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. In present study, bovine serum albumin (BSA) loaded nanoparticles were prepared using novel aminated (aChi) and aminated plus thiolated chitosan (atChi) polymers, to further enhance mucoadhesiveness and adjuvanticity of the vaccine system by improving electrostatic interactions of polymers with negatively charged glycoproteins. Nanocarriers with optimum size and surface charge, high encapsulation efficiency of model antigen and good stability were developed. Negligible toxicity was observed in Calu-3 and A549 cell lines. In vivo studies, revealed high levels of systemic antibodies (IgG, IgG1 and IgG2a) throughout the study and presence of sIgA in vaginal washes showed that common mucosal system was successfully stimulated. Cytokine levels indicated a mixed Th1/Th2 immune response. A shift towards cellular immune responses was observed after nasal immunisation with antigen loaded nanoparticle formulations. These nanoparticles exhibit great potential for nasal application of vaccines.

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. For CCl-CMP and CG-CMP formulations needed a mass ratio of 3:1. Loading efficiency was >90% for all formulations. Nanoparticles' size ranged from 207 to 603 nm. The surface charge of the complexes varied between +14 and +33 mV. SDS-PAGE integrity of the model antigen was also demonstrated. MTT studies showed that nanoparticle composites were less toxic to Calu-3 cells than the particles of cationic polymers alone. FITC-BSA loaded nanoparticles efficiently taken up by J774A.1 macrophages as confirmed by confocal microscopy highlighting the potential of these novel nanoparticulate carriers' use for nasal vaccination.

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. Composite nanoparticles induced higher levels of IgG responses than particles formed with chitosan derivative and antigen. Nasally administered TMC-pullulan composites showed higher DT serum IgG titre when compared with the other composites. Co-encapsulation of CpG ODN within TMC-CMP-DT nanoparticles resulted in a balanced Th1/Th2 response. TMC/pullulan composite nanoparticles also induced highest cytokine levels compared to those of chitosan salts. These findings demonstrated that TMC-CMP-DT composite nanoparticles are promising delivery system for nasal vaccination.

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. The present study of transfection of HEK 293 cells by biolistics technology uses the plasmids gWIZ-luc encoding luciferase and gWIZ-GFP encoding green fluorescence protein (GFP) as model transgenes. This system can be routinely used at varying bombarding conditions that can be adjusted according to experimental requirements and purpose, such as gene gun helium pressure, the sizes and the amount of the gold particles and the length of the spacer. The results obtained show that the Bio-Rad spacer for the gene gun should be optimized for travel distance and spreading of gold particles over a relatively small area, when used for biolistic transfection of cells dispersed in multi-well plate.

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. Sonication was conducted using either phosphate buffered saline (PBS) or an aqueous solution of sodium dodecyl sulphate (SDS) as the coupling medium, and rats were used as the animal models. Liposome application to sonicated skin reduced antigen penetration and transepidermal water loss (TEWL, used as an indication of skin integrity) when the skin had been sonicated using PBS coupling medium. The influence of liposome was evident within 5min of its application, and smaller liposomes were more effective at repairing skin disruption caused by sonication. Such skin repair did not, however, take place when the skin had been sonicated in the presence of SDS (which caused greater skin disruption), and changes in in vitro antigen permeation and in vivo TEWL were negligible. Skin repair by liposomes seems to depend on the extent of the disruption caused by ultrasound application.

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. Mammalian cell lines HEK 293, MCF7 and NIH/3T3 were used in the transfection experiments. Transfection was performed by bombardment of the cells with gene-coated gold particles using the Helios Gene Gun. The technology was assessed by analyzing gene expression and cell damage. Cell damage was evaluated by MTT assay. RESULTS: This technology resulted in efficient in vitro transfection, even in the cells which are difficult to transfect. The gene expression was dependent on the gene gun's helium pressure, the sizes of the gold particles, the amount of the particles and DNA loading, while cell viability was mostly dependent on helium pressure and amount of the gold particles. CONCLUSIONS: This technology was useful to transfection of cells. Optimal transfection conditions were determined to be between 75 and 100 psi of helium pressure, 1.0 to 1.6 mum gold particle size and 0.5 mg of gold particle amount with a loading ratio of 4 microg DNA/mg gold particles. GENERAL SIGNIFICANCE: These findings will be useful in the design of gene gun device, and bring further improvements to the in vitro and in vivo transfection studies including gene therapy and vaccination.

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 2.5 release media, and released antibody was shown to be stable and active by gel electrophoresis, field-flow fractionation and enzyme linked immunosorbent assay. Adding 1% L-leucine to the emulsion formulation, and blending 'SIMANIM' particles with 1% magnesium stearate, achieved a fine particle fraction of approximately 60%, when aerosolised from a simple, capsule-based, dry powder inhaler device. 'SIMANIM' particles could be beneficial for the delivery of antibodies targeted against inhaled pathogens or other extracellular antigens, as well as having potential applications in the delivery of a wide range of other biopharmaceuticals and certain small-molecule drugs.

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. PEI was incorporated into the PLGA particles by spontaneous modified emulsification diffusion method. Incorporation of PEI into PLGA particles with the PLGA to PEI weight ratio 29:1 was found to produce spherical and positively charged nanoparticles where type of polymer, type and concentration of surfactant could affect their physical properties. Particle size of around 100nm was obtained when 5% (m/v) PVA was used as a stabiliser. PLGA-PEI nanoparticles were able to completely bind siRNA at N/P ratio 20:1 and to provide protection for siRNA against nuclease degradation. In vitro cell culture studies subsequently revealed that PLGA-PEI nanoparticles with adsorbed siRNA could efficiently silence the targeted gene in mammalian cells, better than PEI alone, with acceptable cell viability. PLGA-PEI nanoparticles have been found to be superior to its cationising parent compound; PEI polymer.

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. Antibody titres were strongly influenced by experimental conditions. SDS presence in the coupling medium increased antibody titres, though a lower concentration of 0.5% (w/v) generated much higher titres than the commonly used 1% (w/v), despite causing less skin damage. A lower ultrasound duty cycle of 10% generated higher antibody titres than a duty cycle of 20%, also despite causing lower skin damage. Such lack of correlation between skin damage and immune responses indicates that enhancement of skin permeability to topically applied antigen (as indicated by changes in skin integrity) was not the main mechanism of low-frequency ultrasound-assisted skin immunisation.

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. Chitosan not only imparted a positive charge to the surface of the microparticles but was also able to increase the serum specific IgG antibody responses significantly. CONCLUSIONS: The cytokine assays showed that the serum IgG antibody response induced is different according to the formulation, indicated by the differential levels of interleukin 4 (IL-4), interleukin 6 (IL-6) and interferon gamma (IFN-gamma). The microparticles eliciting the highest IgG antibody response did not necessarily elicit the highest levels of the cytokines IL-4, IL-6 and IFN-gamma.

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. Chitosan nanoparticles were prepared by two methods of ionic cross-linking, simple complexation and ionic gelation using sodium tripolyphosphate (TPP). Both methods produced nanosize particles, less than 500 nm depending on type, molecular weight as well as concentration of chitosan. In the case of ionic gelation, two further factors, namely chitosan to TPP weight ratio and pH, affected the particle size. In vitro studies in two types of cells lines, CHO K1 and HEK 293, have revealed that preparation method of siRNA association to the chitosan plays an important role on the silencing effect. Chitosan-TPP nanoparticles with entrapped siRNA are shown to be better vectors as siRNA delivery vehicles compared to chitosan-siRNA complexes possibly due to their high binding capacity and loading efficiency. Therefore, chitosan-TPP nanoparticles show much potential as viable vector candidates for safer and cost-effective siRNA delivery.

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. Both routes of immunisation induced systemic and local immune responses, with high levels of serum IgG being developed in response to both vaccine antigens. In Elispot assays, secretion of cytokines by spleen and draining lymph node cells was demonstrated, revealing activation of both Th1 and Th2 associated cytokines; and spleen cells from animals immunised by either route were found to proliferate in vitro in response to both vaccine antigens. Virulent challenge experiments demonstrated that non-invasive immunisation by intra-nasal instillation can provide strong systemic and local immune responses and protect against high level challenge. Microencapsulation of these vaccine antigens has the added advantage that controlled release of the antigens occurs in vivo, so that protective immunity can be induced after only a single immunising dose.

Diphtheria toxoid loaded poly-(epsilon-caprolactone) nanoparticles as mucosal vaccine delivery systems.

Poly-(epsilon-caprolactone) (PCL), a poly(lactide-co-glycolide) (PLGA)-PCL blend and co-polymer nanoparticles encapsulating diphtheria toxoid (DT) were investigated for their potential as a mucosal vaccine delivery system. The nanoparticles, prepared using a water-in-oil-in-water (w/o/w) double emulsion solvent evaporation method, demonstrated release profiles which were dependent on the properties of the polymers. An in vitro experiment using Caco-2 cells showed significantly higher uptake of PCL nanoparticles in comparison to polymeric PLGA, the PLGA-PCL blend and co-polymer nanoparticles. The highest uptake mediated by the most hydrophobic nanoparticles using Caco-2 cells was mirrored in the in vivo studies following nasal administration. PCL nanoparticles induced DT serum specific IgG antibody responses significantly higher than PLGA. A significant positive correlation between hydrophobicity of the nanoparticles and the immune response was observed following intramuscular administration. The positive correlation between hydrophobicity of the nanoparticles and serum DT specific IgG antibody response was also observed after intranasal administration of the nanoparticles. The cytokine assays showed that the serum IgG antibody response induced is different according to the route of administration, indicated by the differential levels of IL-6 and IFN-gamma. The nanoparticles eliciting the highest IgG antibody response did not necessarily elicit the highest levels of the cytokines IL-6 and IFN-gamma.

Modulating the adjuvanticity of alum by co-administration of muramyl di-peptide (MDP) or Quil-A.

The characterization of the immunological cascades of the innate immune system activated by pathogen associated molecular patterns (PAMP) recognized by pattern recognition receptors (PRR) have allowed the elucidation of the mechanisms underlying the immunomodulatory properties of adjuvants. Thus, the combinatorial use of adjuvants with specific, complementary functions is investigated to achieve tailored immune responses to subunit vaccines. We have previously shown how combinatorial administration of chitosan and cholera toxin B or muramyl-di-peptide (MDP) intranasally, but not intramuscularly, can allow small doses of MDP which, when administered alone cannot adjuvantise Helicobacter pylori urease (rUre), achieve an immunomodulatory effect through the specific physiological effect of chitosan. The aim of this study was to investigate if in the context of rUre the adjuvantising effect of MDP could be realized via the intramuscular route by combination with aluminium hydroxide, as compared with the routinely used veterinary adjuvant combination of alum and Quil-A. Serum IgG kinetics were comparable between the two adjuvant combination groups. However, the alum + MDP combination afforded higher antigen-specific recall responses in splenocyte cultures, associated with elevated release of the type I immune response cytokines IFN-gamma and IL-2. This data suggests that the adjuvanticity of MDP can be modulated in the context of alum in a manner dissimilar to that of Quil-A, achieving a balancing effect on the responses elicited by alum adjuvantisation.

Strategies for DNA vaccine delivery.

Strategies for gene delivery comprise a diverse range of live and synthetic approaches; DNA delivery for the purposes of immunisation in turn comprises a large part of this research. This review mainly discusses synthetic systems for application in the delivery of plasmid DNA vaccines, outlining polylactide-co-glycolide, liposome, chitosan and complex combination delivery systems. Areas of promise for DNA vaccine candidates include immune modulation of allergic responses and veterinarian application. The potential for realistic consideration of DNA vaccines as an alternative to existing approaches is dependent on the development of efficient DNA vaccine vectors and improved systems for DNA vaccine delivery. DNA vaccine technology may yet prove to be an important asset in an environment where there is a critical need for therapeutic and prophylactic strategies to combat a wide range of disease states.

Mechanisms of inactivation of HSV-2 during storage in frozen and lyophilized forms.

The structural integrity of herpes simplex virus 2 (HSV-2) during freezing, thawing, and lyophilization has been studied using scanning and transmission electron microscopy. Viral particles should be thawed quickly from -80 to 37 degrees C to avoid artifacts of thawing. To avoid freezing damage, the virus should be rapidly frozen (>20 K s(-1)) rather than slowly frozen as occurs on the shelf of a lyophilizer (<1 K s(-1)). Fast freezing and thawing allows six cycles of freeze thaw with no loss of viral titer TCID50. Viral particles were characterized using immunogold labeling methods. Freshly thawed virus had 19 +/- 4 polyclonal immunogold particles virus(-1); virus stored at -80 degrees C for at least 4 months had 17 +/- 3 particles virus(-1); virus stored for 1 week at 4 degrees C had 8 +/- 4 particles virus(-1). By bulk lyophilization the number of particles was 4 +/- 4, but by fast freezing and lyophilization the number of gold particles improved to 12 +/- 5. The loss of viral membrane was directly observed, and the in vitro loss was demonstrated to occur through three possible pathways, including (i) simultaneous release of tegument and membrane, (ii) sequential release of membrane and then tegument, and (iii) release like by in vivo infection. The capsids were not further degraded as indicated by the lack of free DNA, which was only released by boiling the viral samples with 1% SDS, followed by a dilution to 0.001% w/v SDS for the real-time PCR reaction.

Comparative immunomodulatory properties of a chitosan-MDP adjuvant combination following intranasal or intramuscular immunisation.

As the precise functions of adjuvants become clearer, opportunities are presented in their complementary use for the induction of tailored immune responses to subunit vaccines. Here we comparatively investigate the immunological outcome following intranasal or intramuscular immunisation with Helicobacter pylori urease admixed to a chitosan and muramyl di-peptide (MDP) combination. MDP appeared to limit the antigenicity of rUre by either administration route. Nasal administration of the combined adjuvant formulation resulted in an up-regulation of type I recall responses in splenocytes as opposed to adjuvantisation with chitosan alone. In contrast, intramuscular immunisation appeared to limit the responsiveness to the antigen when adjuvanted with chitosan and even more so when chitosan was combined with MDP, suggesting that the mechanism of adjuvantisation and adjuvant synergy differed depending on the immunisation route. Recognising the benefit of improved delivery of MDP intranasally due to the specific physiological effects of chitosan, we discuss the impact of the newly identified pathogen associated molecular pattern (PAMP) role of MDP with respect to the adjuvanticity of proposed chemical variants of this peptide adjuvant.

Biodegradable mucoadhesive particulates for nasal and pulmonary antigen and DNA delivery.

Biodegradable polymer and particulate carriers have been shown to be of considerable potential for the delivery of peptides, proteins and DNA in animal models. In the context of vaccine delivery to the upper and lower respiratory tracts, the use of mucoadhesive agents offers a strategy for the facilitation of increased residence time and increased vaccine efficacy. Additional concerns addressed here include the potential of uptake of vaccine formulations by the primary olfactory nerves in the nasal cavity, effective delivery to the lung, strategies to maximise the immunopotentiation of candidate vaccine formulations, as well as the evaluation of animal models and interpretation of engendered immune responses in terms of antigen-specific antibody production. Experimental data are presented that demonstrate the potential of muco- and bioadhesive agents in combination with liposomes for intranasal (i.n.) delivery of tetanus toxoid in mice. A delivery system utilising chitosan for the formulation of microspheres by the spray-drying method is described and assessed for intranasal vaccine delivery, and porous particles with potential for pulmonary administration are also outlined.

Formulation of a microparticle carrier for oral polyplex-based DNA vaccines.

Oral induction of a disseminated mucosal immune response with polyplex-based DNA vaccines requires the delivery of intact polyplexes (polyelectrolyte complexes formed by self-assembly of plasmid DNA with a cationic polymer) to subepithelial lymphoid tissue (e.g. Peyer's patches) within the gastrointestinal tract. This work describes the formulation of a microparticle polyplex carrier allowing the potential of this approach to be realised. PEGylated PEI/DNA polyplexes (DNA concentration 20 microg/ml) formed at N/P 5:0 (defined as the ratio of polycation amino groups to DNA phosphates) were stable to salt-induced aggregation and could be concentrated to a final DNA concentration of 1 mg/ml without polyplex size increase. Polyplexes containing 1:1 polyethylene glycol (PEG)/polyethylenimine (PEI) ratio (mass/mass) gave similar levels of luciferase gene expression in B16F10 cells compared to non-PEG complexes. Poly-(D,L-lactide-co-glycolide) (PLGA) microparticles containing PEGylated polyplexes (approximately 17% DNA encapsulation efficiency) were formulated using a modified double emulsion solvent evaporation method. The microencapsulation and release of intact polyplexes from the microparticle carrier was demonstrated using polyanion (heparin sulfate and poly(aspartic acid) (PAA)) displacement techniques and electron microscopy. Microparticles containing PEGylated polyplexes (24 microg beta-galactosidase DNA) were given orally to Wistar rats. Significant transgene expression (compared to background) was found in peripheral tissue (spleen) 72 h after administration. This work demonstrates the potential application of microparticle carriers for mucosal polyplex-based vaccination.

Sustained expression in mammalian cells with DNA complexed with chitosan nanoparticles.

This work investigates the preparation and in vitro efficiency of chitosan gene transfection systems. Chitosan was used to prepare nanoparticles with a size range of 40-200 nm as determined using photon correlation spectroscopy (PCS) and 40-80 nm as determined using transmission electron microscopy (TEM). The ability of particles to complex DNA was investigated using gel retardation. Plasmid DNA pGL3-Control encoding firefly luciferase and pCH110 encoding beta-galactosidase were used as reporter genes. For transfection 293 human embryonal kidney cells and Chinese hamster ovary (CHO-K1) cells were used. The expression of luciferase was assayed and expressed as relative light units per milligram of protein (RLU/mg protein). Results showed that these chitosan particles have potential as vectors for the transfer of DNA into mammalian cells. Cellular transfection by the chitosan-pGL3-Control particles showed a sustained expression of the luciferase gene for about 10 days. Commercial transfection reagents, SuperFect and Lipofectin were also used. In contrast to chitosan particles, the duration of expression for both SuperFect and Lipofectin was only about 2 days. Agarose gel electrophoresis and displacement experiments using polyaspartic acid indicated a probable multiple interaction between DNA and chitosan whilst the interaction between DNA and the polyamidoamine dendrimer appears to be only ionic interaction. No toxic effect on the mammalian cells was seen with chitosan. SuperFect and Lipofectin however, were observed to engender marked cytotoxicity. Poly-D,L-lactide (PLA) nanoparticles (40-80 nm) and poly-L-lactide (PLLA) lamellae (2-6 microm) were also used to load DNA by an adsorption procedure, but these failed to give good expression data.