Multi-omics analysis of innate and adaptive responses to BCG vaccination reveals epigenetic cell states that predict trained immunity.

Immune responses are tightly regulated yet highly variable between individuals. To investigate human population variation of trained immunity, we immunized healthy individuals with Bacillus Calmette-Guérin (BCG). This live-attenuated vaccine induces not only an adaptive immune response against tuberculosis but also triggers innate immune activation and memory that are indicative of trained immunity. We established personal immune profiles and chromatin accessibility maps over a 90-day time course of BCG vaccination in 323 individuals. Our analysis uncovered genetic and epigenetic predictors of baseline immunity and immune response. BCG vaccination enhanced the innate immune response specifically in individuals with a dormant immune state at baseline, rather than providing a general boost of innate immunity. This study advances our understanding of BCG's heterologous immune-stimulatory effects and trained immunity in humans. Furthermore, it highlights the value of epigenetic cell states for connecting immune function with genotype and the environment.

Reactivity of [Cp*Fe(η5 -As5 )] towards Carbenes, Silylenes and Germylenes.

The reaction behavior of [Cp*Fe(η5 -As5 )] (I) (Cp*=C5 Me5 ) towards carbenes and their heavier analogs was investigated. The reaction of I with NHCs (NHCs=N-heterocyclic carbenes) results in the first substitution products of polyarsenic ligand complexes by NHCs [Cp*Fe(η4 -As5 NHC)] (1 a: NHC=IMe=1,3,4,5-tetramethylimidazolin-2-ylidene, 1 b: NHC=IMes=1,3-bis(2,4,6-trimethylphenyl)-imidazolin-2-ylidene). In contrast, the reaction of I with Et CAAC (Et CAAC=2,6-diisopropylphenyl)-4,4-diethyl-2,2-dimethyl-pyrrolidin-5-ylidene) leads to a fragmentation and the formation of an unprecedented As6 -sawhorse-type compound [As2 (AsEt CAAC)4 ] (2). The reaction of (LE)2 (L=PhC(Nt Bu)2 ; E=Si, Ge) with I resulted in a rearrangement and an insertion of LE fragments, forming unique silicon- (4: [Cp*Fe(η4 -As4 SiL)], 5 a: [Cp*Fe(η4 -As6 SiL)) and germanium-containing (5 b: [Cp*Fe(η4 -As6 GeL)) cyclic polyarsenic ligand complexes.

Elucidating the Impact of Material Properties on Tablet Manufacturability for Binary Paracetamol Blends.

PURPOSE: Although the mechanical properties of paracetamol and MCC are extensively described in literature, there still is a need for a better understanding of the material properties impacting them. Thus, this study systematically analyzed material properties of paracetamol-MCC blends to elucidate their influence on the mechanical tablet properties in roller compaction and direct compression with special focus on surface properties. METHODS: Multiple material characteristics of binary mixtures of paracetamol and MCC with varying drug loads were analyzed, with particular emphasis on specific surface area and surface energy. Subsequently, mechanical tablet properties of the materials in direct compression and after roller compaction were examined. RESULTS: It was demonstrated that the impact of the initial material properties on mechanical tablet properties prevailed over the impact of processing route for paracetamol-MCC blends, underlining the importance of material characterization for tabletability of oral solid dosage forms. By applying bivariate as well as multivariate analysis, key material properties influencing the tabletability of paracetamol, MCC and its mixtures such as surface area, surface energy, effective angle of internal friction and density descriptors were identified. CONCLUSIONS: This study highlighted the importance of comprehensive assessment of different material characteristics leading to a deeper understanding of underlying factors impacting mechanical tablet properties in direct compression and after roller compaction by the example of paracetamol-MCC mixtures with varying drug loads. Furthermore, it was shown that multivariate analysis could be a valuable extension to common bivariate analysis to reveal underlying correlations of material properties.

The Path from Nasal Tissue to Nasal Mucosa on Chip: Part 2-Advanced Microfluidic Nasal In Vitro Model for Drug Absorption Testing.

The nasal mucosa, being accessible and highly vascularized, opens up new opportunities for the systemic administration of drugs. However, there are several protective functions like the mucociliary clearance, a physiological barrier which represents is a difficult obstacle for drug candidates to overcome. For this reason, effective testing procedures are required in the preclinical phase of pharmaceutical development. Based on a recently reported immortalized porcine nasal epithelial cell line, we developed a test platform based on a tissue-compatible microfluidic chip. In this study, a biomimetic glass chip, which was equipped with a controlled bidirectional airflow to induce a physiologically relevant wall shear stress on the epithelial cell layer, was microfabricated. By developing a membrane transfer technique, the epithelial cell layer could be pre-cultivated in a static holder prior to cultivation in a microfluidic environment. The dynamic cultivation within the chip showed a homogenous distribution of the mucus film on top of the cell layer and a significant increase in cilia formation compared to the static cultivation condition. In addition, the recording of the ciliary transport mechanism by microparticle image velocimetry was successful. Using FITC-dextran 4000 as an example, it was shown that this nasal mucosa on a chip is suitable for permeation studies. The obtained permeation coefficient was in the range of values determined by means of other established in vitro and in vivo models. This novel nasal mucosa on chip could, in future, be automated and used as a substitute for animal testing.

The Path from Nasal Tissue to Nasal Mucosa on Chip: Part 1-Establishing a Nasal In Vitro Model for Drug Delivery Testing Based on a Novel Cell Line.

In recent years, there has been a significant increase in the registration of drugs for nasal application with systemic effects. Previous preclinical in vitro test systems for transmucosal drug absorption studies have mostly been based on primary cells or on tumor cell lines such as RPMI 2650, but both approaches have disadvantages. Therefore, the aim of this study was to establish and characterize a novel immortalized nasal epithelial cell line as the basis for an improved 3D cell culture model of the nasal mucosa. First, porcine primary cells were isolated and transfected. The P1 cell line obtained from this process was characterized in terms of its expression of tissue-specific properties, namely, mucus expression, cilia formation, and epithelial barrier formation. Using air-liquid interface cultivation, it was possible to achieve both high mucus formation and the development of functional cilia. Epithelial integrity was expressed as both transepithelial electrical resistance and mucosal permeability, which was determined for sodium fluorescein, rhodamine B, and FITC-dextran 4000. We noted a high comparability of the novel cell culture model with native excised nasal mucosa in terms of these measures. Thus, this novel cell line seems to offer a promising approach for developing 3D nasal mucosa tissues that exhibit favorable characteristics to be used as an in vitro system for testing drug delivery systems.

3D bioprinting of corneal models: A review of the current state and future outlook.

The cornea is the outermost layer of the eye and serves to protect the eye and enable vision by refracting light. The need for cornea organ donors remains high, and the demand for an artificial alternative continues to grow. 3D bioprinting is a promising new method to create artificial organs and tissues. 3D bioprinting offers the precise spatial arrangement of biomaterials and cells to create 3D constructs. As the cornea is an avascular tissue which makes it more attractive for 3D bioprinting, it could be one of the first tissues to be made fully functional via 3D bioprinting. This review discusses the most common 3D bioprinting technologies and biomaterials used for 3D bioprinting corneal models. Additionally, the current state of 3D bioprinted corneal models, especially specific characteristics such as light transmission, biomechanics, and marker expression, and in vivo studies are discussed. Finally, the current challenges and future prospects are presented.

Cyclo-E5-bridged trinuclear triple-decker complexes (E = P, As) containing a triply-bonded Mo2 unit and their isomerisation.

The reaction of the low-coordinate quadruply-bonded dimolybdenum complex Mo2[µ,κ2-PhB(N-2,6-iPr2C6H3)2]2 (1) with Cp*Fe(η5-E5) (E = P, As) gives two trinuclear species Cp*Fe(µ3,η5:2:2-E5)Mo2[µ,κ2-PhB(N-2,6-iPr2C6H3)2]2 (E = P (4) and As (5)). 4 undergoes facile isomerisation upon heating to give Cp*FeMo2[κ2-PhB(N-2,6-iPr2C6H3)2](µ3,κ:κ:η2-P2)[µ3,κ:κ:η3κ-P3PhB(N-2,6-iPr2C6H3)2] (6), where the FeMo2P5 core motif displays a cubane-like structure.

Nucleophilic Attack at Pentaarsaferrocene [Cp*Fe(η5 -As5 )]-The Way to Larger Polyarsenide Ligands.

By the reaction of [Cp*Fe(η5 -As5 )] (I) (Cp*=C5 Me5 ) with main group nucleophiles, unique functionalized products with η4 -coordinated polyarsenide (Asn ) units (n=5, 6, 20) are obtained. With carbon-based nucleophiles such as MeLi or KBn (Bn=CH2 Ph), the anionic organo-substituted polyarsenide complexes, [Li(2.2.2-cryptand)][Cp*Fe(η4 -As5 Me)] (1 a) and [K(2.2.2-cryptand)][Cp*Fe{η4 -As5 (CH2 Ph)}] (1 b), are accessible. The use of KAsPh2 leads to a selective and controlled extension of the As5 unit and the formation of the monoanionic compound [K(2.2.2-cryptand][Cp*Fe(η4 -As6 Ph2 )] (2). When I is reacted with [M]As(SiMe3 )2 (M=Li ⋅ THF; K), the formation of the largest known anionic polyarsenide unit in [M'(2.2.2-cryptand)]2 [(Cp*Fe)4 {µ5 -η4 :η4 :η3 :η3 :η1 :η1 -As20 }] (3) occurred (M'=Li (3 a), K (3 b)).

Controlled introduction of functional groups at one P atom in [Cp*Fe(η5-P5)] and release of functionalised phosphines.

By salt metathesis reactions of the anionic complexes of the type [Cp*Fe(η4-P5R)]- (R = tBu (1a), Me (1b), -C[triple bond, length as m-dash]CPh (1c); Cp* = 1,2,3,4,5-pentamethylcyclopentadienyl) with organic electrophiles (XRFG; X = halogen; RFG = (CH2)3Br, (CH2)4Br, Me) a variety of organo-substituted polyphosphorus ligand complexes of the type [Cp*Fe(η4-P5RRFG)] (2) are obtained. Thereby, organic substituents with different functional groups (FG), such as halogens or nitriles, are introduced. In [Cp*Fe(η4-P5RR')] (2a: R = tBu, R' = (CH2)3Br), the bromine substituent can be easily substituted, leading to functionalized complexes [{Cp*Fe(η4-P5tBu)}(CH2)3{Cp*Fe(η4-P5Me)}] (4) and [Cp*Fe(η4-P5RR')] (5) (R = tBu, R' = (CH2)3PPh2) or by abstraction of a phosphine to the asymmetric substituted phosphine tBu(Bn)P(CH2)3Bn (6). The reaction of the dianionic species [K(dme)2]2[Cp*Fe(η4-P5)] (I') with bromo-nitriles leads to [Cp*Fe{η4-P5((CH2)3CN)2}] (7), allowing the introduction of two functional groups attached to one phosphorus atom. 7 reacts with ZnBr2 in a self-assembly reaction to form the supramolecular compound [Cp*Fe{η4-P5((CH2)3CN)2}ZnBr2]n (8).

Isolation and Cultivation of Porcine Endothelial Cells, Pericytes and Astrocytes to Develop an In Vitro Blood-Brain Barrier Model for Drug Permeation Testing.

The blood-brain barrier (BBB) is the bottleneck in the development of new drugs to reach the brain. Due to the BBB, toxic substances cannot enter the brain, but promising drug candidates also pass the BBB poorly. Suitable in vitro BBB models are therefore of particular importance during the preclinical development process, as they can not only reduce animal testing but also enable new drugs to be developed more quickly. The aim of this study was to isolate cerebral endothelial cells, pericytes, and astrocytes from the porcine brain to produce a primary model of the BBB. Additionally, as primary cells are well suited by their properties but the isolation is complex and better reproducibility with immortalized cells must be ensured, there is a high demand for immortalized cells with suitable properties for use as a BBB model. Thus, isolated primary cells can also serve as the basis for a suitable immortalization technique to generate new cell lines. In this work, cerebral endothelial cells, pericytes, and astrocytes were successfully isolated and expanded using a mechanical/enzymatic method. Furthermore, in a triple coculture model, the cells showed a significant increase in barrier integrity compared with endothelial cell monoculture, as determined by transendothelial electrical resistance measurement and permeation studies using sodium fluorescein. The results demonstrate the opportunity to obtain all three cell types significantly involved in BBB formation from one species, thus providing a suitable tool for testing the permeation properties of new drug candidates. In addition, the protocols are a promising starting point to generate new cell lines of BBB-forming cells as a novel approach for BBB in vitro models.

Correction: Novel synthetic route for (parent) phosphetanes, phospholanes, phosphinanes and phosphepanes.

[This corrects the article DOI: 10.1039/D3SC00580A.].

Novel synthetic route for (parent) phosphetanes, phospholanes, phosphinanes and phosphepanes.

A novel synthetic route for (parent) phosphorus-containing cycloalkanes such as phosphetanes, phospholanes, phosphinanes and phosphepanes is reported. By using [K(dme)2]2[Cp*Fe(η4-P5)] (I) in combination with α,ω-dibromoalkanes C n H2n Br2 [n = 3-6], unique phosphetane, phospholane, phosphinane and phosphepane precursor complexes [Cp*Fe{η4-P5(CH2) n }] [n = 3-6] (2-5) are synthesised. They act as P-atom carriers and the corresponding phosphetane, phospholane, phosphinane and phosphepanes (6-9) can be released by nucleophiles i.e., potassium benzyl (KBn) or LiAlH4. The latter enables the selective synthesis of parent cyclic secondary phosphines (10) in an easy and straightforward way, including the first parent phospholane (10b).

Spray-dried paracetamol/polyvinylpyrrolidone amorphous solid dispersions: Part II - Solubility and in vitro drug permeation behavior.

Amorphous solid dispersions (ASDs) comprising an active pharmaceutical ingredient (API) and polymer are a frequently described approach in the formulation of new drug candidates. This study aimed to evaluate the saturation solubility and dissolution behavior of ASDs consisting of paracetamol (PCM) and polyvinylpyrrolidone/vinyl acetate (PVP/VA) in water and their influence on the transepithelial in vitro permeation of PCM. With increasing amounts of PVP/VA, the water solubility of ASDs containing PCM increased up to six times compared to that of a saturated PCM solution. In the case of preparations with 30 % PCM, two-phase separation was observed in water at room temperature, consisting of a polymer-rich phase with high API loading and an aqueous, polymer-poor phase. This result was attributed to the thermoresponsive behavior of PVP/VA with lower critical solution temperature (LCST). As the PCM content in the ASD increased, the LCST decreased. This behavior was analyzed by measuring the demixing temperature (Tdem) values with differential scanning calorimetry (DSC). Furthermore, the permeation behavior of PCM from these phase-separated preparations through Caco-2 cells was analyzed. Additionally, the effect of these preparations on cell viability was evaluated using the MTT assay. Preparations with relatively high PCM concentrations showed a reduction in cell viability.

Development of In Vitro Dry Eye Models to Study Proliferative and Anti-Inflammatory Effects of Allogeneic Serum Eye Drops.

This study aimed to develop valid in vitro models for preclinical evaluation of proliferative and anti-inflammatory effects of human allogeneic serum eye drops for dry eye disease (DED) treatment. A DED wound healing model was developed by analyzing the influence of coating and serum concentrations on human corneal epithelial (HCE-T) wound closure. Further, intralaboratory variance, freeze-thaw cycle effects, donor variability and stability assays were conducted. Interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNFα) were used to induce the gene expression of matrix metalloproteinase 9 (MMP9), cyclooxygenase 2 (COX2), transforming growth factor-ß (TGFß) and IL-1ß. MMP9 induction was optimized using a design-of-experiments (DoE) approach and applied to examine serum under static and dynamic conditions. MMP9 protein expression was analyzed by ELISA. The DED wound healing model detected proliferative effects of serum down to 1% with a small intralaboratory variance. Serum stability was shown over six months, donor variance could be detected, and freeze-thaw cycle effects did not affect wound closure. Serum decreased MMP9 expression on the gene and protein levels. The induction method was successfully optimized using DoE modeling and transferred to a dynamic setting mimicking tear film fluidics. The DED wound healing and inflammatory DED model present useful in vitro models for the preclinical evaluation of allogeneic serum eye drops without the use of animal experiments.

Understanding the Multidimensional Effects of Polymorphism, Particle Size and Processing for D-Mannitol Powders.

The relevance of the polymorphic form, particle size, and processing of mannitol for the mechanical properties of solid oral dosage forms was examined. Thus, particle and powder properties of spray granulated ß D-mannitol, ß D-mannitol, and δ D-mannitol were assessed in this study with regards to their manufacturability. D-mannitol is a commonly used excipient in pharmaceutical formulations, especially in oral solid dosage forms, and can be crystallized as three polymorphic forms, of which ß is the thermodynamically most stable form and δ is a kinetically stabilized polymorph. A systematic analysis of the powders as starting materials and their respective roller compacted granules is presented to elucidate the multidimensional effects of powder and granules characteristics such as polymorphic form, particle size, and preprocessing on the resulting tablets' mechanical properties. In direct compression and after roller compaction, δ polymorph displayed superior tableting properties over ß mannitol, but was outperformed by spray granulated ß mannitol. This could be primarily correlated to the higher specific surface area, leading to higher bonding area and more interparticle bonds within the tablet. Hence, it was shown that surface characteristics and preprocessing can prevail over the impact of polymorphism on manufacturability for oral solid dosage forms.

Stable Two-Legged Parent Piano-Stool and Mixed Diborabenzene-E4 (E=P, As) Sandwich Complexes of Group 8.

A cyclic alkyl(amino)carbene-stabilized 1,4-diborabenzene (DBB) ligand enables the isolation of 18-electron two-legged parent piano-stool Fe0 and Ru0 complexes, [(η6 -DBB)M(CO)2 ], the ruthenium complex being the first of its kind to be structurally characterized. [(η6 -DBB)Fe(CO)2 ] reacts with E4 (E=P, As) to yield mixed DBB-cyclo-E4 sandwich complexes with planar E4 2- ligands. Computational analyses confirm the strong electron-donating capacity of the DBB ligand and show that the E4 ligand is bound by four equivalent Fe-P σ bonds.

Tissue Barrier-on-Chip: A Technology for Reproducible Practice in Drug Testing.

One key application of organ-on-chip systems is the examination of drug transport and absorption through native cell barriers such the blood-brain barrier. To overcome previous hurdles related to the transferability of existing static cell cultivation protocols and polydimethylsiloxane (PDMS) as the construction material, a chip platform with key innovations for practical use in drug-permeation testing is presented. First, the design allows for the transfer of barrier-forming tissue into the microfluidic system after cells have been seeded on porous polymer or Si3N4 membranes. From this, we can follow highly reproducible models and cultivation protocols established for static drug testing, from coating the membrane to seeding the cells and cell analysis. Second, the perfusion system is a microscopable glass chip with two fluid compartments with transparent embedded electrodes separated by the membrane. The reversible closure in a clamping adapter requires only a very thin PDMS sealing with negligible liquid contact, thereby eliminating well-known disadvantages of PDMS, such as its limited usability in the quantitative measurements of hydrophobic drug molecule concentrations. Equipped with tissue transfer capabilities, perfusion chamber inertness and air bubble trapping, and supplemented with automated fluid control, the presented system is a promising platform for studying established in vitro models of tissue barriers under reproducible microfluidic perfusion conditions.

Dexamethasone-loaded keratin films for ocular surface reconstruction.

Amniotic membrane (AM) is often applied as a substitute material during ocular surface reconstruction. However, since AM has several disadvantages, alternative materials must be considered for this application. Keratin films made from human hair (KFs) have previously been presented as a promising option; they exhibited suitable characteristics and satisfactory biocompatibility in an in vivo rabbit model. Nevertheless, dexamethasone (DEX) eye drops are necessary after surgery to suppress inflammation. Since eye drops must be administered frequently, this might result in poor patient compliance, and the release of DEX at the transplant site would be clinically beneficial. Therefore, we aimed to incorporate DEX into KFs without hindering the positive film characteristics. Drug-loaded KFs were generated either by suspension technique or by the addition of solubilizing agents. The resulting specimens were analyzed regarding appearance, loading capacity, transparency, mechanical characteristics, swelling behavior and in vitro release. Furthermore, biocompatibility was assessed in vitro by determining the cell viability, seeding efficiency and growth behavior of corneal epithelial cells. The amount of incorporated DEX influenced the transparency and biomechanical properties of the films, but even highly loaded films showed properties similar to those of AM. The suspension technique was identified as the best incorporation approach regarding chemical stability and prolonged DEX release. Moreover, suspended DEX in the films did not negatively impact cell seeding efficiencies, and the cell-growth behaviors on the specimens with moderate DEX loads were satisfactory. This suggest that these films could comprise a suitable alternative material with additional anti-inflammatory activity for ocular surface reconstruction. Graphical abstract.

Spray-Dried Paracetamol/Polyvinylpyrrolidone Amorphous Solid Dispersions: Part I-Stability of Powders and Tablets.

The formulation of active pharmaceutical ingredients (APIs) in amorphous solid dispersions (ASDs) is a promising approach to improve the bioavailability of poorly soluble compounds. However, problems often arise in the production of tablets from ASDs regarding the compressibility and recrystallization of the API. In the present study, the preparation of spray-dried ASDs of paracetamol (PCM) and four different types of polyvinylpyrrolidone (PVP) and their further processing into tablets were investigated. The influence of PVP type on the glass transition temperature (Tg) and the physical stability of ASD powders were characterized by differential scanning calorimetry (DSC) and powder X-ray diffraction (XRD). ASD powders with 10 to 30% PCM were stable for at least 48 weeks. PCM contents of 40 to 50% led to recrystallization of the amorphous PCM within a few days or weeks. ASD with PVP/vinyl acetate (VA) copolymer (PVP/VA) was the most unstable and tended to recrystallize in PCM polymorphic form II. This formulation was therefore used for tablet studies. The influence of compression force on recrystallization, crushing strength, and drug release was investigated. Even high compression forces did not affect the stability of the ASD. However, the ASD tablets led to slow release of the API.

Pentaphosphaferrocene-mediated synthesis of asymmetric organo-phosphines starting from white phosphorus.

The synthesis of phosphines is based on white phosphorus, which is usually converted to PCl3, to be afterwards substituted step by step in a non-atomic efficient manner. Herein, we describe an alternative efficient transition metal-mediated process to form asymmetrically substituted phosphines directly from white phosphorus (P4). Thereby, P4 is converted to [Cp*Fe(η5-P5)] (1) (Cp* = η5-C5(CH3)5) in which one of the phosphorus atoms is selectively functionalized to the 1,1-diorgano-substituted complex [Cp*Fe(η4-P5R'R″)] (3). In a subsequent step, the phosphine PR'R″R‴ (R' ≠ R″ ≠ R‴ = alky, aryl) (4) is released by reacting it with a nucleophile R‴M (M = alkali metal) as racemates. The starting material 1 can be regenerated with P4 and can be reused in multiple reaction cycles without isolation of the intermediates, and only the phosphine is distilled off.