Sacroiliac innervation.

PURPOSE: To investigate the innervation pattern of the sacroiliac region, especially with regard to the sacroiliac joint (SIJ). Dorsal SIJ innervation was analyzed and described. Our main hypothesis was that nerves reach the SIJ dorsally, passing ligamental compartments, as this would explain dorsal SIJ pain. METHODS: To examine sacroiliac innervation, we followed the nerves in over 50 specimens over several years. Plastinated slices were evaluated, nerves in the region were stained histologically, and the data were summarized as 3D models. RESULTS: The Rami communicans and posterior branches of the spinal nerves and their branches that form a dorsal sacral plexus and communicating branches, together with corresponding vessels, were observed to form neurovascular bundles embedded by tiny fatty connectives in gaps and tunnels. Branches of L5-S1 pass the inner sacroiliac ligaments (the interosseous sacroiliac ligament and axial interosseous ligament). The outer sacroiliac ligaments (posterior sacroiliac ligaments, long posterior sacroiliac ligament, sacrotuberal ligament, thoracolumbar fascia) are passed by the S1-S4 branches. However, although the paths of these nerves are in the direction of the SIJ, they do not reach it. It is possible that impingement of the neurovascular bundles may result in pain. Moreover, the gaps and tunnels connect to the open dorsal SIJ. CONCLUSION: Our findings suggest that Bogduk's term "sacroiliac pain" correlates to "sacroiliac innervation", which consists of "inner-" and "outer sacroiliac ligament innervation", and to ventral "SIJ pain". The watery gaps and tunnels observed could play a significant role in innervation and thus in the origins of SIJ pain. LEVEL OF EVIDENCE: Individual cross-sectional studies with consistently applied reference standard and blinding.

Coxsackievirus B3 Infection of Human iPSC Lines and Derived Primary Germ-Layer Cells Regarding Receptor Expression.

The association of members of the enterovirus family with pregnancy complications up to miscarriages is under discussion. Here, infection of two different human induced pluripotent stem cell (iPSC) lines and iPSC-derived primary germ-layer cells with coxsackievirus B3 (CVB3) was characterized as an in vitro cell culture model for very early human development. Transcriptomic analysis of iPSC lines infected with recombinant CVB3 expressing enhanced green fluorescent protein (EGFP) revealed a reduction in the expression of pluripotency genes besides an enhancement of genes involved in RNA metabolism. The initial distribution of CVB3-EGFP-positive cells within iPSC colonies correlated with the distribution of its receptor coxsackie- and adenovirus receptor (CAR). Application of anti-CAR blocking antibodies supported the requirement of CAR, but not of the co-receptor decay-accelerating factor (DAF) for infection of iPSC lines. Among iPSC-derived germ-layer cells, mesodermal cells were especially vulnerable to CVB3-EGFP infection. Our data implicate further consideration of members of the enterovirus family in the screening program of human pregnancies. Furthermore, iPSCs with their differentiation capacity into cell populations of relevant viral target organs could offer a reliable screening approach for therapeutic intervention and for assessment of organ-specific enterovirus virulence.

Reversible Fusion Proteins as a Tool to Enhance Uptake of Virus-Functionalized LbL Microcarriers.

For the efficient treatment of an increasing number of diseases the development of new therapeutics as well as novel drug delivery systems is essential. Such drug delivery systems (DDS) must not only consider biodegradability and protective packaging but must also target and control the release of active substances, which is one of the most important points in DDS application. We highlight the improvement of these key aspects, the increased interaction rate of Layer-by-Layer (LbL) designed microcarriers as a promising DDS after functionalization with vesicular stomatitis virus (VSV). We make use of the unique conformational reversibility of the fusion protein of VSV as a surface functionalization of LbL microcarriers. This reversibility allows for VSV to be used both as a tool for assembly onto the DDS and as an initiator for an efficient cellular uptake. We could show that the evolutionary optimized viral fusion machinery can be successfully combined with a biophysical DDS for optimization of its cellular interaction.

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.

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.

Efficient inhibition of human leukocytic elastase by means of α1-antitrypsin/peptide complexes.

α1 -Antitrypsin (AT), a serine protease inhibitor that specifically targets hydrolytic enzymes, plays a significant role in the termination of tissue inflammation and can therefore represent a key factor in chronic incidences as chronic obstructive pulmonary disease (COPD) or chronic hepatitis. A local and low-dose therapy for the treatment of acquired chronic inflammatory processes which are characterized by insufficient AT amounts but also of genetically conditioned AT deficiencies is supposed to be more effective and less cost-intensive compared to current therapies. In this study, a noncovalent complex formation between the cell-penetrating peptide carrier hCT(18-32)-k7 and AT was performed. The complex was applied to HEK293T/17 cells, as proof-of-principle, and polymorphonuclear leukocytes (PMN), which are responsible for tissue destruction and the perpetuation of inflammation in chronic processes. Both cell species show a successful uptake and subsequently both, an intracellular dot-shaped and homogeneous distribution of the complex demonstrating phagolysosomal as well as cytoplasmic availability. Furthermore, a decreased human leukocytic elastase (HLE) activity was observed after the direct complex administration to PMN. Since the application did not cause an enhanced vitality loss, the complex could facilitate an improvement in direct, local and low-dose treatment of chronically proceeding processes in order to attenuate protease-mediated tissue destruction.

Trapping of protein cargo molecules inside DNA origami nanocages.

The development of the DNA origami technique has directly inspired the idea of using three-dimensional DNA cages for the encapsulation and targeted delivery of drug or cargo molecules. The cages would be filled with molecules that would be released at a site of interest upon cage opening triggered by an external stimulus. Though different cage variants have been developed, efficient loading of DNA cages with freely-diffusing cargo molecules that are not attached to the DNA nanostructure and their efficient retention within the cages has not been presented. Here we address these challenges using DNA origami nanotubes formed by a double-layer of DNA helices that can be sealed with tight DNA lids at their ends. In a first step we attach DNA-conjugated cargo proteins to complementary target strands inside the DNA tubes. After tube sealing, the cargo molecules are released inside the cavity using toehold-mediated strand displacement by externally added invader strands. We show that DNA invaders are rapidly entering the cages through their DNA walls. Retention of ∼70 kDa protein cargo molecules inside the cages was, however, poor. Guided by coarse-grained simulations of the DNA cage dynamics, a tighter sealing of the DNA tubes was developed which greatly reduced the undesired escape of cargo proteins. These improved DNA nanocages allow for efficient encapsulation of medium-sized cargo molecules while remaining accessible to small molecules that can be used to trigger reactions, including a controlled release of the cargo via nanocage opening.

Interaction, uptake, and processing of LbL-coated microcarriers by PMNs.

Functionalized microcarriers or hollow capsules transporting active agents offer the opportunity for drug delivery inside cells. A promising application of these drug delivery systems is the direct transport as well as the release of immobilized antiinflammatory substances (AIS) into polymorphonuclear leukocytes (PMNs), which play a key role in the course of inflammatory processes. The intended delivery of AIS into the inflamed tissue could alleviate tissue destruction taking place during chronic inflammation, as well as facilitate the termination of these processes. In this study, the capability of functionalized CaCO(3) microcarriers as AIS transporter system targeted at PMNs is investigated. The time-dependent interaction of protamine sulfate and dextran sulfate multilayer-coated 5 µm ± 1 µm CaCO(3) carriers with PMNs, in comparison with the usage of SiO(2) carriers as monodisperse model system of defined sizes (1, 3, and 5 µm), reveals a sufficient carrier/cell interaction and uptake for coincubation periods between 2 and 24 h. Furthermore, the microcarriers are exposed to an environment simulating primary granule/phagosomal conditions after phagocytosis by means of PMN stimulation. The incubation of CaCO(3) microcarriers with cell supernatant demonstrates a partial multilayer decomposition (three to five layers) within 24 h, allowing the gradual release of AIS within the short PMN life span. This observation suggests a potential application for this drug delivery system inside immunologically active cells and may open the way to new alternatives in the treatment of chronic processes.

Influence of layer-by-layer (LbL) assembled CaCO(3)-carriers on macrophage signaling cascades.

Numerous drawbacks in the current medical treatment of chronic inflammations still require the design of sensitive and gentle methods without side effects. Layer-by-layer (LbL) coated microcarriers loaded with a cocktail of anti-inflammatory substances are supposed to be a new challenge for the medical treatment of immunoreactive cells such as macrophages and polymorphonuclear leukocytes (PMN). Nevertheless, microcarrier application requires biocompatibility of the system itself. Therefore, the aim of this study was to investigate microcarrier CaCO(3) systems LbL coated with biopolymers and a lipid bilayer, respectively, regarding the maintenance of the release of pro-inflammatory cytokines as TNFα and IL1ß at normal levels. Only marginal increases after microcarrier interaction were allowed. The required microcarrier optimization results in the maximum use of a carrier/cell ratio of 1:1 for biopolymer-coated carriers and a carrier/cell ratio up to 5:1 for lipid-bilayer-coated carriers during the coincubation with macrophage-like cells. Low formation of reactive oxygen species (ROS) could not be maintained by either reduced carrier/cell ratios or by a surface lipid bilayer.

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.

Colloidal DNA carriers for direct localization in cell compartments by pH sensoring.

Multifunctional colloidal microparticles allow the integration of various active agents as well as reporter molecules into one system without interfering combining delivery and sensing functions. In this study, calcium carbonate particles were functionalized with fluorescein isothiocyanate-labeled poly(allylamine hydrochloride) (FITC-PAH) allowing particle localization in cell compartments of different pH. Plasmid DNA (pEGFP-C1 and pDsRed1-N1) as a reporter agent for drug release in the cytoplasm and rhodamine-B-isothiocyanate-labeled protamine (RITC-PRM) were integrated into biocompatible and biodegradable PRM/DXS multilayers. The uptake and processing of the particles by HEK293T/17 cells were investigated via flow cytometry and confocal laser scanning microscopy. The presented data show a clear correlation between the fluorescence intensity of the FITC-labeled core, that is, the particle localization after cellular uptake, and the expression of fluorescent proteins by the cells without further cell staining. In conclusion, this particle design allows the simultaneous study of particle location and processing to monitor the transport and release of active agents and should thus be an invaluable tool for the study and design of nano- and microcarrier systems.

Dual Transport of Active Substances with a Layer-by-Layer-Based Drug Delivery System to Terminate Inflammatory Processes.

Conventional therapies for chronic inflammation with high dose application of active agents are often accompanied with severe side effects so that other therapeutical strategies shall be developed to be less physically demanding but still highly efficient. Locally applied Layer-by-Layer (LbL) microcarriers transporting a low, but efficient dosage of active agents directly into the inflamed tissue offer a gentle therapy option. Here, the inhibition of highly degradative enzyme human neutrophile elastase (HNE) is adressed, which is produced and secreted by neutrophile granulocytes (PMNs) in the progress of inflammation. The protected transport and release of its natural inhibitor α1-antitrypsin (AT) as a constituent of the microcarrier's biopolymer multilayer allows for an efficient inhibition of extra- and intracellular elastase. The HOCl scavenger molecule cefoperazone, which preserves AT activity, as an additional multilayer constituent induces a much higher efficacy of the inhibitor. The successful assembly of both agents in different layers of the multilayer and the subsequent HNE inhibition in PMNs is investigated. The parallel application of cefoperazone leads to an enhanced inhibitory effect even with reduced AT amount and reduced carrier:cell ratio. It is demonstrated that the modular assembly strategy of LbL carriers allows for efficient synergistic effect of active agents in inflammatory process.

Enhanced Uptake and Endosomal Release of LbL Microcarriers Functionalized with Reversible Fusion Proteins.

The efficient application of smart drug-delivery systems requires further improvement of their cellular uptake and in particular their release from endolysosomal compartments into the cytoplasm of target cells. The usage of virus proteins allows for such developments, as viruses have evolved efficient entry mechanisms into the cell, mediated by their fusion proteins. In our investigations, the transferability of the glycoprotein G which is a fusion protein of the vesicular stomatitis virus (VSV-G) onto the surface of a layer-by-layer (LbL) designed microcarrier was investigated. The assembly of VSV-G as a reversible viral fusion protein onto LbL microcarriers indeed induced an enhanced uptake rate on Vero cells as well as a fast and efficient release of the intact carriers from endolysosomes into the cytoplasm. Additionally, neither virus-associated effects on cellular viability nor activation of an interferon response were detected. Our study emphasizes the suitability of VSV-G as an efficient surface functionalization of drug-delivery systems.

Non-vital polymorphonuclear leukocytes express myeloperoxidase on their surface.

The heme-containing enzyme myeloperoxidase (MPO) becomes expressed to the cell surface of non-vital polymorphonuclear leukocytes (PMNs) as evidenced by flow cytometry analysis and confocal fluorescence microscopy. While only a very small percentage of freshly isolated cells was able to bind the MPO antibody, PMN suspensions cultured for 36 h or longer time periods contained an increasing number of cells able to interact with these antibodies. Two distinct patterns of fluorescence for the MPO antibodies were observed. Antibodies were localised either in surface patches or distributed over the whole cell body. The latter type dominated in cell samples cultured for more than three days, while the first type was predominantly found in samples cultured for lower time periods. We observed also two peaks for fluorescence distribution by flow cytometry after addition of MPO antibodies to PMNs. Myeloperoxidase was localised at phosphatidylserine epitopes at the surface of non-vital PMNs as evidenced by coincubation with fluorescent MPO antibodies and FITC-labelled annexin V. Myeloperoxidase bound to the outer surface of PMNs uses hydrogen peroxide as a substrate as shown by appearance of an intense chemiluminescence using the impermeable luminescent protein Pholasin. Thus, myeloperoxidase becomes expressed to the surface of non-vital polymorphonuclear leukocytes colocalised with phosphatidylserine that may indicate a role of myeloperoxidase in apoptosis of PMNs.

Destabilization of acrosome and elastase influence mediate the release of secretory phospholipase A2 from human spermatozoa.

AIM: To determine the cellular distribution of secretory phospholipase A(2) (sPLA(2)) in dependence on the acrosomal state and under the action of elastase released under inflammatory processes from leukocytes. METHODS: Acrosome reaction of spermatozoa was triggered by calcimycin. Human leukocyte elastase was used to simulate inflammatory conditions. To visualize the distribution of sPLA(2) and to determine the acrosomal state, immunofluorescence techniques and lectin binding combined with confocal laser scanning fluorescence microscopy and flow cytometry were used. RESULTS: Although sPLA(2) was detected at the acrosome and tail regions in intact spermatozoa, it disappeared from the head region after triggering the acrosome reaction. This release of sPLA(2) was associated with enhanced binding of annexin V-fluoroscein isothiocyanate (FITC) to spermatozoa surfaces, intercalation of ethidium-homodimer I, and binding of FITC-labelled concanavalin A at the acrosomal region. Spermatozoa from healthy subjects treated with elastase were characterized by release of sPLA(2), disturbance of acrosome structure, and loss of vitality. CONCLUSION: The ability of spermatozoa to release secretory phospholipase A(2) is related to the acrosomal state. Premature destabilization of the acrosome and loss of sPLA(2) can occur during silent inflammations in the male genital tract. The distribution pattern of sPLA(2) in intact spermatozoa might be an additional parameter for evaluating sperm quality.

Enhanced cytoplasmic release of drug delivery systems: chloroquine as a multilayer and template constituent of layer-by-layer microcarriers.

Nano- and microcarriers as vehicles for active agents are applied to support them to reach their target in a defined, specific, and protected way. This implies not only a safe transport of agents towards the desired cell type or tissue but also the intracellular processing of the carrier: in particular, release of the incorporated carriers into the cytoplasm is a prerequisite for the successful subsequent delivery of most active agents and is often impeded by endolysosomal degenerative enzymes. We address this issue by using the layer-by-layer strategy of carrier assembly offering the opportunity to independently integrate and carry active agents but also specific agents preventing endolysosomal acidification. The weak base chloroquine (CQ) was investigated as a multilayer, template and capsule constituent regarding its ability to delay endolysosomal acidification and prolong the tolerable time frame in endolysosomes, which allows the carrier to finally escape into the cytoplasm. As a model and reporter active agent, plasmid encoding enhanced green fluorescent protein was used as a multilayer-assembly component to illustrate the cytoplasmic release of the intact carrier by final expression of the green fluorescent protein. Integrating CQ into the carrier, GFP expression could be strongly increased and a transfection efficiency of up to 20% could be obtained. This represents a very high transfection rate for a drug delivery system reached by only one additional reagent that has no further influence on the activity of the transported drug and cell viability, offering a significantly enhanced delivery efficiency.

Cellular interaction of a layer-by-layer based drug delivery system depending on material properties and cell types.

BACKGROUND: Drug delivery systems (DDS) and their interaction with cells are a controversial topic in the development of therapeutic concepts and approaches. On one hand, DDS are very useful for protected and targeted transport of defined dosages of active agents. On the other hand, their physicochemical properties such as material, size, shape, charge, or stiffness have a huge impact on cellular uptake and intracellular processing. Additionally, even identical DDS can undergo a completely diverse interaction with different cell types. However, quite often in in vitro DDS/cell interaction experiments, those aspects are not considered and DDS and cells are randomly chosen. METHODS AND RESULTS: Hence, our investigations provide an insight into layer-by-layer designed microcarriers with modifications of only some of the most important parameters (surface charge, stiffness, and applied microcarrier/cell ratio) and their influence on cellular uptake and viability. We also considered the interaction of these differently equipped DDS with several cell types and investigated professional phagocytes (neutrophil granulocytes; macrophages) as well as non-professional phagocytes (epithelial cells) under comparable conditions. We found that even small modifications such as layer-by-layer (LbL)-microcarriers with positive or negative surface charge, or LbL-microcarriers with solid core or as hollow capsules but equipped with the same surface properties, show significant differences in interaction and viability, and several cell types react very differently to the offered DDS. CONCLUSION: As a consequence, the properties of the DDS have to be carefully chosen with respect to the addressed cell type with the aim to efficiently transport a desired agent.

Fluorescent bead arrays by means of layer-by-layer polyelectrolyte adsorption.

Colloids with graduated fluorescence intensities were fabricated by means of layer-wise adsorption of fluorescein isothiocyanate-labelled poly(allyl amine hydrochloride) (FITC-PAH) together with poly(styrene sulfonate) (PSS) on silica particles. The graduated fluorescence was adjusted by variation of the fluorescent layer number and mixing labelled PAH with unlabelled PAH in one layer. The graduation of fluorescence intensities was adjusted in a geometric progression. It was shown that a proper label content is crucial if self-quenching phenomena are involved. The approach of mixing FITC-PAH with unlabelled polyelectrolyte during adsorption was unsatisfactory since competition in adsorption occurs. The system shows excellent stability at least over a period of two years.

Defoliation and plasmid delivery with layer-by-layer coated colloids.

The uptake of polyelectrolyte multilayer coated colloids into cells, subsequent defoliation and plasmid delivery was studied by means of confocal microscopy and flow cytometry. Silica particles coated layer-wise with protamine and dextran sulfate were given to HEK 293T cells. Optimum uptake was found with protamine as the top layer. The particle uptake likely follows an non-receptor-mediated endocytotic pathway. Defoliation of polyelectrolyte multilayer coated particles within cells was demonstrated by the release of incorporated plasmids as indicated by the expression of plasmid encoded proteins using the enhanced green fluorescence proteine (pEGFP-C1) plasmid and a red fluorescence protein (pDsRed1-N1) plasmid. This proves, together with the direct observation of fluorescent layer debris, the defoliation of coated particles and the release of layer components into the cytoplasm. Particle uptake and GFP expression.

Influence of Growth Characteristics of Induced Pluripotent Stem Cells on Their Uptake Efficiency for Layer-by-Layer Microcarriers.

Induced pluripotent stem cells (iPSCs) have the ability to differentiate into any specialized somatic cell type, which makes them an attractive tool for a wide variety of scientific approaches, including regenerative medicine. However, their pluripotent state and their growth in compact colonies render them difficult to access and, therefore, restrict delivery of specific agents for cell manipulation. Thus, our investigation focus was set on the evaluation of the capability of layer-by-layer (LbL) designed microcarriers to serve as a potential drug delivery system to iPSCs, as they offer several appealing advantages. Most notably, these carriers allow for the transport of active agents in a protected environment and for a rather specific delivery through surface modifications. As we could show, charge and mode of LbL carrier application as well as the size of the iPSC colonies determine the interaction with and the uptake rate by iPSCs. None of the examined conditions had an influence on iPSC colony properties such as colony morphology and size or maintenance of pluripotent properties. An overall interaction rate of LbL carriers with iPSCs of up to 20% was achieved. Those data emphasize the applicability of LbL carriers for stem cell research. Additionally, the potential use of LbL carriers as a promising delivery tool for iPSCs was contrasted to viral particles and liposomes. The identified differences among those delivery tools have substantiated our major conclusion that LbL carrier uptake rate is influenced by characteristic features of the iPSC colonies (most notably colony size) in addition to their surface charges.