Modified chitosan-based nanoadjuvants enhance immunogenicity of protein antigens after mucosal vaccination.

Nasal vaccination is considered to be an effective and convenient way of increasing immune responses both systemically and locally. Although various nanovaccine carriers have been introduced as potential immune adjuvants, further improvements are still needed before they can be taken to clinical usage. Chitosan-based nanovaccine carriers are one of the most widely studiedadjuvants, owing to the abilityof chitosan toopen tight junctions between nasal epithelial cells and enhance particle uptake as well as its inherent immune activating role.

Nasal vaccination with poly(β-amino ester)-poly(d,l-lactide-co-glycolide) hybrid nanoparticles.

Mucosal vaccination stimulates both mucosal and systemic immunity. However, mucosal applications of vaccine antigens in their free form generally result in poor systemic immune responses and need adjuvantation. In this study, bovine serum albumin loaded, new hybridised poly(β-amino ester)-poly(d,l-lactide-co-glycolide) nanoparticles were prepared by double emulsion-solvent evaporation method, characterised and evaluated in vivo as nasal vaccine carriers.

Development of chitosan-pullulan composite nanoparticles for nasal delivery of vaccines: in vivo studies.

Here, we aimed at developing chitosan/pullulan composite nanoparticles and testing their potential as novel systems for the nasal delivery of diphtheria toxoid (DT). All the chitosan derivatives [N-trimethyl (TMC), chloride and glutamate] and carboxymethyl pullulan (CMP) were synthesised and antigen-loaded composites were prepared by polyion complexation of chitosan and pullulan derivatives (particle size: 239-405 nm; surface charge: +18 and +27 mV). Their immunological effects after intranasal administration to mice were compared to intramuscular route.

Development of chitosan-pullulan composite nanoparticles for nasal delivery of vaccines: optimisation and cellular studies.

Nasal immunisation with nanoparticles has already shown promising results. In this study, nanoparticle composites carrying BSA for nasal vaccination prepared using electrostatic interaction process between polycation N-trimethyl chitosan chloride (TMC), chitosan glutamate (CG), chitosan chloride (CCl) and polyanion carboxymethyl pullulan (CMP). A mass ratio of 2:1 for TMC-CMP combination produced stable nanocarriers.

Impact of surfactant selection on the formulation and characterization of microparticles for pulmonary drug delivery.

The effect of suspension stabilizers, internal aqueous phase volume and polymer amount were investigated for the production of protein loaded poly(d,l lactide-co-glycolide) (PLGA) microparticles suitable for pulmonary drug delivery. PLGA microparticles were produced adopting water-in-oil-in-water (W/O/W) solvent evaporation technique and were investigated for surface morphology, particle size, encapsulation efficiency (EE%) and in-vitro release profile. Porous surface morphologies with a narrow size distribution were observed when employing 0.

Influence of suspension stabilisers on the delivery of protein-loaded porous poly (DL-lactide-co-glycolide) (PLGA) microparticles via pressurised metered dose inhaler (pMDI).

Purpose: This work investigates the feasibility of delivering large (≈ 25 μm) porous poly (lactide-co-glycolide) (PLGA) microparticles containing a model protein via pressurised metered dose inhaler (pMDI).

Methods: Porous PLGA microparticles were prepared by modified double emulsion method as pMDI suspension based systems containing suspension stabilisers in 1,1,1,2,3,3,3-heptafluoropropane (HFA 227). Physical suspension stability was assessed by visual and optical suspension techniques.

Bluetongue virus infection induces aberrant mitosis in mammalian cells.

Background: Bluetongue 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.

Inhalable DNase I microparticles engineered with biologically active excipients.

Highly viscous mucus poses a big challenge for the delivery of particulates carrying therapeutics to patients with cystic fibrosis. In this study, surface modifying DNase I loaded particles using different excipients to achieve better lung deposition, higher enzyme stability or better biological activity had been exploited. For the purpose, controlled release microparticles (MP) were prepared by co-spray drying DNase I with the polymer poly-lactic-co-glycolic acid (PLGA) and the biocompatible lipid surfactant 1,2-dipalmitoyl-Sn-phosphatidyl choline (DPPC) using various hydrophilic excipients.

Protection of IFNAR (-/-) mice against bluetongue virus serotype 8, by heterologous (DNA/rMVA) and homologous (rMVA/rMVA) vaccination, expressing outer-capsid protein VP2.

The protective efficacy of recombinant vaccines expressing serotype 8 bluetongue virus (BTV-8) capsid proteins was tested in a mouse model. The recombinant vaccines comprised plasmid DNA or Modified Vaccinia Ankara viruses encoding BTV VP2, VP5 or VP7 proteins. These constructs were administered alone or in combination using either a homologous prime boost vaccination regime (rMVA/rMVA) or a heterologous vaccination regime (DNA/rMVA).

Spray dried inhalable ciprofloxacin powder with improved aerosolisation and antimicrobial activity.

In this study, the spray drying technique was used to prepare ciprofloxacin microparticles (CFX-MPs) for pulmonary administration. By virtue of its amphoteric properties, CFX was dissolved in either a slightly alkaline or acidic solution depending on the used polymer. Dextran and chitosan were used to prepare the MPs and modify the release characteristics of the drug.

Biolistic transfection of human embryonic kidney (HEK) 293 cells.

Due to its excellent transfectability, the human embryonic kidney (HEK) 293 cell line is widely used as an in vitro model system for transfection experiments. Particle bombardment, or biolistics technology, provides a physical transfection approach that can deliver transgene materials efficiently into many different cell lines. Transfection of 293 cells by gene gun, allows examination of transgene expression in epithelial cells, as well as studies concerning a variety of questions in neurobiology.

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.

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.

An investigation into the combination of low frequency ultrasound and liposomes on skin permeability.

Antigen application onto skin that has been pre-treated with low frequency ultrasound leads to immunisation, and it was hypothesised that immunisation could be enhanced if antigens were entrapped within liposomes, the latter being known vaccine adjuvants. However, it has been suggested that liposomes can repair skin damage, which could limit antigen permeation and transcutaneous immunisation. The aim of the present work was therefore to investigate the influence of liposome application on subsequent: (i) in vitro antigen permeation through, and (ii) in vivo barrier properties of, ultrasound-treated skin.

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.

Transfection by particle bombardment: delivery of plasmid DNA into mammalian cells using gene gun.

Background: Recently, particle bombardment has become increasingly popular as a transfection method, because of a reduced dependency on target cell characteristics. In this study, we evaluated in vitro gene transfer by particle bombardment.

Methods: gWIZ luciferase and gWIZ green fluorescent protein (GFP) plasmids were used as reporter genes.

Simultaneously manufactured nano-in-micro (SIMANIM) particles for dry-powder modified-release delivery of antibodies.

Simultaneously Manufactured Nano-In-Micro (SIMANIM) particles for the pulmonary delivery of antibodies have been prepared by the spray-drying of a double-emulsion containing human IgG (as a model antibody), lactose, poly(lactide-co-glycolide) (PLGA) and dipalmitoylphosphatidylcholine (DPPC). The one-step drying process involved producing microparticles of a diameter suitable for inhalation that upon contact with aqueous media, partially dissolved to form nanoparticles, approximately 10-fold smaller than their original diameter. Continuous release of the model antibody was observed for 35 days in pH 2.

Evaluation of different buffers on plasmid DNA encapsulation into PLGA microparticles.

Double emulsion solvent evaporation is a widely used method to prepare poly(dl-lactide-co-glycolide) (PLGA) microparticles encapsulating plasmid DNA. There are inherent problems associated with preparing plasmid DNA in this form, in particular the DNA is liable to degrade during manufacture and the resulting powder has low encapsulation efficiencies. This study compares the use of two buffers, 0.

Development and testing of particulate formulations for the nasal delivery of antibodies.

Therapeutic antibodies offer a potential treatment for, or means of protection against, airborne infections. For this application, it may be desirable to deliver the antibody directly into the nasal cavity, one of its potential sites of action, since this would be more efficient and convenient than systemic administration. Formulations of a model antibody (human IgG) were developed using albumin, sodium chloride and disaccharides.

Transcutaneous immunisation assisted by low-frequency ultrasound.

Low-frequency ultrasound application is known to increase the skin's permeability to large molecules such as vaccines, and to enable transcutaneous immunisation. Sodium dodecyl sulphate (SDS) - a skin irritant - is often included in the coupling medium at 1% (w/v), as this has been found to enhance skin permeability. In this paper we show, for the first time, the feasibility of low-frequency ultrasound-assisted transcutaneous immunisation in the absence of SDS.

Preparation of polyethyleneimine incorporated poly(D,L-lactide-co-glycolide) nanoparticles by spontaneous emulsion diffusion method for small interfering RNA delivery.

Gene therapy based on small interfering RNA (siRNA) has emerged as an exciting new therapeutic approach. However, insufficient cellular uptake and poor stability have limited its usefulness. Polyethyleneimine (PEI) has been extensively studied as a vector for nucleic acids and incorporation of PEI into poly(d,l-lactide-co-glycolide) (PLGA) particles has been shown to be useful in the development of gene delivery.

Effect of preparative variables on small interfering RNA loaded Poly(D,L-lactide-co-glycolide)-chitosan submicron particles prepared by emulsification diffusion method.

In this study, poly(D,L-lactide-co-glycolide) (PLGA)-chitosan particles were investigated as an effective delivery system for small interfering RNA (siRNA) by emulsification diffusion method. The type, molecular weight and concentration of chitosan, PLGA type as well as centrifugation and freeze-drying process were amongst the investigated variables. PLGA-chitosan particles obtained were positively charged with particle size between approximately 0.

Enhancement of immune response of HBsAg loaded poly (L-lactic acid) microspheres against hepatitis B through incorporation of alum and chitosan.

Purpose: Poly (L-lactic acid) (PLA) microparticles encapsulating Hepatitis B surface antigen (HBsAg) with alum and chitosan were investigated for their potential as a vaccine delivery system.

Methods: The microparticles, prepared using a water-in-oil-in-water (w/o/w) double emulsion solvent evaporation method with polyvinyl alcohol (PVA) or chitosan as the external phase stabilising agent showed a significant increase in the encapsulation efficiency of the antigen.

Results: PLA-Alum and PLA-chitosan microparticles induced HBsAg serum specific IgG antibody responses significantly higher than PLA only microparticles and free antigen following subcutaneous administration.

Development and characterisation of chitosan nanoparticles for siRNA delivery.

Gene silencing mediated by double-stranded small interfering RNA (siRNA) has been widely investigated as a potential therapeutic approach for diseases with genetic defects. The use of siRNA, however, is hampered by its rapid degradation and poor cellular uptake into cells in vitro or in vivo. Therefore, we have explored chitosan as a siRNA vector due to its advantages such as low toxicity, biodegradability and biocompatibility.

Protection against bubonic and pneumonic plague with a single dose microencapsulated sub-unit vaccine.

Protection against virulent plague challenge by the parenteral and aerosol routes was afforded by a single administration of microencapsulated Caf1 and LcrV antigens from Yersinia pestis in BALB/c mice. Recombinant Caf1 and LcrV were individually encapsulated in polymeric microspheres, to the surface of which additional antigen was adsorbed. The microspheres containing either Caf1 or LcrV were blended and used to immunise mice on a single occasion, by either the intra-nasal or intra-muscular route.