PEGylated polyethyleneimine-entrapped gold nanoparticles for enhanced and targeted gene delivery applications

Gene therapy, which involves the transfer of nucleic acid into target cells in patients, has become one of the most important and widely explored strategies to treat a variety of diseases, such as cancer, infectious diseases and genetic disorders. Relative to viral vectors that have high immunogenicity, toxicity and oncogenicity, non-viral vectors have gained a lot of interest in recent years. This is largely due to their ability to mimic viral vector features including the capacity to overcome extra- and intra-cellular barriers and to enhance transfection efficiency. Polyethyleneimine (PEI) has been extensively investigated as a non-viral vector. This cationic polymer, which is able to compact nucleic acid through electrostatic interactions and to transport it across the negatively charged cell membranes, has been shown to effectively transfect nucleic acid into different cell lines. Moreover, entrapment of gold nanoparticles (Au NPs) into such an amine-terminated polymer template has been shown to significantly enhance gene transfection efficiency. In this work, a novel non-viral nucleic acid vector system for enhanced and targeted nucleic acid delivery applications was developed. The system was based on the functionalization of PEI with folic acid (FA; for targeted delivery to cancer cells overexpressing FA receptors on their surface) using polyethylene glycol (PEG) as a linker molecule. This was followed by the preparation of PEI-entrapped Au NPs (Au PENPs; for enhancement of transfection efficiency). In the synthesis process, the primary amines of PEI were first partially modified with fluorescein isothiocyanate (FI) using a molar ratio of 1:7. The formed PEI-FI conjugate was then further modified with either PEG or PEGylated FA using a molar ratio of 1:1. This process was finally followed by entrapment of Au NPs into the modified polymers. The resulting conjugates and Au PENPs were characterized by several techniques, namely Nuclear Magnetic Resonance, Dynamic Light Scattering and Ultraviolet-Visible Spectroscopy, to assess their physicochemical properties. In the cell biology studies, the synthesized conjugates and their respective Au PENPs were shown to be non-toxic towards A2780 human ovarian carcinoma cells. The role of these materials as gene delivery agents was lastly evaluated. In the gene delivery studies, the A2780 cells were successfully transfected with plasmid DNA using the different vector systems. However, FA-modification and Au NPs entrapment were not determinant factors for improved transfection efficiency. In the gene silencing studies, on the other hand, the Au PENPs were shown to effectively deliver small interfering RNA, thereby reducing the expression of the B-cell lymphoma 2 protein. Based on these results, we can say that the systems synthesized in this work show potential for enhanced and targeted gene therapy applications.

Sol-gel entrapped cobalt complex

This work describes optimized conditions for preparation of a cobalt complex entrapped in alumina amorphous materials in the form of powder. The hybrid materials, CoNHG, were obtained by a nonhydrolytic sol-gel route through condensation of aluminum chloride with diisopropylether in the presence of cobalt chloride. The materials were calcined at various temperatures. The presence of cobalt entrapped in the alumina matrix is confirmed by ultraviolet visible spectroscopy. The materials have been characterized by X-ray diffraction (XRD), surface area analysis, thermogravimetric analysis (TGA), differential thermal analyses (DTA) and transmission electron microscopy (TEM). The prepared alumina matrix materials are amorphous, even after heat treatment up to 750 degreesC. The XRD, TGA/DTA and TEM data support the increase of sample crystallization with increasing temperature. The specific surface area, pore size and pore diameter changed as a function of the heat treatment temperature employed. Different heat treatment temperatures result in materials with different compositions and structures, and influence their catalytic activity. The entrapped cobalt materials calcined at 750 degreesC efficiently catalyzed the epoxidation of (Z)-cyclooctene using iodozylbenzene as the oxygen donor. (C) 2003 Elsevier B.V. All rights reserved.

H-bonding in entrapped water in poly(o-methoxyaniline): Results from a differential scanning calorimetry study

Differential scanning calorimetry (DSC) was used to investigate entrapped water in poly(o-methoxyaniline) (POMA) in powder form. Two endothermic peaks were attributed to removal of water molecules that were adsorbed with distinct energies. By obtaining thermograms at various heating rates, we succeeded in applying Kissinger's approaches to estimate activation energies for the water adsorbed. The values obtained were ca. 25 and 53 kJ/mol, which correspond to H-bonding interactions, probably at the amine and imine centers of POMA, respectively. (C) 2005 Elsevier B.V. All rights reserved.

Photodynamic inactivation of planktonic cultures and biofilms of Candida albicans mediated by aluminum-chloride-phthalocyanine entrapped in nanoemulsions

New drug delivery systems, such as nanoemulsions (NE), have been developed to allow the use of hydrophobic drugs on the antimicrobial photodynamic therapy. This study evaluated the photodynamic potential of aluminum-chloride- phthalocyanine (ClAlPc) entrapped in cationic and anionic NE to inactivate Candida albicans planktonic cultures and biofilm compared with free ClAlPc. Fungal suspensions were treated with different delivery systems containing ClAlPc and light emitting diode. For planktonic suspensions, colonies were counted and cell metabolism was evaluated by XTT assay. Flow cytometry evaluated cell membrane damage. For biofilms, the metabolic activity was evaluated by XTT and ClAlPc distribution through biofilms was analyzed by confocal laser scanning microscopy (CLSM). Fungal viability was dependent on the delivery system, superficial charge and light dose. Free ClAlPc caused photokilling of the yeast when combined with 100 J cm-2. Cationic NE-ClAlPc reduced significantly both colony counts and cell metabolism (P < 0.05). In addition, cationic NE-ClAlPc and free ClAlPc caused significant damage to the cell membrane (P < 0.05). For the biofilms, cationic NE-ClAlPc reduced cell metabolism by 70%. Anionic NE-ClAlPc did not present antifungal activity. CLSM showed different accumulation on biofilms between the delivery systems. Although NE system showed a lower activity for planktonic culture, cationic NE-ClAlPc showed better results for Candida biofilms. Candida albicans biofilm overview after 30 min of contact with free ClAlPc. This study presents the photodynamic potential of aluminum-chloride-phthalocyanine (ClAlPc) entrapped in cationic and anionic nanoemulsions (NE) to inactivate C. albicans planktonic cultures and biofilm comparing with free ClAlPc. The photodynamic effect was dependent on the delivery system, superficial charge and light dose. Cationic NE-ClAlPc and free ClAlPc caused significant reduction in colony counts, cell metabolism and damage to the cell membrane (P < 0.05). However, only the free ClAlPc was able to cause photokilling of the yeast. The anionic NE-ClAlPc did not present antifungal activity. Although NE system showed a lower activity for planktonic culture, cationic NE-ClAlPc showed better results for Candida biofilms. © 2012 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2012 The American Society of Photobiology.

Antimycobacterial activity of 2-phenoxy-1-phenylethanone, a synthetic analogue of neolignan, entrapped in polymeric microparticles

Conventional treatment of tuberculosis (TB) demands a long course therapy (6 months), known to originate multiple drug resistant strains (MDR-TB), which emphasizes the urgent need for new antituberculous drugs. The purpose of this study was to investigate a novel treatment for TB meant to improve patient compliance by reducing drug dosage frequency. Polymeric microparticles containing the synthetic analogue of neolignan, 1-phenyl-2-phenoxiethanone (LS-2), were obtained by a method of emulsification and solvent evaporation and chemically characterized. Only representative LS-2-loaded microparticles were considered for further studies involving experimental murine TB induced by Mycobacterium tuberculosis H37Rv ATCC 27294. The LS-2-loaded microparticles were spherical in shape, had a smooth wall and showed an encapsulation efficiency of 93% in addition to displaying sustained release. Chemotherapeutic potential of LS-2 entrapped in microparticles was comparable to control groups. These findings are encouraging and indicate that LS-2-loaded microparticles are a potential alternative to conventional chemotherapy of TB.

Molar gas ratios and stable oxygen and nitrogen isotopes of air entrapped in ice, North America

The molar ratios of atmospheric gases change during dissolution in water due to differences in their relative solubilities. We exploited this characteristic to develop a tool to clarify the origin of ice formations in permafrost regions. Extracted from ice, molar gas ratios can distinguish buried glacier ice from intrasedimental ground ice formed by freezing groundwaters. An extraction line was built to isolate gases from ice by melting and trapping with liquid He, followed by analysis of N2, O2, Ar, 18O-O2 and 15N-N2, by continuous flow mass spectrometry. The method was tested using glacier ice, aufeis ice (river icing) and intrasedimental ground ice from sites in the Canadian Arctic. O2/Ar and N2/Ar ratios clearly distinguish between atmospheric gas in glacial ice and gases from intrasedimental ground ice, which are exsolved from freezing water. 615NN2 and 618OO2 in glacier ice, aufeis ice and intrasedimental ground ice do not show clear distinguishing trends as they are affected by various physical processes during formation such as gravitational settling, excess air addition, mixing with snow pack, and respiration.

Liposome-entrapped plasmid DNA:characterisation studies

Plasmid DNA pRc/CMV HBS (5.6 kb) (100 microg) encoding the S (small) region of hepatitis B surface antigen was incorporated by the dehydration-rehydration method into liposomes composed of 16 micromol egg phosphatidylcholine (PC), 8 micromol dioleoylphosphatidylcholine (DOPE) and 1, 2-diodeoyl-3-(trimethylammonium)propane (DOTAP) (cationic liposomes) or phosphatidylglycerol (anionic liposomes) in a variety of molar ratios. The method, entailing mixing of small unilamellar vesicles (SUV) with the DNA, followed by dehydration and rehydration, yielded incorporation values of 95-97 and 48-54% of the DNA used, respectively. Mixing of preformed cationic liposomes with 100 microg plasmid DNA also led to high complexation values of 73-97%. As expected, the association of DNA with preformed anionic liposomes was low (9%). Further work with cationic PC/DOPE/DOTAP liposomes attempted to establish differences in the nature of DNA association with the vesicles after complexation and the constructs generated by the process of dehydration/rehydration. Several lines of evidence obtained from studies on vesicle size and zeta-potential, fluorescent microscopy and gel electrophoresis in the presence of the anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, interaction of DNA with preformed cationic SUV as above, or with cationic SUV made of DOPE and DOTAP (1:1 molar ratio; ESCORT Transfection Reagent), leads to the formation of large complexes with externally bound DNA. For instance, such DNA is accessible to and can be dissociated by competing anionic SDS molecules. However, dehydration of the DNA-SUV complexes and subsequent rehydration, generates submicron size liposomes incorporating most of the DNA in a fashion that prevents DNA displacement through anion competition. It is suggested that, in this case, DNA is entrapped within the aqueous compartments, in between bilayers, presumably bound to the cationic charges.

Developing solid particulate vaccine adjuvants - surface bound antigen favouring a humoural response, whereas entrapped antigen shows a tendency for cell mediated immunity

This present study compares the efficacy of microsphere formulations, and their method of antigen presentation, for the delivery of the TB sub-unit vaccine antigen, Ag85B-ESAT-6. Microspheres based on poly(lactide-co-glycolide) (PLGA) and chitosan incorporating dimethyldioctadecylammonium bromide (DDA) were prepared by either the w/o/w double emulsion method (entrapped antigen) or the o/w single emulsion method (surface bound antigen), and characterised for their physico-chemical properties and their ability to promote an immune response to Ag85B-ESAT-6. The method of preparation, and hence method of antigen association, had a pronounced effect on the type of immune response achieved from the microsphere formulations, with surface bound antigen favouring a humoural response, whereas entrapped antigen favoured a cellular response.

Developing solid particulate vaccine adjuvants:surface bound antigen favouring a humoural response, whereas entrapped antigen shows a tendency for cell mediated immunity

This present study compares the efficacy of microsphere formulations, and their method of antigen presentation, for the delivery of the TB sub-unit vaccine antigen, Ag85B-ESAT-6. Microspheres based on poly(lactide-co-glycolide) (PLGA) and chitosan incorporating dimethyldioctadecylammonium bromide (DDA) were prepared by either the w/o/w double emulsion method (entrapped antigen) or the o/w single emulsion method (surface bound antigen), and characterised for their physico-chemical properties and their ability to promote an immune response to Ag85B-ESAT-6. The method of preparation, and hence method of antigen association, had a pronounced effect on the type of immune response achieved from the microsphere formulations, with surface bound antigen favouring a humoural response, whereas entrapped antigen favoured a cellular response.

CD73 and CD29 concurrently mediate the mechanically induced decrease of migratory capacity of mesenchymal stromal cells

The assumption that mesenchymal stromal cell (MSC)-based therapies are capable of augmenting physiological regeneration processes has fostered intensive basic and clinical research activities. However, to achieve sustained therapeutic success in vivo, not only the biological, but also the mechanical microenvironment of MSCs during these regeneration processes needs to be taken into account. This is especially important for e.g., bone fracture repair, since MSCs present at the fracture site undergo significant biomechanical stimulation. This study has therefore investigated cellular characteristics and the functional behaviour of MSCs in response to mechanical loading. Our results demonstrated a reduced expression of MSC surface markers CD73 (ecto-5’-nucleotidase) and CD29 (integrin β1) after loading. On the functional level, loading led to a reduced migration of MSCs. Both effects persisted for a week after the removal of the loading stimulus. Specifi c inhibition of CD73/CD29 demonstrated their substrate dependent involvement in MSC migration after loading. These results were supported by scanning electron microscopy images and phalloidin staining of actin fi laments displaying less cell spreading, lamellipodia formation and actin accumulations. Moreover, focal adhesion kinase and Src-family kinases were identified as candidate downstream targets of CD73/CD29 that might contribute to the mechanically induced decrease in MSC migration. These results suggest that MSC migration is controlled by CD73 CD29, which in turn are regulated by mechanical stimulation of cells. We therefore speculate that MSCs migrate into the fracture site, become mechanically entrapped, and thereby accumulate to fulfil their regenerative functions.

Local stress-strain distribution and load transfer across cartilage matrix at micro-scale using combined microscopy-based finite element method

Articular cartilage is the load-bearing tissue that consists of proteoglycan macromolecules entrapped between collagen fibrils in a three-dimensional architecture. To date, the drudgery of searching for mathematical models to represent the biomechanics of such a system continues without providing a fitting description of its functional response to load at micro-scale level. We believe that the major complication arose when cartilage was first envisaged as a multiphasic model with distinguishable components and that quantifying those and searching for the laws that govern their interaction is inadequate. To the thesis of this paper, cartilage as a bulk is as much continuum as is the response of its components to the external stimuli. For this reason, we framed the fundamental question as to what would be the mechano-structural functionality of such a system in the total absence of one of its key constituents-proteoglycans. To answer this, hydrated normal and proteoglycan depleted samples were tested under confined compression while finite element models were reproduced, for the first time, based on the structural microarchitecture of the cross-sectional profile of the matrices. These micro-porous in silico models served as virtual transducers to produce an internal noninvasive probing mechanism beyond experimental capabilities to render the matrices micromechanics and several others properties like permeability, orientation etc. The results demonstrated that load transfer was closely related to the microarchitecture of the hyperelastic models that represent solid skeleton stress and fluid response based on the state of the collagen network with and without the swollen proteoglycans. In other words, the stress gradient during deformation was a function of the structural pattern of the network and acted in concert with the position-dependent compositional state of the matrix. This reveals that the interaction between indistinguishable components in real cartilage is superimposed by its microarchitectural state which directly influences macromechanical behavior.

A hybrid of modified PSO and local search on a multi-robot search system

Particle swarm optimization (PSO), a new population based algorithm, has recently been used on multi-robot systems. Although this algorithm is applied to solve many optimization problems as well as multi-robot systems, it has some drawbacks when it is applied on multi-robot search systems to find a target in a search space containing big static obstacles. One of these defects is premature convergence. This means that one of the properties of basic PSO is that when particles are spread in a search space, as time increases they tend to converge in a small area. This shortcoming is also evident on a multi-robot search system, particularly when there are big static obstacles in the search space that prevent the robots from finding the target easily; therefore, as time increases, based on this property they converge to a small area that may not contain the target and become entrapped in that area.Another shortcoming is that basic PSO cannot guarantee the global convergence of the algorithm. In other words, initially particles explore different areas, but in some cases they are not good at exploiting promising areas, which will increase the search time.This study proposes a method based on the particle swarm optimization (PSO) technique on a multi-robot system to find a target in a search space containing big static obstacles. This method is not only able to overcome the premature convergence problem but also establishes an efficient balance between exploration and exploitation and guarantees global convergence, reducing the search time by combining with a local search method, such as A-star.To validate the effectiveness and usefulness of algorithms,a simulation environment has been developed for conducting simulation-based experiments in different scenarios and for reporting experimental results. These experimental results have demonstrated that the proposed method is able to overcome the premature convergence problem and guarantee global convergence.