The effect of complexation on characteristics and drug release from PLGA microspheres loaded by cyclosporine-cyclodextrin complex

The purpose of this study was to evaluate the effect of cyclosporine (CyA)-cyclodextrin (CD) complex incorporated within PLGA inicrospheres on microsphere characteristics, with particular emphasis on drug release kinetics. For this purpose, microspheres encapsulated with CyA and those loaded by CyA-CD complex were prepared by solvent evaporation and multiple emulsification solvent evaporation methods, respectively. Morphology, size, encapsulation efficiency and drug release pattern from microspheres were evaluated. Also, physicochemical properties of drug inside microspheres were characterized by differential scanning calorimetry (DSC) and infrared spectroscopy (IR) studies. Scanning electron microscopy (SEM) studies showed that microspheres encapsulated with CyA had islands on the microsphere surface but the islands were not seen on the surface of microspheres loaded by complex. Size range varied from 1 to 25 mu m for CyA encapsulated microspheres and 1 to 50 mu m for complex loaded microspheres. The release of CyA was biphasic with an initial more rapid release phase followed by a slower phase but drug release was twice as fast for complex loaded microspheres. IR studies did not indicate any chemical interaction between the components of microspheres and DSC thermograms revealed that CyA was present either in its amorphous state in microspheres or the presence of CyA as an inclusion complex within microspheres loaded by complex. In conclusion, using CyA as an inclusion complex with CD within microspheres can affect microsphere characteristics and drug release and it is possible to modify microsphere properties like drug release by incorporating CDs as complexing agents.

Adipose tissue engineering based on the controlled release of fibroblast growth factor-2 in a collagen matrix

Adipose tissue forms when basement membrane extract ( Matrigel (TM)) and fibroblast growth factor-2 (FGF-2) are added to our mouse tissue engineering chamber model. A mouse tumor extract, Matrigel is unsuitable for human clinical application, and finding an alternative to Matrigel is essential. In this study we generated adipose tissue in the chamber model without using Matrigel by controlled release of FGF-2 in a type I collagen matrix. FGF-2 was impregnated into biodegradable gelatin microspheres for its slow release. The chambers were filled with these microspheres suspended in 60 mu L collagen gel. Injection of collagen containing free FGF-2 or collagen containing gelatin microspheres with buffer alone served as controls. When chambers were harvested 6 weeks after implantation, the volume and weight of the tissue obtained were higher in the group that received collagen and FGF-2 impregnated microspheres than in controls. Histologic analysis of tissue constructs showed the formation of de novo adipose tissue accompanied by angiogenesis. In contrast, control groups did not show extensive adipose tissue formation. In conclusion, this study has shown that de novo formation of adipose tissue can be achieved through controlled release of FGF-2 in collagen type I in the absence of Matrigel.

Protease IV production in Pseudomonas aeruginosa from the lungs of adults with cystic fibrosis

Protease IV is important in the pathogenesis of Pseudomonas aeruginosa-induced microbial keratitis, but little is known of its role in cystic fibrosis (CF) lung infection. In this study protease IV production was examined in 43 P. aeruginosa isolates (24 non-clonal and 19 clonal) from the lungs of chronically infected adult patients attending the Royal Prince Alfred Hospital CF Clinic, Sydney, Australia. Overall, 32/43 (74 %) isolates were positive for protease IV protein by Western blotting and 22/43 (51 %) had evidence of active protease IV on gelatin zymography. Clonal strains were 1.6 times more likely than non-clonal strains to produce protease IV [18/19 (95 %) versus 14/24 (58 %), RR=1.6, CI 1.1–2.3, P=0.007] and 3 times more likely to secrete the protein [16/19 (84 %) versus 6/24 (25 %), RR=3.4, CI 1.6–6.9, P

Gravity spun polycaprolactone fibers for applications in vascular tissue engineering:proliferation and function of human vascular endothelial cells

Poly(ε-caprolactone) (PCL) fibers produced by wet spinning from solutions in acetone under low-shear (gravity-flow) conditions resulted in fiber strength of 8 MPa and stiffness of 0.08 Gpa. Cold drawing to an extension of 500% resulted in an increase in fiber strength to 43 MPa and stiffness to 0.3 GPa. The growth rate of human umbilical vein endothelial cells (HUVECs) (seeded at a density of 5 × 104 cells/mL) on as-spun fibers was consistently lower than that measured on tissue culture plastic (TCP) beyond day 2. Cell proliferation was similar on gelatin-coated fibers and TCP over 7 days and higher by a factor of 1.9 on 500% cold-drawn PCL fibers relative to TCP up to 4 days. Cell growth on PCL fibers exceeded that on Dacron monofilament by at least a factor of 3.7 at 9 days. Scanning electron microscopy revealed formation of a cell layer on samples of cold-drawn and gelatin-coated fibers after 24 hours in culture. Similar levels of ICAM-1 expression by HUVECs attached to PCL fibers and TCP were measured using RT-PCR and flow cytometry, indicative of low levels of immune activation. Retention of a specific function of HUVECs attached to PCL fibers was demonstrated by measuring their immune response to lipopolysaccharide. Levels of ICAM-1 expression increased by approximately 11% in cells attached to PCL fibers and TCP. The high fiber compliance, favorable endothelial cell proliferation rates, and retention of an important immune response of attached HUVECS support the use of gravity spun PCL fibers for three-dimensional scaffold production in vascular tissue engineering. © Mary Ann Liebert, Inc.

Novel formulation strategies for the fabrication of lyophilised orally disintegrating tablets

Orally disintegrating Tablets (ODTs), also known as fast-disintegrating, fast-melt or fast-dissolving tablets, are a relatively novel dosage technology that involves the rapid disintegration or dissolution of the dosage form into a solution or suspension in the mouth without the need for water. The solution containing the active ingredients is swallowed, and the active ingredients are then absorbed through the gastrointestinal epithelium to reach the target and produce the desired effect. Formulation of ODTs was originally developed to address swallowing difficulties of conventional solid oral dosage forms (tablets and capsules) experienced by wide range of patient population, especially children and elderly. The current work investigates the formulation and development of ODTs prepared by freeze drying. Initial studies focused on formulation parameters that influence the manufacturing process and performance of lyophilised tablets based on excipients used in commercial products (gelatin and saccharides). The second phase of the work was followed up by comprehensive studies to address the essential need to create saccharide free ODTs using naturally accruing amino acids individually or in combinations. Furthermore, a factorial design study was carried out to investigate the feasibility of delivering multiparticulate systems of challenging drugs using a novel formulation that exploited the electrostatic associative interaction between gelatin and carrageenan. Finally, studies aimed to replace gelatin with ethically and morally accepted components to the end users were performed and the selected binder was used in factorial design studies to investigate and optimise ODT formulations that incorporated drugs with varies physicochemical properties. Our results show that formulation of elegant lyophilised ODTs with instant disintegration and adequate mechanical strength requires carful optimisation of gelatin concentration and bloom strength in addition to saccharide type and concentration. Successful formulation of saccharides free lyophilised ODTs requires amino acids that crystallise in the frozen state or display relatively high Tg', interact and integrate completely with the binder and, also, display short wetting time with the disintegrating medium. The use of an optimised mixture of gelatin, carrageenan and alanine was able to create viscous solutions to suspend multiparticulate systems and at the same time provide tablets with short disintegration times and adequate mechanical properties. On the other hand, gum arabic showed an outstanding potential for use as a binder in the formulation of lyophilised ODTs. Compared to gelatin formulations, the use of gum arabic simplified the formulation stages, shortened the freeze drying cycles and produced tablets with superior performance in terms of the disintegration time and mechanical strength. Furthermore, formulation of lyophilised ODTs based on gum arabic showed capability to deliver diverse range of drugs with advantages over commercial products.

Development and evaluation of a novel intranasal spray for the delivery of amantadine

The aim of this study was to develop and characterize an intranasal delivery system for amantadine hydrochloride (AMT). Optimal formulations consisted of a thermosensitive polymer Pluronic® 127 and either carboxymethyl cellulose or chitosan which demonstrated gel transition at nasal cavity temperatures (34 ± 1°C). Rheologically, the loss tangent (Tan δ) confirmed a 3-stage gelation phenomena at 34 ± 1°C and non-Newtonian behavior. Storage of optimized formulation carboxymethyl cellulose and optimal formulation chitosan at 4°C for 8 weeks resulted in repeatable release profiles at 34°C when sampled, with a Fickian mechanism earlier on but moving toward anomalous transport by week 8. Polymers (Pluronic® 127, carboxymethyl cellulose, and chitosan) demonstrated no significant cellular toxicity to human nasal epithelial cells up to 4 mg/mL and up to 1 mM for AMT (IC50: 4.5 ± 0.05 mM). Optimized formulation carboxymethyl cellulose and optimal formulation chitosan demonstrated slower release across an in vitro human nasal airway model (43%-44% vs 79 ± 4.58% for AMT). Using a human nasal cast model, deposition into the olfactory regions (potential nose-to-brain) was demonstrated on nozzle insertion (5 mm), whereas tilting of the head forward (15°) resulted in greater deposition in the bulk of the nasal cavity.

Administration routes affect the quality of immune responses:a cross-sectional evaluation of particulate antigen-delivery systems

Particulate delivery systems such as liposomes and polymeric nano- and microparticles are attracting great interest for developing new vaccines. Materials and formulation properties essential for this purpose have been extensively studied, but relatively little is known about the influence of the administration route of such delivery systems on the type and strength of immune response elicited. Thus, the present study aimed at elucidating the influence on the immune response when of immunising mice by different routes, such as the subcutaneous, intradermal, intramuscular, and intralymphatic routes with ovalbumin-loaded liposomes, N-trimethyl chitosan (TMC) nanoparticles, and poly(lactide-co-glycolide) (PLGA) microparticles, all with and without specifically selected immune-response modifiers. The results showed that the route of administration caused only minor differences in inducing an antibody response of the IgG1 subclass, and any such differences were abolished upon booster immunisation with the various adjuvanted and non-adjuvanted delivery systems. In contrast, the administration route strongly affected both the kinetics and magnitude of the IgG2a response. A single intralymphatic administration of all evaluated delivery systems induced a robust IgG2a response, whereas subcutaneous administration failed to elicit a substantial IgG2a response even after boosting, except with the adjuvanted nanoparticles. The intradermal and intramuscular routes generated intermediate IgG2a titers. The benefit of the intralymphatic administration route for eliciting a Th1-type response was confirmed in terms of IFN-gamma production of isolated and re-stimulated splenocytes from animals previously immunised with adjuvanted and non-adjuvanted liposomes as well as with adjuvanted microparticles. Altogether the results show that the IgG2a associated with Th1-type immune responses are sensitive to the route of administration, whereas IgG1 response associated with Th2-type immune responses were relatively insensitive to the administration route of the particulate delivery systems. The route of administration should therefore be considered when planning and interpreting pre-clinical research or development on vaccine delivery systems.

Formulation of multiparticulate systems as lyophilised orally disintegrating tablets

The current study aimed to exploit the electrostatic associative interaction between carrageenan and gelatin to optimise a formulation of lyophilised orally disintegrating tablets (ODTs) suitable for multiparticulate delivery. A central composite face centred (CCF) design was applied to study the influence of formulation variables (gelatin, carrageenan and alanine concentrations) on the crucial responses of the formulation (disintegration time, hardness, viscosity and pH). The disintegration time and viscosity were controlled by the associative interaction between gelatin and carrageenan upon hydration which forms a strong complex that increases the viscosity of the stock solution and forms tablet with higher resistant to disintegration in aqueous medium. Therefore, the levels of carrageenan, gelatin and their interaction in the formulation were the significant factors. In terms of hardness, increasing gelatin and alanine concentration was the most effective way to improve tablet hardness. Accordingly, optimum concentrations of these excipients were needed to find the best balance that fulfilled all formulation requirements. The revised model showed high degree of predictability and optimisation reliability and therefore was successful in developing an ODT formulation with optimised properties that were able deliver enteric coated multiparticulates of omeprazole without compromising their functionality.

The role of formulation excipients in the development of lyophilised fast-disintegrating tablets

Despite recent Success, many fast-disintegrating tablets (FDTs) still face problems of low mechanical strength, poor mouth-feel and higher disintegration times. This Study aimed to optimise FDTS using a progressive three-stage approach. A series of hardness, fracturability and disintegration time tests were performed on the formulations at each stage. During Stage 1, tablets were prepared in concentrations between 2% and 5% w/w, and were formulated at each concentration as single and combination bloom strength gelatin (BSG) using 75 and 225 BSGs. Analysis revealed that both hardness and disintegration time increased with an increase in gelatin concentration. A combination (5% gelatin) FDT comprising a 50:50 ratio of 75:225 BSGs (hardness: 13.7 +/- 0.9 N and disintegration time: 24.1 +/- 0.6 s) was judged the most ideal, and was carried forward to Stage II: the addition of the saccharides sorbitol, mannitol and sucrose in concentrations between 10% and 80% w/w. The best properties were exhibited by mannitol-containing formulations (50%-hardness: 30.9 +/- 2.8 N and disintegration time: 13.3 +/- 2.1 s), which were carried forward to the next stage: the addition of viscosity-modifying polymers to improve mouth-feel and aid pre-gastric retention. Addition of carbopol 974P-NF resulted in the enhancement of viscosity with a compromise of the hardness of the tablet, whereas Pluronic F127 (6%) showed an increase in disintegration time and viscosity with retention of mechanical propel-ties. (C) 2008 Elsevier B.V. All rights reserved.

Mechanisms of drying of skin forming materials

The literature relating to evaporation from single droplets of pure liquids, and to the drying of droplets containing solids and of droplet sprays has been reviewed. The heat and mass transfer rates for a single droplet suspended from a nozzle were studied within a 42mm I.D. horizontal wind tunnel designed to supply hot dry air, to simulate conditions encountered in a practical spray dryer. A novel rotating glass nozzle was developed to facilitate direct measurements of droplet weight and core temperature. This design minimised heat conduction through the nozzle. Revised correlations were obtained for heat and mass transfer coefficients, for evaporation from pure water droplets suspended from a rotating nozzle. Nu = 2.0 + 0.27 (l/B)°-18Re°-5Pr°-83 Sh = 2.0 + 0.575 ((T0-T.)/Tomfc) -o.o4Reo.5 ^0.33 Experimental drying studies were carried out on single droplets of different types of skin-forming materials, namely, custard, gelatin, skim milk and fructose at air temperatures ranging from 19°C to 198°C. Dried crusts were recovered and examined by Scanning Electron Microscopy. Skin-forming materials were classified into three types according to the mechanisms of skin formation. In the first type (typified by droplets of custard and starch) skin formed due to gelatinisation at high temperatures. Increasing the drying temperature resulted in increased crust resistance to mass transfer due to increased granule swelling and the crust resistance was completely transferred to a skin resistance at drying temperatures > 150°C. In the second type e.g. gelatin droplets the skin formed immediately drying had taken place at any drying temperature. At drying temperature > 60° C a more resistant skin was formed. In the third type (typified by droplets of skim milk and fructose) the skin appeared on the droplet surface at a certain stage of the drying process under any drying conditions. As the drying temperature was increased the resistance of the skin to mass transfer increased. The drying rate history of any material depended upon the nature of the skin formed which, in turn, depended upon the drying conditions. A mathematical model was proposed for the drying of the first type of skin-forming material. This was based on the assumption that, once all the granules gelatinised at the gelatinisation temperature, a skin appeared instantaneously on the droplet surface. The experimentally-observed times at which the skin appeared on the droplets surfaces were in excellent agreement with those predicted from the model. The work should assist in understanding the fundamentals of paniculate drying processes, particularly when skin-formation occurs and may be a crucial factor in volatiles retention.

Formulation and characterisation of cationic microparticles for the delivery of DNA vaccines

The aim of this research was to formulate a novel biodegradable, biocompatible cationic microparticle vector for the delivery of DNA vaccines. The work builds upon previous research by Singh et al which described the adsorption of DNA to the surface of poly (D,L-lactide-co-glycolide) (PLG) microparticles stabilised with the surfactant cetyltrimethyl ammonium bromide (CT AB). This work demonstrated the induction of antibody and cellular immune responses to HIV proteins encoded on plasmid DNA adsorbed to the particle surface in mice, guinea pigs and non-human primates (Singh et aI, 2000; O'Hagan et aI, 2001). However, the use of surfactants in microparticle formulations for human vaccination is undesirable due to long term safety issues. Therefore, the present research aim was to develop an adsorbed DNA vaccine with enhanced potency and increased safety compared to CTAB stabilised PLG microparticles (PLG/CTAB) by replacement of the surfactant CTAB with an alternative cationic agent. The cationic polymers chitosan and poly (N- vinylpyrrolidone/2-dimethylaminoethyl methacrylate), dimethyl sulfate quaternary (PVP-PDAEMA) were investigated as alternative stabilisers to CTAB. From a variety of initial formulations, the most promising vector(s) for DNA vaccination were selected based on physicochemical data (chapter 3) and in vitro DNA loading and release characteristics (chapter 4). The chosen formulation(s) were analysed in greater depth (chapters 3 and 4), and gene expression was assessed by in vitro cell transfection studies using 293T kidney epithelial and C2C12 myoblast non-phagocytic cell lines (chapter 5). The cytotoxicity of the microparticles and their constituents were also evaluated in vitro (chapter 5). Stability and suitability of the formulation(s) for commercial production were assessed by cryopreparation and lyophilisation studies (chapters 3 and 4). Gene expression levels in cells of the immune response were evaluated by microparticle transfection of the dendritic cell (DC) line 2.4 and primary bone marrow derived DCs (chapter 6). In vivo, mice were injected i.m. with the formulations deemed most promising on the basis of in vitro studies and humoral and cellular immune responses were evaluated (chapter 6).

Novel biodegradable fibres for applications in tissue engineering and drug delivery

The preparation and characterisation of novel biodegradable polymer fibres for application in tissue engineering and drug delivery are reported. Poly(e-caprolactone) (PCL) fibres were produced by wet spinning from solutions in acetone under low shear (gravity flow) conditions. The tensile strength and stiffness of as-spun fibres were highly dependent on the concentration of the spinning solution. Use of a 6% w/v solution resulted in fibres having strength and stiffness of 1.8 MPa and 0.01 GPa respectively, whereas these values increased to 9.9 MPa and 0.1 GPa when fibres were produced from 20% w/v solutions. Cold drawing to an extension of 500% resulted in further increases in fibre strength (up to 50 MPa) and stiffness (0.3 GPa). Hot drawing to 500% further increased the fibre strength (up to 81 MPa) and stiffness (0.5 GPa). The surface morphology of as-spun fibres was modified, to yield a directional grooved pattern by drying in contact with a mandrel having a machined topography characterised by a peak-peak separation of 91 mm and a peak height of 30 mm. Differential scanning calorimetery (DSC) analysis of as-spun fibres revealed the characteristic melting point of PCL at around 58°C and a % crystallinity of approximately 60%. The biocompatibility of as-spun fibres was assessed using cell culture. The number of attached 3T3 Swiss mouse fibroblasts, C2C12 mouse myoblasts and human umbilical vein endothelial cells (HUVECs) on as-spun, 500% cold drawn, and gelatin coated PCL fibres were observed. The results showed that the fibres promoted cell proliferation for 9 days in cell culture and was slightly lower than on tissue culture plastic. The morphology of all cell lines was assessed on the various PCL fibres using scanning electron microscopy. The cell function of HUVECs growing on the as-spun PCL fibres was evaluated. The ability HUVECs to induce an immune response when stimulated with lipopolysaccaride (LPS) and thereby to increase the amount of cell surface receptors was assessed by flow cytometry and reverse transcription-polymerase chain reaction (RT-PCR). The results showed that PCL fibres did not inhibit this function compared to TCP. As-spun PCL fibres were loaded with 1 % ovine albumin (OVA) powder, 1% OVA nanoparticles and 5% OVA nanoparticles by weight and the protein release was assessed in vitro. PCL fibres loaded with 1 % OVA powder released 70%, 1% OVA nanoparticle released 60% and the 5% OVA nanoparticle released 25% of their protein content over 28 days. These release figures did not alter when the fibres were subjected to lipase enzymatic degradation. The OVA released was examined for structural integrity by SDS-PAGE. This showed that the protein molecular weight was not altered after incorporation into the fibres. The bioactivity of progesterone was assessed following incorporation into PCL fibres. Results showed that the progesterone released had a pronounced effect on MCF-7 breast epithelial cells, inhibiting their proliferation. The PCL fibres display high fibre compliance, a potential for controlling the fibre surface architecture to promote contact guidance effects, favorable proliferation rate of fibroblasts, myoblasts and HUVECs and the ability to release pharmaceuticals. These properties recommended their use for 3-D scaffold production in soft tissue engineering and the fibres could also be exploited for controlled presentation and release of biopharmaceuticals such as growth factors.

Controlled release in inhalation

Increasingly complicated medication regimens associated with the necessity of the repeated dosing of multiple agents used in treating pulmonary disease has been shown to compromise both disease management and patient convenience. In this study the viability of spray drying to introduce controlled release vectors into dry powders for inhalation was investigated. The first experimental section highlights the use of leucine in producing highly respirable spray dried powders, with in vitro respirable fractions (Fine particle fraction, FPF: F < 5µm) exceeding 80% of the total dose. The second experimental chapter introduces the biocompatible polymer chitosan (mw 190 – 310 kDa) to formulations containing leucine with findings of increased FPF with increasing leucine concentration (up to 82%) and the prolonged release of the active markers terbulataline sulfate (up to 2 hours) and beclometasone dipropionate (BDP: up to 12 hours) with increasing chitosan molecular weight. Next, the thesis details the use of a double emulsion format in delivering the active markers salbutamol sulfate and BDP at differing rates; using the polymers poly-lactide co-glycolide (PLGA 50:50 and PLGA 75:25) and/or chitosan incorporating leucine as an aerosolisation enhancer the duration of in vitro release of both agents reaching 19 days with FPF exceeding 60%. The final experimental chapter involves dual aqueous and organic closed loop spray drying to create controlled release dry powders for inhalation with in vitro sustained release exceeding 28 days and FPF surpassing 55% of total loaded dose. In conclusion, potentially highly respirable sustained release dry powders for inhalation have been produced by this research using the polymers chitosan and/or PLGA as drug release modifiers and leucine as an aerosolisation enhancer.

Tissue engineering of co-cultured skin substitutes using biocomposite membranes based on collagen and polycaprolactone

The preparation and characterisation of collagen: PCL, gelatin: PCL and gelatin/collagen:PCL biocomposites for manufacture of tissue engineered skin substitutes are reported. Films of collagen: PLC, gelatin: PCL (1:4, 1:8 and 1:20 w/w) and gelatin/collagen:PCL (1:8 and 1:20 w/w) biocomposites were prepared by impregnation of lyophilised collagen and/or gelatin mats by PCL solutions followed by solvent evaporation. In vitro assays of total protein release of collagen:PCL and gelatin: PCL biocomposite films revealed an expected inverse relationship between the collagen release rate and the content of synthetic polymer in the biocomposite samples that may be exploited for controlled presentation and release of biopharmaceuticals such as growth factors. Good compatibility of all biocomposite groups was proven by interaction with 3T3 fibroblasts, normal human epidermal keratinocytes (NHEK), and primary human epidermal keratinocytes (PHEK) and dermal fibroblasts (PHDF) in vitro respectively. The 1:20 collagen: PCL materials exhibiting good cell growth curves and mechanical characteristics were selected for engineering of skin substitutes in this work. The tissue-engineered skin model based on single-donor PHEK and PHDF with differentiated confluent epidermal layer and fibrous porous dermal layer was then developed successfully in vitro proven by SEM and immunohistochemistry assay. The following in vivo animal study on athymic mice revealed early complete wound healing in 10 days and good integration of co-cultured skin substitutes with adjacent mice skin structures. Thus the co-cultured skin substitutes based on 1:20 collagen: PCL biocomposite membranes was proven in principle. The approach to skin modelling reported here may find application in wound treatment, gene therapy and screening of new pharmaceuticals.

Novel bioadhesive formulations for mucosal and parenteral delivery of vaccines

In this work we have established the efficient mucosal delivery of vaccines using absorption enhancers and chitosan. In addition, the use of chitosan was shown to enhance the action of other known adjuvants, such as CTB or Quil-A. Collectively, the results presented herein indicate that chitosan has excellent potential as a mucosal adjuvant. We have evaluated a number of absorption enhancers for their adjuvant activity in vivo. Polyornithine was shown to engender high scrum immune reasons to nasally delivered antigens, with higher molecular weight polyornithine facilitating the best results. We have demonstrated for the first time that vitamin E TPGS can act as mucosal adjuvant. Deoxycholic acid, cyclodextrins and acylcarnitines were also identified as effective mucosal adjuvants and showed enhanced immune responses to nasally delivered TT, DT and Yersinia pestis V and F1 antigens. Previously, none of these agents, common in their action as absorption enhancing agents, have been shown to have immunopotentiating activity for mucosal immunisation. We have successfully developed novel surface modified microspheres using chitosan as an emulsion stabiliser during the preparation of PLA microspheres. It was found that immune responses could be substantially increased, effectively exploiting the immunopenetrating characteristics of both chitosan and PLA microspheres in the same delivery vehicle. In the same study, comparison of intranasal and intramuscular routes of administration showed that with these formulations, the nasal route could be as effective as intramuscular delivery, highlighting the potential of mucosal administration for these particulate delivery systems. Chitosan was co-administered with polymer microspheres. It was demonstrated that this strategy facilitates markedly enhanced immune responses in both magnitude and duration following intramuscular administration. We conclude that this combination shows potential for single dose administration of vaccines. In another study, we have shown that the addition of chitosan to alum adsorbed TT was able to enhance immune responses. PLA micro/nanospheres were prepared and characterised with discreet particle size ranges. A smaller particle size was shown to facilitate higher scrum IgG responses following nasal administration. A lower antigen loading was additionally identified as being preferential for the induction of immune responses in combination with the smaller particle size. This may be due to the fact that the number of particles will be increased when antigen loading is low, which may in turn facilitate a more widespread uptake of particles. PLA lamellar particles were prepared and characterised. Adsorbed TT was evaluated for the potential to engender immune responses in vivo. These formulations were shown to generate effective immune responses following intramuscular administration. Positively charged polyethylcyanoacrylate and PLA nanoparticies were designed and characterised and their potential as delivery vehicles for DNA vaccines was investigated. Successful preparation of particles with narrow size distribution and positive surface charge (imparted by the inclusion of chitosan) was achieved. In the evaluation of antibody responses to DNA encoded antigen in the presence of alum administered intranasally, discrimination between the groups was only seen following intramuscular boosting with the corresponding protein. Our study showed that DNA vaccines in the presence of either alum or Quil-A may advantageously influence priming of the immune system by a mucosal route. The potential for the combination of adjuvants, Quil-A and chitosan, to enhance antibody responses to plasmid encoded antigen co-administered with the corresponding protein antigen was shown and this is worthy of further investigation. The findings here have identified novel adjuvants and approaches to vaccine delivery. In particular, chitosan or vitamin E TPGS are shown here to have considerable promise as non-toxic, safe mucosal adjuvants. In addition, biodegradable mucoadhesive delivery systems, surface modified with chitosan in a single step process, may have application for other uses such as drug and gene delivery.