Lipoplex-based targeted gene therapy for the suppression of tumours with VEGFR expression by producing anti-angiogenic molecules.

BACKGROUND: The anti-angiogenic fusion protein RBDV-IgG1 Fc (RBDV), which comprises the receptor-binding domain of vascular endothelial growth factor-A (VEGF-A), has shown antitumour effects by reducing angiogenesis in vivo. This study used the cationic lipoplex lipo-PEG-PEI-complex (LPPC) to simultaneously encapsulate both the RBDV targeting protein and the RBDV plasmid (pRBDV) without covalent bonds to assess VEGFR targeting gene therapy in mice with melanoma in vivo. RESULTS: LPPC protected the therapeutic transgene from degradation by DNase, and the LPPC/RBDV complexes could specifically target VEGFR-positive B16-F10 cells both in vitro and in vivo. With or without RBDV protein-targeting direction, the pRBDV-expressing RBDV proteins were expressed and reached a maximal concentration on the 7th day in the sera after transfection in vivo and significantly elicited growth suppression against B16-F10 melanoma but not IgG1 control proteins. In particular, LPPC/pRBDV/RBDV treatment with the targeting molecules dramatically inhibited B16-F10 tumour growth in vivo to provide better therapeutic efficacy than the treatments with gene therapy with IgG1 protein targeting or administration of a protein drug with RBDV. CONCLUSIONS: The simultaneous combination of the LPPC complex with pRBDV gene therapy and RBDV protein targeting might be a potential tool to conveniently administer targeted gene therapy for cancer therapy.

Dressing with epigallocatechin gallate nanoparticles for wound regeneration.

Several reagents have been studied to overcome the problems encountered with antiseptic use, such as moderate cutaneous wound cytotoxicity and skin thinning. We successfully prepared a gelatin/chitosan/epigallocatechin gallate nanoparticle incorporated in a poly(γ-glutamic acid)/gelatin hydrogel, which comprised activated carbon fibers with gentamicin, to fabricate a sandwiched dressing to enhance wound regeneration. The inner layer of activated carbon fibers with gentamicin was designed to prevent bacterial infection, and the outer layer of gelatin/chitosan/epigallocatechin gallate nanoparticles incorporated in a poly(γ-glutamic acid)/gelatin hydrogel was designed to prevent inflammation and facilitate reepithelialization. An in vitro study demonstrated that the dressing effectively inhibited target microorganisms, and scanning electron microscope and confocal laser scanning microscope indicated that the nanoparticles were homogeneously dispersed and migrated into the hydrogel. The in vivo study reported that the sandwiched dressing, comprising the poly(γ-glutamic acid)/gelatin hydrogel, was easy to remove from the wound and facilitated wound tissue regeneration and accelerated healing process.

Active Targeted Nanoparticles for Oral Administration of Gastric Cancer Therapy.

Gastric carcinogenesis is a commonly diagnosed type of cancer and has a dismal prognosis because of the rate at which it aggressively spreads and because of the lack of effective therapies to stop its progression. This study evaluated a type of oral drug delivery system of a potential target-activated nanosizer comprising a fucose-conjugated chitosan and polyethylene glycol-conjugated chitosan complex with gelatin containing encapsulated green tea polyphenol extract epigallocatechin-3-gallate, allowing oral administration of the drug through a site-specific release in gastric cancer cells. The results demonstrated that the nanoparticles effectively reduced drug release within gastric acids and that a controlled epigallocatechin-3-gallate release inhibited gastric cancer cell growth, induced cell apoptosis, and reduced vascular endothelial growth factor protein expression. Furthermore, in vivo assay results indicated that the prepared epigallocatechin-3-gallate-loaded fucose-chitosan/polyethylene glycol-chitosan/gelatin nanoparticles significantly affected gastric tumor activity and reduced gastric and liver tissue inflammatory reaction in an orthotopic gastric tumor mouse model.

Synthesis of uniform poly(d,l-lactide) and poly(d,l-lactide-co-glycolide) microspheres using a microfluidic chip for comparison.

Applications of poly(l-lactide) (PLA) and poly(d,l-lactide-co-glycolide) (PLGA) microspheres are widely used in the biomedical and pharmaceutical fields. The effects of PLA/PLGA on microsphere properties when using conventional particulate preparation methods are not easily defined due to the uncontrollable particle size and size distribution. This study was aimed to synthesize uniform PLA and PLGA microspheres using a phenol formaldehyde resin-based microfluidic chip, which has the advantage of being solvent-resistant, flexible, and is readily disassembled for cleaning. The proposed chip can rapidly fabricate reproducible PLA and PLGA microspheres. Uniform emulsion droplets can be achieved by hydrodynamic flow focusing. After solvent evaporation, the free-flowing PLA and PLGA microspheres have a high level of morphological uniformity and size, allowing for a clear comparison of material effects. The results indicate that the sizes of the PLA and PLGA microspheres for the various flow rates of dispersed/continuous phases are very similar. The PLA/PLGA materials do not have a significant effect on particle size, but the particle surface indicates a different morphology. The result of the cytotoxicity evaluation shows no difference between PLA and PLGA and ensures the biocompatibility of both prepared PLA and PLGA microspheres for biomedical and pharmaceutical applications in the future.

A polymer coating applied to Salmonella prevents the binding of Salmonella-specific antibodies.

The use of Salmonella as a potential antitumor agent has been investigated, but innate immunity against this bacterium reduces the efficacy of its tumor-targeting and antitumor activities. The purpose of this study was to investigate the modulation of the tumor-targeting efficiency of Salmonella enterica serovar choleraesuis by modifying the immune response to these bacteria by coating them with poly(allylamine hydrochloride) (PAH), designated PAH-S.C. To evaluate this modulation, we used naïve mice and mice immunized with Salmonella to study the role of the preexisting immune response to the antitumor activity of PAH-S.C. When anti-Salmonella antibodies were present, the invasion activity, cytotoxicity, and gene transfer of Salmonella was significantly decreased, both in vitro and in vivo. Treatment with PAH-S.C. resulted in delayed tumor growth and enhanced survival in immunized mice. Furthermore, immunohistochemical studies of the tumors revealed the infiltration of neutrophils and macrophages in immunized mice treated with PAH-S.C. These results indicate that Salmonella encapsulation effectively circumvented the Salmonella-specific immune response.

A real-time impedance-sensing chip for the detection of emulsion phase separation.

This paper describes a novel real-time impedance chip for the detection of squalene-water emulsion phase separation. Each impedance chip contains eight pairs of indium tin oxide microelectrode arrays for detecting eight samples, and six chips can be connected with the switch relay to measure 48 samples in the system simultaneously. The proposed impedance chip has the advantages of needing only a small sample volume (0.5 mL), and provides parallel, continuous, and real-time detection. The effects of the surfactant concentration on the stability of a squalene/water emulsion were studied by means of a visual inspection, a conductance probe, and by impedance chip. Three different concentrations of Tween 20 surfactant (9, 17, and 29 wt%) were employed for the examinations. The results indicated that the phase separation rate was faster in the lower surfactant concentration. However, the emulsion of 29 wt% Tween 20 was fairly stable for more than 2 days since there were no signal changes according to the three detection methods. The reaction time (TR) for completing the measured phase separation process differed for each of the three methods (measuring aqueous phase height, conductance, and impedance, respectively). For the 9 wt% Tween 20, the reaction times were 24 h, 20 min, and 5 min in the tests using visual inspection, conductance probe, and impedance chip, respectively. For the 17 wt% Tween 20, the TR was also shorter when using the impedance chip method compared to the other two methods. Therefore the proposed impedance chip has a quick reaction response and provides an alternative and effective method to detect emulsion stability.

Development of fibroblast culture in three-dimensional activated carbon fiber-based scaffold for wound healing.

This work developed a novel bi-layer wound dressing composed of 3D activated carbon fibers that allows facilitates fibroblast cell growth and migration to a wound site for tissue reconstruction, and the gentamicin is incorporated into a poly(γ-glutamic acid)/gelatin membrane to prevent bacterial infection. In an in vitro, field emission scanning electron microscopy shows that rat skin fibroblasts appeared and spread on the surface of activated carbon fibers, and penetrated the interior and exterior of the 3D activated carbon fiber construct to a depth of roughly 200 µm. An in vivo analysis shows that fibroblast cells containing the proposed 3D scaffold had the potential of a biologically functionalized dressing to accelerate wound closure. Additionally, fibroblasts migrated to the wound site in a bi-layer wound dressing containing fibroblasts, enhancing fibronectin and type I collagen expression, resulting in faster skin regeneration than that achieved with a Tegaderm™ hydrocolloid dressing or gauze.

Oral delivery of peptide drugs using nanoparticles self-assembled by poly(gamma-glutamic acid) and a chitosan derivative functionalized by trimethylation.

In the study, chitosan (CS) was conjugated with trimethyl groups for the synthesis of N-trimethyl chitosan (TMC) polymers with different degrees of quaternization. Nanoparticles (NPs) self-assembled by the synthesized TMC and poly(gamma-glutamic acid) (gamma-PGA, TMC/gamma-PGA NPs) were prepared for oral delivery of insulin. The loading efficiency and loading content of insulin in TMC/gamma-PGA NPs were 73.8 +/- 2.9% and 23.5 +/- 2.1%, respectively. TMC/gamma-PGA NPs had superior stability in a broader pH range to CS/gamma-PGA NPs; the in vitro release profiles of insulin from both test NPs were significantly affected by their stability at distinct pH environments. At pH 7.0, CS/gamma-PGA NPs became disintegrated, resulting in a rapid release of insulin, which failed to provide an adequate retention of loaded insulin, while the cumulative amount of insulin released from TMC/gamma-PGA NPs was significantly reduced. At pH 7.4, TMC/gamma-PGA NPs were significantly swelled and a sustained release profile of insulin was observed. Confocal microscopy confirmed that TMC40/gamma-PGA NPs opened the tight junctions of Caco-2 cells to allow the transport of insulin along the paracellular pathway. Transepithelial-electrical-resistance measurements and transport studies implied that CS/gamma-PGA NPs can be effective as an insulin carrier only in a limited area of the intestinal lumen where the pH values are close to the p K a of CS. In contrast, TMC40/gamma-PGA NPs may be a suitable carrier for transmucosal delivery of insulin within the entire intestinal tract.

Main pulmonary artery aneurysm.

Pulmonary artery aneurysm is a rare lesion of the thoracic cavity. Different etiologies have been reviewed, but idiopathic lesions without other symptoms are seldom reported. Usually, surgical interventions are suggested, but the long-term outcomes are not well established. Here, we report a 24-year-old man with main pulmonary artery aneurysm who successfully underwent aneurysmectomy and polytetrafluoroethylene vascular graft replacement. The postoperative course was uneventful, and the following image study revealed normal size of the great vessels.

Development of chitosan/poly-γ-glutamic acid/pluronic/curcumin nanoparticles in chitosan dressings for wound regeneration.

The hydrophobic polyphenol curcumin has anti-inflammatory, antimicrobial, and wound-healing properties that warrant its pharmacological consideration. We report a curcumin nanoparticle with a tripolymeric composite that can be used as a delivery device for wound healing. The present composite nanoparticles were prepared with three biocompatible polymers of chitosan, poly-γ-glutamic acid, and pluronic using a simple ionic gelation technology. Pluronic was used to enhance the solubility of curcumin in chitosan/poly-γ-glutamic acid nanoparticles, leading to the incorporation of chitosan/poly-γ-glutamic acid/pluronic/curcumin nanoparticles into chitosan membranes, and reduced inflammation and bacterial infection during wound regeneration. Nanoparticles were of 193.1 ± 8.9 nm and had a zeta potential of 20.6 ± 2.4 mV. Moreover, in vitro analyses indicated controlled curcumin release in a simulated skin tissue model. Subsequent in vivo studies show that chitosan wound dressing containing chitosan/poly-γ-glutamic acid/pluronic/curcumin nanoparticles promoted neocollagen regeneration and tissue reconstruction. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 81-90, 2017.

A novel method for the preparation of nanoaggregates of methoxy polyethyleneglycol linked chitosan.

In the study, methoxy polyethyleneglycol (MPEG) linked chitosan (PLC) with a different degrees of substitution were prepared using a novel yet simple method in the presence of formaldehyde in a solvent of formic acid and dimethylsulfoxide (DMSO). The obtained PLC was verified by the Fourier transformed infrared (FT-IR) and carbon nuclear magnetic resonance (13C-NMR) spectroscopy and by the gel permeation chromatography (GPC). The aqueous solubility of chitosan increased after chemically linking with MPEG and was found to depend on its degree of substitution. With a proper degree of substitution of MPEG on chitosan, PLC may undergo inter- and/or intra-molecular entanglements to produce nanoaggregates. The critical aggregation concentration (CAC) of PLC was determined by the fluorescence emission spectra of pyrene and was found to be 0.003 mg/ml. Measurements of the size distribution and zeta potential of the prepared nanoaggregates were carried out using a Zetasizer. The results suggested that as the degree of MPEG substitution increased, the size and polydispersity index of the prepared nanoaggregates decreased. The prepared nanoaggregates showed a pH-sensitive property and thus may be suitable for the development of drug delivery devices for tumors.

Evaluation of silver-containing activated carbon fiber for wound healing study: In vitro and in vivo.

Silver has antiseptic properties, anti-inflammatory properties, and is a broad-spectrum antibiotic for multidrug-resistant strains of bacteria. The commercially available product, Silverlon®, is a silver-plated three-dimensional polyamide fabric with a high silver concentration of 546 mg/100 cm(2). Thus, fibroblast cell growth is affected when exposed to the Silverlon® treated cell medium. Our study produced an activated carbon fiber wound dressing that incorporated various silver concentrations (in cooperation of Bio-Medical Carbon Technology) to examine antimicrobial properties and determine fibroblast cell viability upon exposure to the silver impregnated dressing material as compared to other commercially available products such as calcium alginate dressing, Sorbalgon®, and silver-polyamide fabric dressing, Silverlon®. The silver impregnated activated carbon fiber dressing induced less damage to fibroblast cells compared to the effect produced by Silverlon® and exhibited similar antibacterial abilities in vitro. An in vivo analysis showed that various silver concentrations impregnated activated carbon fiber dressings promoted tissue reconstruction for wound healing in rats with Pseudomonas aeruginosa infected wounds.

Development of pH-responsive chitosan/heparin nanoparticles for stomach-specific anti-Helicobacter pylori therapy.

The microorganism now known as Helicobacter pylori is considered to be an important factor in the etiology of peptic ulcers. It can secrete urease enzyme and buffer gastric acids to survive in the stomach. H. pylori can colonize the gastric mucosa and preferentially adheres near the cell-cell junctions of the gastric mucous cells. In this study, pH-responsive nanoparticles were produced instantaneously upon the addition of heparin solution to a chitosan solution with magnetic stirring at room temperature. The nanoparticles appeared to have a particle size of 130-300 nm, with a positive surface charge, and were stable at pH 1.2-2.5, allowing them to protect an incorporated drug from destructive gastric acids. We also demonstrated that the prepared nanoparticles can adhere to and infiltrate cell-cell junctions and interact locally with H. pylori infection sites in intercellular spaces.