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.

Dual-Targeted Therapy in HER2-Overexpressing Breast Cancer with Trastuzumab and Novel Cholesterol-Based Nioplexes Silencing Mcl-1.

The challenge in HER2-overexpressing breast cancer therapy lies in creating an effective target therapy to overcome treatment resistance. Monoclonal antibodies and target gene silencing by siRNA are two potential strategies that have been widely developed for treating HER2-positive breast cancer. The siRNA delivery system is a crucial factor that influences siRNA therapy's success. In this study, lipid-based nanoparticles (cationic niosomes) composed of different cholesterol-based cationic lipids were formulated and characterized for delivering siRNA into HER2-overexpressing breast cancer cells. Niosomes containing a trimethylammonium headgroup showed the highest siRNA delivery efficiency with low toxicity. The myeloid cell leukemia-1 (Mcl-1) siRNA nioplex treatment significantly decreased mRNA expression and breast cancer cell growth. Dual-targeted therapy, consisting of treatment with an Mcl-1 siRNA nioplex and trastuzumab (TZ) solution, noticeably promoted cell-growth inhibition and apoptosis. The synergistic effect of dual therapy was also demonstrated by computer modeling software (CompuSyn version 1.0). These findings suggest that the developed cationic niosomes were effective nanocarriers for siRNA delivery in breast cancer cells. Furthermore, the Mcl-1 nioplex/TZ dual treatment establishes a synergistic outcome that may have the potential to treat HER2-overexpressing breast cancer.

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.

In vitro transfection efficiencies of T-shaped spermine-based cationic lipids with identical and nonidentical tails under high serum conditions.

T-shaped spermine-based cationic lipids with identical and nonidentical hydrophobic tails having variable carbon lengths (from C10 to C18) were designed and synthesized. These lipids were characterized, and their structure-activity relationships were determined for DNA binding and transfection ability of these compounds when formulated as cationic liposomes. These liposomes were then applied as non-viral vectors to transfect HEK293T, HeLa, PC3, H460, HepG2, and Calu'3 cell lines with plasmid DNA encoding the green fluorescent protein. ST9, ST12 and ST13 with nonidentical tails could deliver DNA into HEK293T cells up to 60% under serum-free conditions. The lipid ST15 bearing nonidentical tails was found to be a potent gene transfer agent under 40% serum conditions in HEK293T and HeLa cells. Besides their low cytotoxicity, these lipoplexes also exhibited greater transfection efficiency than the commercially available transfection agent, Lipofectamine 3000.

The Oral Wound Healing Potential of Thai Propolis Based on Its Antioxidant Activity and Stimulation of Oral Fibroblast Migration and Proliferation.

Introduction: Propolis has demonstrated wound healing effects. Propolis' effects vary based on its composition and geographical origin. However, there are few reports on the effects of propolis on oral wound healing. The aim of this study was to evaluate the antioxidant and in vitro gingival wound healing effects of the n-hexane extract of propolis (HEP), ethyl acetate extract of propolis (EEP), and aqueous extract of propolis (AEP) fractions of the ethanol extract of Thai propolis. Materials and Methods: The crude ethanol extract of propolis was obtained by maceration with 95% ethanol that was sequentially fractionated with hexane, ethyl acetate, and distilled water. The chemical profiles of the samples were assessed by thin-layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS). Antioxidant activity was determined using DPPH and FRAP assays. The effects of the propolis fractions on human gingival fibroblast (HGF) proliferation, migration, and in vitro wound healing were determined by MTT, modified Boyden chamber, and scratch assay, respectively. Results: We found that solvent polarity greatly affected the extract yield and TLC profiles. The highest extract yield was found in HEP (38.88%), followed by EEP (19.8%) and AEP (1.42%). TLC revealed 7 spots in the crude ethanol extract (Rf 0.36-0.80), 6 spots in HEP (Rf 0.42-0.80) and EEP (Rf 0.36-0.72), and 4 spots in AEP (Rf 0.17-0.79). GC-MS analysis revealed a high amount of triterpenoids in HEP (82.97%) compared with EEP (28.96%). However, no triterpenoid was found in AEP. The highest antioxidant activity and stimulation of HGF proliferation were observed in HEP, followed by EEP and AEP. HEP and EEP, but not AEP, enhanced HGF migration. However, all propolis fractions induced wound closure. Conclusions: HEP contained a large amount of triterpenoids. Antioxidant and in vitro wound closure effects were found in HEP, EEP, and AEP fractions.

siRNA Targeting Mcl-1 Potentiates the Anticancer Activity of Andrographolide Nanosuspensions via Apoptosis in Breast Cancer Cells.

Breast cancer is the second leading cause of cancer-related death in the US. However, recurrence is frequently found despite adjuvant therapy being available. Combination therapy with cytotoxic drugs and gene therapy is being developed to be a new promising cancer treatment strategy. Introducing substituted dithiocarbamate moieties at the C12 position of andrographolide (3nAG) could improve its anticancer selectivity in the MCF-7 breast cancer cell line. However, its hydrophobicity is one of its main drawbacks. This work successfully prepared 3nAG nanosuspension stabilized with the chitosan derivative NSC (3nAGN-NSC) to increase solubility and pharmacological effectiveness. siRNAs have emerged as a promising therapeutic alternative for interfering with particular mRNA. The 3nAGN-NSC had also induced Mcl-1 mRNA expression in MCF-7 human breast cancer cells at 8, 12, and 24 h. This indicates that, in addition to Mcl-1 silencing by siRNA (siMcl-1) in MCF-7 with substantial Mcl-1 reliance, rationally devised combination treatment may cause the death of cancer cells in breast cancer. The Fa-CI analysis showed that the combination of 3nAGN-NSC and siMcl-1 had a synergistic effect with a combination index (CI) value of 0.75 (CI < 1 indicating synergistic effects) at the fractional inhibition of Fa 0.7. The synergistic effect was validated by flow cytometry, with the induction of apoptosis as the mechanism of reduced cell viability. Our findings suggested the rational use of 3nAGN-NSC in combination with siMcl-1 to kill breast cancer cells.

Enhancing Anticancer Potency of a 13-Substituted Berberine Derivative with Cationic Liposomes.

Cationic liposomal formulations of the telomeric G-quadruplex stabilizing ligand, 13-(2-naphthylmethoxy)berberine bromide (1), have been developed with the purpose of delivering 1 into the nucleus of cancer cells for potential telomere targeting. Berberine derivative 1 was encapsulated in various cationic lipids 2-4 by the thin film evaporation method; these lipids are cationic after amine protonation. The most appropriate liposomal berberine formulation was that of 1 and the cholesterol derived cationic lipid 4 in a weight ratio of 1 : 20 with 76.5% encapsulation efficiency of 1. Cellular uptake studies in the HeLa and HT-29 cancer cells lines showed that the liposomal berberine derivative uptake in the cells was higher and more stable than for berberine derivative 1 alone while free 1 was completely decomposed in the cells within 60 min exposure to the cells. Anticancer activity of the liposomal berberine derivative 1 based on 4 was greater than that for the free berberine derivative 1 in the MCF-7, HeLa and HT-29 cell line by 2.3-, 4.9- and 5.3-fold, respectively, and also, interestingly, superior to the anticancer drug doxorubicin against the HT29 cancer cell line.

Serum Compatible Spermine-Based Cationic Lipids with Nonidentical Hydrocarbon Tails Mediate High Transfection Efficiency.

Cationic lipids are widely used as nonviral synthetic vectors for gene delivery as a safer alternative to viral vectors. In this work, a library of L-shaped spermine-based cationic lipids with identical and nonidentical hydrophobic chains having variable carbon lengths (from C10 to C18) was designed and synthesized. These lipids were characterized and the structure-activity relationships of these compounds were determined for DNA binding and transfection ability when formulated as cationic liposomes. The liposomes were then used successfully for the transfection of HEK293T, HeLa, PC3, H460, HepG2, SH-SY5Y and Calu'3 cell lines. The transfection efficiency of lipids with nonidentical hydrocarbon chains was greater than the identical analogue. These reagents exhibited superior efficiency to the commercial reagent, Lipofectamine3000, under both serum-free and 10-40 % serum conditions in HEK293T, HeLa and H460 cell lines. The lipids were not toxic to the tested cell line. The results suggest that L-shaped spermine-based cationic lipids with nonidentical hydrocarbon tails could serve as efficient and safe nonviral vector gene carriers in further in vivo studies.

Delivery of small interfering RNAs by nanovesicles for cancer therapy.

Small interfering ribonucleic acids (siRNAs) are originally recognized as an intermediate of the RNA interference (RNAi) pathway. They can inhibit or silence various cellular pathways by knocking down specific messenger RNA molecules. In cancer cells, siRNAs can suppress the expression of several multidrug-resistant genes, leading to the increased deposition of chemotherapeutic drugs at the tumor site. siRNA therapy can be used to selectively increase apoptosis of cancer cells or activate an immune response to the cancer. However, delivering siRNAs to the targeted location is the main limitation in achieving safe and effective delivery of siRNAs. This review highlights some representative examples of nonviral delivery systems, especially nanovesicles such as exosomes, liposomes, and niosomes. Nanovesicles can improve the delivery of siRNAs by increasing their intracellular delivery, and they have demonstrated excellent potential for cancer therapy. This review focuses on recent discoveries of siRNA targets for cancer therapy and the use of siRNAs to successfully silence these targets. In addition, this review summarizes the recent progress in designing nanovesicles (liposomes or niosomes) for siRNA delivery to cancer cells and the effects of a combination of anticancer drugs and siRNA therapy in cancer therapy.

Apis mellifera propolis enhances apoptosis and invasion inhibition in head and neck cancer cells.

BACKGROUND: Propolis is a resinous product accumulated from several plant sources that possess a wide range of therapeutic properties, including anti-cancer activities. However, the role of honeybee-produced propolis on head and neck squamous carcinoma (HNSCC) is not well understood. The aim of this study was to investigate the effects of Apis mellifera propolis on apoptosis and invasiveness in HNSCC cell lines. METHODS: Ethyl acetate extract of propolis (EAEP) was prepared from A. mellifera beehives using liquid-liquid extraction. High-performance liquid chromatography coupled with electrospray ionization-time of flight-mass spectrometry (HPLC-ESI-TOF-MS) was used to determine the flavonoids in EAEP. Isogenic HNSCC cell lines derived from primary (HN30 and HN4) and metastatic site (HN31 and HN12) were used in this study. The cytotoxicity, apoptosis, invasion, and MMP activity of EAEP on HNSCC cells were determined using an MTT assay, flow cytometry, Matrigel invasion assay, and gelatinase zymography, respectively. RESULTS: We found that EAEP exhibited cytotoxic activity and induced apoptosis in the HNSCC cell lines. Furthermore, EAEP significantly decreased HNSCC cell invasion by reducing MMP-2 and MMP-9 activity. Two flavonoids, galangin and apigenin, were identified in EAEP by HPLC-ESI-TOF-MS. The results suggest that EAEP promotes apoptosis and exerts anti-invasion potential by inhibiting MMP-2 and MMP-9 activity in HNSCC cell lines. These inhibitory effects may be mediated by galangin and apigenin.

Synergistic Effect of Doxorubicin and siRNA-Mediated Silencing of Mcl-1 Using Cationic Niosomes against 3D MCF-7 Spheroids.

Chemotherapy is a vital option for cancer treatment; however, its therapeutic outcomes are limited by dose-dependent toxicity and the occurrence of chemoresistance. siRNAs have emerged as an attractive therapeutic option enabling specific interference with target genes. Combination therapy using chemotherapeutic agents along with gene therapy could be a potential strategy for cancer management, which not only improves therapeutic efficacy but also decreases untoward effects from dose reduction. In this study, a cationic niosome containing plier-like cationic lipid B was used to convey siRNA against anti-apoptotic mRNA into MCF-7 and MDA-MB-231 cells. Mcl-1 silencing markedly decreased the viability of MCF-7 cells and triggered apoptosis. Moreover, computer modeling suggested that the combination of doxorubicin (Dox) and Mcl-1 siRNA exhibited a synergistic relationship and enabled a dose reduction of each agent at 1.71 and 3.91 folds, respectively, to reach a 90% inhibitory effect when compared to single-agent treatments. Synergistic antitumor activity was further verified in a 3D spheroid culture which revealed, in contrast to single-agent treatment, the combination markedly decreased spheroid volume over time. Together, the combination therapy between Mcl-1 silencing and Dox exhibits a synergistic effect that may be exploited for novel breast cancer treatment.

Ethanolic Extract of Ocimum sanctum Leaves Reduced Invasion and Matrix Metalloproteinase Activity of Head and Neck Cancer Cell Lines.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) has a yearly incidence of 600,000 cases worldwide with a low survival rate. Ocimum sanctum L. or Ocimum tenuiflorum L. (Holy basil; Tulsi in Hindi), is a traditional medicine herb that demonstrates numerous effects including anti-oxidant, anti-microbial, and anti-tumor effects. The aim of this study was to evaluate the anti-invasive effect of O. sanctum leaf extract on HNSCC cell lines. METHODS: Ethanolic extract of O. sanctum leaf (EEOS) was prepared and the phenolic compounds were identified using high-performance liquid chromatography-electrospray ionization-time of flight-mass spectrometry. Genetically matched HNSCC cell lines derived from primary (HN30 and HN4) and metastatic sites (HN31 and HN12) from the same patient were used in this study. The EEOS cytotoxicity to the cell lines was determined using an MTT assay. The invasion and matrix metalloproteinase (MMP)-2 and -9 activity of EEOS-treated cells were tested using a modified Boyden chamber assay and zymography, respectively. RESULTS: We found that EEOS significantly inhibited the invasion and MMP-2 and MMP-9 activity of HN4 and HN12 cells, but not HN30 and HN31 cells. Rosmarinic acid, caffeic acid, and apigenin were detected in EEOS. Moreover, rosmarinic acid was found as the major phenolic compound. CONCLUSION: EEOS exerted its anti-invasive effect on HNSCC cells by attenuating MMP activity. The active compounds identified in EEOS might be promising as an alternative therapeutic agent for HNSCC.

The effect of polar headgroups and spacer length on the DNA transfection of cholesterol-based cationic lipids.

This article is related to the effects of the headgroups and spacer length of cationic lipids on transfection efficiency. To develop highly potent cationic lipids, a series of divalent lysine-diamine conjugated cholesterol-based cationic lipids with three different headgroups (ammonium, trimethyl ammonium, and guanidinium) were synthesized. The newly synthesized cationic lipids (1-6)A formed cationic liposomes in the presence and absence of a zwitterionic helper lipid, DOPE (dioleoylphosphatidylethanolamine). A gel retardation assay showed that most of the prepared lipoplexes could retard DNA migration in the presence of DOPE. We attempted to modify the diverse cationic headgroups to improve the transfection efficiency. However, the lysine-1,3-diaminopropane-conjugated cholesterol-based lipid 4A, having divalent ammonium of unmodified lysine headgroup, exhibited high relative transfection efficiency in HEK293. When the transfection efficiency of 4A was formulated with DOPE (1 : 1 weight ratio), it produced the same range in comparison with that of a commercially available transfection agent, Lipofectamine™ 2000 (L2k). The lipid 4A was studied to optimize the conditions with respect to the lipid/DOPE and DNA/lipid ratios and the amount of DNA. The transfection efficiency of the highly potent lipid 4A was also studied to determine the transfection efficiency of HeLa, PC3, and HC-04 cell lines. This lipid also protected the DNA from a serum and had low toxicity. Lipoplexes 4A with DOPE had the particle size of around 300-600 nm and the zeta potential of around 0-45 mV. In summary, cationic liposomes 4A demonstrated a high performance as DNA carriers.

Effect of hydrophobic tails of plier-like cationic lipids on nucleic acid delivery and intracellular trafficking.

In the optimization of transfection efficacy, one of the crucial barriers to effective gene delivery is in fact the intracellular trafficking of nucleic acids, besides the first and the last steps of gene transfer, i.e., delivery to the cell and transcription. Modifications of cationic lipid structure have been reported to have a significant effect on gene delivery. Therefore, the plier-like cationic lipids (PCLs) have been synthesized and the effect of the different types of hydrophobic tails (chain length and unsaturated hydrocarbon) on physicochemical properties, cellular uptake, trafficking process, transfection, and silencing efficiency has been investigated. In this study, the plier-like cationic niosomes (PCNs) containing PCL (A, B, and C) were evaluated their performance to deliver pDNA and siRNA to HeLa cells. Among the PCNs, PCN-B with saturated asymmetric hydrocarbon tails (C18 and C12) provided the highest efficiency for pDNA and siRNA delivery. Furthermore, the results revealed that the structure of the cationic lipids affected the internalization pathway and the intracellular trafficking. PCL-B and PCL-C with asymmetric tails preferred clathrin- and caveolae-mediated endocytosis as the predominant internalization pathways and were also involved in the polymerization process for transfection. However, PCL-A with symmetry hydrocarbon tails (C12) was predominantly taken up via macropinocytosis. All PCNs were able to escape from endosomal-lysosomal systems through facilitation of acidification. Results obtained from the cytotoxicity test revealed that the PCNs were safe in vitro. Therefore, PCNs provide a great prospect as an alternative effective gene delivery system.

Anti-biofilm, nitric oxide inhibition and wound healing potential of purpurin-18 phytyl ester isolated from Clinacanthus nutans leaves.

AIMS: Clinacanthus nutans (C. nutans) has demonstrated anti-inflammatory activity, however, the active compound generating this activity remains unknown. The aim of this study was to identify the bioactive compound in C. nutans responsible for its anti-inflammatory, in-vitro wound healing, and anti-biofilm activities. MAIN METHODS: A pure compound was isolated from the chloroform extract (CE) of C. nutans leaves by chromatographic techniques and bioassay-guided fractionation. This compound's structure was determined by spectroscopic analyses (FTIR/NMR/HRES-MS). Biological activities were evaluated using cytotoxicity, nitric oxide (NO), wound scratch, anti-microbial activity, and anti-biofilm assays; and the compound's bactericidal depth into the biofilm was visualized by confocal laser scanning microscopy. KEY FINDINGS: CE and its pure isolated compound, purpurin-18 phytyl ester (P18PE), significantly inhibited lipopolysaccharide (LPS)-induced NO production in RAW 264.7 cells at concentrations of 100 µg/ml and 10-100 µg/ml, respectively. These concentrations significantly induced wound closure by human gingival fibroblasts. CE (100-1000µg/ml) and P18PE (1-500 µg/ml) did not inhibit Streptococcus (S.) mutans growth. However, these concentrations significantly reduced S. mutans biofilm formation below 50% at 250 µg/ml for CE, and 25 µg/ml for P18PE (p<0.05). SIGNIFICANCE: C. nutans contains a bioactive compound, P18PE, which exhibits anti-inflammatory, in-vitro wound healing, and anti-biofilm activities.

Cationic Niosomes for Enhanced Skin Immunization of Plasmid DNA-Encoding Ovalbumin via Hollow Microneedles.

The purpose of the present study was to evaluate the use of cationic niosomes composed of Span20:cholesterol:cationic lipid (N 1,N 1-dimyristeroyloxyethyl-spermine) at the molar ratio of 2.5:2.5:0.5 mM combined with hollow microneedle (MN) devices for in vivo skin immunization of plasmid DNA-encoding ovalbumin (pOVA). The results revealed that using hollow MNs with cationic niosomes for pOVA penetration successfully induced both humoral and cell-mediated immune responses including immunoglobulin G (IgG) antibody responses, interleukin-4 (IL-4), and interferon gamma (IFN-γ) cytokine secretion. When using hollow MNs with cationic niosome/pOVA complexes, the immune response was superior to naked pOVA, which testifies the increased amount of IgG antibody responses and cytokine secretion. In comparison with conventional subcutaneous (SC) injections, using hollow MNs with cationic niosome/pOVA complexes induced a higher level of both IgG immune response and cytokine release. Moreover, a group of mice immunized with hollow MNs did not show infection or bleeding on the skin. Consequently, targeted delivery of pOVA using cationic niosomes combined with hollow MNs might prove a promising vaccination method for skin vaccination.

Synergistic Inhibition of Human Carcinoma Cell Growth via Co-Delivery of p53 Plasmid DNA and bcl-2 Antisense Oligodeoxyribonucleotide by Cholic Acid-modified Polyethylenimine.

BACKGROUND/AIM: This study investigated the co-delivery of plasmid DNA and antisense oligodeoxyribonucleotide (AS ODN) into carcinoma cells by cholic acid-modified polyethylenimine (PEI-CA). MATERIALS AND METHODS: PEI-CA/plasmid DNA and AS ODN complexes were formulated and evaluated for delivery of plasmid DNA and AS ODN in HeLa cells. The efficiency of co-delivery of plasmid DNA and AS ODN was evaluated by cell growth inhibition using p53 and bcl-2 AS ODN. RESULTS: AS ODN intracellular delivery and green fluorescent protein expression upon cellular transfection were greater than in cells treated with uncomplexed nucleic acids. Treatment of the cells with PEI-CA/p53 plasmid DNA and bcl-2 AS ODN complexes resulted in cell growth inhibition that was greater than that of either PEI-CA/p53 plasmid DNA complexes or PEI-CA/bcl-2 AS ODN complexes alone. CONCLUSION: The co-delivery of p53 plasmid DNA and bcl-2 AS ODN in PEI-CA complexes enhanced therapeutic activities of both p53 plasmid DNA and bcl-2 AS ODN.

Conessine as a novel inhibitor of multidrug efflux pump systems in Pseudomonas aeruginosa.

BACKGROUND: Holarrhena antidysenterica has been employed as an ethnobotanical plant for the treatment of dysentery, diarrhoea, fever, and bacterial infections. Biological activities of the principle compound, conessine including anti-diarrhoea and anti-plasmodial effects were documented. Our previous study reported potency of Holarrhena antidysenterica extract and conessine as resistance modifying agents against extensively drug-resistant Acinetobacter baumannii. This study aimed to investigate (i) whether conessine, a steroidal alkaloid compound, could act as a resistance modifying agent against multidrug-resistant Pseudomonas aeruginosa, and (ii) whether MexAB-OprM efflux pump involved in the mechanism. METHODS: Conessine combined with various antibiotics were determined for synergistic activity against P. aeruginosa PAO1 strain K767 (wild-type), K1455 (MexAB-OprM overexpressed), and K1523 (MexB deletion). H33342 accumulation assay was used to evaluate efflux pump inhibition while NPN uptake assay was assessed membrane permeabilization. RESULTS: Conessine significantly reduced MICs of all antibiotics by at least 8-fold in MexAB-OprM overexpressed strain. The levels were comparable to those obtained in wild-type strain for cefotaxime, levofloxacin, and tetracycline. With erythromycin, novobiocin, and rifampicin, MICs were 4- to 8-fold less than MICs of the wild-type strain. Loss of MexAB-OprM due to deletion of mexB affected susceptibility to almost all antibiotics, except novobiocin. Synergistic activities between other antibiotics (except novobiocin) and conessine observed in MexB deletion strain suggested that conessine might inhibit other efflux systems present in P. aeruginosa. Inhibition of H33342 efflux in the tested strains clearly demonstrated that conessine inhibited MexAB-OprM pump. In contrast, the mode of action as a membrane permeabilizer was not observed after treatment with conessine as evidenced by no accumulation of 1-N-phenylnaphthylamine. CONCLUSIONS: The results suggested that conessine could be applied as a novel efflux pump inhibitor to restore antibiotic activity by inhibiting efflux pump systems in P. aeruginosa. The findings speculated that conessine may also have a potential to be active against homologous resistance-nodulation-division (RND) family in other Gram-negative pathogens.

Cationic niosomes an effective gene carrier composed of novel spermine-derivative cationic lipids: effect of central core structures.

CONTEXT: Cationic niosomes formulated from Span 20, cholesterol (Chol) and novel spermine-based cationic lipids of multiple central core structures (di(oxyethyl)amino, di(oxyethyl)amino carboxy, 3-amino-1,2-dioxypropyl and 2-amino-1,3-dioxypropyl) were successfully prepared for improving transfection efficiency in vitro. The niosomes composed of spermine cationic lipid with central core structure of di(oxyethyl)amino revealed the highest gene transfection efficiency. OBJECTIVES: To investigate the factors affecting gene transfection and cell viability including differences in the central core structures of cationic lipids, the composition of vesicles, molar ratio of cationic lipids in formulations and the weight ratio of niosomes to DNA. METHODS: Cationic niosomes composed of nonionic surfactants (Span20), cholesterol and spermine-based cationic lipids of multiple central core structures were formulated. Gene transfection and cell viability were evaluated on a human cervical carcinoma cell line (HeLa cells) using pDNA encoding green fluorescent protein (pEGFP-C2). The morphology, size and charge were also characterized. RESULTS AND DISCUSSION: High transfection efficiency was obtained from cationic niosomes composed of Span20:Chol:cationic lipid at the molar ratio of 2.5:2.5:0.5 mM. Cationic lipids with di(oxyethyl)amino as a central core structure exhibited highest transfection efficiency. In addition, there was also no serum effect on transfection efficiency. CONCLUSIONS: These novel cationic niosomes may constitute a good alternative carrier for gene transfection.

Cholesterol-based cationic liposome increases dsRNA protection of yellow head virus infection in Penaeus vannamei.

Protection of shrimp from yellow head virus (YHV) infection has been demonstrated by injection and oral delivery of dsRNA-YHV protease gene (dsYHV) or shrimp endogenous gene (dsRab7). However, to achieve complete viral suppression and to prolong dsRNA activity, the development of an effective dsRNA delivery system is required. In this study, four cationic liposomes were synthesized and tested for their ability to increase dsRNA efficiency. The results demonstrated that entrapping dsYHV in a cholesterol-based cationic liposome gave the best protection against YHV infection when compared with other cationic lipids. The cholesterol-based cationic liposome-dsYHV (Chol-dsYHV) complex conferred YHV protection in a dose-dependent manner. Injection with Chol-dsYHV at 0.05µg dsYHV/g shrimp could give comparable level of YHV protection to the injection with 1.25µg naked dsYHV/g shrimp. The shrimp injected with Chol- dsYHV at 1.25µg dsRNA/g shrimp showed only 50% mortality at 60days post injection whereas the naked dsYHV at the same concentration gave 90% mortality. Thus, the liposome-entrapped dsYHV could lower an effective dsRNA concentration in viral protection and prolong dsRNA activity. In addition, encapsulating dsRab7 in the cholesterol-based cationic liposome could protect the dsRab7 from enzymatic digestion, and continuous feeding the shrimp with the diet formulated with the liposome-entrapped dsRab7 for 4days in the total of 960µg dsRab7/g shrimp could enhance YHV protection efficiency compared with the naked dsRab7. Our studies reveal that cholesterol-based cationic liposome is a promising dsRNA carrier to enhance dsRNA efficiency in both injection and oral delivery systems.