Intra-articular injection of an extended-release flavopiridol formulation represents a potential alternative to other intra-articular medications for treating equine joint disease.

OBJECTIVE: To establish the pharmacokinetics of the cyclin-dependent kinase-9 inhibitor flavopiridol in equine middle carpal joints, using an extended-release poly lactic-co-glycolic acid (PLGA) microparticle formulation. ANIMALS: 4 healthy horses without evidence of forelimb lameness. METHODS: A 6-week longitudinal pharmacokinetic study was conducted in 2 phases (6 weeks each) in 4 healthy horses. The PLGA microparticles containing 122 µg flavopiridol in 3 mL saline were administered by intra-articular injection into 1 middle carpal joint, with empty PLGA microparticles injected into the contralateral joint as a control. Synovial fluid and plasma were collected at time points out to 6 weeks, and drug concentrations in synovial fluid and plasma were determined using validated protocols. Synovial fluid total protein and total nucleated cell count and differential, CBC, serum biochemistry, and lameness exams were performed at each of the time points. RESULTS: Synovial fluid flavopiridol averaged 19 nM at week 1, gradually reduced to 1.4 nM by 4 weeks, and was generally below the detection limit at 5 and 6 weeks. There was no detectable flavopiridol in the plasma samples, and no adverse effects were observed at any time point. CLINICAL RELEVANCE: Intra-articular injection of PLGA microparticle-encapsulated flavopiridol was well tolerated in horses, with detectable levels of flavopiridol in the synovial fluid out to 4 weeks with negligible systemic exposure. Flavopiridol is a cyclin-dependent kinase-9 inhibitor with potent anti-inflammatory and analgesic activity. The extended-release microparticle formulation promotes intra-articular retention of the drug and it may be an alternative to other intra-articular medications for treatment of equine joint disease.

Surfactant-Mediated Structural Modulations to Planar, Amphiphilic Multilamellar Stacks.

The hydrophobic effect, a ubiquitous process in biology, is a primary thermodynamic driver of amphiphilic self-assembly. It leads to the formation of unique morphologies including two highly important classes of lamellar and micellar mesophases. The interactions between these two types of structures and their involved components have garnered significant interest because of their importance in key biochemical technologies related to the isolation, purification, and reconstitution of membrane proteins. This work investigates the structural organization of mixtures of the lamellar-forming phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and two zwitterionic micelle-forming surfactants, being n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent 3-12 or DDAPS) and 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (O-Lyso-PC), when assembled by water vapor hydration with X-ray diffraction measurements, brightfield optical microscopy, wide-field fluorescence microscopy, and atomic force microscopy. The results reveal that multilamellar mesophases of these mixtures can be assembled across a wide range of POPC to surfactant (POPC:surfactant) concentration ratios, including ratios far surpassing the classical detergent-saturation limit of POPC bilayers without significant morphological disruptions to the lamellar motif. The mixed mesophases generally decreased in lamellar spacing (D) and headgroup-to-headgroup distance (Dhh) with a higher concentration of the doped surfactant, but trends in water layer thickness (Dw) between each bilayer in the stack are highly variable. Further structural characteristics including mesophase topography, bilayer thickness, and lamellar rupture force were revealed by atomic force microscopy (AFM), exhibiting homogeneous multilamellar stacks with no significant physical differences with changes in the surfactant concentration within the mesophases. Taken together, the outcomes present the assembly of unanticipated and highly unique mixed mesophases with varied structural trends from the involved surfactant and lipidic components. Modulations in their structural properties can be attributed to the surfactant's chemical specificity in relation to POPC, such as the headgroup hydration and the hydrophobic chain tail mismatch. Taken together, our results illustrate how specific chemical complexities of surfactant-lipid interactions can alter the morphologies of mixed mesophases and thereby alter the kinetic pathways by which surfactants dissolve lipid mesophases in bulk aqueous solutions.

Production of Lipid Constructs by Design via Three-Dimensional Nanoprinting.

Atomic force microscopy (AFM) in conjunction with microfluidic delivery was utilized to produce three-dimensional (3D) lipid structures following a custom design. While AFM is well-known for its spatial precision in imaging and 2D nanolithography, the development of AFM-based nanotechnology into 3D nanoprinting requires overcoming the technical challenges of controlling material delivery and interlayer registry. This work demonstrates the concept of 3D nanoprinting of amphiphilic molecules such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Various formulations of POPC solutions were tested to achieve point, line, and layer-by-layer material delivery. The produced structures include nanometer-thick disks, long linear spherical caps, stacking grids, and organizational chiral architectures. The POPC molecules formed stacking bilayers in these constructions, as revealed by high-resolution structural characterizations. The 3D printing reached nanometer spatial precision over a range of 0.5 mm. The outcomes reveal the promising potential of our designed technology and methodology in the production of 3D structures from nanometer to continuum, opening opportunities in biomaterial sciences and engineering, such as in the production of 3D nanodevices, chiral nanosensors, and scaffolds for tissue engineering and regeneration.

Mechanical Cues for Triggering and Regulating Cellular Movement Selectively at the Single-Cell Level.

Cell motility plays important roles in many biophysical and physiological processes ranging from in vitro biomechanics, wound healing, to cancer metastasis. This work introduces a new means to trigger and regulate motility individually using transient mechanical stimulus applied to designated cells. Using BV2 microglial cells, our investigations indicate that motility can be reproducibly and reliably initiated using mechanical compression of the cells. The location and magnitude of the applied force impact the movement of the cell. Based on observations from this investigation and current knowledge of BV2 cellular motility, new physical insights are revealed into the underlying mechanism of force-induced single cellular movement. The process involves high degrees of myosin activation to repair actin cortex breakages induced by the initial mechanical compression, which leads to focal adhesion degradation, lamellipodium detachment, and finally, cell polarization and movement. Modern technology enables accurate control over force magnitude and location of force delivery, thus bringing us closer to programming cellular movement at the single-cell level. This approach is of generic importance to other cell types beyond BV2 cells and has the intrinsic advantages of being transient, non-toxic, and non-destructive, thus exhibiting high translational potentials including mechano-based therapy.

Production of Inhalable Ultra-Small Particles for Delivery of Anti-Inflammation Medicine via a Table-Top Microdevice.

A table-top microdevice was introduced in this work to produce ultrasmall particles for drug delivery via inhalation. The design and operation are similar to that of spray-drying equipment used in industry, but the device itself is much smaller and more portable in size, simpler to operate and more economical. More importantly, the device enables more accurate control over particle size. Using Flavopiridol, an anti-inflammation medication, formulations have been developed to produce inhalable particles for pulmonary delivery. A solution containing the desired components forms droplets by passing through an array of micro-apertures that vibrate via a piezo-electrical driver. High-purity nitrogen gas was introduced and flew through the designed path, which included the funnel collection and cyclone chamber, and finally was pumped away. The gas carried and dried the micronized liquid droplets along the pathway, leading to the precipitation of dry solid microparticles. The formation of the cyclone was essential to assure the sufficient travel path length of the liquid droplets to allow drying. Synthesis parameters were optimized to produce microparticles, whose morphology, size, physio-chemical properties, and release profiles met the criteria for inhalation. Bioactivity assays have revealed a high degree of anti-inflammation. The above-mentioned approach enabled the production of inhalable particles in research laboratories in general, using the simple table-top microdevice. The microparticles enable the inhalable delivery of anti-inflammation medicine to the lungs, thus providing treatment for diseases such as pulmonary fibrosis and COVID-19.

Intra-articular injection of flavopiridol-loaded microparticles for treatment of post-traumatic osteoarthritis.

Rapid joint clearance of small molecule drugs is the major limitation of current clinical approaches to osteoarthritis and its subtypes, including post-traumatic osteoarthritis (PTOA). Particulate systems such as nano/microtechnology could provide a potential avenue for improved joint retention of small molecule drugs. One drug of interest for PTOA treatment is flavopiridol, which inhibits cyclin-dependent kinase 9 (CDK9). Herein, polylactide-co-glycolide microparticles encapsulating flavopiridol were formulated, characterized, and evaluated as a strategy to mitigate PTOA-associated inflammation through the inhibition of CDK9. Characterization of the microparticles, including the drug loading, hydrodynamic diameter, stability, and release profile was performed. The mean hydrodynamic diameter of flavopiridol particles was ∼15 µm, indicating good syringeability and low potential for phagocytosis. The microparticles showed no cytotoxicity in-vitro, and drug activity was maintained after encapsulation, even after prolonged exposure to high temperatures (60 °C). Flavopiridol-loaded microparticles or blank (unloaded) microparticles were administered by intraarticular injection in a rat knee injury model of PTOA. We observed significant joint retention of flavopiridol microparticles compared to the soluble flavopiridol, confirming the sustained release behavior of the particles. Matrix metalloprotease (MMP) activity, an indicator of joint inflammation, was significantly reduced by flavopiridol microparticles 3 days post-injury. Histopathological analysis showed that flavopiridol microparticles reduced PTOA severity 28 days post-injury. Taken altogether, this work demonstrates a promising biomaterial platform for sustained small molecule drug delivery to the joint space as a therapeutic measure for post-traumatic osteoarthritis. STATEMENT OF SIGNIFICANCE: Post-traumatic osteoarthritis (PTOA) begins with the deterioration of subchondral bone and cartilage after acute injuries. In spite of the prevalence of PTOA and its associated financial and psychological burdens, therapeutic measures remain elusive. A number of small molecule drugs are now under investigation to replace FDA-approved palliative measures, including cyclin-dependent kinase 9 (CDK9) inhibitors which work by targeting early inflammatory programming after injury. However, the short half-life of these drugs is a major hurdle to their success. Here, we show that biomaterial encapsulation of Flavopiridol (CDK9 inhibitor) in poly (lactic-co-glycolic acid) microparticles is a promising route for direct delivery and improved drug retention time in the knee joint. Moreover, administration of the flavopiridol microparticles reduced the severity of PTOA.

Impact of Surface Polarity on Lipid Assembly under Spatial Confinement.

Molecular dynamics (MD) simulations in the MARTINI model are used to study the assembly of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) molecules under spatial confinement, such as during solvent evaporation from ultrasmall (femtoliter quantity) droplets. The impact of surface polarity on molecular assembly is discussed in detail. To the best of our knowledge, this work represents the first of its kind. Our results reveal that solvent evaporation gives rise to the formation of well-defined stacks of lipid bilayers in a smectic alignment. These smectic mesophases form on both polar and nonpolar surfaces but with a notable distinction. On polar surfaces, the director of the stack is oriented perpendicular to the support surface. By contrast, the stacks orient at an angle on the nonpolar surfaces. The packing of head groups on surfaces and lipid molecular mobility exhibits significant differences as surface polarity changes. The role of glycerol in the assembly and stability is also revealed. The insights revealed from the simulation have a significant impact on additive manufacturing, biomaterials, model membranes, and engineering protocells. For example, POPC assemblies via evaporation of ultrasmall droplets were produced and characterized. The trends compare well with the bilayer stack models. The surface polarity influences the local morphology and structures at the interfaces, which could be rationalized via the molecule-surface interactions observed from simulations.

3D nanoprinting via spatially controlled assembly and polymerization.

Macroscale additive manufacturing has seen significant advances recently, but these advances are not yet realized for the bottom-up formation of nanoscale polymeric features. We describe a platform technology for creating crosslinked polymer features using rapid surface-initiated crosslinking and versatile macrocrosslinkers, delivered by a microfluidic-coupled atomic force microscope known as FluidFM. A crosslinkable polymer containing norbornene moieties is delivered to a catalyzed substrate where polymerization occurs, resulting in extremely rapid chemical curing of the delivered material. Due to the living crosslinking reaction, construction of lines and patterns with multiple layers is possible, showing quantitative material addition from each deposition in a method analogous to fused filament fabrication, but at the nanoscale. Print parameters influenced printed line dimensions, with the smallest lines being 450 nm across with a vertical layer resolution of 2 nm. This nanoscale 3D printing platform of reactive polymer materials has applications for device fabrication, optical systems and biotechnology.

Production of Organizational Chiral Structures by Design.

Organizational chirality on surfaces has been of interest in chemistry and materials science due to its scientific importance as well as its potential applications. Current methods for producing organizational chiral structures on surfaces are primarily based upon the self-assembly of molecules. While powerful, the chiral structures are restricted to those dictated by surface reaction thermodynamics. This work introduces a method to create organizational chirality by design with nanometer precision. Using atomic force microscopy-based nanolithography, in conjunction with chosen surface chemistry, various chiral structures are produced with nanometer precision, from simple spirals and arrays of nanofeatures to complex and hierarchical chiral structures. The size, geometry, and organizational chirality is achieved in deterministic fashion, with high fidelity to the designs. The concept and methodology reported here provide researchers a new and generic means to carry out organizational chiral chemistry, with the intrinsic advantages of chiral structures by design. The results open new and promising applications including enantioselective catalysis, separation, and crystallization, as well as optical devices requiring specific polarized radiation and fabrication and recognition of chiral nanomaterials.

Nanoscale Raman Characterization of a 2D Semiconductor Lateral Heterostructure Interface.

The nature of the interface in lateral heterostructures of 2D monolayer semiconductors including its composition, size, and heterogeneity critically impacts the functionalities it engenders on the 2D system for next-generation optoelectronics. Here, we use tip-enhanced Raman scattering (TERS) to characterize the interface in a single-layer MoS2/WS2 lateral heterostructure with a spatial resolution of 50 nm. Resonant and nonresonant TERS spectroscopies reveal that the interface is alloyed with a size that varies over an order of magnitude─from 50 to 600 nm─within a single crystallite. Nanoscale imaging of the continuous interfacial evolution of the resonant and nonresonant Raman spectra enables the deconvolution of defect activation, resonant enhancement, and material composition for several vibrational modes in single-layer MoS2, MoxW1-xS2, and WS2. The results demonstrate the capabilities of nanoscale TERS spectroscopy to elucidate macroscopic structure-property relationships in 2D materials and to characterize lateral interfaces of 2D systems on length scales that are imperative for devices.

Direct Observations of Silver Nanowire-Induced Frustrated Phagocytosis among NR8383 Lung Alveolar Macrophages.

The interaction of long nanowires and living cells is directly related to nanowires' nanotoxicity and health impacts. Interactions of silver nanowires (AgNWs) and macrophage cell lines (NR8383) were investigated using laser scanning confocal microscopy and single cell compression (SCC). With high-resolution imaging and mechanics measurement of individual cells, AgNW-induced frustrated phagocytosis was clearly captured in conjunction with structural and property changes of cells. While frustrated phagocytosis is known for long microwires and long carbon nanotubes, this work reports first direct observations of frustrated phagocytosis of AgNWs among living cells in situ. In the case of partial penetration of AgNWs into NR8383 cells, confocal imaging revealed actin participation at the entry sites, whose behavior differs from microwire-induced frustrated phagocytosis. The impacts of frustrated phagocytosis on the cellular membrane and cytoskeleton were also quantified by measuring the mechanical properties using SCC. Taken collectively, this study reveals the structural and property characteristics of nanowire-induced frustrated phagocytosis, which deepens our understanding of nanowire-cell interactions and nanocytotoxicity.

Direct Visualization of the Binding of Transforming Growth Factor Beta 1 with Cartilage Oligomeric Matrix Protein via High-Resolution Atomic Force Microscopy.

This work reports the first direct observations of binding and complex formation between transforming growth factor beta 1 (TGF-ß1) and cartilage oligomeric matrix protein (COMP) using high-resolution atomic force microscopy (AFM). Each COMP molecule consists of pentamers whose five identical monomeric units bundle at N-termini. From this central point, the five monomers' flexible arms extend outward with C-terminal domains at the distal ends, forming a bouquet-like structure. In commonly used buffer solutions, TGF-ß1 molecules typically form homodimers (majority), double dimers (minority), and aggregates (trace amount). Mixing TGF-ß1 and COMP leads to rapid binding and complex formation. The TGF-ß1/COMP complexes contain one to three COMP and multiple TGF-ß1 molecules. For complexes with one COMP, the structure is more compact and less flexible than that of COMP alone. For complexes with two or more COMP molecules, the conformation varies to a large degree from one complex to another. This is attributed to the presence of double dimers or aggregates of TGF-ß1 molecules, whose size and multiple binding sites enable binding to more than one COMP. The number and location of individual TGF-ß1 dimers are also clearly visible in all complexes. This molecular-level information provides a new insight into the mechanism of chondrogenesis enhancement by TGF-ß1/COMP complexes, i.e., simultaneous and multivalent presentation of growth factors. These presentations help explain the high efficacy in sustained activation of the signaling pathway to augment chondrogenesis.

High Mannose N-Glycans Promote Migration of Bone-Marrow-Derived Mesenchymal Stromal Cells.

For hundreds of indications, mesenchymal stromal cells (MSCs) have not achieved the expected therapeutic efficacy due to an inability of the cells to reach target tissues. We show that inducing high mannose N-glycans either chemically, using the mannosidase I inhibitor Kifunensine, or genetically, using an shRNA to silence the expression of mannosidase I A1 (MAN1A1), strongly increases the motility of MSCs. We show that treatment of MSCs with Kifunensine increases cell migration toward bone fracture sites after percutaneous injection, and toward lungs after intravenous injection. Mechanistically, high mannose N-glycans reduce the contact area of cells with its substrate. Silencing MAN1A1 also makes cells softer, suggesting that an increase of high mannose N-glycoforms may change the physical properties of the cell membrane. To determine if treatment with Kifunensine is feasible for future clinical studies, we used mass spectrometry to analyze the N-glycan profile of MSCs over time and demonstrate that the effect of Kifunensine is both transitory and at the expense of specific N-glycoforms, including fucosylations. Finally, we also investigated the effect of Kifunensine on cell proliferation, differentiation, and the secretion profile of MSCs. Our results support the notion of inducing high mannose N-glycans in MSCs in order to enhance their migration potential.

Determination of the Maturation Status of Dendritic Cells by Applying Pattern Recognition to High-Resolution Images.

The maturation or activation status of dendritic cells (DCs) directly correlates with their behavior and immunofunction. A common means to determine the maturity of dendritic cells is from high-resolution images acquired via scanning electron microscopy (SEM) or atomic force microscopy (AFM). While direct and visual, the determination has been made by directly looking at the images by researchers. This work reports a machine learning approach using pattern recognition in conjunction with cellular biophysical knowledge of dendritic cells to determine the maturation status of dendritic cells automatically. The determination from AFM images reaches 100% accuracy. The results from SEM images reaches 94.9%. The results demonstrate the accuracy of using machine learning for accelerating data analysis, extracting information, and drawing conclusions from high-resolution cellular images, paving the way for future applications requiring high-throughput and automation, such as cellular sorting and selection based on morphology, quantification of cellular structure, and DC-based immunotherapy.

Daylight-Active Cellulose Nanocrystals Containing Anthraquinone Structures.

Antimicrobial and antiviral materials have attracted significant interest in recent years due to increasing occurrences of nosocomial infections and pathogenic microbial contamination. One method to address this is the combination of photoactive compounds that can produce reactive oxygen species (ROS), such as hydrogen peroxide and hydroxyl radicals to disinfect microbes, with carrier materials that meet the application requirements. Using anthraquinone (AQ) and cellulose nanocrystals (CNCs) as the photoactive and carrier components, respectively, this work demonstrated the first covalent incorporation of AQ onto CNCs. The morphology and the photoactive properties were investigated, revealing the structural integrity of the CNCs and the high degree of photoactivity of the AQ-CNC materials upon UVA exposure. The AQ-CNCs also exhibited an unexpected persistent generation of ROS under darkness, which adds advantages for antimicrobial applications.

Tunneling nanotubes mediate the expression of senescence markers in mesenchymal stem/stromal cell spheroids.

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is limited by acquired senescence following prolonged culture expansion and high-passage numbers. However, the degree of cell senescence is dynamic, and cell-cell communication is critical to promote cell survival. MSC spheroids exhibit improved viability compared with monodispersed cells, and actin-rich tunneling nanotubes (TNTs) may mediate cell survival and other functions through the exchange of cytoplasmic components. Building upon our previous demonstration of TNTs bridging MSCs within these cell aggregates, we hypothesized that TNTs would influence the expression of senescence markers in MSC spheroids. We confirmed the existence of functional TNTs in MSC spheroids formed from low-passage, high-passage, and mixtures of low- and high-passage cells using scanning electron microscopy, confocal microscopy, and flow cytometry. The contribution of TNTs toward the expression of senescence markers was investigated by blocking TNT formation with cytochalasin D (CytoD), an inhibitor of actin polymerization. CytoD-treated spheroids exhibited decreases in cytosol transfer. Compared with spheroids formed solely of high-passage MSCs, the addition of low-passage MSCs reduced p16 expression, a known genetic marker of senescence. We observed a significant increase in p16 expression in high-passage cells when TNT formation was inhibited, establishing the importance of TNTs in MSC spheroids. These data confirm the restorative role of TNTs within MSC spheroids formed with low- and high-passage cells and represent an exciting approach to use higher-passage cells in cell-based therapies.

New Means to Control Molecular Assembly.

While self-assembly of molecules is relatively well-known and frequently utilized in chemical synthesis and material science, controlled assembly of molecules represents a new concept and approach. The present work demonstrates the concept of controlled molecular assembly using a non-spherical biomolecule, heparosan tetrasaccharide (MW = 1.099 kD). The key to controlled assembly is the fact that ultra-small solution droplets exhibit different evaporation dynamics from those of larger ones. Using an independently controlled microfluidic probe in an atomic force microscope, sub-femtoliter aqueous droplets containing designed molecules produce well-defined features with dimensions as small as tens of nanometers. The initial shape of the droplet and the concentration of solute within the droplet dictate the final assembly of molecules due to the ultrafast evaporation rate and dynamic spatial confinement of the droplets. The level of control demonstrated in this work brings us closer to programmable synthesis for chemistry and materials science which can be used to develop vehicles for drug delivery three-dimensional nanoprinting in additive manufacturing.

Dry Transfer of van der Waals Crystals to Noble Metal Surfaces To Enable Characterization of Buried Interfaces.

Two-dimensional (2D) transition-metal dichalcogenides (TMDCs) have been explored for many optoelectronic applications. Most of these applications require them to be on insulating substrates. However, for many fundamental property characterizations, such as mapping surface potential or conductance, insulating substrates are nonideal as they lead to charging and doping effects or impose the inhomogeneity of their charge environment on the atomically thin 2D layers. Here, we report a simple method of residue-free dry transfer of 2D TMDC crystal layers. This method is enabled via noble-metal (gold, silver) thin films and allows comprehensive nanoscale characterization of transferred TMDC crystals with multiple scanning probe microscopy techniques. In particular, intimate contact with underlying metal allows efficient tip-enhanced Raman scattering characterization, providing high spatial resolution (<20 nm) for Raman spectroscopy. Further, scanning Kelvin probe force microscopy allows high-resolution mapping of surface potential on transferred crystals, revealing their spatially varying structural and electronic properties. The layer-dependent contact potential difference is clearly observed and explained by charge transfer from contacts with Au and Ag. The demonstrated sample preparation technique can be generalized to probe many different 2D material surfaces and has broad implications in understanding of the metal contacts and buried interfaces in 2D material-based devices.