The impact of carboxyalkylation of branched polyethylenimine on effectiveness in small interfering RNA delivery

Background

Carboxyalkylation of branched 25 kDa polyethylenimine (PEI) was considered to reduce the positive surface charge of the polymer without reducing its ‘proton sponge’ buffering capacity, and to provide alkylene domains for hydrophobic interactions, thus generating optimized novel PEI carriers for efficient delivery of small interfering RNA (siRNA).

Methods

Substitution of PEI was evaluated in the range of 6 to > 50 mole percentage of primary amines. Additionally, variation of the carboxyalkyl chain (one to 15 methylene groups) was explored to modulate the carrier hydrophobicity. Carriers were characterized in their buffering capacity, capability of siRNA polyplex formation, and cytotoxicity. Marker gene‐silencing efficacy was evaluated using Neuro2A‐eGFPLuc neuroblastoma cells.

Results

Carboxyalkylation strongly reduced cytotoxicity of PEI and improved siRNA mediated luciferase gene knockdown. An optimum silencing activity was observed at an alkylcarboxylation degree of 6–9 mole percentage of primary amines and with a broad range of carboxyalkylene chains (containing one to 15 methylene groups). Strongly enhanced gene‐silencing efficacy also was observed when the biocompatible polymers were separately added at 1 h after transfection with tolerated doses of standard PEI25/siRNA polyplexes.

Conclusions

Carboxyalkylation of branched 25 kDa PEI resulted in polymers with strongly reduced cytotoxicity and improved silencing efficacy. Mechanistic studies demonstrated that the presence of a surplus of free carboxyalkylated polymer is responsible for the improved siRNA delivery. Copyright © 2010 John Wiley & Sons, Ltd.     

Designing Alginate/Chitosan Nanoparticles Containing Echinacea angustifolia: A Novel Candidate for Combating Multidrug-Resistant Staphylococcus aureus

Nanoparticles (NPs) may help treat multidrug-resistant Staphylococcus aureus (MDR). This study prepared and evaluated chitosan/alginate-encapsulated Echinacea angustifolia extract against MDR strains. Evaluating synthesized NPs with SEM, DLS, and FT-IR. Congo red agar and colorimetric plate techniques examined isolate biofilm formation. NP antibacterial power was assessed using well diffusion. Real-time PCR assessed biofilm-forming genes. MTT assessed the synthesized NPs′ toxicity. According to DLS measurements, spherical E. angustifolia NPs had a diameter of 335.3±1.43 nm. The PDI was 0.681, and the entrapment effectiveness (EE%) of the E. angustifolia extract reached 83.45 %. Synthesized NPs were most antimicrobial. S. aureus resistant to several treatments was 80 percent of 100 clinical samples. Biofilm production was linked to MDR in all strains. The ALG/CS-encapsulated extract had a 4 to 32-fold lower MIC than the free extract, which had no bactericidal action. They also significantly decreased the expression of genes involved in biofilm formation. E. angustifolia-encapsulated ALG/CS decreased IcaD, IcaA, and IcaC gene expression in all MDR strains (***p<0.001). Free extract, free NPs, and E. angustifolia-NPs had 57.5 %, 85.5 %, and 90.0 % cell viability at 256 μg/ml. These discoveries could assist generate stable plant extracts by releasing natural-derived substances under controlled conditions.     

The application of electrosprayed minocycline-loaded PLGA microparticles for the treatment of glioblastoma

The survival of patients with glioblastoma multiforme (GBM), the most common and invasive form of malignant brain tumors, remains poor despite advances in current treatment methods including surgery, radiotherapy, and chemotherapy. Minocycline is a semi-synthetic tetracycline derivative that has been widely used as an antibiotic and more recently, it has been utilized as an antiangiogenic factor to inhibit tumorigenesis. The objective of this study was to investigate the utilization of electrospraying process to fabricate minocycline-loaded poly(lactic-co-glycolic acid) (PLGA) microparticles with high drug loading and loading efficiency and to evaluate their ability to induce cell toxicity in human glioblastoma (i.e., U87-MG) cells. The results from this study demonstrated that solvent mixture of dicholoromethane (DCM) and methanol is the optimal solvent combination for minocycline and larger amount of methanol (i.e., 70:30) resulted in a higher drug loading. All three solvent ratios of DCM:methanol tested produced microparticles that were both spherical and smooth, all in the micron size range. The electrosprayed microparticles were able to elicit a cytotoxic response in U87-MG glioblastoma cells at a lower concentration of drug compared to the free drug. This work provides proof of concept to the hypothesis that electrosprayed minocycline-loaded PLGA microparticles can be a promising agent for the treatment of GBM and could have potential application for cancer therapies.     

Transient expression of visible marker genes in meristem cells of wheat embryos after ballistic micro-targeting

Seven days after anthesis, the shoot apical meristem of immature embryos of wheat (Triticum aestivum L.) is not yet covered by the coleoptile or leaf primordia and provides an optimal target for ballistic micro-targeting. Gold particles 1.2 μm in diameter at a concentration of 5·10⁵ particles per μl and propelled by 110-bar nitrogen penetrated up to four cell layers into embryo apical meristems but produced no deleterious effects on germination. The use of diaphragms with internal diameters of 100 or 200 μm restricted bombardment to meristem cells or also included surrounding tissues, respectively. The results of transient-expression experiments indicated successful delivery of foreign DNA into meristem cells. Cells of the central zone of the meristem or pro-meristem transiently expressed foreign genes driven by the Cauliflower mosaic virus (CaMV) 35S and rice actin1-D constitutive promoters. Partial plasmolysis before bombardment and slow recovery of normal turgor pressure increased transient-expression frequencies. Meristem cells transiently expressed foreign genes at frequencies 10-fold less than surrounding tissues under identical conditions. Transgenic sectors were observed in both coleoptiles and leaf primordia.     

Transformation efficiencies and progeny analysis after varying different parameters of direct gene transfer of Nicotiana tabacum protoplasts

Nicotiana tabacum protoplasts were transformed by polyethylene glycol (PEG)-mediated uptake and electroporation, with circular and linear DNA, and with or without X-ray irradiation. We investigated the influence on the transient expression by these parameters as well as on the frequencies for stable transformation. Plants were regenerated and selfed, and the progenies of the transformed plants were analysed and used to compare the pattern of gene integration by these different variations in transformation methods. The results from the transient expression as judged by glucuronidase (GUS) activity, showed electroporation to give higher and more reproducible results than PEG-mediated uptake. Using linear instead of circular DNA increased the rate of stable transformation about 3 times. Including a mild X-ray treatment gave an increase in the same range. When the inheritance of the transferred trait was investigated, it was found that protoplasts transformed with linear DNA resulted in the highest number of plants with single-copy insertions. Protoplasts transformed with circular DNA showed the highest incidence of losing the trait, while plants in which the transformation included an X-ray treatment, had the highest frequency of multicopy insertion events.     

Histaminergic H2 Action Protects Hippocampal CA1 Neurons by Prolonging the Onset of the Anoxic Depolarization in Gerbils

Abstract: The central histaminergic action on ischemia-induced neuronal damage was examined by evaluating the histological outcome and the direct current (DC) potential shift in the hippocampal CA1 region in gerbils. An intracerebroventricular administration of histamine (10–100 nmol) improved the delayed ischemic damage in hippocampal CA1 pyramidal cells produced by 3 min of transient forebrain ischemia. A high dose (75 nmol) of mepyramine, an H₁ antagonist, aggravated ischemia-induced neuronal damage, but not a low dose (0.75 nmol). Administration of cimetidine (4 nmol) and ranitidine (3 nmol), H₂ antagonists, aggravated the neuronal damage. An injection of histamine (100 nmol) prolonged the onset time of the ischemia-induced sudden shift in the extracellular DC potential (anoxic depolarization; AD) to 133% of that in control animals. Administration of mepyramine (75 nmol) did not markedly change the AD, whereas injections of cimetidine (40 nmol) and ranitidine (3 nmol) reduced the onset latency to 47 and 45%, respectively. These findings suggest that the central H₂ action serves to protect neurons by delaying the onset of AD in gerbils.     

Tat-mediated protein delivery can facilitate MHC class I presentation of antigens

We have previously shown that the tat protein of HIV-1 can be used as a carrier to promote the intracellular delivery of heterologous proteins. Here we have tested if the tat-delivery technology can be used to direct MHC class I presentation of native protein, using ovalbumin (OVA) as a model system. We show that a tat-ovalbumin conjugate (tatOVA) can be delivered into cells and that subsequent processing and presentation occurs, resulting in effective and specific killing of these target cells by an OVA specific cytotoxic T-lymphocyte (CTL) line. Comparison with the E.G7 line that expresses the OVA gene indicates that tat-mediated delivery is as efficient as endogenous expression in this system. Tat-mediated antigenic protein delivery may be useful both as a research technique and, potentially, as a therapeutic or prophylactic vaccine.     

Species turnover and diversity patterns along an evergreen broad-leaved forest coenocline

Direct gradient analysis was applied to the evergreen broad-leaved forest coenocline in the Tatera Forest Reserve, Japan. 10 quadrats of 0.1 -0.05 ha were laid out from 140 m to 560 m above sea level at intervals of 25–70 m. Gradient analysis revealed that distributions of many species terminated or started at ca. 400 m. Community similarity, calculated in Percentage Similarity (PS) and Community Coefficient (CC), changed abruptly below and above the 400 m contour, suggesting a change of vegetation structure at this altitude, which was also clear from population distributions. The spatial turnover rate of species along the altitudinal gradient was calculated in two ways: as the Average turnover rate along the whole range of the gradient, and as the Zone turnover rate at individual altitudes. The overall rates calculated for five categories of populations: DBH > 10 cm, DBH >3 cm, all woody species, herb-layer, and total vegetation, were- 0.0011 to- 0.0021 for PS, and - 0.0009 to- 0.0019 for CC. The calculated rates (PS basis) indicate that a 95% change in species composition is reached at 1120 to 620 m altitude. Similarly, the rates -0.0009 to - 0.0019 (CC) correspond to 1410 - 680 m. The altitudinal range expected here for a 95% change agrees with the actual elevation of forest zonation in northwestern Kyushu. The average rate of both PS and CC in the herb-layer population was 1.56 times higher than the rate in the woody species population, showing a more rapid change in herb-layer population than in the woody ones along the gradient. The Zone turnover rates were higher at the 370–440 m belt than those below and above the belt. This coincided with the interchanging pattern in population distributions and the abrupt change in similarity at about 400 m above sea level. This may be due to the change in environmental conditions such as physiography and air humidity. In the diversity measurements, the species density per 100 m² showed a gradual increase in the DBH >3 cm population but a constant level in the DBH >10 cm population along the whole range of the forest coenocline studied, while index values of S₍₁₀₀₎ and Shannon's H showed decreasing trends in the same gradient with a few exceptionally high and low values.     

Direct gene transfer to protoplasts: Fate of the transferred genes

Direct gene transfer to protoplasts is one of several methods developed for the production of transgenic plants. This method utilizes the efficient uptake of DNA from the surrounding medium by protoplasts (cell wall-less plant cells). Where a suitable protoplast system exists large numbers of transformant clones can be efficiently produced and often regenerated to normal fertile plants. This review concentrates on the fate of the DNA which is taken up into the protoplasts. Particular emphasis is given to the factors which can influence the integration and form of the transferred DNA, the expression of transferred genes, and the inheritance in further generations of those genes. The information available suggests (1) that DNA is taken up by a large proportion of the cells in a transformation mixture, (2) that this DNA forms complexes sometimes involving carrier DNA, (3) that fewer cells actually take up DNA into the nucleus, and (4) that the complex may be rearranged and/or amplified and then integrated into the genome. If the DNA is arranged in such a way that a gene can be expressed it does so in a normal manner and is stably inherited both mitotically and meiotically.