Production of nabumetone nanoparticles: Effect of molecular weight, concentration and nature of cellulose ether stabiliser.

The ability of a range of hydrophilic nonionic cellulose ethers (CEs) (namely methylhydroxethylcellulose, hydroxypropylmethylcellulose, ethylhydroxyethylcellulose, hydroxyethylcellulose and hydroxypropylcellulose) to prepare stable nabumetone nanoparticles (<1000nm, as measured by laser diffraction) using wet-bead milling has been investigated. Due to the limited range of CE molecular weights commercially available, the CEs were degraded using ultrasonication for varying lengths of time to yield CEs of lower molecular weight. Of the CEs tested, only hydroxyethylcellulose was found not to stabilise the production of nabumetone nanoparticles at any of the molecular weights tested, namely viscosity average molecular weights (Mv) in the range of 236-33kg/mol. All other CEs successfully stabilised nabumetone nanoparticles, with the lower molecular weight/viscosity polymers within a series being more likely to result in nanoparticle production than their higher molecular weight counterparts. Unfortunately due to the nature of the ultrasonication process, it was not possible to compare the size of nabumetone particles produced using polymers of identical Mv. There was, however, enough similarity in the Mv of the various polymers to draw the general conclusion that there was no strong correlation between the Mv of the various polymers and their ability to produce nanoparticles. For example hydroxypropylcellulose of 112.2kg/mol or less successfully produced nanoparticles while only ethylhydroxyethylcellulose and hydroxypropylmethyl polymers of 52 and 38.8kg/mol or less produced nanoparticles. These results suggest that polymer molecular weight is not the only determinant of nanoparticle production and that structure of the polymer is at least as important as its molecular weight. In particular the hydrophobic nature of the CE was thought to be an important factor in the production of nabumetone nanoparticles: the more hydrophobic the polymer, the stronger its interaction with nabumetone and the greater its ability to produce nanoparticles. In this context HPC was the most hydrophobic polymer and HEC the least hydrophobic.

Effect of polymer molecular weight on the production of drug nanoparticles.

Stable, polymer-coated nanoparticles of two hydrophobic drugs, namely nabumetone and halofantrine, have been prepared by a wet-bead milling process performed in the presence of a stabilizing homopolymer, either hydroxypropylmethylcellulose (HPMC) or polyvinylpyrrolidone (PVP), of differing molecular weights and concentrations. Although nabumetone nanoparticles could only be produced when HPMC was used as stabilizing polymer, halofantrine nanoparticles could be prepared using either HPMC or PVP. Stable nanoparticles of nabumetone could be produced using a HPMC solution of viscosity average molecular weight, M(v), of 5 kg/mol over an approximate four fold polymer concentration range (0.63-2.5% w/w) when a drug loading of 20% w/w was used. Increasing the molecular weight of HPMC up to a limiting M(v) of 89 kg/mol did not result in the formation of nanoparticles at any of the polymer concentrations examined. The amount of polymer absorbed onto the nanoparticles was determined by measuring the depletion of polymer from solution based on either an ultra-violet (PVP) or optical rotatory dispersion (ORD) (HPMC) assay. The slightly lower concentration of HMPC found to be present on the surface of the halofantrine nanoparticles compared with the nabumetone nanoparticles suggested a differing affinity of the polymer for the surface of the two drugs.

Herpesvirus saimiri open reading frame 50 (Rta) protein reactivates the lytic replication cycle in a persistently infected A549 cell line.

Herpesviruses occur in two distinct forms of infection, lytic replication and latent persistence. In this study, we investigated the molecular mechanisms that govern the latent-lytic switch in the prototype gamma-2 herpesvirus, herpesvirus saimiri (HVS). We utilized a persistently HVS-infected A549 cell line, in which HVS DNA is stably maintained as nonintegrated circular episomes, to assess the role of the open reading frame 50 (ORF 50) (Rta) proteins in the latent-lytic switch. Northern blot analysis and virus recovery assays determined that the ORF 50a gene product, when expressed under the control of a constitutively active promoter, was sufficient to reactivate the entire lytic replication cycle, producing infectious virus particles. Furthermore, although the ORF 50 proteins of HVS strains A11 and C488 are structurally divergent, they were both capable of inducing the lytic replication cycle in this model of HVS latency.

A gamma-2 herpesvirus nucleocytoplasmic shuttle protein interacts with importin alpha 1 and alpha 5.

Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus. This is an increasing important subfamily of herpesviruses due to the identification of the first human gamma-2 herpesvirus, Kaposi's sarcoma-associated herpesvirus. The HVS open reading frame (ORF) 57 protein is a multifunctional trans-regulatory protein homologous to genes identified in all classes of herpesviruses. Recent analysis has demonstrated that ORF 57 has the ability to bind viral RNA and to shuttle between the nucleus and cytoplasm, and is required for efficient nuclear export of viral transcripts. Here we have investigated the nucleocytoplasmic shuttling mechanism utilized by the ORF 57 protein. The yeast two-hybrid system was employed to identify interacting cellular proteins using ORF 57 as bait. We demonstrate that ORF 57 interacts with importin alpha isoforms 1 and 5. In addition, the binding of ORF 57 to importin alpha was mediated by the importin alpha hydrophobic internal armadillo repeats. An ORF 57 amino-terminal arginine-rich sequence, which functions as a nuclear localization sequence, was also required for this interaction. Furthermore, the ORF 57 protein is responsible for the redistribution of importin alpha into the nucleoli. These results identify novel cellular interactions essential for the functioning of this important herpesvirus regulatory protein.

Specific oncolytic activity of herpesvirus saimiri in pancreatic cancer cells.

The potential use of oncolytic viruses in the treatment of cancer has been investigated for some time. A variety of agents have been studied, including some which appear to be selectively replication-competent in cancer cell lines. In this study, we have investigated the ability of herpesvirus saimiri to specifically lyse selected human cancer cell lines. Upon infection with a replication-competent virus carrying the EGFP reporter gene and a neomycin resistance marker, the pancreatic cancer lines MIAPACA and PANC-1 exhibited definite cytopathic effects. In contrast, the colonic carcinoma cell lines SW480 and HCT116 were phenotypically unaltered. In addition, stable cell lines could not be generated from PANC-1 infected cultures, in marked contrast to cultures of cells from other human tissues. Virus recovery assays demonstrated that all of the cell lines produced a small amount of virus post-infection, but that virus replication was minimal after 1 week in culture. In addition, treatment with acyclovir inhibited virus replication but paradoxically increased cytopathic effect. These data suggest that herpesvirus saimiri may have potential as an oncolytic agent for the treatment of pancreatic cancer.

Analysis of gene expression in a human cell line stably transduced with herpesvirus saimiri.

Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus; it has significant homology to the human gammaherpesviruses Kaposi's sarcoma-associated virus and Epstein-Barr virus and the murine gammaherpesvirus murine herpesvirus 68. HVS causes a persistent asymptomatic infection in its natural host, the squirrel monkey. Both subgroups A and C possess the ability to immortalize common marmoset T lymphocytes to interleukin-2-independent proliferation. However, only subgroup C is capable of transforming human, rabbit, and rhesus monkey lymphocytes in vitro. In addition, HVS can stably transduce a variety of human cell lines where the virus persists as a nonintegrating circular episome. In this study, we have developed a system in which the HVS DNA is stably maintained as a nonintegrated circular episome in the human lung carcinoma cell line A549. Virus production can be reactivated using chemical inducing agents, including tetradecanoyl phorbol acetate and n-butyrate, suggesting that the infection in human A549 cells is latent. To analyze virus gene expression in these stably transduced cells, Northern blot analysis was performed using a series of probes produced from restriction fragments spanning the entire coding region of the HVS genome. This demonstrated that an adjacent set of genes containing open reading frames (ORFs) 71 to 73 are expressed in this stably transduced cell line. Moreover, these genes are transcribed as a polycistronic mRNA species produced from a common promoter upstream of ORF 73. This model may serve as a useful tool in the further analysis of the role of ORFs 71 to 73 in gamma-2 herpesvirus latency.