Particulate delivery systems for vaccines

This review focuses on the use of particulate delivery systems for the purposes of immunization. This includes poly(lactide-co-glycolide) (PLGA), ISCOMs, liposomes, niosomes, virosomes, chitosan, and other biodegradable polymers. These systems are evaluated in terms of their use as carriers for protein subunit and DNA vaccines. There is an extensive focus on recent literature, the understanding of biological interactions, and relation of this to our present understanding of immunological mechanisms of action. In addition, there is consideration of formulation techniques including emulsification, solvent diffusion, DNA complexation, and entrapment. The diversity of formulation strategies presented is a testament to the exponential growth and interest in the area of vaccine delivery systems. A case study for the application of particulate vaccine carriers is assessed in terms of vaccine development and recent insights into the possible design and application of vaccines against two of the most important pathogens that threaten mankind and for which there is a significant need: Mycobacterium tuberculosis and human immunodeficiency virus. This review addresses the rationale for the use of particulate delivery systems in vaccine design in the context of the diversity of carriers for DNA- and protein-based vaccines and their potential for application in terms of the critical need for effective vaccines. © 2005 by Begell House, Inc.

The rational design of vaccines

This review provides an insight into the various opportunities for vaccine intervention, analysis of strategies for vaccine development, vaccine ability to modulate immune responses and resultant rational vaccine design. In addition, wider aspects are considered, such as biotechnological advances, advances in immunological understanding and host-pathogen interactions. The key question addressed here is, with all our research and understanding, have we reached a new echelon in vaccine development, that of rational design? ©2005 Elsevier Ltd. All rights reserved.

A comparative study of cationic liposome and niosome-based adjuvant systems for protein subunit vaccines:characterisation, environmental scanning electron microscopy and immunisation studies in mice

Vesicular adjuvant systems composing dimethyldioctadecylammonium (DDA) can promote both cell-mediated and humoral immune responses to the tuberculosis vaccine fusion protein in mice. However, these DDA preparations were found to be physically unstable, forming aggregates under ambient storage conditions. Therefore there is a need to improve the stability of such systems without undermining their potent adjuvanticity. To this end, the effect of incorporating non-ionic surfactants, such as 1-monopalmitoyl glycerol (MP), in addition to cholesterol (Chol) and trehalose 6,6′-dibehenate (TDB), on the stability and efficacy of these vaccine delivery systems was investigated. Differential scanning calorimetry revealed a reduction in the phase transition temperature (T c) of DDA-based vesicles by ∼12°C when MP and cholesterol (1:1 molar ratio) were incorporated into the DDA system. Transmission electron microscopy (TEM) revealed the addition of MP to DDA vesicles resulted in the formation of multi-lamellar vesicles. Environmental scanning electron microscopy (ESEM) of MP-Chol-DDA-TDB (16:16:4:0.5 μmol) indicated that incorporation of antigen led to increased stability of the vesicles, perhaps as a result of the antigen embedding within the vesicle bilayers. At 4°C DDA liposomes showed significant vesicle aggregation after 28 days, although addition of MP-Chol or TDB was shown to inhibit this instability. Alternatively, at 25°C only the MP-based systems retained their original size. The presence of MP within the vesicle formulation was also shown to promote a sustained release of antigen in-vitro. The adjuvant activity of various systems was tested in mice against three subunit antigens, including mycobacterial fusion protein Ag85b-ESAT-6, and two malarial antigens (Merozoite surface protein 1, MSP1, and the glutamate rich protein, GLURP). The MP- and DDA-based systems induced antibody responses at comparable levels whereas the DDA-based systems induced more powerful cell-mediated immune responses. © 2006 The Authors.

Particulate delivery systems for vaccines:what can we expect?

In our attempts to thwart the unwanted attentions of microbes by prophylactic and therapeutic vaccination, the knowledge of interactions at the molecular level may prove to be an invaluable asset. This article examines how particulate delivery systems such as liposomes and polymer microspheres can be applied in the light of recent advances in immunological understanding. Some of the biological interactions of these delivery systems are discussed with relevance for antigen trafficking and molecular pathways of immunogenicity and emphasis on the possible interaction of liposomal components. In particular, traditional concepts such as antigen protection, delivery to antigen presenting cells and depot formation remain important aspects, whilst the inclusion of selected co-adjuvants and enhanced delivery of these moieties in conjunction with antigen now has a firm rationale. © 2006 The Authors.

Liposome-entrapped plasmid DNA:characterisation studies

Plasmid DNA pRc/CMV HBS (5.6 kb) (100 microg) encoding the S (small) region of hepatitis B surface antigen was incorporated by the dehydration-rehydration method into liposomes composed of 16 micromol egg phosphatidylcholine (PC), 8 micromol dioleoylphosphatidylcholine (DOPE) and 1, 2-diodeoyl-3-(trimethylammonium)propane (DOTAP) (cationic liposomes) or phosphatidylglycerol (anionic liposomes) in a variety of molar ratios. The method, entailing mixing of small unilamellar vesicles (SUV) with the DNA, followed by dehydration and rehydration, yielded incorporation values of 95-97 and 48-54% of the DNA used, respectively. Mixing of preformed cationic liposomes with 100 microg plasmid DNA also led to high complexation values of 73-97%. As expected, the association of DNA with preformed anionic liposomes was low (9%). Further work with cationic PC/DOPE/DOTAP liposomes attempted to establish differences in the nature of DNA association with the vesicles after complexation and the constructs generated by the process of dehydration/rehydration. Several lines of evidence obtained from studies on vesicle size and zeta-potential, fluorescent microscopy and gel electrophoresis in the presence of the anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, interaction of DNA with preformed cationic SUV as above, or with cationic SUV made of DOPE and DOTAP (1:1 molar ratio; ESCORT Transfection Reagent), leads to the formation of large complexes with externally bound DNA. For instance, such DNA is accessible to and can be dissociated by competing anionic SDS molecules. However, dehydration of the DNA-SUV complexes and subsequent rehydration, generates submicron size liposomes incorporating most of the DNA in a fashion that prevents DNA displacement through anion competition. It is suggested that, in this case, DNA is entrapped within the aqueous compartments, in between bilayers, presumably bound to the cationic charges.

Vaccine entrapment in liposomes

The use of liposomes as carriers of peptide, protein, and DNA vaccines requires simple, easy-to-scale-up technology capable of high-yield vaccine entrapment. Work from this laboratory has led to the development of techniques that can generate liposomes of various sizes, containing soluble antigens such as proteins and particulate antigens (e.g., killed or attenuated bacteria or viruses), as well as antigen-encoding DNA vaccines. Entrapment of vaccines is carried out by the dehydration-rehydration procedure which entails freeze-drying of a mixture of "empty" small unilamellar vesicles and free vaccines. On rehydration, the large multilamellar vesicles formed incorporate up to 90% or more of the vaccine used. When such liposomes are microfluidized in the presence of nonentrapped material, their size is reduced to about 100 nm in diameter, with much of the originally entrapped vaccine still associated with the vesicles. A similar technique applied for the entrapment of particulate antigens (e.g., Bacillus subtilis spores) consists of freeze-drying giant vesicles (4-5 microm in diameter) in the presence of spores. On rehydration and sucrose gradient fractionation of the suspension, up to 30% or more of the spores used are associated with generated giant liposomes of similar mean size.

Lyophilisation and sterilisation of liposomal vaccines to produce stable and sterile products

The advantages of liposomes as delivery systems for peptide, protein and DNA vaccines is well-recognised, unfortunately their application has been stinted by their instability during storage and their limited shelf-life. Further, sterilisation of these systems has been problematic, with degradation of the liposomes being reported after sterilisation using the various techniques available. Work form our laboratory has investigated techniques that can be applied to particulate liposomal vaccines such that they can be prepared in a freeze-dried and sterile format. In this article, we describe techniques for the lyophilisation, cryoprotection and sterilisation of liposomal vaccines. Applying these methods allows for the retention of both the chemical integrity of the lipids and the key physico-chemical characteristics of the liposomes (e.g., particle size, zeta potential, and dynamic viscosity), thus supporting the enhanced transition of liposomal vaccines from the bench to the clinic. © 2006 Elsevier Inc. All rights reserved.

Entrapment of plasmid DNA vaccines into liposomes by dehydration/rehydration

Intramuscular injection of naked plasmid DNA is known (1-3) to elicit humoral and cell-mediated immune responses against the encoded antigen. It is thought (2,3) that immunity follows DNA uptake by muscle cells, leading to the expression and extracellular release of the antigen which is then taken up by antigen presenting cells (APC). In addition, it is feasible that some of the injected DNA is taken up directly by APC. Disadvantages (1-3) of naked DNA vaccination include: uptake of DNA by only a minor fraction of muscle cells, exposure of DNA to deoxyribonuclease in the interstitial fluid thus necessitating the use of relatively large quantities of DNA, and, in some cases, injection into regenerating muscle in order to enhance immunity. We have recently proposed (1,4) that DNA immunization via liposomes (phospholipid vesicles) could circumvent the need of muscle involvement and instead facilitate (5) uptake of DNA by APC infiltrating the site of injection or in the lymphatics, at the same time protecting DNA from nuclease attack (6). Moreover, transfection of APC with liposomal DNA could be promoted by the judicial choice of vesicle surface charge, size and lipid composition, or by the co-entrapment, together with DNA, of plasmids expressing appropriate cytokines (e.g., interleukin 2), or immunostimulatory sequences.

Characterization of cationic liposomes based on dimethyldioctadecylammonium and synthetic cord factor from M. tuberculosis (trehalose 6,6′- dibehenate) - A novel adjuvant inducing both strong CMI and antibody responses:a novel adjuvant inducing both strong CMI and antibody responses

Incorporation of the glycolipid trehalose 6,6′-dibehenate (TDB) into cationic liposomes composed of the quaternary ammonium compound dimethyldioctadecylammonium (DDA) produce an adjuvant system which induces a powerful cell-mediated immune response and a strong antibody response, desirable for a high number of disease targets. We have used differential scanning calorimetry (DSC) to investigate the effect of TDB on the gel-fluid phase transition of DDA liposomes and to demonstrate that TDB is incorporated into DDA liposome bilayers. Transmission Electron Microscopy (TEM) and cryo-TEM confirmed that liposomes were formed when a lipid film of DDA containing small amounts of TDB was hydrated in an aqueous buffer solution at physiological pH. Furthermore, time development of particle size and zeta potential of DDA liposomes incorporating TDB during storage at 4°C and 25°C, indicates that TDB effectively stabilizes the DDA liposomes. Immunization of mice with the mycobacterial fusion protein Ag85B-ESAT-6 in DDA-TDB liposomes induced a strong, specific Th1 type immune response characterized by substantial production of the interferon-γ cytokine and high levels of IgG2b isotype antibodies. The lymphocyte subset releasing the interferon-γ was identified as CD4 T cells.

Precipitation casting of drug-loaded microporous PCL matrices:incorporation of progesterone by co-dissolution

Microporous, poly(ε-caprolactone) (PCL) matrices were loaded with progesterone by precipitation casting using co-solutions of PCL and progesterone in acetone. Progesterone loadings up to 32% w/w were readily achieved by increasing the drug content of the starting PCL solution. The kinetics of steroid release in PBS at 37°C over 10 days could be described effectively by a diffusional release model although the Korsmeyer-Peppas model indicated the involvement of multiple release phenomena. The diffusion rate constant (D) increased from 8 to 24 μg/mg matrix/day0.5 as the drug loading increased from 3.6 to 12.4% w/w. A total cumulative release of 75%-95% indicates the high efficiency of steroid delivery. Increasing the matrix density from 0.22 to 0.39 g/cm3, by increasing the starting PCL solution concentration, was less effective in changing drug release kinetics. Retention of anti-proliferative activity of released steroid was confirmed using cultures of breast cancer epithelial (MCF-7) cells. Progesterone released from PCL matrices into PBS at 37°C over 14 days retarded the growth of MCF-7 cells by a factor of at least 3.5 compared with progesterone-free controls. These findings recommend further investigation of precipitation-cast PCL matrices for delivery of bioactive molecules such as anti-proliferative agents from implanted, inserted or topical devices.

3D culture of bone-derived cells immobilised in alginate following light-triggered gelation

Photoreactive liposomes have been exploited as a means of developing 3D tissue constructs. Liposomes formulated using the photosensitive lipid 1,2-bis(4-(n-butyl)phenylazo-4′-phenylbutyroyl)phosphatidylcholine (Bis Azo PC), which undergoes conformational change on stimulation with long wavelength ultraviolet light, were prepared with entrapped CaCl2 before being incorporated into a 4% alginate solution. It was shown that stimulation of the photosensitive lipid using a light emitting diode (LED) (peak emission at 385 nm, dose equivalent to 9 mJ/cm2) caused the release of liposome-entrapped CaCl2, resulting in cross-linking of the alginate solution and immobilisation of bone-derived cells over a range of seeding densities, approximately 97% of which remained viable for periods of up to 14 days in culture. Entrapment volumes of a variety of liposome types were evaluated and interdigitating fusion vesicles were identified as having the highest payload (24%), however the inclusion of cholesterol as a means of shifting Bis Azo PC sensitivity into the visible light wavelengths resulted in an approximately 10-fold reduction in calcium entrapment. This application of light-sensitised liposomes offers the potential to create complex tissue engineering substrates containing cells immobilised in precise locations, in contrast with substrates onto which cells are seeded post-production. © 2007 Elsevier B.V. All rights reserved.

Comparison of vesicle based antigen delivery systems for delivery of hepatitis B surface antigen

There is a clinical need for a more effective vaccine against hepatitis B, and in particular vaccines that may be suitable for therapeutic administration. This study assesses the potential of cationic surfactant vesicle based formulations using two agents; the cationic amine containing [N-(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) or dimethyl dioctadecylammonium bromide (DDA) with hepatitis B surface antigen (HBsAg). Synthetic mycobacterial cord factor, trehalose 6,6′-dibehenate (TDB) has been used as an adjuvant and the addition of 1-monopalmitoyl glycerol (C16:0) (MP) and cholesterol (Chol) to DDA-TDB is assessed for its potential to facilitate formation of dehydration-rehydration vesicles (DRV) at room temperature, and the effect of this on immune responses. A DRV formulation is directly compared to an adsorbed formulation of the same composition and preparation protocol (MP:dioleoyl phosphoethanolamine (DOPE):Chol:DC-Chol) and the direct substitution of MP with phosphatidylcholine (PC) is also compared in DRV antigen-entrapped formulations. MP and Chol were shown to facilitate the use of DDA-TDB in DRV formulations prepared at room temperature, whilst there was marginal alteration of immunogenicity (a reduction in HBsAg-specific IL-2). The HBsAg adsorbed DRV formulation was not significantly different from the HBsAg entrapped DRV formulation. Overall, DDA formulations incorporating TDB showed markedly increased antigen specific splenocyte proliferation and elicited cytokine production concomitant with a strong T cell driven response, delineating formulations that may be useful for further evaluation of their clinical potential. © 2007 Elsevier B.V. All rights reserved.

Effects of fibre reinforcement on the mechanical properties of brushite cement

In this study the effect of structure and amount of polyglactin fibre incorporation into a brushite forming calcium phosphate cement system and the effect of mechanical compaction on the fibre modified system were investigated. In comparison the effect of resorbable polycaprolactone surface coating of cement specimens was investigated. The results showed that, apart from the mechanical properties of the reinforcing material, the structure of the incorporated fibres, regular or random, is crucial for the resulting flexural strength and modulus of elasticity. Fibre reinforcement could also be combined with mechanical compaction of the cement/fibre composite paste leading to a possible 7-fold increase in flexural strength or an almost 5-fold increase in modulus of elasticity. Reinforcement of the tensile surface of cement grafts may ultimately improve strength where required, especially in conjunction with bone fixation devices. © 2005 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

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.

Recent developments in particulate-based vaccines

Vaccines remain a key tool in the defence against major diseases. However, in the development of vaccines a trade off between safety and efficacy is required with newer vaccines, based on sub-unit proteins and peptides, displaying improved safety profiles yet suffering from low efficacy. Adjuvants can be employed to improve their potency, but currently there are only a limited number of adjuvant systems licensed for clinical use. Of the new adjuvants being investigated, particulate systems offer several advantages including: passive targeting to the antigen-presenting cells within the immune system, protection against adjuvant degradation, and ability for sustained antigen release. There has been a range of particulate vaccine delivery systems outlined in recent patents including polymer-based microspheres (which are generally more focused on the use of synthetic polymers, in particular the polyesters) and surfactant-based vesicles. Within these formulations, several patented systems are exploiting the use of cationic lipids which, despite their limitations in gene therapy, clearly offer strong potential as adjuvants. Within this review, the current range of particulate system technologies being investigated as potential adjuvants are discussed with regard to both their respective advantages and the potential hurdles which must be overcome for such systems to be converted into successful pharmaceutical products.