Oya Alpar

TMC–MCC (N-trimethyl chitosan–mono-N-carboxymethyl chitosan) nanocomplexes for mucosal delivery of vaccines

In this study, for the first time, TMC/MCC complex nanoparticles as a delivery system and as an adjuvant were developed and evaluated to obtain systemic and mucosal immune responses against nasally administered tetanus toxoid (TT). Nanoparticles were developed by complexation between the oppositely charged chitosan derivatives, N-trimethyl chitosan (TMC, polycationic) and mono-N-carboxymethyl chitosan (MCC, polyampholytic) without using any crosslinker for mucosal vaccination. The cellular viability was found to be higher with TMC/MCC complex compared to that of MCC and TMC alone. Size, zeta potential and morphology of the nanoparticles were investigated as a function of preparation method. Nanoparticles with high loading efficacy (95%) and positively charged surface were obtained with an average particle size of 283+/-2.5 nm. The structural integrity of the TT in the nanoparticles was confirmed by SDS-PAGE electrophoresis analysis. Cellular uptake studies indicated that FITC-BSA loaded nanoparticles were effectively taken up into the mouse Balb/c monocyte macrophages. Mice were nasally immunized with TT loaded TMC/MCC complex nanoparticles and compared to that of TMC and MCC nanoparticles. TMC/MCC complex nanoparticles were shown to induce both the mucosal and systemic immune response indicating that this newly developed system has potential for mucosal administration of vaccines.

An information rich biomedical polymer library

We have designed and prepared a library of polymers with the aim to use a minimum number of polymers to obtain relevant information about structure–property correlations. An active ester homopolymer precursor and two blocked copolymer active ester precursor polymers were used to prepare a library of 16 cationic functionalised polymethacrylamides. These precursor polymers are narrow molecular weight distribution (MWD) polymers prepared by copper mediated polymerisation. This allows a precursor polymer to be used to generate a family of functionalised polymers for study, all with the same molecular weight characteristics. The polycations were then prepared by the sequential conjugation of two amines from the following set: (2-aminoethyl)trimethylammonium chloride hydrochloride (TMA), 3-(dimethylamino)propylamine (DMA), histamine (His) and 1-amino-2-propanol (AP). The conjugates contained different amounts of tertiary amine, quaternary ammonium, imidazole and hydroxypropyl pendent moieties that were selected to provide differing degrees of charge density along the polymer main chain. The molecular weight characteristics, viscosities and aqueous solubility of the polycations were evaluated at pH 4 and 7 to determine property trends. These conjugates were assessed for their ability to complex plasmid DNA at pH 7.4 and 4.5 by agarose gel electrophoresis. Triple detection GPC analysis at different pH values relevant to the cellular environment and photon correlation spectroscopy results indicated that some polycations formed interpolyelectrolyte complexes of under 100 nm. Preliminary titration and cellular biocompatibility studies are also described.

Antibody and cytokine-associated immune responses to S. equi antigens entrapped in PLA nanospheres

Strangles is an infectious disease caused by Streptococcus equi subspecies equi that affects the upper respiratory tract of the Equidae. The control of this disease seems to be dependent on its earlier detection and prevention, but prolonged animal protection without development of strong and severe side effects has not yet been achieved. Convalescent horses exhibit a protective immune response, mainly against SeM (58 kDa), an antiphagocytic and opsonogenic S. equi M-like protein, known as the major protective antigen against strangles. Purified recombinant SeM and S. equi protein extract-entrapped poly(lactic acid) (PLA) nanospheres were developed and their adjuvant potential was studied via the intramuscular route. The effect including molecules with adjuvant properties such as spermine, oleic acid, alginate and glycol-chitosan was also evaluated. Spherical nanometric particles <500 nm containing the protein antigen were prepared by the solvent evaporation method and protein structure was not affected throughout preparation. The humoral immune response induced by nanospheres was markedly higher than that elicited by soluble antigens, isolated or co-admixed with CpG. The IgG and IgG subtypes, along with cytokine titres, indicated that nanospheres composed by glycolchitosan developed a more balanced Th1/Th2 response for both purified SeM and S. equi enzymatic extract proteins, although those induced by the pure antigen-entrapped particles were higher than the S. equi tested vaccines composed by total antigens entrapped in polymeric nanospheres.

The compression properties of lactose

The tabletting characteristics of crystalline and spray dried lactose under direct compression have been examined together with the effect of particle size, shape and storage of the powders. The results indicated that the particle size had little effect, although there was a general tendency for the compact strength to increase as the particle size decreased. On the other hand, the particle shape and storage of the powder influenced the strength of the tablets and the force lost to the die wall. The 22 μm and 35 μm fractions of spray dried lactose—especially the latter—were almost entirely very regular spherical particles which resulted in the strongest tablets, whereas fractions below or above contained many more angular particles resulting in weaker tablets. Storage of the small particle size fraction of the crystalline material appeared to induce aggregation, and on compaction, a harder tablet was formed, accompanied by a decrease in the force lost to the die wall.

Enhanced properties of discrete pulmonary deoxyribonuclease I (DNaseI) loaded PLGA nanoparticles during encapsulation and activity determination

In the present work, DNaseI loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) for pulmonary delivery were prepared using emulsion solvent evaporation. The effects of the various formulation and experimental variables on the size and morphological characteristics of the particles as well as on the encapsulation efficiency were investigated. The stability of the encapsulated DNaseI was evaluated and the respirable fraction was determined. Cytotoxicity of the NPs was evaluated on lung epithelial cells. The results showed that by using leucine and dipalmito-phosphatidyl-choline (DPPC), discrete NPs with 76% retained biological activity were prepared. A high respirable fraction (particles below 6 μm) reaching 71.3% was achieved after nebulization of the NP suspension. The results revealed the suitability of the prepared particles for pulmonary delivery and highlighted the role of excipients in the stabilization of DNaseI against the stresses encountered during preparation.

Bluetongue virus infection induces aberrant mitosis in mammalian cells.

BackgroundBluetongue virus (BTV) is an arbovirus that is responsible for ‘bluetongue’, an economically important disease of livestock. Although BTV is well characterised at the protein level, less is known regarding its interaction with host cells. During studies of virus inclusion body formation we observed what appeared to be a large proportion of cells in mitosis. Although the modulation of the cell cycle is well established for many viruses, this was a novel observation for BTV. We therefore undertook a study to reveal in more depth the impact of BTV upon cell division.MethodsWe used a confocal microscopy approach to investigate the localisation of BTV proteins in a cellular context with their respective position relative to cellular proteins. In addition, to quantitatively assess the frequency of aberrant mitosis induction by the viral non-structural protein (NS) 2 we utilised live cell imaging to monitor HeLa-mCherry tubulin cells transfected with a plasmid expressing NS2.ResultsOur data showed that these ‘aberrant mitoses’ can be induced in multiple cell types and by different strains of BTV. Further study confirmed multiplication of the centrosomes, each resulting in a separate mitotic spindle during mitosis. Interestingly, the BTV NS1 protein was strongly localised to the centrosomal regions. In a separate, yet related observation, the BTV NS2 protein was co-localised with the condensed chromosomes to a region suggestive of the kinetochore. Live cell imaging revealed that expression of an EGFP-NS2 fusion protein in HeLa-mCherry tubulin cells also results in mitotic defects.ConclusionsWe hypothesise that NS2 is a microtubule cargo protein that may inadvertently disrupt the interaction of microtubule tips with the kinetochores during mitosis. Furthermore, the BTV NS1 protein was distinctly localised to a region encompassing the centrosome and may therefore be, at least in part, responsible for the disruption of the centrosome as observed in BTV infected mammalian cells.