Development and optimization of an ocular hydrogel adhesive patch using definitive screening design (DSD).

Adhesive hydrogels based on chemically modified photocrosslinkable polymers with specific physicochemical properties are frequently utilized for sealing wounds or incisions. These adhesive hydrogels offer tunable characteristics such as tailorable tissue adhesion, mechanical properties, swelling ratios, and enzymatic degradability. In this study, we developed and optimized a photocrosslinkable adhesive patch, GelPatch, with high burst pressure, minimal swelling, and specific mechanical properties for application as an ocular (sclera and subconjunctival) tissue adhesive. To achieve this, we formulated a series of hydrogel patches composed of different polymers with various levels of methacrylation, molecular weights, and hydrophobic/hydrophilic properties. A computerized multifactorial definitive screening design (DSD) analysis was performed to identify the most prominent components impacting critical response parameters such as adhesion, swelling ratio, elastic modulus, and second order interactions between applied components. These parameters were mathematically processed to generate a predictive model that identifies the linear and non-linear correlations between these factors. In conclusion, an optimized formulation of GelPatch was selected based on two modified polymers: gelatin methacryloyl (GelMA) and glycidyl methacrylated hyaluronic acid (HAGM). The ex vivo results confirmed adhesion and retention of the optimized hydrogel subconjunctivally and on the sclera for up to 4 days. The developed formulation has potential to be used as an ocular sealant for quick repair of laceration type ocular injuries.

Engineering a drug eluting ocular patch for delivery and sustained release of anti-inflammatory therapeutics.

Ocular inflammation is commonly associated with eye disease or injury. Effective and sustained ocular delivery of therapeutics remains a challenge due to the eye physiology and structural barriers. Herein, we engineered a photocrosslinkable adhesive patch (GelPatch) incorporated with micelles (MCs) loaded with Loteprednol etabonate (LE) for delivery and sustained release of drug. The engineered drug loaded adhesive hydrogel, with controlled physical properties, provided a matrix with high adhesion to the ocular surfaces. The incorporation of MCs within the GelPatch enabled solubilization of LE and its sustained release within 15 days. In vitro studies showed that MC loaded GelPatch supported cell viability and growth. In addition, subcutaneous implantation of the MC loaded GelPatch in rats confirmed its in vivo biocompatibility and stability within 28 days. This non-invasive, adhesive, and biocompatible drug eluting patch can be used as a matrix for the delivery and sustained release of hydrophobic drugs.

Engineering elastic sealants based on gelatin and elastin-like polypeptides for endovascular anastomosis.

Cerebrovascular ischemia from intracranial atherosclerosis remains difficult to treat. Although current revascularization procedures, including intraluminal stents and extracranial to intracranial bypass, have shown some benefit, they suffer from perioperative and postoperative morbidity. To address these limitations, here we developed a novel approach that involves gluing of arteries and subsequent transmural anastomosis from the healthy donor into the ischemic recipient. This approach required an elastic vascular sealant with distinct mechanical properties and adhesion to facilitate anastomosis. We engineered two hydrogel-based glues: an elastic composite hydrogel based on methacryloyl elastin-like polypeptide (mELP) combined with gelatin methacryloyl (GelMA) and a stiff glue based on pure GelMA. Two formulations with distinct mechanical characteristics were necessary to achieve stable anastomosis. The elastic GelMA/mELP composite glue attained desirable mechanical properties (elastic modulus: 288 ± 19 kPa, extensibility: 34.5 ± 13.4%) and adhesion (shear strength: 26.7 ± 5.4 kPa) to the blood vessel, while the pure GelMA glue exhibited superior adhesion (shear strength: 49.4 ± 7.0 kPa) at the cost of increased stiffness (elastic modulus: 581 ± 51 kPa) and reduced extensibility (13.6 ± 2.5%). The in vitro biocompatibility tests confirmed that the glues were not cytotoxic and were biodegradable. In addition, an ex vivo porcine anastomosis model showed high arterial burst pressure resistance of 34.0 ± 7.5 kPa, which is well over normal (16 kPa), elevated (17.3 kPa), and hypertensive crisis (24 kPa) systolic blood pressures in humans. Finally, an in vivo swine model was used to assess the feasibility of using the newly developed two-glue system for an endovascular anastomosis. X-ray imaging confirmed that the anastomosis was made successfully without postoperative bleeding complications and the procedure was well tolerated. In the future, more studies are required to evaluate the performance of the developed sealants under various temperature and humidity ranges.

Advanced nanodelivery platforms for topical ophthalmic drug delivery.

Conventional eye drops have several limitations, including the need for multiple applications per dose, hourly based dosage regiments, and suboptimal ocular bioavailability (<5%). The efficacy of topical ophthalmic medications can be significantly improved by controlling their contact time with the adherent mucin layer and by inducing sustained release properties, thus allowing for a prolonged contact time of the drug with the ocular tissues, which eventually will lead to improved drug bioavailability and a significant decrease in the frequency of eyedrop instillation. In this review, we critically highlight recent and innovative nanodrug delivery platforms, with a primary focus on the integration of nanotechnology, biomaterials, and polymer chemistry to facilitate precise spatial and temporal control over sustained drug release to the cornea.

Advances and limitations of drug delivery systems formulated as eye drops.

Topical instillation of eye drops remains the most common and easiest route of ocular drug administration, representing the treatment of choice for many ocular diseases. Nevertheless, low ocular bioavailability of topically applied drug molecules can considerably limit their efficacy. Over the last several decades, numerous drug delivery systems (DDS) have been developed in order to improve drug bioavailability on the ocular surfaces. This review systematically covers the most recent advances of DDS applicable by topical instillation, that have shown better performance in in vivo models compared to standard eye drop formulations. These delivery systems are based on in situ forming gels, nanoparticles and combinations of both. Most of the DDS have been developed using natural or synthetic polymers. Polymers offer many advantageous properties for designing advanced DDS including biocompatibility, gelation properties and/or mucoadhesiveness. However, despite the high number of studies published over the last decade, there are several limitations for clinical translation of DDS. This review article focuses on the recent advances for the development of ocular drug delivery systems. In addtion, the potential challenges for commercialization of new DDS are presented.

Anti-GD2 Immunoliposomes for Targeted Delivery of the Survivin Inhibitor Sepantronium Bromide (YM155) to Neuroblastoma Tumor Cells.

PURPOSE: Sepantronium bromide (YM155) is a hydrophilic quaternary compound that cannot be administered orally due to its low oral bioavailability; it is furthermore rapidly eliminated via the kidneys. The current study aims at improving the pharmacokinetic profile of YM155 by its formulation in immunoliposomes that can achieve its enhanced delivery into tumor tissue and facilitate uptake in neuroblastoma cancer cells. METHODS: PEGylated YM155 loaded liposomes composed of DPPC, cholesterol and DSPE-PEG2000 were prepared via passive film-hydration and extrusion method. Targeted (i.e. immuno-)liposomes were prepared by surface functionalization with SATA modified monoclonal anti-disialoganglioside (GD2) antibodies. Liposomes were characterized based on their size, charge, antibody coupling and YM155 encapsulation efficiency, and stability. Flow cytometry analysis and confocal microscopy were performed on IMR32 and KCNR neuroblastoma cell lines. The efficacy of developed formulations were assessed by in-vitro toxicity assays. A pilot pharmacokinetic analysis was performed to assess plasma circulation and tumor accumulation profiles of the developed liposomal formulations. RESULTS: YM155 loaded immunoliposomes had a size of 170 nm and zeta potential of -10 mV, with an antibody coupling efficiency of 60% andYM155 encapsulation efficiency of14%. Targeted and control liposomal formulations were found to have similar YM155 release rates in a release medium containing 50% serum. An in-vitro toxicity study on KCNR cells showed less toxicity for immunoliposomes as compared to free YM155. In-vivo pharmacokinetic evaluation of YM155 liposomes showed prolonged blood circulation and significantly increased half-lives of liposomal YM155 in tumor tissue, as compared to a bolus injection of free YM155. CONCLUSIONS: YM155 loaded immunoliposomes were successfully formulated and characterized, and initial in-vivo results show their potential for improving the circulation time and tumor accumulation of YM155.

E-selectin targeted immunoliposomes for rapamycin delivery to activated endothelial cells.

Activated endothelial cells play a pivotal role in the pathology of inflammatory disorders and thus present a target for therapeutic intervention by drugs that intervene in inflammatory signaling cascades, such as rapamycin (mammalian target of rapamycin (mTOR) inhibitor). In this study we developed anti-E-selectin immunoliposomes for targeted delivery to E-selectin over-expressing tumor necrosis factor-α (TNF-α) activated endothelial cells. Liposomes composed of 1,2-dipalmitoyl-sn-glycero-3.;hosphocholine (DPPC), Cholesterol, and 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]-maleimide (DSPE-PEG-Mal) were loaded with rapamycin via lipid film hydration, after which they were further functionalized by coupling N-succinimidyl-S-acetylthioacetate (SATA)-modified mouse anti human E-selectin antibodies to the distal ends of the maleimidyl (Mal)-PEG groups. In cell binding assays, these immunoliposomes bound specifically to TNF-α activated endothelial cells. Upon internalization, rapamycin loaded immunoliposomes inhibited proliferation and migration of endothelial cells, as well as expression of inflammatory mediators. Our findings demonstrate that rapamycin-loaded immunoliposomes can specifically inhibit inflammatory responses in inflamed endothelial cells.

PLGA-PEG nanoparticles for targeted delivery of the mTOR/PI3kinase inhibitor dactolisib to inflamed endothelium.

Dactolisib (NVP-BEZ235, also referred to as: 'BEZ235' or 'BEZ') is a dual mTOR/PI3K inhibitor that is of potential interest in the treatment of inflammatory disorders. This work focuses on formulation of BEZ-loaded polymeric nanoparticles composed of a blend of poly(D,L-lactide-co-glycolide) (PLGA) and poly(D,L-lactide-co-glycolide)-poly(ethylene glycol)-2000 (PLGA-PEG). The nanoparticles were prepared by an oil/water emulsion solvent evaporation method, and were subsequently characterized for yield, encapsulation efficiency, morphology, particle size, drug-polymer interaction and in vitro drug release profiles. A targeted formulation was developed by conjugation of a S-acetyl-thioacetyl (SATA)-modified mouse-anti human E-selectin antibody to the distal end of PLGA-PEG-SPDP containing nanoparticles. Our results show the successful preparation of spherical PLGA/PLGA-PEG nanoparticles loaded with BEZ. The particle size distribution showed a range from 250 to 360nm with a high (>75%) BEZ encapsulation efficiency. Approximately 35% of the loaded BEZ was released within 10days at 37°C in a medium containing 5% bovine serum albumin (BSA). Evaluation of efficacy of anti-E-selectin decorated BEZ-loaded nanoparticles was carried out in tumor necrosis factor-α (TNF-α) activated endothelial cells. Confocal microscopy analysis showed that cellular uptake of the targeted nanoparticles and subsequent internalization. Cell functional assays, including migration assay and phosphowestern blot analysis of the mTOR and pI3K signaling pathways, revealed that the E-selectin targeted nanoparticles loaded with BEZ had a pronounced effect on inflammation-activated endothelial cells as compared to the non-targeted BEZ-loaded nanoparticles. In conclusion, E-selectin targeted nanoparticles have a high potential in delivering the potent mTOR/pI3K inhibitor dactolisib to inflamed endothelial cells and are an interesting nanomedicine for anti-inflammatory therapy.

Microfluidic approaches for isolation, detection, and characterization of extracellular vesicles: Current status and future directions.

Extracellular vesicles (EVs) are cell-derived vesicles present in body fluids that play an essential role in various cellular processes, such as intercellular communication, inflammation, cellular homeostasis, survival, transport, and regeneration. Their isolation and analysis from body fluids have a great clinical potential to provide information on a variety of disease states such as cancer, cardiovascular complications and inflammatory disorders. Despite increasing scientific and clinical interest in this field, there are still no standardized procedures available for the purification, detection, and characterization of EVs. Advances in microfluidics allow for chemical sampling with increasingly high spatial resolution and under precise manipulation down to single molecule level. In this review, our objective is to give a brief overview on the working principle and examples of the isolation and detection methods with the potential to be used for extracellular vesicles. This review will also highlight the integrated on-chip systems for isolation and characterization of EVs.

Targeting Rapamycin to Podocytes Using a Vascular Cell Adhesion Molecule-1 (VCAM-1)-Harnessed SAINT-Based Lipid Carrier System.

Together with mesangial cells, glomerular endothelial cells and the basement membrane, podocytes constitute the glomerular filtration barrier (GFB) of the kidney. Podocytes play a pivotal role in the progression of various kidney-related diseases such as glomerular sclerosis and glomerulonephritis that finally lead to chronic end-stage renal disease. During podocytopathies, the slit-diaphragm connecting the adjacent podocytes are detached leading to severe loss of proteins in the urine. The pathophysiology of podocytopathies makes podocytes a potential and challenging target for nanomedicine development, though there is a lack of known molecular targets for cell selective drug delivery. To identify VCAM-1 as a cell-surface receptor that is suitable for binding and internalization of nanomedicine carrier systems by podocytes, we investigated its expression in the immortalized podocyte cell lines AB8/13 and MPC-5, and in primary podocytes. Gene and protein expression analyses revealed that VCAM-1 expression is increased by podocytes upon TNFα-activation for up to 24 h. This was paralleled by anti-VCAM-1 antibody binding to the TNFα-activated cells, which can be employed as a ligand to facilitate the uptake of nanocarriers under inflammatory conditions. Hence, we next explored the possibilities of using VCAM-1 as a cell-surface receptor to deliver the potent immunosuppressant rapamycin to TNFα-activated podocytes using the lipid-based nanocarrier system Saint-O-Somes. Anti-VCAM-1-rapamycin-SAINT-O-Somes more effectively inhibited the cell migration of AB8/13 cells than free rapamycin and non-targeted rapamycin-SAINT-O-Somes indicating the potential of VCAM-1 targeted drug delivery to podocytes.

Diverse origins of the myofibroblast­implications for kidney fibrosis.

Fibrosis is the common end point of chronic kidney disease. The persistent production of inflammatory cytokines and growth factors leads to an ongoing process of extracellular matrix production that eventually disrupts the normal functioning of the organ. During fibrosis, the myofibroblast is commonly regarded as the predominant effector cell. Accumulating evidence has demonstrated a diverse origin of myofibroblasts in kidney fibrosis. Proposed major contributors of myofibroblasts include bone marrow-derived fibroblasts, tubular epithelial cells, endothelial cells, pericytes and interstitial fibroblasts; the published data, however, have not yet clearly defined the relative contribution of these different cellular sources. Myofibroblasts have been reported to originate from various sources, irrespective of the nature of the initial damage responsible for the induction of kidney fibrosis. Here, we review the possible relevance of the diversity of myofibroblast progenitors in kidney fibrosis and the implications for the development of novel therapeutic approaches. Specifically, we discuss the current status of preclinical and clinical antifibrotic therapy and describe targeting strategies that might help support resident and circulating cells to maintain or regain their original functional differentiation state. Such strategies might help these cells resist their transition to a myofibroblast phenotype to prevent, or even reverse, the fibrotic state.

Status quo of annotation of human disease variants.

BACKGROUND: The ever on-going technical developments in Next Generation Sequencing have led to an increase in detected disease related mutations. Many bioinformatics approaches exist to analyse these variants, and of those the methods that use 3D structure information generally outperform those that do not use this information. 3D structure information today is available for about twenty percent of the human exome, and homology modelling can double that fraction. This percentage is rapidly increasing so that we can expect to analyse the majority of all human exome variants in the near future using protein structure information. RESULTS: We collected a test dataset of well-described mutations in proteins for which 3D-structure information is available. This test dataset was used to analyse the possibilities and the limitations of methods based on sequence information alone, hybrid methods, machine learning based methods, and structure based methods. CONCLUSIONS: Our analysis shows that the use of structural features improves the classification of mutations. This study suggests strategies for future analyses of disease causing mutations, and it suggests which bioinformatics approaches should be developed to make progress in this field.

Idiopathic hypercalciuria in children with vesico ureteral reflux and recurrent urinary tract infection.

PURPOSE: Our aim was to determine association of vesicoureteral reflux (VUR) and idiopathic hypercalciuria in children with recurrent and single episode of urinary tract infection (UTI). MATERIALS AND METHODS: The study group consisted of 45 children with VUR and recurrent UTI, and 2 control groups: 45 normal healthy children (control group 1) and 45 children with VUR and single episode of UTI (control group 2). Idiopathic hypercalciuria was defined as urine calcium to creatinine ratio more than 0.8 (mg/mg) in infants younger than 1 year old, and more than 0.2 (mg/mg) in older children (without any detectable causes for hypercalciuria). RESULTS: The study group consisted of 26 (57.8%) girls and 19 (42.2%) boys, with the mean age of 41.14 +/- 22.1 months. Nine (20%) subjects had hypercalciuria. The control group 1 composed of 22 (48.9%) girls and 23 (51.1%) boys, with the mean age of 43.98 +/- 16.23 months. In this group, 6 subjects (13.3%) with hypercalciuria were detected. The control group 2 composed of 23 (51.1%) girls and 22 (48.9%) boys, with the mean age of 39.96 +/- 24.2 months. In group 2, 7 subjects (15.6%) with hypercalciuria were detected. CONCLUSION: Comparison between such results was not statistically significant. Despite reports of different studies about accompanying of hypercalciuria with recurrent UTI with or without anatomical abnormalities, according to the present study, idiopathic hypercalciuria is not a major contributing factor to recurrent UTI in children with VUR.