules) enhances the microcarrier functionality even more. However, a homogeneously assembled supported lipid bilayer is a prerequisite for a specific binding of functional components. Our investigations show that a tightly packed SLB improves the efficiency of functional components attached to the microcarrier's surface, as illustrated with specific antibodies in cellular application. Only a low quantity of antibodies is needed to obtain improved cellular uptake rates independent from cell type as compared to an antibody-functionalized loosely packed lipid bilayer or directly assembled antibody onto the multilayer. A fast disassembly of the lipid bilayer within endolysosomes exposing the underlying drug delivering multilayer structure demonstrates the suitability of LbL-microcarriers as a multifunctional drug delivery system.

No abstract

The Dna2 helicase-nuclease functions in concert with the replication protein A (RPA) in DNA double-strand break repair. Using ensemble and single-molecule biochemistry, coupled with structure modeling, we demonstrate that the stimulation of S. cerevisiae Dna2 by RPA is not a simple consequence of Dna2 recruitment to single-stranded DNA. The large RPA subunit Rfa1 alone can promote the Dna2 nuclease activity, and we identified mutations in a helix embedded in the N-terminal domain of Rfa1 that specifically disrupt this capacity. The same RPA mutant is instead fully functional to recruit Dna2 and promote its helicase activity. Furthermore, we found residues located on the outside of the central DNA-binding OB-fold domain Rfa1-A, which are required to promote the Dna2 motor activity. Our experiments thus unexpectedly demonstrate that different domains of Rfa1 regulate Dna2 recruitment, and its nuclease and helicase activities. Consequently, the identified separation-of-function RPA variants are compromised to stimulate Dna2 in the processing of DNA breaks. The results explain phenotypes of replication-proficient but radiation-sensitive RPA mutants and illustrate the unprecedented functional interplay of RPA and Dna2.

Key challenges in the development of drug delivery systems are the prevention of serum compartment interaction and the targeted delivery of the cargo. Layer-by-Layer microcarriers offer many advantages due to various options in drug assembly and multifunctional design. Surface modification with a supported lipid membrane enhances biocompatibility, drug protection ability, and specific functionality. However, the integration of functionalized lipids strongly influences the membrane formation and is often accompanied by submicrometer irregularities: The accessibility of underlying polymers to serum components may change the carrier's properties and enhances the susceptibility to opsonization. Therefore, the formation of a tightly assembled multifunctional lipid membrane has been emphasized. A phosphatidylserine/phosphatidylcholine (POPS/POPC) bilayer equipped with phosphatidylethanolamine-polyethylene glycol-biotin (PE-PEG-Biotin) was used to facilitate a biotin/streptavidin binding site for a variable attachment of an additional function, such as antibodies for specific targeting. Thus, a prefunctionalized carrier where only the outer functionality needs to be replaced without disturbing the underlying structure could be created.