Liposomal vaccine delivery systems

INTRODUCTION: Liposomes remain at the forefront of drug and vaccine design owing to their well-documented abilities to act as delivery vehicles. Nevertheless, the concept of liposomes as delivery vehicles is not a new one, with most works focusing on their use for the delivery of genes and drugs. However, in the last 10 years a significant amount of research has focused on using liposomes as vaccine adjuvants, not only as an antigen delivery vehicle but also as a tool to increase the immunogenicity of peptide and protein antigens. AREAS COVERED: This paper reviews liposomal adjuvants now in vaccine development, with particular emphasis on their adjuvant mechanism and how specific physicochemical characteristics of liposomes affect the immune response. The inclusion of immunomodulators is also discussed, with prominence given to Toll-like receptor ligands. EXPERT OPINION: The use of liposomes as vaccine delivery systems is evolving rapidly owing to the combined increase in technological advances and understanding of the immune system. Liposomes that contain and deliver immunostimulators and antigens are now being developed to target diseases that require stimulation of both humoral and cell-mediated immune responses. The CAF liposomal system, described in detail in this review, is one liposomal model that shows such flexibility.

Investigation of liquid sodium alginates as mucoadhesive bandages coating the oesophageal mucosa and protecting it from gastric reflux

Gastro-oesophageal Reflux Disease (GORD), is generally caused by excess gastric reflux back to the oesophagus where damage to the mucosa results in injury. GORD is a very common disease in western countries, more than a quarter of western people are suffering from this disease and there is a trend that the percentage population in eastern countries who are diagnosed as GORD is increasing. GORD and its complications damage the quality of life and can lead to serious oesophageal diseases including Barrett’s disease and oesophageal carcinoma. Sodium alginate dissolved in water forms a viscous liquid and can coat on oesophageal mucosa for a period of time. In this study the ability of the liquid alginate to adhere to the oesophageal mucosa was investigated and the factors that affect this retention were examined. The potential of this liquid alginate as a drug delivery vehicle to extend the duration of contact with the oesophageal mucosa was confirmed by this study. The capacity of an alginate coating to retard acid and pepsin diffusion, the two main aggressive factors in gastric reflux, was investigated. A significant reduction in acid and pepsin diffusion by alginate gel layer was demonstrated in this project, indicating that alginate has great potential to protect against damage caused by acidic reflux. A novel method was introduced using an independent score system to assess the protection of oesophageal tissue by a coating of liquid alginate using microscopy as a technique. This technique demonstrated that alginate can protect the oesophageal epithelial tissue from the damage caused by gastric acid and pepsin. Many techniques were used in this study. The experimental results suggested that liquid sodium alginate is a very promising candidate in treating local oesophageal diseases through forming a coating on the oesophageal mucosal surface, retarding the diffusion of components of gastric refluxate and thus reducing the contact of these noxious factors with the epithelium and minimising injury.

Oral delivery strategies for the controlled release of cimetidine

It is advantageous to develop controlled release dosage forms utilising site-specific delivery or gastric retention for those drugs with frequent or high dosing regimes. Cimetidine is a potent and selective H2 -reception antagonist used in the treatment of various gastrointestinal disorders and localisation in the upper gastrointestinal tract could significantly improve the drug absorption. Three strategies were undertaken to prepare controlled release systems for the delivery of cimetidine to the GI tract. Firstly, increasing the contact time of the dosage form with the mucus layer which coats the gastrointestinal tract, may lead to increased gastric residence times. Mucoadhesive microspheres, by forming a gel-like structure in contact with the mucus, should prolong the contact between the delivery system and the mucus layer, and should have the potential for releasing the drug in sustained and controlled manner. Gelatin microspheres were prepared, optimised and characterised for their physicochemical properties. Crosslinking concentration, particle size and cimetidine loading influenced drug release profiles. Particle size was influenced by surfactant concentration and stirring speed. Mucoadheisve polymers such as alginates, chitosans, carbopols and polycarbophil were incorporated into the microspheres using different strategies. The mucoadhesion of the microspheres was determined using in vitro surface adsorption and ex vivo rat intestine models. The surface-modification strategy resulted in highest levels of microsphere adhesion, with chitosan, carbopols and polycarbophil as the most successful candidates for improvement of adhesion, with over 70% of the microspheres retained ex vivo. Specific targeting agent UEA I lectin was conjugated to the surface of gelatin microspheres, which enhanced the adhesion of the microspheres. Alginate raft systems containing antacids have been used extensively in the treatment of gastro-oesophageal disease and protection of the oesophageal mucosa from acid reflux by forming a viscous raft layer on the surface of the stomach content, and could be an effective delivery system for controlled release of cimetidine.

Use of amino acids as counterions improves the solubility of the BCS II model drug, indomethacin

The number of new chemical entities (NCE) is increasing every day after the introduction of combinatorial chemistry and high throughput screening to the drug discovery cycle. One third of these new compounds have aqueous solubility less than 20µg/mL [1]. Therefore, a great deal of interest has been forwarded to the salt formation technique to overcome solubility limitations. This study aims to improve the drug solubility of a Biopharmaceutical Classification System class II (BCS II) model drug (Indomethacin; IND) using basic amino acids (L-arginine, L-lysine and L-histidine) as counterions. Three new salts were prepared using freeze drying method and characterised by FT-IR spectroscopy, proton nuclear magnetic resonance ((1)HNMR), Differential Scanning Calorimetry (DSC) and Thermogravimetric analysis (TGA). The effect of pH on IND solubility was also investigated using pH-solubility profile. Both arginine and lysine formed novel salts with IND, while histidine failed to dissociate the free acid and in turn no salt was formed. Arginine and lysine increased IND solubility by 10,000 and 2296 fold, respectively. An increase in dissolution rate was also observed for the novel salts. Since these new salts have improved IND solubility to that similar to BCS class I drugs, IND salts could be considered for possible waivers of bioequivalence.

Poly (D,L-Lactide) based microspheres for pulmonary delivery of proteins

Initial work focused on the preparation, optimisation and characterisation of poly (D,L-lactide) (PLA) microspheres with the aim of optimising their formulation based on minimizing the particle size into the range suitable for pulmonary delivery to alveoli. In order to produce dry powders and to enhance their long-term physico-chemical stability, microspheres were prepared as a dry powder via freeze-drying. Optimisation studies showed that using appropriate concentrations of polymer 3% (w/v) in organic phase and emulsifier 10% (w/v) in external aqueous phase, the double solvent evaporation method produced high protein loading microspheres (72 ± 0.5%) with an appropriate particle size for pulmonary drug delivery. Combined use of trehalose and leucine as cyroprotectants (6% and 1% respectively, w/v) produced freeze-dried powders with the best aerosolisation profile among those tested. Although the freeze-dried PLA microsphere powders were not particularly respirable in dry powder inhalation, nebulisation of the rehydrated powders using an ultrasonic nebuliser resulted in improved aerosilisation performance compared to the air-jet nebuliser. When tested in vitro using a macrophage cell line, the PLA microspheres system exhibited a low cytotoxicity and the microspheres induced phagocytic activity in macrophages. However, interestingly, the addition of an immunomodulator to the microsphere formulations (4%, w/w of polymer) reduced this phagocytic activity and macrophage activation compared to microspheres formulated using PLA alone. This suggested that the addition of trehalose dibehenate may not enhance the ability of these microspheres to be used as vaccine delivery systems.

Microspheres as a vehicle for biomolecule delivery to neural stem cells

Neural stem cells (NSC) are a valuable model system for understanding the intrinsic and extrinsic controls for self-renewal and differentiation choice. They also offer a platform for drug screening and neurotoxicity studies, and hold promise for cell replacement therapies for the treatment of neurodegenerative diseases. Fully exploiting the potential of this experimental tool often requires the manipulation of intrinsic cues of interest using transfection methods, to which NSC are relatively resistant. In this paper, we show that mouse and human NSC readily take up polystyrene-based microspheres which can be loaded with a range of chemical or biological cargoes. This uptake can take place in the undifferentiated stage without affecting NSC proliferation and their capacity to give rise to neurons and glia. We demonstrate that ß-galactosidase-loaded microspheres could be efficiently introduced into NSC with no apparent toxic effect, thus providing proof-of-concept for the use of microspheres as an alternative biomolecule delivery system.

Amino acids as cryoprotectants for liposomal delivery systems

Liposomes provide an efficient delivery system for solubilisation and delivery of both small and macro molecules. However, they suffer from the disadvantage of instability when stored as aqueous dispersions. Cryoprotection of the liposomal systems provides an effective approach to overcome poor stability whilst maintaining formulation characteristics, although, the formulation of a freeze-dried product requires the consideration of not only the selection of an appropriate cryoprotectant, but also needs careful consideration of the processing parameters including pre-freezing conditions, primary and secondary drying protocols along with optimisation of cryoprotectant concentration. This current work investigates the application of amino acids as potential cryoprotectants for the stabilisation of liposomes, and results indicate that amino acids show biphasic nature of stabilisation with 4 mol of cryoprotectant per mole of the lipid exhibiting optimum cryoprotection. The investigations of process parameters showed that the pre-freezing temperatures below the glass transition of the amino acids followed by drying for over 6 h resulted in stable formulations. Studies investigating the efficiency of drug retention showed that the cryoprotection offered by lysine was similar to that shown by trehalose, suggesting that amino acids act as effective stabilisers. ESEM analysis was carried out to monitor morphology of the rehydrated liposomes. © 2007 Elsevier B.V. All rights reserved.

Characterization and optimization of bilosomes for oral vaccine delivery

Oral vaccines offer significant benefits due to the ease of administration, better patient compliance and non-invasive, needle-free administration. However, this route is marred by the harsh gastro intestinal environment which is detrimental to many vaccine formats. To address this, a range of delivery systems have been considered including bilosomes; these are bilayer vesicles constructed from non-ionic surfactants combined with the inclusion of bile salts which can stabilize the vesicles in the gastro intestinal tract by preventing membrane destabilization. The aim of this study was to investigate the effect of formulation parameters on bilosome carriers using Design of Experiments to select an appropriate formulation to assess in vivo. Bilosomes were constructed from monopalmitoylglycerol, cholesterol, dicetyl phosphate and sodium deoxycholate at different blends ratios. The optimized bilosome formulation was identified and the potential of this formulation as an oral vaccine delivery system were assessed in biodistribution and vaccine efficacy studies. Results showed that the larger bilosomes vesicles (~6 µm versus 2 µm in diameter) increased uptake within the Peyer's patches and were able to reduce median temperature differential change and promote a reduction in viral cell load in an influenza challenge study. © 2013 Informa UK, Ltd.

Consideration of the efficacy of non-ionic vesicles in the targeted delivery of oral vaccines

The fundamentals of this research were to exploit non-ionic surfactant technology for delivery and administration of vaccine antigens across the oral route and to gain a better understanding of vaccine trafficking. Using a newly developed method for manufacture of non-ionic surfactant vesicles (niosomes and bilosomes) lower process temperatures were adopted thus reducing antigen exposure to potentially damaging conditions. Vesicles prepared by this method offered high protection to enzymatic degradation, with only ~10 % antigen loss measured when vesicles incorporating antigen were exposed to enzyme digestion. Interestingly, when formulated using this new production method, the addition of bile salt to the vesicles offered no advantage in terms of stability within simulated gastro-intestinal conditions. Considering their ability to deliver antigen to their target site, results demonstrated that incorporation of antigen within vesicles enhanced delivery and targeting of the antigen to the Peyer's Patch, again with niosomes and bilosomes offering similar efficiency. Delivery to both the Peyer's patches and mesentery lymphatics was shown to be dose dependent at lower concentrations, with saturation kinetics applying at higher concentrations. This demonstrates that in the formulation of vaccine delivery systems, the lipid/antigen dose ratio is not only a key factor in production cost, but is equally a key factor in the kinetics of delivery and targeting of a vaccine system. © 2013 Controlled Release Society.

Consideration of the efficacy of non-ionic vesicles in the targeted delivery of oral vaccines

The fundamentals of this research were to exploit non-ionic surfactant technology for delivery and administration of vaccine antigens across the oral route and to gain a better understanding of vaccine trafficking. Using a newly developed method for manufacture of non-ionic surfactant vesicles (niosomes and bilosomes) lower process temperatures were adopted thus reducing antigen exposure to potentially damaging conditions. Vesicles prepared by this method offered high protection to enzymatic degradation, with only ~10 % antigen loss measured when vesicles incorporating antigen were exposed to enzyme digestion. Interestingly, when formulated using this new production method, the addition of bile salt to the vesicles offered no advantage in terms of stability within simulated gastro-intestinal conditions. Considering their ability to deliver antigen to their target site, results demonstrated that incorporation of antigen within vesicles enhanced delivery and targeting of the antigen to the Peyer's Patch, again with niosomes and bilosomes offering similar efficiency. Delivery to both the Peyer's patches and mesentery lymphatics was shown to be dose dependent at lower concentrations, with saturation kinetics applying at higher concentrations. This demonstrates that in the formulation of vaccine delivery systems, the lipid/antigen dose ratio is not only a key factor in production cost, but is equally a key factor in the kinetics of delivery and targeting of a vaccine system. © 2013 Controlled Release Society.

Improving brain drug targeting through exploitation of the nose-to-brain route:a physiological and pharmacokinetic perspective

With an ageing population and increasing prevalence of central-nervous system (CNS) disorders new approaches are required to sustain the development and successful delivery of therapeutics into the brain and CNS. CNS drug delivery is challenging due to the impermeable nature of the brain microvascular endothelial cells that form the blood-brain barrier (BBB) and which prevent the entry of a wide range of therapeutics into the brain. This review examines the role intranasal delivery may play in achieving direct brain delivery, for small molecular weight drugs, macromolecular therapeutics and cell-based therapeutics, by exploitation of the olfactory and trigeminal nerve pathways. This approach is thought to deliver drugs into the brain and CNS through bypassing the BBB. Details of the mechanism of transfer of administrated therapeutics, the pathways that lead to brain deposition, with a specific focus on therapeutic pharmacokinetics, and examples of successful CNS delivery will be explored. © 2014 Bentham Science Publishers.