Transfection agents "from traditional to minaturised screening"

Three libraries of cationic lipids were synthesised and their transfection efficiency in vitro on the HEK293T cell line compared to available commercial compounds.  Two compounds were shown to be more effective and showed minimal cytotoxicity by both MTT and trypan blue assays showing the value of this novel class of compounds for gene delivery in vitro. Single bead screening was evaluated.  A small library of Arginine containing cationic lipids was synthesised on high-loading beads to assess the possibility of screening the material cleaved from single beads.  One compound was more efficient than EffecteneTM and seven was demonstrated transfection up to 80% efficiency.  These studies showed that single  bead screening was viable but further studies with accurate quantitation need to be undertaken to realise maximum activity. Finally, several compounds from the synthesised libraries were tested for their transfection activity using a microarray setup.  Poor transfection was observed, however this technique is very rapid, miniaturised and fully automated, and with further research and optimisation will offer an ideal screening technique.</p

DNA transfection screening from single beads

The solid-phase synthesis of a library of arginine-containing lipid transfection agents on high-loading beads is described. The transfection activity of the cationic lipids was determined using compound cleaved from single beads (single-bead screening) and showed, in some cases, comparable or higher DNA transfection activities as compared to commercially available reagents. Lipids with one arginine headgroup and a cholesterol tail were found to be the most active, even though their DNA binding strength (retardation assays) was relatively weak. Single-bead screening of transfection activity facilitates the rapid analysis of libraries of transfection reagents and will allow the rapid optimization of gene delivery into cells, both in culture and in vivo

Solid-phase synthesis of 89 polyamine-based cationic lipids for DNA delivery to mammalian cells

The ability of non-viral gene delivery systems to overcome extracellular and intracellular barriers is a critical issue for future clinical applications of gene therapy. In recent years much effort has been focused on the development of a variety of DNA carriers, and cationic liposomes have become the most common non-viral gene delivery system. Solid-phase synthesis was used to produce three libraries of polyamine-based cationic lipids with diverse hydrophobic tails. These were characterised, and structure-activity relationships were determined for DNA binding and transfection ability of these compounds when formulated as cationic liposomes. Two of the cationic lipids produced high-efficiency transfection of human cells. Surprisingly, these two compounds were from the library with two headgroups and one aliphatic tail, a compound class regarded as detergent-like and little investigated for transfection. These cationic lipids are promising reagents for gene delivery and illustrate the potential of solid-phase synthesis methods for lipoplex discovery

Ethanolic extract of Ocimum sanctum leaf modulates oxidative stress, cell cycle and apoptosis in head and neck cancer cell lines

Ocimum sanctum Linn. is a medicinal herb that has cytotoxic effects by inducing oxidative stress in some carcinomas. This study aimed to examine the impact of O. sanctum leaf extract on oxidative stress, cell cycle progression, and apoptosis in cell lines of head and neck squamous cell carcinoma (HNSCC). Isogenic primary (HN18/HN30) and metastatic (HN17/HN31) HNSCC cell lines were used. Preparation of the ethanolic extract of O. sanctum leaf (EEOS) was carried out. HNSCC cell lines were exposed to varying concentrations (0.1–0.8 mg/ml) of EEOS for a duration of 72 h, and the MTT assay was utilized to determine the cytotoxic doses. To assess the impact of EEOS on HNSCC cells, the levels of reactive oxygen species (ROS) and malondialdehyde were measured using a fluorometric method. Flow cytometry was utilized to evaluate effects of EEOS on the cell cycle, DNA damage, and apoptosis in HNSCC cells. Caspase-3 and -9 levels in the EEOS-treated HNSCC cells were measured by ELISA. The chemical components in EEOS were detected using high-performance liquid chromatography-electrospray ionization-time of flight-mass spectrometry. EEOS exhibited cytotoxicity against the HN18, HN17, HN30 and HN31 cells at minimum concentrations of 0.1, 0.3, 0.2 and 0.2 mg/ml, respectively. Treatment with EEOS resulted in a significant increase in ROS levels in HN18 and HN17 cells. Additionally, EEOS significantly induced the levels of malondialdehyde in HN18 and HN31 cells. Moreover, EEOS arrested the cell cycle in HN30 and HN31 cells, and significantly induced DNA damage and apoptosis in the HN18, HN30, and HN31 cells. EEOS selectively increased caspase-9 in the HN18 cells. However, caspase-3 was activated without apoptosis in the EEOS-treated HN17 cells. The constituents of EEOS were identified as rosmarinic acid, caffeic acid, and apigenin. In conclusion, EEOS exhibits various prooxidative and apoptotic effects between HNSCC cells

Anti-Acne Vulgaris Potential of the Ethanolic Extract of <i>Mesua ferrea</i> L. Flowers

Acne vulgaris is a common chronic inflammatory skin disease. In the present study, we reported the anti-acne vulgaris effect of the Mesua ferrea (M. ferrea) flower extract. The extract was evaluated for three anti-acne-causing bacteria properties including Cutibacterium acnes (C. acnes), Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus (S. aureus). The results indicated that the M. ferrea flower extract could be considered as the bactericidal agent against S. epidermidis and S. aureus with MIC values of 0.78 and 6.25 mg mL−1 and MBC values of 1.56 and 12.50 mg mL−1 and the bacteriostatic agent against C. acnes with MIC and MBC values of 3.12 and 25.00 mg mL−1, respectively. The extract at a concentration of 25 µg mL−1 also presented potent anti-inflammatory activity with a significant decrease of nitric oxide (NO) and tumor necrosis factor (TNF)-α productions in RAW 264.7 macrophage cells stimulated by LPS. In addition, the extract showed moderate to weak anti-oxidative capacities against DPPH, ABTS, FRAP and NO assays and also showed weak anti-tyrosinase activity. M. ferrea flower extract may serve as the alternative natural anti-acne formulations

Anti-Acne Vulgaris Potential of the Ethanolic Extract of Mesua ferrea L. Flowers

Acne vulgaris is a common chronic inflammatory skin disease. In the present study, we reported the anti-acne vulgaris effect of the Mesua ferrea (M. ferrea) flower extract. The extract was evaluated for three anti-acne-causing bacteria properties including Cutibacterium acnes (C. acnes), Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus (S. aureus). The results indicated that the M. ferrea flower extract could be considered as the bactericidal agent against S. epidermidis and S. aureus with MIC values of 0.78 and 6.25 mg mL−1 and MBC values of 1.56 and 12.50 mg mL−1 and the bacteriostatic agent against C. acnes with MIC and MBC values of 3.12 and 25.00 mg mL−1, respectively. The extract at a concentration of 25 µg mL−1 also presented potent anti-inflammatory activity with a significant decrease of nitric oxide (NO) and tumor necrosis factor (TNF)-α productions in RAW 264.7 macrophage cells stimulated by LPS. In addition, the extract showed moderate to weak anti-oxidative capacities against DPPH, ABTS, FRAP and NO assays and also showed weak anti-tyrosinase activity. M. ferrea flower extract may serve as the alternative natural anti-acne formulations

A Novel Antibiotic, Rhodomyrtone: Pharmacokinetic Studies in a Murine Model and Optimization and Validation of High-Performance Liquid Chromatographic Method for Plasma Analysis

Rhodomyrtone has indisputable and undeniable potential as a new antibiotic for antibiotic-resistant Gram-positive bacteria. Therefore, the main objective of this study was to determine the pharmacokinetics profiles of orally administered rhodomyrtone in rats. A reverse-phase HPLC-UV method was developed, optimized and validated for the analysis of rhodomyrtone concentrations in rat plasma. The retention time of papaverine and rhodomyrtone was 3.928 and 5.937 min, with no interference with the excipients used. The lower limit of quantification (LLOQ) of rhodomyrtone in the plasma sample was 0.04 μg/mL, the accuracy of rhodomyrtone at the LLOQ level ranged from 93.64 to 106.36%, precision was 6.59%, 80–120% for accuracy and <20% CV for precision. The calibration curve was linear at concentrations ranging from 0.04 to 128 µg/mL with a correlation coefficient (r) value of equal to or greater than 0.999. Sprague Dawley rats received a single dose of rhodomyrtone at 50 and 100 mg/kg. Blood samples were collected from tail veins. The peak plasma concentration was observed at 2 h, and the area under the curve of rhodomyrtone at 50 mg/kg and 100 mg/kg was 3.41 ± 1.04 and 7.82 ± 1.53 μg·h/mL, respectively. The results demonstrated linear pharmacokinetics characteristics at the studied dosage range. The plasma concentration of rhodomyrtone was above the minimal inhibition concentrations of several common pathogenic bacteria of medical importance. The proposed HPLC-UV method is fast, cost-effective, reliable and reproducible, and it is proposed for the routine analysis of rhodomyrtone

Influence of serum on DNA protection ability and transfection efficiency of cationic lipid-based nanoparticles for gene delivery

Cationic lipid-based nanoparticulate systems are delivery systems that has been widely used in pharmaceutical field including gene delivery. There are many barriers obstructing genetic materials and their delivery systems to reach the target. Serum is one of the imperative factor that should be investigated. Therefore, the aim of this study was to examine the effect of serum on DNA protection ability of spermine-liposomes and niosomes by evaluating the percentage of transfection efficiency in Hela cell and observing the DNA degradation band using agarose gel electrophoresis in the presence of serum. The results showed that the percentage of transfection efficiency of spermine-liposomes was dramatically decreased when serum is presented (p< 0.05). In contrast, whether or not the serum is presented, the spermine-niosomes showed no significant difference in transfection efficiency. Concisely, liposomes could slightly protect DNA from DNase in the serum, whereas, niosomes had potential ability to protect DNA from the enzymes in serum. This result revealed an advantage of the cationic niosomes system as a gene carrier over the cationic liposomes