Phragmites australis (Cav.) Trin. ex Steud. Extract Induces Apoptosis-like Programmed Cell Death in Acanthamoeba castellanii Trophozoites.

Acanthamoeba keratitis (AK) is an infectious ocular disease which is difficult to diagnose correctly and cure. Development of an effective and safe therapeutic drug for AK is needed. Our preliminary screening of more than 200 extracts from wild plants collected in Korea suggested the potential amoebicidal activity of Phragmites australis (Cav.) Trin. ex Steud. extract (PAE) against Acanthamoeba species. Here, we aimed to analyze the amoebicidal activity of PAE on Acanthamoeba and its underlying amoebicidal mechanism. PAE induced amoebicidal activity against both A. castellanii and A. polyphaga trophozoites, while it showed low cytotoxicity in human corneal epithelial cells (HCE-2) and human retinal pigment epithelial cells (ARPE-19). Transmission electron microscopy analysis showed subcellular morphological changes, such as increased granules, abnormal mitochondria, and atypical cyst wall formation, in the PAE-treated A. castellanii. Fluorometric apoptosis assay and TUNEL assay revealed apoptosis-like programmed cell death (PCD) in the PAE-treated A. castellanii. The PAE treatment increased reactive oxygen species production and reduced mitochondrial membrane potential in the amoeba. The enhanced expression of autophagy-associated genes was also detected. These results suggested that PAE exerted a promising amoebicidal effect on A. castellanii trophozoites via the PCD pathway. PAE could be a potential candidate for developing a therapeutic drug for AK.

In situ facile-forming PEG cross-linked albumin hydrogels loaded with an apoptotic TRAIL protein.

The key to making a practicable hydrogel for pharmaceutical or medical purposes is to endow it with relevant properties, i.e., facile fabrication, gelation time-controllability, and in situ injectability given a firm basis for safety/biocompatibility. Here, the authors describe an in situ gelling, injectable, albumin-cross-linked polyethylene glycol (PEG) hydrogel that was produced using a thiol-maleimide reaction. This hydrogel consists of two biocompatible components, namely, thiolated human serum albumin and 4-arm PEG20k-maleimide, and can be easily fabricated and gelled in situ within 60s by simply mixing its two components. In addition, the gelation time of this system is controllable in the range 15s to 5min. This hydrogel hardly interacted with an apoptotic TRAIL protein, ensuring suitable release profiles that maximize therapeutic efficacy. Specifically, tumors (volume: 278.8mm(3)) in Mia Paca-2 cell-xenografted BALB/c nu/nu mice treated with the TRAIL-loaded HSA-PEG hydrogel were markedly smaller than mice treated with the hydrogel prepared via an amine-N-hydroxysuccinimide reaction or non-treated mice (1275.5mm(3) and 1816.5mm(3), respectively). We believe that this hydrogel would be a new prototype of locally injectable sustained-release type anti-cancer agents, and furthermore offers practical convenience for a doctor and universal applicability for a variety of therapeutic proteins.

PEGylated apoptotic protein-loaded PLGA microspheres for cancer therapy.

The aim of the current study was to investigate the antitumor potential of poly (D,L-lactic-co-glycolic acid) microspheres (PLGA MSs) containing polyethylene glycol (PEG)-conjugated (PEGylated) tumor necrosis factor-related apoptosis-inducing ligand (PEG-TRAIL). PEG-TRAIL PLGA MSs were prepared by using a water-in-oil-in-water double-emulsion method, and the apoptotic activities of supernatants released from the PLGA MSs at days 1, 3, and 7 were examined. The antitumor effect caused by PEG-TRAIL PLGA MSs was evaluated in pancreatic Mia Paca-2 cell-xenografted mice. PEG-TRAIL PLGA MS was found to be spherical and 14.4±1.06 µm in size, and its encapsulation efficiency was significantly greater than that of TRAIL MS (85.7%±4.1% vs 43.3%±10.9%, respectively). The PLGA MS gradually released PEG-TRAIL for 14 days, and the released PEG-TRAIL was shown to have clear apoptotic activity in Mia Paca-2 cells, whereas TRAIL released after 1 day had a negligible activity. Finally, PEG-TRAIL PLGA MS displayed remarkably greater antitumor efficacy than blank or TRAIL PLGA MS in Mia Paca-2 cell-xenografted mice in terms of tumor volume and weight, apparently due to increased stability and well-retained apoptotic activity of PEG-TRAIL in PLGA MS. We believe that this PLGA MS system, combined with PEG-TRAIL, should be considered a promising candidate for treating pancreatic cancer.

Inhalable self-assembled albumin nanoparticles for treating drug-resistant lung cancer.

Direct pulmonary delivery of anti-cancer agents is viewed as an effective way of treating lung cancer. Here, we fabricated inhalable nanoparticles made of human serum albumin (HSA) conjugated with doxorubicin and octyl aldehyde and adsorbed with apoptotic TRAIL protein (TRAIL/Dox HSA-NP). The octyl aldehyde and doxorubicin endowed HSA with significant hydrophobicity that facilitated self-assembly. TRAIL/Dox HSA-NP was found to have excellent particle size (~340nm), morphology, dispersability, and aerosolization properties. TRAIL/Dox HSA-NP displayed synergistic cytotoxicity and apoptotic activity in H226 lung cancer cells vs. HSA-NP containing TRAIL or Dox alone. TRAIL/Dox HSA-NP was well deposited in the mouse lungs using an aerosolizer, and TRAIL and Dox-HSA were found to be gradually released over 3days. The anti-tumor efficacy of pulmonary administered TRAIL/Dox HSA-NP was evaluated in BALB/c nu/nu mice bearing H226 cell-induced metastatic tumors. It was found that the tumors of H226-implanted mice treated with TRAIL/Dox HSA-NP were remarkably smaller and lighter than those of mice treated with TRAIL or Dox HSA-NP alone (337.5±7.5; 678.2±51.5; and 598.9±24.8mg, respectively). Importantly, this improved anti-tumor efficacy was found to be due to the synergistic apoptotic effects of Dox and TRAIL. In the authors' opinion, TRAIL/Dox HSA-NP offers a potential inhalable anti-lung cancer drug delivery system. Furthermore, the synergism displayed by combined use of Dox and TRAIL could be used to markedly reduce doxorubicin doses and minimize its side effects.

Apoptotic activity and antitumor efficacy of PEGylated TNF-related apoptosis-inducing ligand (TRAIL) in a Mia Paca-2 cell-xenografted mouse model.

The purpose of this study was to demonstrate the apoptotic activity and antitumor effect of PEGylated tumor necrosis factor-related apoptosis-inducing ligand (PEG-TRAIL) in pancreatic carcinoma Mia Paca-2 cells and in Mia Paca-2 cell-xenografted mice. PEG-TRAIL was prepared using mPEG-aldehyde (Mw 5 kDa). The apoptosis induced by PEG-TRAIL in Mia Paca-2 cells and in the tumors of Mia Paca-2 cell-xenografted mice was quantified by FACS analysis and using a TUNEL assay. Mia Paca-2 cell-xenografted BALB/c nu/nu mice were administered intratumoral injections of PEG-TRAIL (50 µg/mouse/injection) every 3 days from day 0 (~4 weeks after xenografting) to day 15. Tumor volumes were measured every 3 days from day 0 to day 27. PEG-TRAIL displayed obvious apoptotic activity in Mia Paca-2 cells; the FACS signal was shifted to the apoptotic area and the cells exhibited green fluorescence indicating apoptosis in the TUNEL assay. Furthermore, PEG-TRAIL was found to suppress tumors in Mia Paca-2 cell-xenografted mice (tumor volumes: 183.9±134.1 for PEG-TRAIL vs. 1827.3±264.5 mm(3) for saline control). In addition, in vivo TUNEL assays of tumor tissues showed that the antitumor effect of PEG-TRAIL was due apoptosis. Our findings provide clear in vivo evidence of the antitumor potential of PEG-TRAIL in a Mia Paca-2 cell-xenografted mouse model based of pancreatic cancer.

Four-arm PEG cross-linked hyaluronic acid hydrogels containing PEGylated apoptotic TRAIL protein for treating pancreatic cancer.

Four-arm polyethylene glycol (PEG) cross-linked hyaluronic acid (HA) hydrogels containing PEGylated tumor necrosis factor-related apoptosis-inducing ligand (PEG-TRAIL) were fabricated, and their antitumor effects were evaluated in pancreatic cell (Mia Paca-2)-xenografted mice. HA was conjugated with 4-arm PEG(10k)-amine (a cross-linker) at ratios of 100:1 and 100:2 using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride as a cross-linker, and TRAIL or PEG-TRAIL was incorporated into these HA hydrogels. HA hydrogels at a 100:1 ratio were prepared in good yields (>88%), were moderately stiff, and gradually released PEG-TRAIL over ~14 days in vitro and over ~7 days in vivo (as determined by high-pressure liquid chromatography and infrared imaging). The released PEG-TRAIL was found to have obvious apoptotic activity in Mia Paca-2 cells. PEG-TRAIL HA hydrogels displayed remarkably more antitumor efficacy than TRAIL HA hydrogels in Mia Paca-2 cell-xenografted mice in terms of tumor volumes (size) and weights (453.2mm(3) and 1.03 g vs. 867.5mm(3) and 1.86 g). Furthermore, this improved antitumor efficacy was found to be due to the apoptotic activity of PEG-TRAIL in vivo (determined by a TUNEL assay) despite its substantially lower cytotoxicity than native TRAIL (IC50 values: 71.8 and 202.5 ng ml(-1), respectively). This overall enhanced antitumor effect of PEG-TRAIL HA hydrogels appeared to be due to the increased stability of PEGylated TRAIL in HA hydrogels. These findings indicate that this HA hydrogel system combined with PEG-TRAIL should be considered a potential candidate for the treatment of pancreatic cancer.

Decanoic acid-modified glycol chitosan hydrogels containing tightly adsorbed palmityl-acylated exendin-4 as a long-acting sustained-release anti-diabetic system.

Decanoic acid-modified glycol chitosan (DA-GC) hydrogels containing tightly adsorbed palmitic acid-modified exendin-4 (Ex4-C16) were prepared, and their pharmaceutical abilities as a long-acting sustained-release exendin-4 system for the treatment of diabetes were evaluated. Glycol chitosan (GC) was conjugated with N-hydroxysuccinimide-activated decanoic acid (DA) in anhydrous 0.4% dimethylaminopyridine/dimethylsulfoxide at different feed ratios. DA-GC hydrogels formed by physical self-assembly during dialysis vs. deionized water, and their inner network structures, swelling or gel-forming abilities and release properties were examined. The hypoglycemia caused by Ex4-C16-loaded DA-GC hydrogels was evaluated by subcutaneous administration in type 2 diabetic db/db mice. The results obtained showed that GC prepared at a DA:GC feed ratio of 1:100 had optimal properties with respect to hydrogel swelling, stiffness and Ex4-C16 incorporation, whereas DA-GC hydrogels prepared at a feed ratio of greater than 1:100 formed gels that were too stiff. The in vitro and in vivo release of Ex4-C16 from DA-GC hydrogels was dramatically delayed compared with native Ex4 probably due to strong hydrophobic interactions. In particular, Ex4-C16 in DA-GC hydrogels was found to be present around the injection site up to 10 days after subcutaneous administration, whereas Ex4 in DA-GC hydrogels was cleared from injection sites in ≈ 2 days in ICR mice. Finally, the hypoglycemia induced by Ex4-C16 DA-GC hydrogels was maintained for >7 days. Our findings demonstrate that Ex4-C16 DA-GC hydrogels offer a potential delivery system for the long-term treatment of type 2 diabetes.

Long-acting inhalable chitosan-coated poly(lactic-co-glycolic acid) nanoparticles containing hydrophobically modified exendin-4 for treating type 2 diabetes.

Inhalable glycol chitosan-coated poly(lactic-co-glycolic acid) (PLGA) nanoparticles containing palmitic acid-modified exendin-4 (Pal-Ex4) (chitosan Pal-Ex4 PLGA NPs) were prepared and characterized. The surface morphology, particle size, and zeta potential of chitosan Pal-Ex4 PLGA NPs were investigated, and the adsorption and cytotoxicity of chitosan Pal-Ex4 PLGA NPs were evaluated in human lung epithelial cells (A549). Finally, the lung deposition characteristics and hypoglycemia caused by chitosan Pal-Ex4 PLGA NPs were evaluated after pulmonary administration in imprinting control region (ICR) and type 2 diabetic db/db mice. Results showed that chitosan Pal-Ex4 PLGA NPs were spherical, compact and had a diameter of ~700 nm and a positive surface charge of +28.5 mV Chitosan-coated PLGA NPs were adsorbed onto A549 cells much more so than non-coated PLGA NPs. Pal-Ex4 release from chitosan-coated PLGA NPs was delayed by as much as 1.5 days as compared with chitosan-coated Ex4 PLGA NPs. In addition, chitosan-coated PLGA NPs remained in the lungs for ~72 hours after pulmonary administration, whereas most non-coated PLGA NPs were lost at 8 hours after administration. Furthermore, the hypoglycemic efficacy of inhaled chitosan Pal-Ex4 PLGA NPs was 3.1-fold greater than that of chitosan Ex4 PLGA NPs in db/db mice. The authors believe chitosan Pal-Ex4 PLGA NPs have considerable potential as a long-acting inhalation delivery system for the treatment of type 2 diabetes.