A Study of the Role of Gata6 in Definitive Endoderm Specification and Β-Cell Functionality by Genome Engineering of Pluripotent Stem Cells

Human pluripotent stem cells (PSCs) provide a powerful model system for the study of early human development, disease modeling and physiology. We chose to focus our studies on monogenic diabetes using this model system. Within the pancreas, β cells are one of the most critical endocrine cells as loss of this cell type disrupts blood glucose homeostasis, leading to diabetes. Due to the limited availability of primary human cells it is difficult to study them in vitro, especially in the context of genetic disease where patient material is even more difficult to obtain. Here, we characterize endodermal progenitor (EP) derived β-like cells as a model system for studying β cell development and function in vitro. EP cells are a population of endodermal stem cells, which can self-renew and be differentiated into multiple endodermal cell lineages. EP derived β-like cells are mono-hormonal for insulin and express a number of genes important for insulin processing and secretion. By use of both static stimulations and perifusion assays we show that EP derived β-like cells are responsive to both glucose and a number of other know secretagogues. Next, we demonstrate that genome editing with a zinc finger nuclease at the AAVS1 safe harbor locus can generate stable gene expression in PSCs during differentiation. We then combine these tools and describe the use of PSCs as an in vitro model system to study GATA6. Heterozygous mutations in this gene are the leading cause of pancreas agenesis while studies in mice do not replicate the human phenotype. Induced pluripotent stem cells were created from a pancreas agenesis patient with a heterozygous mutation in GATA6. Using genome editing technology, additional stem cell lines with mutations in both GATA6 alleles were generated and demonstrate a severe block in definitive endoderm induction. Re-expression of GATA6 or other GATA family members can rescue this endoderm phenotype. Partial rescue could also be achieved by treatment with a basic fibroblast growth factor. Using the EP cell culture system to bypass the developmental block at the endoderm stage, cell lines with mutations in one or both GATA6 alleles were differentiated into β-like cells. The mutant cells were shown to be functionally defective by failure to secrete insulin upon glucose stimulation. Decrease in retinoic acid concentrations used during the differentiation lead to decreased β-like cell differentiation efficiency of the heterozygous GATA6 mutants suggesting a possible mechanism for the patient phenotypes. These data show that GATA6 plays a critical role in endoderm specification and β-like cell functionality in humans while in mice it is dispensable, highlighting the importance of studying a human system

DEVELOPMENT AND CHARACTERIZATION OF CREAM CONTAINING NANOSTRUCTURED LIPID CARRIERS FOR HAIR SPLIT-END REPAIRING

Objective: This study aimed to develop the nanostructured lipid carriers (NLCs) for repairing the hair split-end and to compare the influence of NLCs charges on hair binding effects. Methods: NLCs was prepared by a high-pressure homogenization technique. The difference solid lipids were selected to obtain the negatively charged NLCs (N-NLCs) and positively charged NLCs (P-NLCs). The physical characterizations of both NLCs were examined. The NLCs cream was prepared by mixing NLCs into a cream base. Then, the hair split-end binding effects of the NLCs cream were assessed. The physical stability of both NLCs were investigated by kept at room temperature for 6 mo. Results: The N-NLCs and P-NLCs were successfully prepared with a zeta potential of -44.4 mV and 32.3 mV, respectively. They were a spherical shape with a similar mean size of ~150-160 nm. The hair split-end evaluation showed that both NLCs illustrated similar hair split-end binding time of more than 3 d, suggesting no extra benefit from the positively charged of P-NLCs. On the contrary, the cream base and commercial product showed a short binding time of <8 h. Furthermore, after 6-mo storage, N-NLCs demonstrated good physical stability without particle aggregation, while P-NLCs exhibited a phase separation. Conclusion: The developed NLCs is a novel alternative for the leave-on product, which would be highly benefits for hair split-end repairing

DEVELOPMENT AND CHARACTERIZATION OF PIPER RETROFRACTUM EXTRACT LOADED MUCOADHESIVE NANOSTRUCTURED LIPID CARRIERS FOR TOPICAL ORAL DRUG DELIVERY

Objective: To develop and characterize Piper retrofractum extract loaded nanostructured lipid carriers (PRE loaded NLCs) for topical oral cavity administration to enhance bioavailability and stability of piperine.Methods: PRE loaded NLCs were prepared with a hot high-pressure homogenization technique followed by coating the particle surface with mucoadhesive polymers; polyethene glycol 400 (PEG) and polyvinyl alcohol (PVA). The physicochemical properties in terms of particle size, polydispersity index, zeta potential, drug entrapment efficiency, in vitro drug release profile and antimicrobial activities were examined. In vitro, mucoadhesion studies were assessed by the wash-off test. The physicochemical stabilities of mouth spray containing PRE loaded NLCs were investigated by kept at room temperature and 4 °C for 6 mo.Results: The PRE loaded NLCs showed spherical shape with a mean particle size of ~100-120 nm and zeta potential of ~-24 mV. Up to 90% drug entrapment efficiency was achieved. PEG-NLCs and PVA-NLCs showed a strong interaction with porcine buccal mucosa than uncoated-NLCs. All PRE loaded NLCs formulations revealed fast release characteristics and effective against Streptococcus mutans and S. sanguinis. The mouth spray containing PRE loaded NLCs showed good physical stability without particle aggregation. In addition, the chemical stability of piperine in NLCs was significantly improved during storage at both storage conditions compared to its solution form.Conclusion: The developed PRE loaded polymer coated-NLCs showed high potential to use as a local drug delivery system for reducing the bacterial growth in the oral cavity

PREPARATION AND CHARACTERIZATION OF CHITOSAN/REGENERATED SILK FIBROIN (CS/RSF) FILMS AS A BIOMATERIAL FOR CONTACT LENSES-BASED OPHTHALMIC DRUG DELIVERY SYSTEM

Objective: The aim of this study was to develop chitosan/regenerated silk fibroin (CS/RSF) films as a biomaterial for contact lenses-based ophthalmic drug delivery system. Methods: CS/RSF films were prepared with polyethylene glycol 400 as a plasticizer by using a film casting technique. Their physicochemical properties were investigated by measuring various properties such as thickness, morphology, chemical interaction, light transparency, mechanical properties, water content, oxygen permeability, thermal properties and enzyme degradation. In addition, cytotoxicity was also studied. Results: At optimal preparation conditions, CS/RSF films showed smooth surfaces with highly visible light transparency of >90%, which meet the visual requirement. CS/RSF films showed high water content, 59-65% by weight, and their Young’s modulus and elongation at break was in the range of 3.8-6 N/mm2 and 113-135%, respectively. The CS/RSF films also could be sterilized by autoclave method as they possessed high thermal decomposition temperature of >260 °C which can be confirmed by both differential scanning calorimetry and thermogravimetric analysis. In addition, CS/RSF films showed no degradation in stimulated tear fluid containing lysozyme for 7 d and showed no cytotoxicity by MTT assay. Conclusion: CS/RSF films showed excellent physicochemical properties and non-cytotoxicity indicating their promising potential use as a biomaterial for contact lenses-based ophthalmic drug delivery system

DEVELOPMENT AND CHARACTERIZATION OF TOPICAL OPHTHALMIC FORMULATIONS CONTAINING LUTEIN-LOADED MUCOADHESIVE NANOPARTICLES

Objective: To develop and characterize a topical ophthalmic formulation containing chitosan-dextran sulfate nano particles (CDNs) for enhanced ocular bioavailability and stability of lutein.Methods: Lutein-loaded CDNs (LCDNs) were prepared by polyelectrolyte complexation employing oppositely charged polymers, chitosan and dextran sulfate. Effects of the polymer mass ratios, the total amount of polymers, and the amount of EDC and PEG400 on their physicochemical properties as well as the drug release profiles were investigated. The physicochemical stability of LCDNs dispersed in various ophthalmic vehicles and the accompanying microbial contamination were also evaluated.Results: LCDNs possessed a mean size of ~400 nm with a positive surface charge of+30 mV and entrapment efficiency up to 75%. Dissolution profiles followed a Higuchi's square root model, indicating a diffusional release mechanism. LCDNs dispersed in Feldman ophthalmic buffer showed good physical stability with no microbial contamination. The chemical stability of lutein was significantly improved in LCDNs and further improved by the addition of antioxidant in the ophthalmic vehicle.Conclusion: The ophthalmic formulation containing LCDNs, developed in this work, has characteristics suitable for application in ocular surface drug delivery systems.Keywords: Chitosan, dextran sulfate, Nanoparticles, Ophthalmic vehicle, Lutein

DEVELOPMENT AND CHARACTERIZATION OF INDOMETHACIN-LOADED MUCOADHESIVE NANOSTRUCTURED LIPID CARRIERS FOR TOPICAL OCULAR DELIVERY

Objective: To develop and characterize indomethacin loaded-nanostructured lipid carriers (IND-NLCs) for topical ophthalmic delivery with different particle sizes and polymer coating to improve the mucoadhesive property on the ocular surface.Methods: Nanostructured lipid carriers (NLCs) with different solid lipids and surfactants were prepared by the high-pressure homogenization technique. The optimized IND-NLCs was coated with polyethylene glycol 400 (PEG). The physicochemical properties and entrapment efficacy (EE) were examined. In vitro release studies were investigated using the shake-flask method. Ex vivo mucoadhesive studies were assessed by the wash-off test. In addition, the cytotoxicity was assessed by the short time exposure test.Results: IND-NLCs of ~300 and ~40 nm in diameter were successfully produced with a zeta potential of -30 mV and EE of 60–70 %. IND-NLCs prepared with Tween 80 as surfactant could be sterilized by autoclaving. The PEG coating of IND-NLCs did not affect either the particle size or EE. In vitro release showed a prolonged release for 360 min with a burst release of 50-60% occurring within 5 min. The smaller-sized IND-NLCs showed slightly faster release rates and better mucoadhesion to cornea compared to the larger IND-NLCs. PEG-coated IND-NLCs showed the highest mucoadhesion. In addition, IND-NLCs showed less cytotoxicity compared to IND alone. Conclusion: The small and PEG-coated NLCs represents a potentially useful carrier for safe delivery of indomethacin to the ocular surface with increased residence time

DESIGN OF EXPERIMENTS MODEL FOR THE OPTIMIZATION OF SILK FIBROIN BASED NANOPARTICLES

Objective: Silk fibroin based nanoparticles have been utilized extensively in biomedical fields. Amongst many preparation methods, desolvation is a favorable one. However, this method yields nanoparticles with unpredictable parameters. Thus, this investigation aimed to systematically study the effects of three independent variables including fibroin concentration (% w/v, X1), volume ratio between fibroin solution and ethanol (X2), formulation time (h, X3) on three main responses, particle size (nm, Y1), polydispersity index (Y2), zeta potential (mV, Y3).Methods: Fibroin was extracted from degummed Bombyx mori silk. The fibroin calibration curve was constructed by UV-spectrophotometer at 276 nm. The nanoparticles were prepared using the desolvation method of aqueous fibroin solution in ethanol. Design Expert® software was used to design the model. The mean particle size, polydispersity index and zeta potential were determined using ZetaPALS®analyzer.Results: By using D-optimal design with the quadratic model, the results showed that all X1, X2, and X3 variables had significant impacts on the fibroin nanoparticles characteristics Y1, Y2, and Y3. The generated model was also validated and demonstrated to be solid and reliable. The obtained optimal nanoparticles possessed Y1 of 238.1 nm, Y2 of 0.12, and Y3of-21.78 mV, which were in agreement with the predicted values, 224.8 nm, 0.13 and-19.31 mV, respectively. The optimal actual and theoretical particle characteristics were correlated with a desirable value of R2 = 0.8770. Conclusion: The D-optimal design proved its effectiveness in the prediction and optimization of fibroin nanoparticle properties

PENETRATION OF HYDROPHILIC SULFORHODAMINE B ACROSS THE PORCINE CORNEA EX-VIVO

Objective: Sulforhodamine B (SRB) is a hydrophilic tracer whose fluorescence is unaffected by pH unlike that of carboxyfluorescein. Therefore, SRB may serve as a better tracer when there are significant changes in pH. Thus, in this study, the suitability of SRB to assess the barrier properties of the cellular layers of the cornea was examined using a custom-built confocal scanning micro-fluorometer (CSMF). Methods: The dye solution (0.1% SRB) was prepared in PBS-Ca2+and three experiments were performed ex vivo using freshly isolated porcine eyes. First, we investigated the penetration of SRB across the endothelium by injection of the dye into the anterior chamber. Next, we measured the penetration of SRB across the epithelium after exposing the ocular surface to the dye. Finally, we examined the penetration after exposure to the dye with detergent (Tween 20) and exposure to the dye concomitant with microneedle injuries. The dye concentration profiles across the cornea were measured using CSMF.Results: SRB penetrated the corneal endothelium readily into the stroma following injection into the anterior chamber in a time-dependent manner. Despite accumulation in the stroma, SRB did not partition into the epithelium. In agreement with these findings, the dye did not cross the epithelium after topical administration. Co-administration with Tween 20 and injury to the epithelium with microneedles, however, led to penetration of the dye into the stroma.Conclusions: SRB is a hydrophilic dye that can be used as an alternative fluorescent tracer to assess the barrier function of the cellular layers of the cornea

DEVELOPMENT, CHARACTERIZATION AND SKIN IRRITATION OF MANGOSTEEN PEEL EXTRACT SOLID DISPERSION CONTAINING CLAY FACIAL MASK

Objective: To develop a clay facial mask containing mangosteen peel extract solid dispersion (MPESD) for enhancing α-mangostin bioavailability and to determine suitable clay-based facial mask.Methods: The MPESD were prepared by a melting-solvent method employing PVP K30 and poloxamer 188 as a carrier. The water solubility was determined by HPLC method. The in vitro skin permeability was examined using porcine ear epidermis. The effects of clay types on the physical stability of MPESD and α-mangostin adsorption capacity were evaluated. The skin irritation was determined by 4 h human patch test.Results: After dissolved optimal formulation of MPESD in water, the spherical micelle was observed with a mean size of ~150 nm and showed significantly α-mangostin water solubility enhancement of ~7 mg/ml, 700 times greater than MPE. Upon mixing the MPESD with clays, a dry powder was obtained. In vitro permeation studies of the MPESD mixed with titanium dioxide showed lowest α-mangostin permeation, while MPESD mixed with mica or talcum showed similar permeation profile as free MPESD solutions. No sign of skin irritation was observed in volunteers after application of the MPESD-based clay facial mask patch on the inner forearm skin for 4 h.Conclusion: MPESD demonstrates a promising technique for improving water solubility and permeation of α-mangostin which reducing the staining effect. In addition, it is safe for topical application and cosmetically acceptable

INTERACTION OF NANOSTRUCTURED LIPID CARRIERS WITH HUMAN MEIBUM

Objective: This study aimed to determine the possibility of nanostructured lipid carriers (NLCs) as a bionic tear film by determining the surface activities of the developed NLCs and their interaction with human meibomian lipid films. Methods: NLCs with different types of solid lipids and surfactants were prepared by a high-pressure homogenizer. The particle size was determined by dynamic light scattering. The surface activities of the NLCs and NLCs mixed with meibomian lipids were measured using a Langmuir trough and the resulting surface pressure area (Π-A) profiles were compared. These lipid films were further analyzed using fluorescence microscopy and scanning electron microscopy (SEM). Results: The particle size of prepared NLCs varied from 38–280 nm based on types of solid lipid and surfactant. All NLCs were highly surface active as indicated by their maximum surface pressure (Πmax). The Π-A profiles of meibum seeded with NLCs showed higher surface pressure than meibum alone and the shape of profiles were dominated by the meibomian lipids. These findings were in agreement with fluorescence and SEM micrographs, which revealed that the NLCs could adsorb and integrate to the meibomian lipid films as well as diffuse from the subphase to the lipid films. Conclusion: NLCs are surface active and can integrate with meibomian lipid films formed stable films. The type of interaction can be tailored by altering the solid lipids used in the formulation of the NLCs which could provide the means to develop efficient formulations for targeting dry eye disease related to a non-functional tear film lipid layer