Shell properties and concentration stability of acoustofluidic delivery agents
Alsadiq, Hussain, Karnaker Tupally, Robert Vogel, Ganesh Kokil, Harendra S. Parekh, and Martin Veidt. "Shell properties and concentration stability of acoustofluidic delivery agents." Physical and Engineering Sciences in Medicine 44, no. 1 (2021): 79-91.
This paper investigates the shell elastic properties and the number-concentration stability of a new acoustofluidic delivery agent liposome in comparison to Definity™, a monolayer ultrasonic contrast agent microbubble. The frequency dependent attenuation of an acoustic beam passing through a microbubble suspension was measured to estimate the shell parameters. The excitation voltage was adjusted to ensure constant acoustic pressure at all frequencies. The pressure was kept at the lowest possible magnitude to ensure that effects from nonlinear bubble behaviour which are not considered in the analytical model were minimal. The acoustofluidic delivery agent shell stiffness Sp and friction Sf parameters were determined as (Sp = 0.11 N/m, Sf = 0.31 × 10−6 Kg/s at 25 °C) in comparison to the Definity™ monolayer ultrasound contrast agent which were (Sp = 1.53 N/m, Sf = 1.51 × 10−6 Kg/s at 25 °C). When the temperature was raised to physiological levels, the friction coefficient Sf decreased by 28% for the monolayer microbubbles and by only 9% for the liposomes. The stiffness parameter Sp of the monolayer microbubble decreased by 23% while the stiffness parameter of the liposome increased by a similar margin (27%) when the temperature was raised to 37 °C. The size distribution of the bubbles was measured using Tunable Resistive Pulse Sensing (TRPS) for freshly prepared microbubbles and for bubble solutions at 6 h and 24 h after activation to investigate their number-concentration stability profile. The liposome maintained >80% of their number-concentration for 24 h at physiological temperature, while the monolayer microbubbles maintained only 27% of their number-concentration over the same period. These results are important input parameters for the design of effective acoustofluidic delivery systems using the new liposomes.
Investigation into the Photochemical Properties of Methylene Blue-Immobilized Hydroxyapatite Nanoparticles for Theranostic Application
In the biomedical field, there has been a requirement for developing theranostic nanomaterials with higher biosafety, leading to both diagnosis and therapy. Methylene blue (MB+) is an organic dye with both photoluminescence (PL) and photosensitization abilities to generate singlet oxygen (1O2). However, MB+ easily loses its generation ability by hydrogen reduction in vivo or by forming aggregates. In this study, MB+ immobilized on biocompatible hydroxyapatite (HA) nanoparticles was applied for the bifunctions of efficient PL and photosensitization. The MB+-immobilized HA nanoparticles (MH) formed aggregates with sizes of 80–100 nm in phosphate buffer (PB). The generation amount and efficiency of 1O2 from the nanoparticles in PB seem to depend on the immobilized MB+ amount and the percentage of the monomer, respectively. Considering the larger immobilized amount and percentage of the MB+ monomer, it was found that there was MH with the lower generation amount and efficiency of 1O2 to exhibit the highest PL intensity. The photofunctional measurement of MB+ revealed the state of MB+ molecules on the HA surface, and it was suggested that the MB+ molecules immobilized on the MH surface would form more hydrogen bonds to change their excitation states. In the cellular experiments, the Hela cancer cells reacted with the nanoparticles and showed red-color PL, indicating cellular imaging. Furthermore, the adherent cell coverage decreased by 1O2 generation, indicating the importance of the immobilization amount of the MB+ monomer. Therefore, theranostic nanomaterials with biosafety were successfully synthesized to show two photofunctions, which provide both cellular imaging and photodynamic therapy by the nanohybrid system between HA and MB+.
A novel surface functionalization platform to prime extracellular vesicles for targeted therapy and diagnostic imaging
Extracellular vesicles (EVs), nanovesicles released by cells to effectively exchange biological information, are gaining interest as drug delivery system. Yet, analogously to liposomes, they show short blood circulation times and accumulation in the liver and the spleen. For tissue specific delivery, EV surfaces will thus have to be functionalized. We present a novel platform for flexible modification of EVs with target-specific ligands based on the avidin-biotin system. Genetic engineering of donor cells with a glycosylphosphatidylinositol-anchored avidin (GPI-Av) construct allows the isolation of EVs displaying avidin on their surface, functionalized with any biotinylated ligand. For proof of concept, GPI-Av EVs were modified with i) a biotinylated antibody or ii) de novo designed and synthesized biotinylated ligands binding carbonic anhydrase IX (CAIX), a membrane associated enzyme overexpressed in cancer. Functionalized EVs showed specific binding and uptake by CAIX-expressing cells, demonstrating the power of the system to prepare EVs for cell-specific drug delivery.
Human corneal stromal stem cells express anti-fibrotic microRNA-29a and 381-5p – A robust cell selection tool for stem cell therapy of corneal scarring
Introduction Corneal blindness due to scarring is treated with corneal transplantation. However, a global problem is the donor material shortage. Preclinical and clinical studies have shown that cell-based therapy using corneal stromal stem cells (CSSCs) suppresses corneal scarring, potentially mediated by specific microRNAs transported in extracellular vesicles (EVs). However, not every CSSC batch from donors achieves similar anti-scarring effects. Purpose To examine miRNA profiles in EVs from human CSSCs showing “healing” versus “non-healing” effects on corneal scarring and to design a tool to select CSSCs with strong healing potency for clinical applications. Methods Small RNAs from CSSC-EVs were extracted for Nanostring nCounter Human miRNA v3 assay. MicroRNAs expressed > 20 folds in “healing” EVs (P < 0.05) were subject to enriched gene ontology (GO) term analysis. MiRNA groups with predictive regulation on inflammatory and fibrotic signalling were studied by mimic transfection to (1) mouse macrophages (RAW264.7) for M1 phenotype assay; (2) human corneal keratocytes for cytokine-induced fibrosis, and (3) human CSSCs for corneal scar prevention in vivo. The expression of miR-29a was screened in additional CSSC batches and the anti-scarring effect of cells was validated in mouse corneal wounds. Results Twenty-one miRNAs were significantly expressed in “healing” CSSC-EVs and 9 miRNA groups were predicted to associate with inflammatory and fibrotic responses, and tissue regeneration (P <10−6). Overexpression of miR-29a and 381-5p significantly prevented M1 phenotype transition in RAW264.7 cells after lipopolysaccharide treatment, suppressed transforming growth factor β1-induced fibrosis marker expression in keratocytes, and reduced scarring after corneal injury. High miR-29a expression in EV fractions distinguished human CSSCs with strong healing potency, which inhibited corneal scarring in vivo. Conclusion We characterized the anti-inflammatory and fibrotic roles of miR-29a and 381-5p in CSSCs, contributing to scar prevention. MiR-29a expression in EVs distinguished CSSCs with anti-scarring quality, identifying good quality cells for a scarless corneal healing.