Han Hsiang Huang

Improvement and enhancement of antibladder carcinoma cell effects of heteronemin by the nanosized hyaluronan aggregation

The effects against tumors exerted by marine active compounds have been highlighted and investigated. Polymeric nanoparticles made from biodegradable and biocompatible molecules such as hyaluronan (HA) and chitosan (CHI) are able to aggregate the compounds to enhance their activities against tumor cells and reduce the toxicity on normal cells. Here, we extensively examined the antitumor activities and the mechanisms of HA/CHI nanoparticles-aggregated heteronemin (HET) extracted from the sponge Hippospongia sp. The half-maximal inhibitory concentration (IC50) of pure HET toward T24 bladder carcinoma cells is ~0.28 µg/mL. Pure HET from 0.2 to 0.8 µg/mL and HA nanoparticles-aggregated HET at 0.1 and 0.2 µg/mL significantly reduced T24 cell viability. Compared to pure HET, HA nanoparticles/HET aggregates showed much weaker viability-inhibitory effects on L929 normal fibroblasts. HET dose-dependently suppressed cancer cell migration as HA/CHI nanoparticles-aggregated HET displayed stronger migration-inhibitory effects than pure HET. Flow cytometric analysis showed that pure HET increased early/total apoptosis and JC-1 monomer fluorescence, while HA/CHI nanoparticles-aggregated HET induced higher apoptosis and JC-1 monomer rates than pure HET, suggesting that aggregation of HA nanoparticles offers HET stronger apoptosis-inducing capacity through mitochondrial depolarization. Western blot analysis showed that HA nanoparticles-aggregated HET further increased mitochondrial-associated, caspase-dependent and caspase-independent, as well as endoplasmic reticulum stress-related factors in comparison with pure HET. These data indicated that pure HET possesses cytotoxic, antimigratory, and apoptosis-inducing effects on bladder cancer cells in vitro, and its induction of apoptosis in bladder carcinoma cells is mainly caspase dependent. Moreover, HA nanoparticle aggregation reinforced the cytotoxic, antimigratory, and apoptosis-inducing activities against bladder carcinoma cells and attenuated the viability-inhibitory effects on normal fibroblasts. This aggregation reinforces antibladder carcinoma effects of HET via diverse routes, including mitochondrial-related/caspase-dependent, caspase-independent, and endoplasmic reticulum stress-related pathways. The current data also strongly suggested that HA/CHI nanoparticles-aggregated HET would be a potential treatment for urothelial cancer in vivo.

Liposomal Encapsulation for Systemic Delivery of Propranolol via Transdermal Iontophoresis Improves Bone Microarchitecture in Ovariectomized Rats

The stimulatory effects of liposomal propranolol (PRP) on proliferation and differentiation of human osteoblastic cells suggested that the prepared liposomes-encapsulated PRP exerts anabolic effects on bone in vivo. Iontophoresis provides merits such as sustained release of drugs and circumvention of first pass metabolism. This study further investigated and evaluated the anti-osteoporotic effects of liposomal PRP in ovariectomized (OVX) rats via iontophoresis. Rats subjected to OVX were administered with pure or liposomal PRP via iontophoresis or subcutaneous injection twice a week for 12 weeks. Changes in the microarchitecture at the proximal tibia and the fourth lumbar spine were assessed between pure or liposomal PRP treated and non-treated groups using micro-computed tomography. Administration of liposomal PRP at low dose (0.05 mg/kg) via iontophoresis over 2-fold elevated ratio between bone volume and total tissue volume (BV/TV) in proximal tibia to 9.0% whereas treatment with liposomal PRP at low and high (0.5 mg/kg) doses via subcutaneous injection resulted in smaller increases in BV/TV. Significant improvement of BV/TV and bone mineral density (BMD) was also found in the fourth lumbar spine when low-dose liposomal PRP was iontophoretically administered. Iontophoretic low-dose liposomal PRP also elevated trabecular numbers in tibia and trabecular thickness in spine. Enhancement of bone microarchitecture volumes has highlighted that liposomal formulation with transdermal iontophoresis is promising for PRP treatment at the lower dose and with longer duration than its clinical therapeutic range and duration to exhibit optimal effects against bone loss in vivo.

Anticancer Effects of Sinulariolide-Conjugated Hyaluronan Nanoparticles on Lung Adenocarcinoma Cells

Lung cancer is one of the most clinically challenging malignant diseases worldwide. Sinulariolide (SNL), extracted from the farmed coral species Sinularia flexibilis, has been used for suppressing malignant cells. For developing anticancer therapeutic agents, we aimed to find an alternative for non-small cell lung cancer treatment by using SNL as the target drug. We investigated the SNL bioactivity on A549 lung cancer cells by conjugating SNL with hyaluronan nanoparticles to form HA/SNL aggregates by using a high-voltage electrostatic field system. SNL was toxic on A549 cells with an IC50 of 75 µg/mL. The anticancer effects of HA/SNL aggregates were assessed through cell viability assay, apoptosis assays, cell cycle analyses, and western blotting. The size of HA/SNL aggregates was approximately 33–77 nm in diameter with a thin continuous layer after aggregating numerous HA nanoparticles. Flow cytometric analysis revealed that the HA/SNL aggregate-induced apoptosis was more effective at a lower SNL dose of 25 µg/mL than pure SNL. Western blotting indicated that caspases-3, -8, and -9 and Bcl-xL and Bax played crucial roles in the apoptotic signal transduction pathway. In summary, HA/SNL aggregates exerted stronger anticancer effects on A549 cells than did pure SNL via mitochondria-related pathways.

Alternative approach of cell encapsulation by Volvox spheres

Volvox sphere is a bio-mimicking concept of a biomaterial structure design able to encapsulate chemicals, drugs and/or cells. The aim of this study was to prepare Volvox spheres encapsulating AML12 liver cells and mesenchymal stem cells (MSCs) via a high voltage electrostatic field system. The results demonstrated that AML12 liver cells and MSCs could be successfully encapsulated into the inner spheres and the outer sphere of the Volvox spheres. The improved cell viability of MSCs was achieved by the addition of collagen and polyethylene glycol into the preparation components of the Volvox spheres. Collagen material potentially provides extracellular matrix-like structure for cell adhesion while polyethylene glycol provides a void/loose space for permeability of metabolites. The encapsulated MSCs were able to differentiate into hepatocytes or hepatocyte-like cells and express liver cell markers including albumin, alpha feto-protein and cytokeratin 18. The encapsulated cells secreted albumin to about 140 ng on day 14. Based on these observations, we conclude that Volvox spheres can be used as an alternative approach to encapsulate multiple types of cells, here AML12 hepatocyte cell line and MSCs. Nevertheless, efforts are still needed to improve the viability of the encapsulated cells and increase the differentiation of MSCs into functional liver cells.

Effects of culturing media on hepatocytes differentiation using Volvox sphere as co-culturing vehicle.

Volvox sphere is a unique design to mimic natural volvox consists of a large outer-sphere that contains smaller inner-spheres, which provide three-dimensional (3D) environment to culture cells. The purpose of this study is to co-culture mesenchymal stem cells (MSCs) and AML12 liver cells in Volvox spheres and to evaluate the effects of two media, DMEM and DMEM/F12 on the cultured cells. The results of this study shows that the 3D Volvox sphere can successfully be applied for co-culture of MSCs and AML12 liver cells, and the MSCs are able to differentiate into hepatocyte-like cells expressing hepatocyte-specific markers including albumin (ALB), alpha feto-protein (AFP) and cytokeratin 18 (CK18) mRNA expressions and producing CK18 and ALB proteins. Interestingly, the MSCs expressed higher ALB, AFP and CK18 mRNA expression at the initial 7-day culture by using DMEM, whereas, the MSCs expressed more mRNA expressions from 7-day to 14-day by the usage of DMEM/F12. The result demonstrated that DMEM and DMEM/F12 media could affect MSCs behaviors during a 14-day culture.

In vitro and in vivo study of the application of volvox spheres to co-culture vehicles in liver tissue engineering

Volvox sphere is a biomimetic concept of a natural Volvox, wherein a large outer sphere contains smaller inner spheres, which can encapsulate cells and provide a double-layer three-dimensional environment for culturing cells. This study simultaneously encapsulated rat mesenchymal stem cells (MSCs) and AML12 hepatocytes in volvox spheres and extensively evaluated the effects of various culturing modes on cell functions and fates. The results showed that compared with a static flask culture, MSCs encapsulated in volvox spheres differentiated into hepatocyte-like cells with a 2-fold increase in albumin (ALB) expression and a 2.5-fold increase in cytokeratin 18 expression in a dynamic bioreactor. Moreover, the restorative effects of volvox spheres encapsulating cells on retrorsine-exposed CCl4-induced liver injuries in rats were evaluated. The data presented significant reductions in AST and ALT levels after the implantation of volvox spheres encapsulating both MSCs and AML12 hepatocytes in vivo. In contrast to the negative control group, histopathological analysis demonstrated liver repair and formation of the new liver tissue in groups implanted with volvox spheres containing cells. These results demonstrate that liver cells implanted with volvox spheres encapsulating both MSCs and AML12 hepatocytes promote liver repair and liver tissue regeneration in liver failure caused by necrotizing agents such as retrorsine and CCl4. Hence, volvox spheres encapsulating MSCs and liver cells can be a promising and clinically effective therapy for liver injury. STATEMENT OF SIGNIFICANCE In this study, we used a volvox sphere, which is a unique design that mimics the natural Volvox, that consists of a large outer sphere that contains smaller inner spheres, which provide a three-dimensional environment to culture cells. The purpose of this study is to co-culture mesenchymal stem cells (MSCs) and AML12 liver cells in volvox spheres and evaluate two different culture methods, dynamic bioreactor and static culture flask,on the cultured cells. In addition, we aimed to evaluate the restorative effects of volvox spheres encapsulating MSCs and/or AML12 liver cells on rats with retrorsine-exposed CCl4-induced liver injuries. The results showed that MSCs encapsulated in volvox spheres differentiated into hepatocyte-like cells with a 2-fold increase in albumin expression and a 2.5-fold increase in cytokeratin 18 expression ina dynamic bioreactor. Moreover, the data presented significant reductions in AST and ALT levels after the implantation of volvox spheres encapsulating both MSCs and AML12 hepatocytes in vivo. In contrast to the negative control group, histopathological analysis demonstrated liver repair and formation of new liver tissue in groups implanted with volvox spheres containing cells. These results demonstrate that liver cells implanted with volvox spheres encapsulating both MSCs and AML12 hepatocytes promote liver repair and liver tissue regeneration in liver failure caused by necrotizing agents such as retrorsine and CCl4. Hence, volvox spheres encapsulating MSCs and liver cells can be a promising and clinically effective therapy for liver injury.

Characterization and human osteoblastic proliferation- and differentiation-stimulatory effects of phosphatidylcholine liposomes-encapsulated propranolol hydrochloride

DMPC and DSPC liposomes were prepared via thin film hydration method followed by sonication. Propranolol solution was incorporated into liposomes at hydration stage. TEM images showed the sizes of DSPC and DMPC were around 88 and 137 nm, respectively. The highest encapsulation ratio of propranolol was approximately 70% using DSPC/CHO/OCT liposomes, which release the drug over 60% in 24 h and reached 100% in 48 h. Both propranolol (10⁻⁸-10⁻⁶ M) and DSCP liposomes-encapsulated propranolol showed over 1.5-fold increases in the proliferation of human osteoblastic cells hFOB1.19 while differentiation of the cells was approximately doubled by plain and liposomal propranolol, indicating that the stimulatory effects of liposomal propranolol are similar with those of propranolol on human osteoblastic hFOB1.19 cells. The phosphatidylcholine liposomes-encapsulated propranolol prepared in this study potentially possesses anabolic effects in vivo and is also a promising anti-osteoporotic agent in future.

Reparative Effects of Astaxanthin-Hyaluronan Nanoaggregates against Retrorsine-CCl4-Induced Liver Fibrosis and Necrosis

Astaxanthin (Asta), a xanthophyll carotenoid, has been reported to be a strong antioxidative agent and has anti-inflammatory, antitumor and free radical-scavenging activities. However, inadequate stability and water solubility results in its low bioavailability. This study incorporated Asta into hydrophilic hyaluronan nanoparticles (HAn) to produce Asta-HAn aggregates (AHAna) using an electrostatic field system and investigated the restorative effects of AHAna on retrorsine-CCl4-induced liver fibrosis in rats in vivo. Transmission electron microscopy (TEM) revealed that the prepared HAn were approximately 15 ± 2.1 nm in diameter and after the incorporation of Asta into HAn, the size increased to 210–500 nm. The incorporation efficiency of Asta was approximately 93% and approximately 54% of Asta was released after incubation for 18 h. Significant reductions in alanine aminotransferase and aspartate aminotransferase levels were observed after the rats were intraperitoneally injected with AHAna. Histopathological findings revealed the greatest reduction in hepatic fibrosis and hepatocyte necrosis in the rats after 2 weeks of intraperitoneal injection with AHAna, which is consistent with the data acquired from serum biochemical analysis. The restorative effects on liver damage displayed by AHAna in vivo demonstrated that Asta aggregated through HAn incorporation exerts therapeutic effects on liver fibrosis and necrosis.

Evaluation of the UVB-screening capacity and restorative effects exerted by farnesol gel on UVB-caused sunburn

Farnesol, a natural 15‐carbon organic compound, has various microbiological and cellular activities. It has been found to exert apoptosis‐inducing effects against carcinoma cells as well as antiallergic and anti‐inflammatory effects in vivo. In the current study, a series of formulations composed of various concentrations of hydroxypropyl methylcellulose (HPMC) with the addition of hyaluronan (HA) and xanthan gum (XG) was designed to evaluate the UVB‐screening and H2O2‐eliminating effects of farnesol in normal fibroblasts. Farnesol at 0.005, 0.0075, and 0.01% exhibited significant capacity for H2O2 scavenging; at 0.0025%, it showed insignificant effects. Under 120‐min UVB exposure, screening with plural gel composed of 0.0025% farnesol, 0.5% HA, and 0.5% XG containing 1.5% or 2% HPMC retained normal fibroblast viability. After 60‐min exposure to UVB, screening with plural gel composed of farnesol, HA, XG, and 0.5%, 1.0%, 1.5%, or 2% HPMC decreased the ratio of the G1 phase and increased ratio of the S phase in comparison with the accumulated cell cycle of the normal fibroblasts without screening. The gel with 2% HPMC displayed the strongest cell cycle‐reversal ability. In vivo histopathological results showed that the prepared plural gels with 0.5% or 2% HPMC and farnesol, HA, and XG had greater antiphotoaging and reparative effects against UVB‐induced changes and damage in the skin. In conclusion, the current in vitro and in vivo results demonstrated that the prepared plural composed of 0.0025% farnesol, 0.5% HA, 0.5% XG, and 2% HPMC possessed the greatest UVB‐screening capacity and the strongest restorative effects on UVB‐induced sunburned skin.

In vitro and in vivo evaluation of the effect of nano-sized collagen molecules and nicotinamide on mesenchymal stem cell differentiation

Advances and improvements in mesenchymal stromal/stem cells (MSCs) and cell replacement therapies have been promising approaches to treat diabetes mellitus (DM) since their potent capacities for differentiation into various functional cells match the demands of tissue repair and regeneration. The aim of this study is to examine the effects of nano-sized type I collagen molecules in combination with nicotinamide (NCT) and exendin-4 (EX4) on MSC differentiation into insulin-secreting cells in vitro and to evaluate their reparative effects against type 2 diabetes mellitus (T2DM) in vivo. Differentiation of MSCs in the presence of NCT, nano-sized type I collagen molecules and EX4 was represented with insulin production and Nkx6.1/PDX-1 mRNA expression assessed by insulin secretion assay and quantitative RT-PCR. Histopathological and glycosylated haemoglobin (HbA1) analysis was performed to assess reparative effects against T2DM in the rat model. The results revealed that MSCs showed increased differentiation into insulin-secreting cells with higher mRNA expression for Nkx6.1 and early PDX-1 in the presence of NCT and nano-sized type I collagen molecules. Addition of nano-sized type I collagen fibrils increased morphologically islet-like clusters in differentiated cells. T2DM rats reverted to their normal HbA1 values and exhibited structurally repaired islets in the pancreas implanted with NCT/nano-sized collagen I molecule/EX4-incubated differentiated cells. In short, the combined recipe showed reparative actions on the destructive islet of Langerhans in the pancreas coupled with glucoregulatory effects in T2DM rats in vivo. Therefore, MSCs incubated with NCT/EX4 and nano-sized collagen I molecules could be a potential therapy for retrieval of destructed islets and could efficiently regulate blood glucose in T2DM.