Cubosomes enhance drug permeability across the blood-brain barrier in zebrafish.

The potential of cubosomes to improve delivery of incorporated cargo to the brain was explored in zebrafish. Cubosomes were formulated with one of three stabilisers, Pluronic F68, Pluronic F127 or Tween 80, with the hypothesis that coating with Tween 80 will enable brain targeting of cubosomes as has been previously shown for polymeric nanoparticles. The physiochemical properties and the ability of the cubosomes to facilitate delivery of the model drug lissamine rhodamine (RhoB) into the brain was investigated. Distribution of cubosomes in the midbrain was also investigated by ultrastructural analysis via incorporation of octanethiol-functionalized gold nanoparticles. Cubosomes were typically 165-195 nm in size with a Pn3m (Pluronics) or Im3m (Tween 80) cubic phase internal structure. Cubosomes were injected intravenously into zebrafish larvae (12-14 days post fertilization) and the concentration of RhoB in the midbrain was determined by quantifying its fluorescence intensity. Uptake of RhoB was significantly greater in larvae injected with Tween 80 stabilized cubosomes as compared to a control suspension of RhoB or cubosomes stabilized with Pluronics. Collectively, we show for the first time that cubosomes can be functionalized to deliver drug across the BBB, offering new opportunities to overcome drug delivery issues across this formidable biological barrier.

Utilization of Microfluidics for the Preparation of Polymeric Nanoparticles for the Antioxidant Rutin: A Comparison with Bulk Production.

OBJECTIVE: To compare the characteristics of rutin-loaded PLGA (poly(lactic-coglycolic acid)) nanoparticles prepared using a single emulsion evaporation method (bulk method) and a nanoprecipitation method using microfluidics. METHODS: Rutin-loaded PLGA nanoparticles were produced using different methods and characterized for size, zeta potential, entrapment efficiency (EE) and drug loading (DL). A design of experiments approach was used to identify the effect of method parameters to optimize the formulation. DSC was used to investigate the solid-state characteristics of rutin and PLGA and identify any interactions in the rutin-loaded PLGA nanoparticles. The release of rutin from PLGA nanoparticles was examined in biorelevant media and phosphate buffer (PBS). RESULTS: The optimal formulation of rutin-loaded PLGA nanoparticles produced using a microfluidics method resulted in a higher entrapment efficiency of 34 ± 2% and a smaller size of 123 ± 4 nm compared to a bulk method (EE 27 ± 1%, size 179 ± 13 nm). The solidstate of rutin and PLGA changed from crystalline to amorphous with the preparation of rutin- loaded PLGA nanoparticles. More importantly, using microfluidics, rutin released faster from rutin-loaded PLGA nanoparticles in biorelevant media and PBS with higher burst release compared to the rutin release from the nanoparticles prepared by using the bulk method. CONCLUSION: Rutin can be encapsulated in nanoparticles formulated with different methods with mean sizes of less than 200 nm. Microfluidics produced more uniform rutin-loaded PLGA nanoparticles with a higher EE, DL and faster release compared to a bulk production method.

Are phytosomes a superior nanodelivery system for the antioxidant rutin?

Rutin, a strong antioxidant, has been implicated in the prevention of liver inflammation. However, low solubility and permeability through the gut wall limit development of rutin as a therapeutic agent for oral administration. Phytosomes are described as lipid nanocarriers with a complexation between the phospholipid headgroups and entrapped phytochemicals. The aim of this research was to compare the structure of rutin liposomes to rutin phytosomes. FT-IR, DSC and NMR were employed to investigate the presence of any molecular interactions between the formulation components. The FT-IR spectra showed that a new -OH bond had formed in the rutin phytosomes, suggesting the formation of a molecular complex. 31P NMR experiments revealed that the DPPC molecule is altered when formulated as liposomes but that these changes were greater for samples from the phytosome formulation. DSC data revealed that when rutin was added to DPPC there was a significant shift in the transition temperature of DPPC. Further, the shift was greater in the THF solvent used to produce phytosomes compared to CHCl3 used to produce liposomes. 1H NMR spectra of the phytosome samples indicated three additional peaks that were greater than in the liposome formulation. ROESY NMR spectra provided evidence supporting the interaction between rutin and DPPC in both liposomes and phytosomes. The apparent differences in molecular interaction between liposomes and phytosomes did not however impact rutin release in biorelevant media or during in vitro small intestinal lipolysis.

Improved Antitumor Activity of a Therapeutic Melanoma Vaccine through the Use of the Dual COX-2/5-LO Inhibitor Licofelone.

Immune-suppressive cell populations impair antitumor immunity and can contribute to the failure of immune therapeutic approaches. We hypothesized that the non-steroidal anti-inflammatory drug licofelone, a dual cyclooxygenase-2/5-LO inhibitor, would improve therapeutic melanoma vaccination by reducing immune-suppressive cell populations. Therefore, licofelone was administered after tumor implantation, either alone or in combination with a peptide vaccine containing a long tyrosinase-related protein 2-peptide and the adjuvant α-galactosylceramide, all formulated into cationic liposomes. Mice immunized with the long-peptide vaccine and licofelone showed delayed tumor growth compared to mice given the vaccine alone. This protection was associated with a lower frequency of immature myeloid cells (IMCs) in the bone marrow (BM) and spleen of tumor-inoculated mice. When investigating the effect of licofelone on IMCs in vitro, we found that the prostaglandin E2-induced generation of IMCs was decreased in the presence of licofelone. Furthermore, pre-incubation of BM cells differentiated under IMC-inducing conditions with licofelone reduced the secretion of cytokines interleukin (IL)-10 and -6 upon lipopolysaccharides (LPS) stimulation as compared to untreated cells. Interestingly, licofelone increased IL-6 and IL-10 secretion when administered after the LPS stimulus, demonstrating an environment-dependent effect of licofelone. Our findings support the use of licofelone to reduce tumor-promoting cell populations.

Chitosan gel vaccine protects against tumour growth in an intracaecal mouse model of cancer by modulating systemic immune responses.

BACKGROUND: Vaccination generating a robust memory population of CD8+ T cells may provide protection against cancer. However, immune therapies for cancer are influenced by the local tumour immune microenvironment. An infiltrate of T cells into tumours of people with colorectal cancer has proven to be a significant indicator of good prognosis. METHODS: We used an intracaecal mouse model of cancer to determine whether a protective immune response against a mucosal gut tumour could be generated using a systemic intervention. We investigated the generation of murine memory CD8+ T cells using a sustained antigen release vaccine vehicle (chitosan gel; Gel + OVA) containing the model antigen ovalbumin, chitosan gel alone (Gel) or conventional dendritic cell vaccination (DC + OVA) using the same protein antigen. RESULTS: Following vaccination with Gel + OVA, CD8+ T cell memory populations specific for ovalbumin protein were detected. Only vaccination with Gel + OVA gave decreased tumour burden compared to unvaccinated or DC + OVA-vaccinated mice in the intracaecal cancer challenge model. CONCLUSION: These results indicate that subcutaneous vaccination with Gel + OVA generates a population of functional CD8+ memory T cells in lymphoid tissue able to protect against intracaecal tumour challenge. Vaccination with chitosan gel may be valuable in anti-cancer treatment at both peripheral and mucosal sites.

Reasons for use and non-use of the pertussis vaccine during pregnancy: an interview study.

INTRODUCTION In New Zealand, pertussis vaccination is recommended and government-funded during every pregnancy to protect the infant after birth. However, uptake is low and needs to be increased. AIM To investigate enablers and barriers for uptake of the pertussis vaccination by pregnant women in New Zealand, and explore the acceptability of provision in pharmacies. METHODS Women with infants were recruited in selected pharmacies and interviewed using abrief structured interview. Transcripts were analysed using a framework approach. RESULTS Thirty-seven women aged 18-43 years provided data for analysis. Seventeen women reported receiving a pertussis vaccination during their pregnancy. Information from health professionals appeared important to encourage vaccination, but other sources of information (eg antenatal groups and media) were also cited. Non-vaccination arose from being unaware of the need for pertussis vaccination during pregnancy, concerns about safety, and misinformation. Participants supported pertussis vaccination in pharmacies to help access or increase the opportunity for health professionals to inform women. DISCUSSION The information received by participants affected their uptake of the pertussis vaccine during pregnancy. Education of the public and health professionals about the pertussis vaccine during pregnancy is necessary.

Smad2: a candidate gene for the murine autoimmune diabetes locus Idd21.1.

CONTEXT: Congenic NOD.ABH(D18Mit8-D18Mit214) mice, which contain greater than 12.8 Mb of DNA encompassing Idd21.1 from diabetes-resistant Biozzi/ABH mice, have a lower frequency of diabetes compared with the parental nonobese diabetic (NOD) strain, possibly due to reduced pathogenicity of ß-islet-infiltrating immune cells. OBJECTIVE: The objective of the study was to identify an Idd21.1 candidate gene. METHODS: The methods used in the study were adoptive transfer into scid mice lacking an adaptive immune system; dendritic cell phenotyping and gene expression analysis; and fine-mapping Idd21.1 by congenic mapping. RESULTS: Diabetes incidences of NOD.scid.ABH(D18Mit8-D18Mit214) mice receiving splenocytes from NOD and NOD.ABH(D18Mit8-D18Mit214) were similar to that previously observed in NOD.scid recipients, suggesting that the diabetes resistance in NOD.ABH(D18Mit8-D18Mit214) is primarily mediated by the adaptive immune system, findings supported by adoptive transfer of CD4(+) T cells. In activated dendritic cells, there were no conclusive differences in cytokine profiles and activation marker expression. However, microarray analysis comparing gene expression between activated dendritic cells from NOD and NOD.ABH (D18Mit8-D18Mit214) revealed that Smad2, in a maximal 6.5-Mb region to which Idd21.1 was further resolved by congenic mapping, was differentially expressed (increased in NOD). Quantitative real-time PCR confirmed the differential expression of Smad2, and other genes in the TGF-ß signaling pathway, in activated dendritic cells. CONCLUSIONS: These results implicate Smad2 as an Idd21.1 candidate and Smad2 and the TGF-ß signaling pathway in activated dendritic cells in diabetogenesis. With suggestive evidence from human genome-wide association studies supporting a role for SMAD7 in human type 1 diabetes, a comprehensive genetic investigation of the SMAD genes in type 1 diabetes is warranted.

Mannosylated saponins based on oleanolic and glycyrrhizic acids. Towards synthetic colloidal antigen delivery systems.

Immunostimulatory saponin based colloidal antigen delivery systems show promise as adjuvants for subunit vaccines. For this reason, allyl oleanolate was glycosylated at the 3-position using trichloroacetimidate donors to give monodesmodic saponins following deprotection. Bisdesmodic saponins were synthesized by double glycosylation at the 3- and 28-positions of oleanolic acid. When formulated together with cholesterol and phospholipids, ring-like, helical and rod-like nanostructures were formed depending on the saponin concentrations used. As an indication of adjuvant activity, the ability of these formulations, and the saponins by themselves, to induce dendritic cell maturation was measured, but no significant activity was observed.

Oral insulin delivery using nanoparticles based on microemulsions with different structure-types: optimisation and in vivo evaluation.

The purpose of this study was to optimise entrapment of insulin in poly(alkylcyanoacrylate) nanoparticles prepared from microemulsions with different microstructure containing isopropyl myristate, caprylocaproyl macrogolglycerides, polyglyceryl oleate and insulin solution and to investigate the in vitro release and bioactivity of insulin in nanoparticles dispersed in the microemulsion templates. Entrapment efficiency and release of insulin were studied using a reverse-phase HPLC assay. Morphology of the nanoparticles was examined with scanning electron microscopy. Bioactivity of insulin was studied using a streptozotocin-diabetic rat model. Nanoparticles were spherical with 200-400 nm in size without significant difference between different microemulsion templates, types and amounts of monomer. Entrapment efficiency increased significantly with increasing monomer concentration but decreased with increasing aqueous fraction in the microemulsion template. Insulin loading however, showed an opposite trend. In vitro release profiles of insulin from the nanoparticles dispersed in the microemulsion templates were controlled by the monomer concentration only. In vivo, a consistent and significant hypoglycemic effect over controls was found for up to 36 h depending on the type of monomer. No significant serum insulin levels were detectable. This study showed that the strategy of delivering insulin orally, entrapped in nanoparticles and dispersed in a biocompatible microemulsion is promising and highlights the importance of optimisation studies in combination with in vivo experiments.

Distribution of fibroblast growth factor-2 (FGF-2) within model excisional wounds following topical application.

OBJECTIVE: To characterise the magnitude and distribution of fibroblast growth factor-2 (FGF-2) following topical application in hypromellose gel and film formulations or a solution in an animal wound model, in order to assess the potential of this route for treatment of chronic wounds. METHOD: Topical formulations of FGF-2 were applied to punch biopsy wounds, and FGF-2 levels within the wound measured. Each 12 mm diameter wound received 0.3 microg FGF-2 in solution, a 7% (w/w) hypromellose gel, a dried hypromellose film on Melolin-backing or a saline control. After 2, 5 or 8 h the wounds were horizontally dissected into four sections (surface granulation, subcutaneous fat, superficial muscle and deep muscle) which were then analysed for FGF-2 concentration using ELISA. Confocal microscopy was used to evaluate the distribution of FGF-2 within the wound. KEY FINDINGS: There were significant differences in the mean FGF-2 levels with respect to formulation and time following application (P < 0.05). FGF-2 penetrated faster into tissue when formulated as a solution than as a gel or a film. There did not appear to be a significant difference between the gel and the film with respect to total concentrations achieved in the tissue, although confocal microscopy showed differences in FGF-2 distribution within the wound. CONCLUSIONS: Delivery of FGF-2 to wounds in a solution gave the greatest increase in tissue FGF-2 concentration when measured by ELISA and visualised using confocal microscopy. Gel and film formulations prolonged the release of FGF-2 into the wound, although FGF-2 levels were not significantly different from controls when measured by ELISA. Confocal microscopy highlighted the differences in the penetration and distribution of the FGF-2 within the wound when released from different formulations.

Protein delivery using nanoparticles based on microemulsions with different structure-types.

Poly(alkylcyanoacrylate) nanoparticles based on microemulsions with different structure-types and containing insulin as a model protein were prepared and characterised in this study. A phase diagram of the pseudoternary system isopropyl myristate, caprylocaproyl macrogolglycerides, polyglycerol oleate and water was established. All compounds used in this study were pharmaceutically acceptable and biocompatible. The area in the phase diagram containing optically isotropic, monophasic systems was designated as the microemulsion region. Systems within this region were identified as water-in-oil (w/o), bicontinuous and oil-in-water (o/w) microemulsions with viscosity, conductivity, differential scanning calorimetry and self-diffusion NMR. The size distributions of the resulting nanoparticles prepared by interfacial polymerisation from selected microemulsions using ethyl (2) cyanoacrylate and butyl (2) cyanoacrylate were unimodal but template- and monomer-dependent and ranged from 160 to 400 nm. Entrapment and release of insulin were also studied. Entrapment ranged from 11.5 to 20.9% and a near zero-order release was observed after an initial burst. Release of insulin was monitored for 6h. Insulin-loaded nanoparticles were 320-350 nm in size. The microemulsion-structure was retained during the polymerisation process as determined by NMR. This study showed that these microemulsions with flexible formulation possibilities for the solubilisation of peptides and proteins depending on their microstructure could serve well as a platform for designing encapsulation processes for oral delivery of insulin.

Preparation of poly (alkylcyanoacrylate) nanoparticles by polymerization of water-free microemulsions.

Phase diagrams of the pseudoternary systems ethyloleate, polyoxyethylene 20 sorbitan mono-oleate/sorbitan monolaurate and propylene glycol with and without butanol as a co-surfactant were prepared. Areas containing optically isotropic, one-phase systems were identified and samples therein designated as droplet, bicontinuous or solution type microemulsions using conductivity, viscosity and self-diffusion NMR. Nanoparticles were prepared by polymerization of selected microemulsions with ethyl-2-cyanoacrylate and the morphology of the particles was investigated. Addition of monomer to all types of microemulsions led to the formation of nanoparticles, which had an average size of 244 +/- 25 nm, an average polydispersity index of 0.15 +/- 0.04 and a zeta-potential of -17 +/- 3 mV. The formation of particles from water-free microemulsions of different types is surprising, particularly considering that polymerization is expected to occur at a water-oil interface by base-catalysed polymerization. It would appear that propylene glycol is sufficiently nucleophilic to initiate the polymerization. The use of water-free microemulsions as templates for the preparation of poly (alkylcyanoacrylate) nanoparticles opens up interesting opportunities for the encapsulation of bioactives which do not have suitable properties for encapsulation on the basis of water-containing microemulsions.

Rotavirus hospitalisation in New Zealand children under 3 years of age.

OBJECTIVE: To describe the epidemiology of severe rotavirus gastroenteritis and to estimate the hospitalisation rates of this illness in New Zealand children under 3 years of age. METHODS: Children under 3 years of age with acute diarrhoea admitted to 1 of 8 study hospitals between 1 May 1998 and 30 April 2000 were surveyed. Their socio-demographic, treatment and length-of-stay data were recorded and stool samples tested by a rotavirus-specific enzyme-linked immunoassay. National hospital discharge data for infectious diarrhoea (International Classification of Diseases, ninth revision, 003-009) were reviewed, allowing population-based estimates for rotavirus-related hospitalisation in New Zealand. RESULTS: Of 2019 enrolled children, 1138 (56.4%) provided stools for testing, and of these 485 (42.6%) tested rotavirus positive. Rotavirus detection varied significantly by age (26.8% for 0 to 5 months, 42.5% for 6 to 11 months and 52.1% for children aged 12 to 35 months; P < 0.001), and by season (51.2% in winter/spring vs. 24.5% in summer/autumn; P < 0.001). While those infected with rotavirus were more likely to be dehydrated (50.6% vs. 37.4%; P < 0.001), their median hospital stay was similar (1.0 vs. 2.0 days; P = 0.09) to other children with acute gastroenteritis. The estimated national hospitalisation rate for rotavirus diarrhoea in children under 3 years, standardised for age and season, was 634 (95% CI 597, 672) per 100,000. In New Zealand, rotaviruses result in 1 in 52 children being hospitalised by 3 years of age. CONCLUSIONS: Rotavirus diarrhoea is an important, potentially vaccine-preventable cause of hospitalisation in New Zealand children, especially during winter and spring seasons.

Lower airway inflammation in infants with cystic fibrosis detected by newborn screening.

Controversy exists over whether the lower airway inflammation that characterizes cystic fibrosis (CF) is initiated primarily by the genetic defect. To determine if inflammation precedes infection, we examined bronchoalveolar lavage (BAL) fluid cytology, cytokines (interleukin (IL)-1beta, IL-4, IL-5, IL-6, IL-8, IL-10, and tumor necrosis factor-alpha), and free neutrophil elastase activity from 70 CF (aged 1.5-71 months) children detected by newborn screening and 19 (aged 2.0-48 months) controls with chronic stridor. CF subjects were selected and categorized as pristine (13 aged /= 10(5) colony-forming units/ml of pathogenic bacteria in BAL), and uninfected (15 aged > 6 months, asymptomatic, not taking antibiotics at bronchoscopy, and free of pathogens in their BAL). To further resolve if inflammation develops without infection, inflammatory mediators in paired annual BAL samples from 38 CF subjects were measured, and results were grouped according to whether BAL showed persistence (n = 6), acquisition (n = 8), clearance (n = 13), or absence (n = 11) of infection. While pristine, uninfected, and control subjects had similar BAL profiles, infected patients showed elevated inflammatory indices, including increased IL-10 (P < 0.001). Pristine subjects had the fewest signs of inflammation. Analysis of BAL pairs found differences between the four infection groups for changes in neutrophil percentages, IL-8 (P < 0.001), and free neutrophil elastase (P = 0.009). Infection was associated with elevated inflammatory mediators in BAL fluid. In contrast, minimal or reduced signs of inflammation accompanied absence of eradication of infection from BAL fluid. We conclude that in CF, infection initiates and sustains airway inflammation.

Colocalization of mouse autoimmune diabetes loci Idd21.1 and Idd21.2 with IDDM6 (human) and Iddm3 (rat).

Comparative mapping between the human and rodent genomes is one approach for positional cloning of complex disease loci. The human type 1 diabetes susceptibility locus IDDM6 has orthology with distal rodent chromosome 18, to which Iddm3 has been mapped in rat. Previously, we mapped Idd21 to mouse chromosome 18. Here, the primary aim was to determine whether Idd21 mapped to distal mouse chromosome 18. We constructed novel congenic strains from the consomic NOD-Chr 18(ABH) strain and mapped two loci (Idd21.1 and Idd21.2) to the distal 29.3-Mb portion of mouse chromosome 18, orthologous to IDDM6 (human) and Iddm3 (rat). Idd21.3 was mapped to proximal mouse chromosome 18 (0-21.9 Mb). Although Idd21.1 did not influence beta-islet inflammation, splenocytes from pre-diabetic Idd21.1-congenic mice were less efficient at transferring diabetes to immunodeficient NOD-scid mice. This suggests that Idd21.1 may act by reducing the pathogenicity of islet-infiltrating immune cells. For the first time, the presence of a non-major histocompatibility complex autoimmune diabetes locus colocalizing in three species has been demonstrated; IDDM6 (human), Iddm3 (rat), and now Idd21.1-21.2 in mouse. Further genetic localization of Idd21.1 and Idd21.2 could expedite characterization of the human IDDM6 region.

Bystander suppression of allergic airway inflammation by lung resident memory CD8+ T cells.

CD8+ memory T cells have recently been recognized as playing a key role in natural immunity against unrelated viral infections, a phenomenon referred to as "heterologous antiviral immunity." We now provide data that the cellular immunological interactions that underlie such heterologous immunity can play an equally important role in regulating T helper 2 immune responses and protecting mucosal surfaces from allergen-induced inflammation. Our data show that CD8+ T cells, either retained in the lung after infection with influenza virus, or adoptively transferred via the intranasal route can suppress allergic airway inflammation. The suppression is mediated by IFN-gamma, which acts to reduce the activation level, T helper 2 cytokine production, airways hyperresponsiveness, and migration of allergen-specific CD4+ T cells into the lung, whereas the systemic and draining lymph node responses remain unchanged. Of note, adoptive transfer of previously activated transgenic CD8+ T cells conferred protection against allergic airway inflammation, even in the absence of specific-antigen. Airway resident CD8+ T cells produced IFN-gamma when directly exposed to conditioned media from activated dendritic cells or the proinflammatory cytokines IL-12 and IL-18. Taken together these data indicate that effector/memory CD8+ T cells present in the airways produce IFN-gamma after inflammatory stimuli, independent of specific-antigen, and as a consequence play a key role in modifying the degree and frequency of allergic responses in the lung.