Induction of cell size vesicles from human lymphoma cell lines and their application to drug carriers

Sodium butyrate (NaB) induced the membrane enclosed cell size vesicles from several IgM producing cell lines. We considered the application of the cell-derived vesicles (CDVs) to drug delivery system (DDS) using the lung cancer specific IgM producing AE6 cell line. Microscopic observation showed that the DiI fluorescence labeled AE6 vesicles were incorporated into the lung cancer cell line A549. The anticancer drug, actinomycin D (actD), contained in AE6 and Ramos vesicles decreased the A549 cell viability to 46 and 62% of control without actD, respectively. The cytotoxic effect in AE6 vesicles was superior to that in the Ramos vesicles that have the lung cancer non-specific IgM on their surfaces. However, the result of the Ramos vesicles suggests that the surface molecules other than IgM may interact with the A549 cells. In our method for vesicle production, more specific and abundant antibodies mounted vesicles can be generated by transfection of their genes into cells followed by NaB treatment. These suggest that the CDVs may be useful for the development of a drug carrier for DDS.     

Investigation of archaeosomes as carriers for oral delivery of peptides

Oral administration of peptide and protein drugs faces a big challenge partly due to the hostile gastrointestinal (GI) environment. Lipid-based delivery systems are attractive because they offer some protection for peptides and proteins. In this context, we prepared a special lipid-based oral delivery system: archaeosomes, made of the polar lipid fraction E (PLFE) extracted from Sulfolobus acidocaldarius, and explored its potential as an oral drug delivery vehicle. Our study demonstrates that archaeosomes have superior stability in simulated GI fluids, and enable fluorescent labeled peptides to reside for longer periods in the GI tract after oral administration. Although archaeosomes have little effect on the transport of insulin across the Caco-2 cell monolayers, the in vivo experiments indicated that archaeosomes containing insulin induced lower levels of blood glucose than a conventional liposome formulation. These data indicate that archaeosomes could be a potential carrier for effective oral delivery of peptide drugs.     

Sites in the AAV5 capsid tolerant to deletions and tandem duplications

Gene therapy vectors based on adeno-associated virus (AAV) have shown much promise in clinical trials for the treatment of a variety of diseases. However, the ability to manipulate and engineer the viral surface for enhanced efficiency is necessary to overcome such barriers as pre-existing immunity and transduction of non-target cells that currently limit AAV applications. Although single amino acid changes and peptide insertions at select sites have been explored previously, the tolerance of AAV to small deletions and tandem duplications of sequence has not been globally addressed. Here, we have generated a large, diverse library of >10⁵ members containing deletions and tandem duplications throughout the viral capsid of AAV5. Four unique mutants were identified that maintain the ability to form viral particles, with one showing improved transduction on both 293T and BEAS-2B cells. This approach may find potential use for the generation of novel variants with improved and altered properties or in the identification of sites that are tolerant to insertions of targeting ligands.     

Targeted delivery of NRASQ61R and Cre-recombinase to post-natal melanocytes induces melanoma in Ink4a/Arflox/lox mice

We have developed a somatic cell gene delivery mouse model of melanoma that allows for the rapid validation of genetic alterations identified in this disease. A major advantage of this system is the ability to model the multi-step process of carcinogenesis in immune-competent mice without the generation and cross breeding of multiple strains. We have used this model to evaluate the role of RAS isoforms in melanoma initiation in the context of conditional Ink4a/Arf loss. Mice expressing the tumor virus A (TVA) receptor specifically in melanocytes under control of the dopachrome tautomerase (DCT) promoter were crossed to Ink4a/Arf^lox/lox mice and newborn DCT-TVA/Ink4a/Arf^lox/lox mice were injected with retroviruses containing activated KRAS, NRAS and/or Cre-recombinase. No mice injected with viruses containing KRAS and Cre or NRAS alone developed tumors; however, more than one-third of DCT-TVA/Ink4a/Arf^lox/lox mice injected with NRAS and Cre viruses developed melanoma and two-thirds developed melanoma when NRAS and Cre expression was linked.     

The Boar-Operated-System: a Novel Method to Deliver Baits to Wild Pigs

ABSTRACT Bait-delivered pharmaceuticals, increasingly used to manage populations of wild boar (Sus scrofa) and feral pigs, may be ingested by nontarget species. Species-specificity could be achieved through a delivery system. We designed the BOS^TM (Boar-Operated-System) as a device to deliver baits to wild pigs. The BOS^TM consists of a metal pole onto which a round perforated base is attached. A metal cone with a wide rim slides up and down the pole and fully encloses the base onto which the baits are placed. We conducted a pilot, captive trial and found that captive wild boar fed from the BOS^TM either directly, by lifting the cone, or indirectly, by feeding once another animal had lifted the cone. Thus, we tested whether free-living wild boar fed from the BOS^TM and whether the BOS^TM could prevent bait uptake by nontarget species. We observed that free-living wild boar fed regularly from the BOS^TM and that the device successfully prevented bait uptake by nontarget species. The BOS^TM should be trialed more extensively to confirm its effectiveness and species-specificity to distribute pharmaceuticals to wild suids. If successful, the BOS^TM could be used to deliver vaccines in disease control programs as well as contraceptives to manage overabundant populations of wild suids.     

卡巴胆碱改善犬50%TBSA烧伤胃内补液时氧动力学指标

目的:研究卡巴胆碱(CAR)对50%TBSA烧伤休克期胃内补液时氧动力学指标的影响。方法:成年雄性Beagle犬12只,先期无菌手术行颈动、静脉置管和肠造口术,24h后用凝固汽油燃烧法造成50%体表面积Ⅲ度烧伤。随机分为2组(n=6):胃内补液组和胃内补液+CAR组。伤后第一个24h从胃内分别输注葡萄糖-电解质溶液(GES)和含CAR的GES液(20μg/kgCAR溶于GES);伤后24h起实施静脉延迟补液,补液量和速率均根据Park-land公式确定。测定动物非麻醉状态下的平均动脉压(MAP)、肠粘膜血流量(IMBF)和血乳酸(LAC)含量,通过血气分析测定动、静脉氧分压和血氧含量、计算氧供量(DO2)、氧耗量(VO2)和氧摄取(Oext),并统计动物72h死亡数。结果:两组犬MAP和IMBF伤后均显著降低,LAC显著升高;伤后72hMAP回升至0h水平,但IMBF和LAC仍低于或高于0h水平。伤后2h胃内补液+CAR组MAP显著高于胃内补液组(P0.01),但之后两组MAP水平无统计学差别。胃内补液/CAR组IMBF伤后高于胃内补液组,伤后8h起LAC也显著低于胃内补液组(P0.05或P0.01)。伤后两组犬DO、VO2和Oext水平较伤前均显著降低(P0.01),伤后72hDO2恢复至0h水平,但VO2和Oext仍显著低于0h。胃内补液/CAR组DO2、VO2和Oext水平伤后8h起始终高于胃内补液组(P0.05或P0.01)。伤后72h胃内补液组死亡数为3/6,胃内补液/CAR为2/6。结论:50%TBSA烧伤胃内补液时加入卡巴胆碱能显著改善氧动力学指标,降低高乳酸血症,提高口服液体复苏的疗效。     

Ultrasound, microbubbles and the blood-brain barrier

The blood–brain barrier (BBB) is a specialized system of capillary endothelial cells that protects the brain from harmful substances in the blood stream, while supplying the brain with the required nutrients for proper function. The BBB controls transport through both tight junctions and metabolic barriers and is often a rate-limiting factor in determining permeation of therapeutic drugs into the brain. It is a significant obstacle for delivery of both small molecules and macromolecular agents. Although many drugs could be potentially used to treat brain disease, there has been no method that allows non-invasive-targeted delivery through the BBB. Recently, promising studies indicate that ultrasound can be used to locally deliver a drug or gene to a specific region of interest in the brain. If microbubbles are combined with ultrasound exposure, the effects of ultrasound can be focused upon the vasculature to reduce the acoustic intensity needed to produce BBB opening. Several avenues of transcapillary passage after ultrasound sonication have been identified including transcytosis, passage through endothelial cell cytoplasmic openings, opening of tight junctions and free passage through injured endothelium. This article reviews the topic of transient disruption of the BBB with ultrasound and microbubbles and addresses related safety issues. It also discusses possible roles of the BBB in brain disease and potential interactions with ultrasound and microbubbles in such disease states.     

Recombinant protein production by large-scale transient gene expression in mammalian cells: state of the art and future perspectives

The expansion of the biologics pipeline depends on the identification of candidate proteins for clinical trials. Speed is one of the critical issues, and the rapid production of high quality, research-grade material for preclinical studies by transient gene expression (TGE) is addressing this factor in an impressive way: following DNA transfection, the production phase for TGE is usually 2-10 days. Recombinant proteins (r-proteins) produced by TGE can therefore enter the drug development and screening process in a very short time--weeks. With "classical" approaches to protein expression from mammalian cells, it takes months to establish a productive host cell line. This article summarizes efforts in industry and academia to use TGE to produce tens to hundreds of milligrams of r-proteins for either fundamental research or preclinical studies.     

Synthesis and characterization of semi-interpenetrating polymer network microspheres of acrylamide grafted dextran and chitosan for controlled release of acyclovir

Semi-interpenetrating polymer network (IPN) microspheres of acrylamide grafted on dextran (AAm-g-Dex) and chitosan (CS) were prepared by emulsion-crosslinking method using glutaraldehyde (GA) as a crosslinker. The grafting efficiency was found to be 94%. Acyclovir, an antiviral drug with limited water solubility, was successfully encapsulated into IPN microspheres by varying the ratio of AAm-g-Dex and CS, % drug loading and amount of GA. Microspheres were characterized by FT-IR spectroscopy to assess the formation of IPN structure and to confirm the absence of chemical interactions between drug, polymer and crosslinking agent. Particle size was measured using laser light scattering technique. Microspheres with average particle sizes in the range of 265–388 μm were obtained. Differential scanning calorimetry (DSC) and X-ray diffraction (X-RD) studies were performed to understand the crystalline nature of drug after encapsulation into IPN microspheres. Acyclovir encapsulation of up to 79.6% was achieved as measured by UV spectroscopy. Both equilibrium and dynamic swelling studies were performed in 0.1 N HCl. Diffusion coefficients (D) and diffusional exponents (n) for water transport were determined using an empirical equation. In vitro release studies indicated the dependence of drug release rates on both the extent of crosslinking and amount of AAm-g-Dex used in preparing microspheres; the slow release was extended up to 12 h. The release rates were fitted to an empirical equation to compute the diffusional exponent (n), which indicated non-Fickian trend for the release of acyclovir.