'IntraCell' plugin for assessment of intracellular localization of nano-delivery systems and their targeting to the individual organelles

Efficient intracellular targeting of drugs and drug delivery systems (DDSs) is a major challenge that should be overcome to enhance the therapeutic efficiency of biopharmaceuticals and other intracellularly-acting drugs. Studies that quantitatively assess the mechanisms, barriers, and efficiency of intracellular drug delivery are required to determine the therapeutic potential of intracellular targeting of nano-delivery systems. In this study we report development and application of a novel ‘IntraCell’ plugin for ImageJ that is useful for quantitative assessment of uptake and intracellular localization of the drug/DDS and estimation of targeting efficiency. The developed plugin is based on threshold-based identification of borders of cell and of the individual organelles on confocal images and pixel-by-pixel analysis of fluorescence intensities.We applied the developed ‘IntraCell’ plugin to investigate uptake and intracellular targeting of novel endoplasmic reticulum (ER)-targeted delivery system based on PLGA nanoparticles decorated with ER-targeting or control peptides and encapsulating antigenic peptide and fluorescent marker. Decoration of the nanoparticles with peptidic residues affected their uptake and intracellular trafficking in HeLa cells, indicating that the targeting peptide was identified as ER-targeting signal by the intracellular trafficking mechanisms in HeLa cells and that these mechanisms can handle nano-DDS of the size comparable to some intracellular vesicles (hundreds of nanometers in diameter).We conclude that decoration of nanoparticles with peptidic residues affects their intracellular localization and trafficking and can be potentially used for intracellularly-targeted drug delivery. ‘IntraCell’ plugin is an useful tool for quantitative assessment of efficiency of uptake and intracellular drug targeting. In combination with other experimental approaches, it will be useful for the development of intracellularly-targeted formulations with enhanced and controlled drug pharmacological activities, such as delivery of antigenic peptides for anticancer vaccination and for other applications.     

Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis

BACKGROUND: The relatively high transfection efficiency of polyethylenimine (PEI) vectors has been hypothesized to be due to their ability to avoid trafficking to degradative lysosomes. According to the proton sponge hypothesis, the buffering capacity of PEI leads to osmotic swelling and rupture of endosomes, resulting in the release of the vector into the cytoplasm. METHODS: The mechanism of PEI-mediated DNA transfer was investigated using quantitative methods to study individual steps in the overall transfection process. In addition to transfection efficiency, the cellular uptake, local pH environment, and stability of vectors were analyzed. N-Quaternized (and therefore non-proton sponge) versions of PEI and specific cell function inhibitors were used to further probe the proton sponge hypothesis. RESULTS: Both N-quaternization and the use of bafilomycin A1 (a vacuolar proton pump inhibitor) reduced the transfection efficiency of PEI by approximately two orders of magnitude. Chloroquine, which buffers lysosomes, enhanced the transfection efficiency of N-quaternized PEIs and polylysine by 2-3-fold. In contrast, chloroquine did not improve the transfection efficiency of PEI. The measured average pH environment of PEI vectors was 6.1, indicating that they successfully avoid trafficking to acidic lysosomes. Significantly lower average pH environments were observed for permethyl-PEI (pH 5.4), perethyl-PEI (pH 5.1), and polylysine (pH 4.6) vectors. Cellular uptake levels of permethyl-PEI and perethyl-PEI vectors were found to be 20 and 90% higher, respectively, than that of parent PEI vectors, indicating that the reduction in transfection activity of the N-quaternized PEIs is due to a barrier downstream of cellular uptake. A polycation/DNA-binding affinity assessment showed that the more charge dense N-quaternized PEIs bind DNA less tightly than PEI, demonstrating that poor vector unpackaging was not responsible for the reduced transfection activity of the N-quaternized PEIs. CONCLUSIONS: The results obtained are consistent with the proton sponge hypothesis and strongly suggest that the transfection activity of PEI vectors is due to their unique ability to avoid acidic lysosomes.     

层状双金属氢氧化物及其在药物传递系统中的应用研究现状

层状双金属氢氧化物作为一种新型无机纳米载体材料,具有独特优势,近年来其在各类药物传递系统中的应用已成为研究热点。介绍层状双金属氢氧化物的制备与修饰,分类综述其在不同药物传递系统中的应用研究。     

A proteomic and phosphoproteomic landscape of KRAS mutant cancers identifies combination therapies

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Odoriferous Therapy: A Review Identifying Essential Oils against Pathogens of the Respiratory Tract

This review explores the body of scientific information available on the antimicrobial properties of essential oils against pathogens responsible for respiratory infections and critically compares this to what is recommended in the Layman's aroma‐therapeutic literature. Essential oils are predominantly indicated for the treatment of respiratory infections caused by bacteria or viruses (total 79.0 %), the efficacy of which has not been confirmed through clinical trials. When used in combination, they are often blended for presumed holistic synergistic effects. Of the essential oils recommended, all show some degree of antioxidant activity, 50.0 % demonstrate anti‐inflammatory effects and 83.3 % of the essential oils showed antihistaminic activity. Of the essential oils reviewed, 43.8 % are considered non‐toxic while the remaining essential oils are considered slightly to moderately toxic (43.7 %) or the toxicity is unknown (12.5 %). Recommendations are made for further research into essential oil combinations.     

Gene transfer to skeletal muscle using hydrodynamic limb vein injection: current applications,hurdles and possible optimizations

Hydrodynamic limb vein injection is an in vivo locoregional gene delivery method. It consists of administrating a large volume of solution containing nucleic acid constructs in a limb with both blood inflow and outflow temporarily blocked using a tourniquet. The fast, high pressure delivery allows the musculature of the whole limb to be reached. The skeletal muscle is a tissue of choice for a variety of gene transfer applications, including gene therapy for Duchenne muscular dystrophy or other myopathies, as well as for the production of antibodies or other proteins with broad therapeutic effects. Hydrodynamic limb vein delivery has been evaluated with success in a large range of animal models. It has also proven to be safe and well‐tolerated in muscular dystrophy patients, thus supporting its translation to the clinic. However, some possible limitations may occur at different steps of the delivery process. Here, we have highlighted the interests, bottlenecks and potential improvements that could further optimize non‐viral gene transfer following hydrodynamic limb vein injection.     

Biotin and glucose dual-targeting,ligand-modified liposomes promote breast tumor-specific drug delivery

Breast cancer is the second leading cause of cancer-related deaths in women. Ligand-modified liposomes are used for breast tumor-specific drug delivery to improve the efficacy and reduce the side effects of chemotherapy; however, only a few liposomes with high targeting efficiency have been developed because the mono-targeting, ligand-modified liposomes are generally unable to deliver an adequate therapeutic dose. In this study, we designed biotin-glucose branched ligand-modified, dual-targeting liposomes (Bio-Glu-Lip) and evaluated their potential as a targeted chemotherapy delivery system in vitro and in vivo. When compared with the non-targeting liposome (Lip), Bio-Lip, and Glu-Lip, Bio-Glu-Lip had the highest cell uptake in 4T1 cells (3.00-fold, 1.60-fold, and 1.95-fold higher, respectively) and in MCF-7 cells (2.63-fold, 1.63-fold, and 1.85-fold higher, respectively). The subsequent cytotoxicity and in vivo assays further supported the dual-targeting liposome is a promising drug delivery carrier for the treatment of breast cancer.     

Glutaminyl‐tRNA Synthetase from Pseudomonas aeruginosa: Characterization,structure, and development as a screening platform

Pseudomonas aeruginosa has a high potential for developing resistance to multiple antibiotics. The gene (glnS) encoding glutaminyl‐tRNA synthetase (GlnRS) from P. aeruginosa was cloned and the resulting protein characterized. GlnRS was kinetically evaluated and the K_M and k_cat^obs, governing interactions with tRNA, were 1.0 μM and 0.15 s^?1, respectively. The crystal structure of the α₂ form of P. aeruginosa GlnRS was solved to 1.9 Å resolution. The amino acid sequence and structure of P. aeruginosa GlnRS were analyzed and compared to that of GlnRS from Escherichia coli. Amino acids that interact with ATP, glutamine, and tRNA are well conserved and structure overlays indicate that both GlnRS proteins conform to a similar three‐dimensional structure. GlnRS was developed into a screening platform using scintillation proximity assay technology and used to screen ~2,000 chemical compounds. Three inhibitory compounds were identified and analyzed for enzymatic inhibition as well as minimum inhibitory concentrations against clinically relevant bacterial strains. Two of the compounds, BM02E04 and BM04H03, were selected for further studies. These compounds displayed broad‐spectrum antibacterial activity and exhibited moderate inhibitory activity against mutant efflux deficient strains of P. aeruginosa and E. coli. Growth of wild‐type strains was unaffected, indicating that efflux was likely responsible for the lack of sensitivity. The global mode of action was determined using time‐kill kinetics. BM04H03 did not inhibit the growth of human cell cultures at any concentration and BM02E04 only inhibit cultures at the highest concentration tested (400 μg/ml). In conclusion, GlnRS from P. aeruginosa is shown to have a structure similar to that of E. coli GlnRS and two natural product compounds were identified as inhibitors of P. aeruginosa GlnRS with the potential for utility as lead candidates in antibacterial drug development in a time of increased antibiotic resistance.