Muco-Adhesive and Muco-Penetrative Formulations for the Oral Delivery of Insulin.

Insulin, a pivotal anabolic hormone, regulates glucose homeostasis by facilitating the conversion of blood glucose to energy or storage. Dysfunction in insulin activity, often associated with pancreatic ß cells impairment, leads to hyperglycemia, a hallmark of diabetes. Type 1 diabetes (T1D) results from autoimmune destruction of ß cells, while type 2 diabetes (T2D) stems from genetic, environmental, and lifestyle factors causing ß cell dysfunction and insulin resistance. Currently, insulin therapy is used for most of the cases of T1D, while it is used only in a few persistent cases of T2D, often supplemented with dietary and lifestyle changes. The key challenge in oral insulin delivery lies in overcoming gastrointestinal (GI) barriers, including enzymatic degradation, low permeability, food interactions, low bioavailability, and long-term safety concerns. The muco-adhesive (MA) and muco-penetrative (MP) formulations aim to enhance oral insulin delivery by addressing these challenges. The mucus layer, a hydrogel matrix covering epithelial cells in the GI tract, poses significant barriers to oral insulin absorption. Its structure, composition, and turnover rate influence interactions with insulin and other drug carriers. Some of the few factors that influence mucoadhesion and mucopenetration are particle size, surface charge distribution, and surface modifications. This review discusses the challenges associated with oral insulin delivery, explores the properties of mucus, and evaluates the strategies for achieving excellent MA and MP formulations, focusing on nanotechnology-based approaches. The development of effective oral insulin formulations holds the potential to revolutionize diabetes management, providing patients with a more convenient and patient-friendly alternative to traditional insulin administration methods.

A thiazole-based colorimetric and photoluminescent chemosensors for As3+ ions detection: Density functional theory, test strips, real samples, and bioimaging applications.

A Schiff-base Ethyl (E)-2-(3-((2-carbamothioylhydrazono)methyl)-4-hydroxyphenyl)-4-methylthiazole-5-carboxylate (TZTS) dual functional colorimetric and photoluminescent chemosensor which includes thiazole and thiosemicarbazide has been synthesized to detect arsenic (As3+) ions selectively in DMSO: H2O (7:3, v/v) solvent system. The molecular structure of the probe was characterized via FT-IR, 1H, and 13C NMR & HRMS analysis. Interestingly, the probe exhibits a remarkable and specific colorimetric and photoluminescence response to As3+ ions when exposed to various metal cations. The absorption spectral changes of TZTS were observed upon the addition of As3+ ions, with a naked eye detectable color change from colorless to yellow color. Additionally, the chemosensor (TZTS) exhibited a new absorption band at 412 nm and emission enhancements in photoluminescence at 528 nm after adding As3+ ions. The limit of detection (LOD) for As3+ ions was calculated to be 16.5 and 7.19 × 10-9 M by the UV-visible and photoluminescent titration methods, respectively. The underlying mechanism and experimental observations have been comprehensively elucidated through techniques such as Job's plot, Benesi-Hildebrand studies, and density functional theory (DFT) calculations. For practical application, the efficient determination of As3+ ions were accomplished using a spike and recovery approach applied to real water samples. In addition, the developed probe was successfully employed in test strip applications, allowing for the naked-eye detection of arsenic ions. Moreover, fluorescence imaging experiments of As3+ ions in the breast cancer cell line (MCF-7) demonstrated their practical applications in biological systems. Consequently, these findings highlight the significant potential of the TZTS sensor for detecting As3+ ions in environmental analysis systems.

Efficient photocatalytic degradation of textile dye pollutants using thermally exfoliated graphitic carbon nitride (TE-g-C3N4).

Graphitic carbon nitride (g-C3N4), an organic photocatalyst was reported to have beneficial properties to be used in wastewater treatment applications. However, g-C3N4, in its bulk form was found to have poor photocatalytic degradation efficiency due to its inherent limitations such as poor specific surface area and fast electron-hole pair recombination rate. In this study, we have tuned the physiochemical properties of bulk g-C3N4 by direct thermal exfoliation (TE-g-C3N4) and examined their photocatalytic degradation efficiency against abundant textile dyes such as methylene blue (MB), methyl orange (MO), and rhodamine B (RhB). The degradation efficiencies for MB, MO, and RhB dyes are 92 ± 0.18%, 93 ± 0.31%, and 95 ± 0.4% respectively in 60 min of UV light irradiation. The degradation efficiency increased with an increase in the exfoliation temperature. The prepared catalysts were characterized using FTIR, XRD, FE-SEM, EDAX, BET, and UV-DRS. In BET analysis, TE-g-C3N4 samples showed improved surface area (48.20 m2/g) when compared to the bulk g-C3N4 (5.03 m2/g). Further, the TE-g-C3N4 had 2.98 times higher adsorption efficiency than the bulk ones. The free radicals scavenging studies revealed that the superoxide radicals played an important role in the photodegradation for dyes, when compared to the hydroxyl radical (.OH) and the photo-induced holes (h+), Photoluminescence (PL) emission and electrochemical impedance spectroscopy (EIS) spectra of TE-g-C3N4 indicated a lowered electron-hole pairs' recombination rate and an increased photo-induced charge transfer respectively. Further, the TE-g-C3N4 were found to have excellent stability for up to 5 cycles with only a minor decrease in the activity from 92% to 86.2%. These findings proved that TE-g-C3N4 was an excellent photocatalyst for the removal and degradation of textile dyes from wastewater.

Development and optimization of starch-based biomaterial inks and the effect of infill patterns on the mechanical, physicochemical, and biological properties of 3D printed scaffolds for tissue engineering.

Plant-based hydrogels have wide application as scaffolds in tissue engineering and regenerative medicine due to their low cost and excellent biocompatibility. Scaffolds act as vehicles for cell-based therapeutics and regenerating diseased tissue. While there is a plethora of methods to generate hydrogels with tunable properties to mimic the tissue of interest, 3D bioprinting is a novel emerging technology with the capability to generate versatile patient-specific scaffolds typically embedded with tissue specific cells. Starch-based hydrogels are garnering attention in extrusion-based 3D printing, however owing to their poor mechanical strength and degradation render this material inefficient in its native form. Additionally, the effect of various printing process parameters on mechanical strength and bioactivity is poorly understood. In the present study, we investigate the use of starch and gelatin as composite biomaterial ink and its effect on mechanical, physical and biological properties. We also investigated printability of composite hydrogels with the aim to understand the correlation between two infill patterns and its effect on mechanical, physicochemical, and biological properties. Our results showed that the composite hydrogels had competent mechanical properties and suitable bioactivity when seeded with H9C2 cardiomyocytes. Rheometric analyses provided a broader insight into the required viscosity for printing and has a direct correlation with the composition of the hydrogel. Thus, the composite materials are found to have tissue-specific mechanical properties and may serve as a better, cheaper and personalized alternative to existing scaffolds for the fabrication of engineered cardiac tissue.

In vitro Cytotoxicity Evaluation of Flowable Hyaluronic Acid-Acellular Stromal Vascular Fraction (HA-aSVF) Mixture for Tissue Engineering Applications.

Background: The stromal vascular fraction (SVF) is an aqueous fraction isolated from the adipose tissue that constitutes different kinds of cells and extracellular matrix components. Hyaluronic acid (HA) is a linear polysaccharide in vertebrate tissues and is considered a potential tissue engineering scaffold due to its biocompatible nature. In this study, we have evaluated the cytotoxicity of xenofree HA in combination with an acellular component of adipose SVF (HA-aSVF) to propose it as a candidate biomaterial for future applications. Materials and Methods: 3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide assay of L-929 cells treated with HA-aSVF was used in our study. Data were normalized to cell control (untreated) and extracts of copper and ultra-high molecular weight polyethylene were used as positive (PC) and negative controls (NC). Results: Fibroblast cells retained the morphology after 24 h of treatment with HA-aSVF mixture and exhibited a similar percentage of cell activity compared to NC. PC showed a positive cytotoxic response as expected. The cells incubated with HA-aSVF showed a linear increase in cell activity indicating proliferation. Conclusion: The mixture of HA and acellular SVF in its flowable form is non-cytotoxic and showed improved cell proliferation. Hence the mixture can be proposed as a biomaterial and can be further explored for specific tissue engineering applications.

Evaluation of Non-Cross Linked Xeno-Free Hyaluronic Acid Solutions as Visco Elastic Biomaterials.

Background: Hyaluronic acid (HA) is a naturally occurring biodegradable, high molecular weight, non-sulfated glycosaminoglycan (GAG) polymer known for its excellent biocompatibility. HA-based products are widely used as viscosupplements, dermal fillers, and ophthalmic lubricants in clinical settings. Although animal and bacterial-derived HA are commonly reported, plant-sourced HA is not frequently reported. In this study, we have evaluated various viscoelastic properties of one such plant-based HA solution and propose them as an alternative to existing animal/bacteria-sourced HA. Materials and Methods: The viscoelastic properties of plant-sourced HA solution of various concentrations (0.1%, 0.5%, 1%, and 2% in PBS) were studied using a rheometer at 37°C. Flow curves, amplitude sweep studies, and frequency sweep studies were performed and compared for all HA solutions. Results: The HA solutions displayed shear-thinning behavior, which is an important characteristic of an injectable biomaterial. The 0.1 and 0.5% HA were found to have viscoelastic properties appropriate for eye lubricants, while 1 and 2% HA solutions showed properties suitable for soft tissue fillers. Frequency sweep studies indicated that all the samples are typically viscoelastic liquids with a loss modulus (G″) higher than the storage modulus (G'). This indicated that the samples needed further processing like crosslinking of HA or using higher molecular weight HA to be suitable as viscosupplements. However, the frequency sweep studies also indicated that these solutions can be used as soft tissue fillers of any type based on the G' and tan δ values. Conclusion: The plant-sourced HA solutions are found to exhibit good shear-thinning properties with viscoelastic properties suitable for eye lubricants and soft tissue fillers. However, to be used as viscosupplements, the viscoelastic properties of HA solutions have to be further modified through non-toxic crosslinking strategies, and hydrophobic derivatives as well as by using high molecular weight HAs.

Recent Advances in Two-Dimensional MXene-Based Electrochemical Biosensors for Sweat Analysis.

Sweat, a biofluid secreted naturally from the eccrine glands of the human body, is rich in several electrolytes, metabolites, biomolecules, and even xenobiotics that enter the body through other means. Recent studies indicate a high correlation between the analytes' concentrations in the sweat and the blood, opening up sweat as a medium for disease diagnosis and other general health monitoring applications. However, low concentration of analytes in sweat is a significant limitation, requiring high-performing sensors for this application. Electrochemical sensors, due to their high sensitivity, low cost, and miniaturization, play a crucial role in realizing the potential of sweat as a key sensing medium. MXenes, recently developed anisotropic two-dimensional atomic-layered nanomaterials composed of early transition metal carbides or nitrides, are currently being explored as a material of choice for electrochemical sensors. Their large surface area, tunable electrical properties, excellent mechanical strength, good dispersibility, and biocompatibility make them attractive for bio-electrochemical sensing platforms. This review presents the recent progress made in MXene-based bio-electrochemical sensors such as wearable, implantable, and microfluidic sensors and their applications in disease diagnosis and developing point-of-care sensing platforms. Finally, the paper discusses the challenges and limitations of MXenes as a material of choice in bio-electrochemical sensors and future perspectives on this exciting material for sweat-sensing applications.

Molecularly Imprinted Polymer-Based Biomimetic Systems for Sensing Environmental Contaminants, Biomarkers, and Bioimaging Applications.

Molecularly imprinted polymers (MIPs), a biomimetic artificial receptor system inspired by the human body's antibody-antigen reactions, have gained significant attraction in the area of sensor development applications, especially in the areas of medical, pharmaceutical, food quality control, and the environment. MIPs are found to enhance the sensitivity and specificity of typical optical and electrochemical sensors severalfold with their precise binding to the analytes of choice. In this review, different polymerization chemistries, strategies used in the synthesis of MIPs, and various factors influencing the imprinting parameters to achieve high-performing MIPs are explained in depth. This review also highlights the recent developments in the field, such as MIP-based nanocomposites through nanoscale imprinting, MIP-based thin layers through surface imprinting, and other latest advancements in the sensor field. Furthermore, the role of MIPs in enhancing the sensitivity and specificity of sensors, especially optical and electrochemical sensors, is elaborated. In the later part of the review, applications of MIP-based optical and electrochemical sensors for the detection of biomarkers, enzymes, bacteria, viruses, and various emerging micropollutants like pharmaceutical drugs, pesticides, and heavy metal ions are discussed in detail. Finally, MIP's role in bioimaging applications is elucidated with a critical assessment of the future research directions for MIP-based biomimetic systems.

TiO2 nanostructures - a double edged sword: current progress on their role in stem cells' differentiation, cancer therapy, and their toxicity issues.

Titanium dioxide (TiO2)-based nanostructures have wide applications in cosmetics, toothpastes, pharmaceuticals, coatings, papers, inks, plastics, food products, textiles, and many others. Recently, they have also been found to have huge potential as stem cells' differentiation agents as well as stimuli-responsive drug delivery systems in cancer therapy. In this review, we present some of the recent progress in the role of TiO2-based nanostructures toward the above-mentioned applications. We also present recent studies on the toxicity issues of these nanomaterials and the mechanisms behind the toxicity issues. We have reviewed the recent progress of TiO2-based nanostructures on their stem cells' differentiation potentials, their photo- and sono-dynamic capabilities, as stimuli-responsive drug delivery systems, and finally their toxicity issues with mechanistic understanding on the same. We believe that this review will help researchers be aware of the latest progress in the applications as well as few toxicity issues associated with TiO2-based nanostructures, which will help them design better nanomedicine for future applications.

Fabrication and Characterization Techniques of In Vitro 3D Tissue Models.

The culturing of cells in the laboratory under controlled conditions has always been crucial for the advancement of scientific research. Cell-based assays have played an important role in providing simple, fast, accurate, and cost-effective methods in drug discovery, disease modeling, and tissue engineering while mitigating reliance on cost-intensive and ethically challenging animal studies. The techniques involved in culturing cells are critical as results are based on cellular response to drugs, cellular cues, external stimuli, and human physiology. In order to establish in vitro cultures, cells are either isolated from normal or diseased tissue and allowed to grow in two or three dimensions. Two-dimensional (2D) cell culture methods involve the proliferation of cells on flat rigid surfaces resulting in a monolayer culture, while in three-dimensional (3D) cell cultures, the additional dimension provides a more accurate representation of the tissue milieu. In this review, we discuss the various methods involved in the development of 3D cell culture systems emphasizing the differences between 2D and 3D systems and methods involved in the recapitulation of the organ-specific 3D microenvironment. In addition, we discuss the latest developments in 3D tissue model fabrication techniques, microfluidics-based organ-on-a-chip, and imaging as a characterization technique for 3D tissue models.

Terminalia arjuna, a Cardioprotective Herbal Medicine-Relevancy in the Modern Era of Pharmaceuticals and Green Nanomedicine-A Review.

Herbal medicines were the main source of therapeutic agents in the ancestral era. Terminalia arjuna (TA) is one such medicinal plant widely known for its several medicinal properties, especially its cardiovascular properties. They have several phytochemicals, such as flavonoids, polyphenols, triterpenoids, tannins, glycosides, and several minerals, proteins, and others that are responsible for the above-mentioned medicinal properties. In this review, we have first elaborated on the various processes and their parameters for the efficient extraction of relevant phytochemicals from TA extracts. Secondly, the mechanisms behind the various medicinal properties of TA extracts are explained. We have also highlighted the role of TA extracts on the green synthesis of metallic nanoparticles, especially silver and gold nanoparticles, with an elucidation on the mechanisms behind the synthesis of nanoparticles. Finally, TA extracts-based polymeric formulations are discussed with limitations and future perspectives. We believe that this review could help researchers understand the importance of a well-known cardioprotective medicinal plant, TA, and its biomedical properties, as well as their role in green nanotechnology and various formulations explored for encapsulating them. This review will help researchers design better and greener nanomedicines as well as better formulations to improve the stability and bioavailability of TA extracts.

Influence of Graphene Nanoplatelets on the Performance of Axial Suspension Plasma-Sprayed Hydroxyapatite Coatings.

Axial suspension plasma spraying (ASPS) is an alternative technique to atmospheric plasma spraying (APS), which uses a suspension of much finer powders (<5-micron particle size) as the feedstock. It can produce more refined microstructures than APS for biomedical implants. This paper highlights the influence of incorporated graphene nanoplatelets (GNPs) on the behavior of ASPS hydroxyapatite (HAp) coatings. The characterization of the ASPS coatings (HAp + varying GNP contents) was carried out using scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), confocal Raman microscopy (CRM), white light interferometry (WLI), and contact angle measurements. The evaluation of the mechanical properties such as the hardness, roughness, adhesion strength, and porosity was carried out, along with a fretting wear performance. Additionally, the biocompatibility of the Hap + GNP coatings was evaluated using cytotoxicity testing which revealed a decrease in the cell viability from 92.7% to 85.4%, with an increase in the GNP wt.%. The visualization of the cell's components was carried out using SEM and Laser Scanning Microscopy. Furthermore, the changes in the genetic expression of the various cellular markers were assessed to analyze the epigenetic changes in human mesenchymal stem cells. The gene expression changes suggested that GNPs upregulated the proliferation marker and downregulated the pluripotent markers by a minimum of three folds.

Chitosan as Functional Biomaterial for Designing Delivery Systems in Cardiac Therapies.

Cardiovascular diseases are a leading cause of mortality across the globe, and transplant surgeries are not always successful since it is not always possible to replace most of the damaged heart tissues, for example in myocardial infarction. Chitosan, a natural polysaccharide, is an important biomaterial for many biomedical and pharmaceutical industries. Based on the origin, degree of deacetylation, structure, and biological functions, chitosan has emerged for vital tissue engineering applications. Recent studies reported that chitosan coupled with innovative technologies helped to load or deliver drugs or stem cells to repair the damaged heart tissue not just in a myocardial infarction but even in other cardiac therapies. Herein, we outlined the latest advances in cardiac tissue engineering mediated by chitosan overcoming the barriers in cardiac diseases. We reviewed in vitro and in vivo data reported dealing with drug delivery systems, scaffolds, or carriers fabricated using chitosan for stem cell therapy essential in cardiac tissue engineering. This comprehensive review also summarizes the properties of chitosan as a biomaterial substrate having sufficient mechanical stability that can stimulate the native collagen fibril structure for differentiating pluripotent stem cells and mesenchymal stem cells into cardiomyocytes for cardiac tissue engineering.

Self-Healing Hydrogels: Preparation, Mechanism and Advancement in Biomedical Applications.

Polymeric hydrogels are widely explored materials for biomedical applications. However, they have inherent limitations like poor resistance to stimuli and low mechanical strength. This drawback of hydrogels gave rise to ''smart self-healing hydrogels'' which autonomously repair themselves when ruptured or traumatized. It is superior in terms of durability and stability due to its capacity to reform its shape, injectability, and stretchability thereby regaining back the original mechanical property. This review focuses on various self-healing mechanisms (covalent and non-covalent interactions) of these hydrogels, methods used to evaluate their self-healing properties, and their applications in wound healing, drug delivery, cell encapsulation, and tissue engineering systems. Furthermore, composite materials are used to enhance the hydrogel's mechanical properties. Hence, findings of research with various composite materials are briefly discussed in order to emphasize the healing capacity of such hydrogels. Additionally, various methods to evaluate the self-healing properties of hydrogels and their recent advancements towards 3D bioprinting are also reviewed. The review is concluded by proposing several pertinent challenges encountered at present as well as some prominent future perspectives.

In situ differentiation of human-induced pluripotent stem cells into functional cardiomyocytes on a coaxial PCL-gelatin nanofibrous scaffold.

Human-induced pluripotent stem cells (hiPSCs) derived cardiomyocytes (hiPSC-CMs) have been explored for cardiac regeneration and repair as well as for the development of in vitro 3D cardiac tissue models. Existing protocols for cardiac differentiation of hiPSCs utilize a 2D culture system. However, the efficiency of hiPSC differentiation to cardiomyocytes in 3D culture systems has not been extensively explored. In the present study, we investigated the efficiency of cardiac differentiation of hiPSCs to functional cardiomyocytes on 3D nanofibrous scaffolds. Coaxial polycaprolactone (PCL)-gelatin fibrous scaffolds were fabricated by electrospinning and characterized using scanning electron microscopy (SEM) and fourier transform infrared (FTIR) spectroscopy. hiPSCs were cultured and differentiated into functional cardiomyocytes on the nanofibrous scaffold and compared with 2D cultures. To assess the relative efficiencies of both the systems, SEM, immunofluorescence staining and gene expression analyses were performed. Contractions of differentiated cardiomyocytes were observed in 2D cultures after 2 weeks and in 3D cultures after 4 weeks. SEM analysis showed no significant differences in the morphology of cells differentiated on 2D versus 3D cultures. However, gene expression data showed significantly increased expression of cardiac progenitor genes (ISL-1, SIRPA) in 3D cultures and cardiomyocytes markers (TNNT, MHC6) in 2D cultures. In contrast, immunofluorescence staining showed no substantial differences in the expression of NKX-2.5 and α-sarcomeric actinin. Furthermore, uniform migration and distribution of the in situ differentiated cardiomyocytes was observed in the 3D fibrous scaffold. Overall, our study demonstrates that coaxial PCL-gelatin nanofibrous scaffolds can be used as a 3D culture platform for efficient differentiation of hiPSCs to functional cardiomyocytes.

Electrospun Aligned Coaxial Nanofibrous Scaffold for Cardiac Repair.

Cardiovascular diseases (CVDs) are one of the leading causes of mortality worldwide and a number one killer in the USA. Cell-based approaches to treat CVDs have only shown modest improvement due to poor survival, retention, and engraftment of the transplanted cells in the ischemic myocardium. Recently, tissue engineering and the use of 3D scaffolds for culturing and delivering stem cells for ischemic heart disease are gaining rapid potential. Here, we describe a protocol for the fabrication of aligned coaxial nanofibrous scaffold comprising of a polycaprolactone (PCL) core and gelatin shell. Furthermore, we describe a detailed protocol for the efficient seeding and maintenance of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) on these nanofibrous scaffolds, which could have a potential application in the generation of functional "cardiac patch" for myocardial repair applications as well as an in vitro 3D cardiac tissue model to evaluate the efficacy of cardiovascular drugs and cardiac toxicities.

Scalable Biomimetic Coaxial Aligned Nanofiber Cardiac Patch: A Potential Model for "Clinical Trials in a Dish".

Recent advances in cardiac tissue engineering have shown that human induced-pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) cultured in a three-dimensional (3D) micro-environment exhibit superior physiological characteristics compared with their two-dimensional (2D) counterparts. These 3D cultured hiPSC-CMs have been used for drug testing as well as cardiac repair applications. However, the fabrication of a cardiac scaffold with optimal biomechanical properties and high biocompatibility remains a challenge. In our study, we fabricated an aligned polycaprolactone (PCL)-Gelatin coaxial nanofiber patch using electrospinning. The structural, chemical, and mechanical properties of the patch were assessed by scanning electron microscopy (SEM), immunocytochemistry (ICC), Fourier-transform infrared spectroscopy (FTIR)-spectroscopy, and tensile testing. hiPSC-CMs were cultured on the aligned coaxial patch for 2 weeks and their viability [lactate dehydrogenase (LDH assay)], morphology (SEM, ICC), and functionality [calcium cycling, multielectrode array (MEA)] were assessed. Furthermore, particle image velocimetry (PIV) and MEA were used to evaluate the cardiotoxicity and physiological functionality of the cells in response to cardiac drugs. Nanofibers patches were comprised of highly aligned core-shell fibers with an average diameter of 578 ± 184 nm. Acellular coaxial patches were significantly stiffer than gelatin alone with an ultimate tensile strength of 0.780 ± 0.098 MPa, but exhibited gelatin-like biocompatibility. Furthermore, hiPSC-CMs cultured on the surface of these aligned coaxial patches (3D cultures) were elongated and rod-shaped with well-organized sarcomeres, as observed by the expression of cardiac troponin-T and α-sarcomeric actinin. Additionally, hiPSC-CMs cultured on these coaxial patches formed a functional syncytium evidenced by the expression of connexin-43 (Cx-43) and synchronous calcium transients. Moreover, MEA analysis showed that the hiPSC-CMs cultured on aligned patches showed an improved response to cardiac drugs like Isoproterenol (ISO), Verapamil (VER), and E4031, compared to the corresponding 2D cultures. Overall, our results demonstrated that an aligned, coaxial 3D cardiac patch can be used for culturing of hiPSC-CMs. These biomimetic cardiac patches could further be used as a potential 3D in vitro model for "clinical trials in a dish" and for in vivo cardiac repair applications for treating myocardial infarction.

In vitro characterization of sonothrombolysis and echocontrast agents to treat ischemic stroke.

The development of adjuvant techniques to improve thrombolytic efficacy is important for advancing ischemic stroke therapy. We characterized octafluoropropane and recombinant tissue plasminogen activator (rt-PA)-loaded echogenic liposomes (OFP t-ELIP) using differential interference and fluorescence microscopy, attenuation spectroscopy, and electrozone sensing. The loading of rt-PA in OFP t-ELIP was assessed using spectrophotometry. Further, it was tested whether the agent shields rt-PA against degradation by plasminogen activator inhibitor-1 (PAI-1). An in vitro system was used to assess whether ultrasound (US) combined with either Definity or OFP t-ELIP enhances rt-PA thrombolysis. Human whole blood clots were mounted in a flow system and visualized using an inverted microscope. The perfusate consisted of either (1) plasma alone, (2) rt-PA, (3) OFP t-ELIP, (4) rt-PA and US, (5) OFP t-ELIP and US, (6) Definity and US, or (7) rt-PA, Definity, and US (n = 16 clots per group). An intermittent US insonation scheme was employed (220 kHz frequency, and 0.44 MPa peak-to-peak pressures) for 30 min. Microscopic imaging revealed that OFP t-ELIP included a variety of structures such as liposomes (with and without gas) and lipid-shelled microbubbles. OFP t-ELIP preserved up to 76% of rt-PA activity in the presence of PAI-1, whereas only 24% activity was preserved for unencapsulated rt-PA. The use of US with rt-PA and Definity enhanced lytic efficacy (p < 0.05) relative to rt-PA alone. US combined with OFP t-ELIP enhanced lysis over OFP t-ELIP alone (p < 0.01). These results demonstrate that ultrasound combined with Definity or OFP t-ELIP can enhance the lytic activity relative to rt-PA or OFP t-ELIP alone, respectively.

Characterization and Imaging of Lipid-Shelled Microbubbles for Ultrasound-Triggered Release of Xenon.

Xenon (Xe) is a bioactive gas capable of reducing and stabilizing neurologic injury in stroke. The goal of this work was to develop lipid-shelled microbubbles for xenon loading and ultrasound-triggered release. Microbubbles loaded with either xenon (Xe-MB) or xenon and octafluoropropane (Xe-OFP-MB) (9:1 v/v) were synthesized by high-shear mixing. The size distribution and the frequency-dependent attenuation coefficient of Xe-MB and Xe-OFP-MB were measured using a Coulter counter and a broadband acoustic attenuation spectroscopy system, respectively. The Xe dose was evaluated using gas chromatography/mass spectrometry. The total Xe doses in Xe-MB and Xe-OFP-MB were 113.1 ± 13.5 and 145.6 ± 25.5 µl per mg of lipid, respectively. Co-encapsulation of OFP increased the total xenon dose, attenuation coefficient, microbubble stability (in an undersaturated solution), and shelf life of the agent. Triggered release of gas payload was demonstrated with 6-MHz duplex Doppler and 220-kHz pulsed ultrasound. These results constitute the first step toward the use of lipid-shelled microbubbles for applications such as neuroprotection in stroke.

Bactericidal Activity of Lipid-Shelled Nitric Oxide-Loaded Microbubbles.

The global pandemic of antibiotic resistance is an ever-burgeoning public health challenge, motivating the development of adjunct bactericidal therapies. Nitric oxide (NO) is a potent bioactive gas that induces a variety of therapeutic effects, including bactericidal and biofilm dispersion properties. The short half-life, high reactivity, and rapid diffusivity of NO make therapeutic delivery challenging. The goal of this work was to characterize NO-loaded microbubbles (MB) stabilized with a lipid shell and to assess the feasibility of antibacterial therapy in vitro. MB were loaded with either NO alone (NO-MB) or with NO and octafluoropropane (NO-OFP-MB) (9:1 v/v and 1:1 v/v). The size distribution and acoustic attenuation coefficient of NO-MB and NO-OFP-MB were measured. Ultrasound-triggered release of the encapsulated gas payload was demonstrated with 3-MHz pulsed Doppler ultrasound. An amperometric microelectrode sensor was used to measure NO concentration released from the MB and compared to an NO-OFP-saturated solution. The effect of NO delivery on the viability of planktonic (free living) Staphylococcus aureus (SA) USA 300, a methicillin-resistant strain, was evaluated in a 96 well-plate format. The co-encapsulation of NO with OFP increased the total volume and attenuation coefficient of MB. The NO-OFP-MB were destroyed with a clinical ultrasound scanner with an output of 2.48 MPa peak negative pressure (in situ MI of 1.34) but maintained their echogenicity when exposed to 0.02 MPa peak negative pressure (in situ MI of 0.01. The NO dose in NO-MB and NO-OFP-MB was more than 2-fold higher than the NO-OFP-saturated solution. Delivery of NO-OFP-MB increased bactericidal efficacy compared to the NO-OFP-saturated solution or air and OFP-loaded MB. These results suggest that encapsulation of NO with OFP in lipid-shelled MB enhances payload delivery. Furthermore, these studies demonstrate the feasibility and limitations of NO-OFP-MB for antibacterial applications.