The Metabolic Response of Various Cell Lines to Microtubule-Driven Uptake of Lipid- and Polymer-Coated Layer-by-Layer Microcarriers

Lipid structures, such as liposomes or micelles, are of high interest as an approach to support the transport and delivery of active agents as a drug delivery system. However, there are many open questions regarding their uptake and impact on cellular metabolism. In this study, lipid structures were assembled as a supported lipid bilayer on top of biopolymer-coated microcarriers based on the Layer-by-Layer assembly strategy. The functionalized microcarriers were then applied to various human and animal cell lines in addition to primary human macrophages (MΦ). Here, their influence on cellular metabolism and their intracellular localization were detected by extracellular flux analysis and immunofluorescence analysis, respectively. The impact of microcarriers on metabolic parameters was in most cell types rather low. However, lipid bilayer-supported microcarriers induced a decrease in oxygen consumption rate (OCR, indicative for mitochondrial respiration) and extracellular acidification rate (ECAR, indicative for glycolysis) in Vero cells. Additionally, in Vero cells lipid bilayer microcarriers showed a more pronounced association with microtubule filaments than polymer-coated microcarrier. Furthermore, they localized to a perinuclear region and induced nuclei with some deformations at a higher rate than unfunctionalized carriers. This association was reduced through the application of the microtubule polymerization inhibitor nocodazole. Thus, the effect of respective lipid structures as a drug delivery system on cells has to be considered in the context of the respective target cell, but in general can be regarded as rather low

The Metabolic Response of Various Cell Lines to Microtubule-Driven Uptake of Lipid- and Polymer-Coated Layer-by-Layer Microcarriers

Lipid structures, such as liposomes or micelles, are of high interest as an approach to support the transport and delivery of active agents as a drug delivery system. However, there are many open questions regarding their uptake and impact on cellular metabolism. In this study, lipid structures were assembled as a supported lipid bilayer on top of biopolymer-coated microcarriers based on the Layer-by-Layer assembly strategy. The functionalized microcarriers were then applied to various human and animal cell lines in addition to primary human macrophages (MΦ). Here, their influence on cellular metabolism and their intracellular localization were detected by extracellular flux analysis and immunofluorescence analysis, respectively. The impact of microcarriers on metabolic parameters was in most cell types rather low. However, lipid bilayer-supported microcarriers induced a decrease in oxygen consumption rate (OCR, indicative for mitochondrial respiration) and extracellular acidification rate (ECAR, indicative for glycolysis) in Vero cells. Additionally, in Vero cells lipid bilayer microcarriers showed a more pronounced association with microtubule filaments than polymer-coated microcarrier. Furthermore, they localized to a perinuclear region and induced nuclei with some deformations at a higher rate than unfunctionalized carriers. This association was reduced through the application of the microtubule polymerization inhibitor nocodazole. Thus, the effect of respective lipid structures as a drug delivery system on cells has to be considered in the context of the respective target cell, but in general can be regarded as rather low

The Metabolic Response of Various Cell Lines to Microtubule-Driven Uptake of Lipid- and Polymer-Coated Layer-by-Layer Microcarriers

Lipid structures, such as liposomes or micelles, are of high interest as an approach to support the transport and delivery of active agents as a drug delivery system. However, there are many open questions regarding their uptake and impact on cellular metabolism. In this study, lipid structures were assembled as a supported lipid bilayer on top of biopolymer-coated microcarriers based on the Layer-by-Layer assembly strategy. The functionalized microcarriers were then applied to various human and animal cell lines in addition to primary human macrophages (MΦ). Here, their influence on cellular metabolism and their intracellular localization were detected by extracellular flux analysis and immunofluorescence analysis, respectively. The impact of microcarriers on metabolic parameters was in most cell types rather low. However, lipid bilayer-supported microcarriers induced a decrease in oxygen consumption rate (OCR, indicative for mitochondrial respiration) and extracellular acidification rate (ECAR, indicative for glycolysis) in Vero cells. Additionally, in Vero cells lipid bilayer microcarriers showed a more pronounced association with microtubule filaments than polymer-coated microcarrier. Furthermore, they localized to a perinuclear region and induced nuclei with some deformations at a higher rate than unfunctionalized carriers. This association was reduced through the application of the microtubule polymerization inhibitor nocodazole. Thus, the effect of respective lipid structures as a drug delivery system on cells has to be considered in the context of the respective target cell, but in general can be regarded as rather low

Flow cytometry of HEK 293T cells interacting with polyelectrolyte multilayer capsules containing fluorescein-labeled poly(acrylic acid) as a pH sensor

Polyelectrolyte multilayer sensor capsules, 5 m in diameter, which contained fluorescein-labeled poly(acrylic acid) (PAAAF) as pH-sensitive reporter molecules, were fabricated and employed to explore their endocytotic uptake into HEK 293T cells by flow cytometry. The percentage of capsules residing in the endolysosomal compartment was estimated from the fluorescence intensity decrease caused by acidification. Capsules attached to the extracellular surface of the plasma membrane were identified by trypan blue quenching. The number of capsules in the cytoplasm was rather small, being below the detection limit of the method. The advantages of polyelectrolyte multilayer capsules are that the fluorophore is protected from interaction with cellular compartments and that the multilayer can be equipped with additional functions

The Metabolic Response of Various Cell Lines to Microtubule-Driven Uptake of Lipid- and Polymer-Coated Layer-by-Layer Microcarriers

Lipid structures, such as liposomes or micelles, are of high interest as an approach to support the transport and delivery of active agents as a drug delivery system. However, there are many open questions regarding their uptake and impact on cellular metabolism. In this study, lipid structures were assembled as a supported lipid bilayer on top of biopolymer-coated microcarriers based on the Layer-by-Layer assembly strategy. The functionalized microcarriers were then applied to various human and animal cell lines in addition to primary human macrophages (MΦ). Here, their influence on cellular metabolism and their intracellular localization were detected by extracellular flux analysis and immunofluorescence analysis, respectively. The impact of microcarriers on metabolic parameters was in most cell types rather low. However, lipid bilayer-supported microcarriers induced a decrease in oxygen consumption rate (OCR, indicative for mitochondrial respiration) and extracellular acidification rate (ECAR, indicative for glycolysis) in Vero cells. Additionally, in Vero cells lipid bilayer microcarriers showed a more pronounced association with microtubule filaments than polymer-coated microcarrier. Furthermore, they localized to a perinuclear region and induced nuclei with some deformations at a higher rate than unfunctionalized carriers. This association was reduced through the application of the microtubule polymerization inhibitor nocodazole. Thus, the effect of respective lipid structures as a drug delivery system on cells has to be considered in the context of the respective target cell, but in general can be regarded as rather low

Design of a Homogeneous Multifunctional Supported Lipid Membrane on Layer-by-Layer Coated Microcarriers

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

Specific Uptake of Lipid-Antibody-Functionalized LbL Microcarriers by Cells

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

Inhibition of human neutrophil elastase by α 1-antitrypsin functionalized colloidal microcarriers

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 α1-antitrypsin (AT) by colloidal microcarriers. AT is an anti-inflammatory agent and shows inhibitory effects toward its pro-inflammatory 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 intra- and 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