Studies on the lipase-induced degradation of lipid-based drug delivery systems. Part II - Investigations on the mechanisms leading to collapse of the lipid structure.

It has recently been found that lipid composition appears to have a major influence on the rate of lipase-induced degradation of lipid-based extended release drug delivery systems (microparticles, compressed implants and extrudated implants). Previously, we have found that during lipase incubation, depending on the lipid used, lipidic extrudates can lose their physical strength and collapse generating lipid particles in the µm-range. The aim of this study was to characterise the processes leading to collapse of solid lipid-based drug delivery systems during in vitro lipase incubation. Compressed lipid implants were used as model systems. Free fatty acids (FFA) generated in the incubation experiments were derivatised and subsequently analysed via reversed phase-HPLC in order to characterise the degradation behaviour of single lipid components (glyceryltrilaurate (D112), glyceryltrimyristate (D114), glyceryltripalmitate (D116) and glyceryltristearate (D118)) used for the preparation of compressed lipid implants. Further, Raman spectroscopy/microscopy, differential scanning calorimetry, scanning electron and light microscopy were used to investigate the physical and chemical changes in the implants upon lipase incubation. This study revealed that the lipid component D112 plays a major role in the degradation and erosion processes occurring during lipase incubation of lipid implants. The production of D112/lauric acid mixtures, with a melting point below human body temperature, leads to lipid matrices melting and losing their physical integrity.

Generation of hypoallergenic neoglycoconjugates for dendritic cell targeted vaccination: a novel tool for specific immunotherapy.

The incidence of allergic disorders and asthma continuously increased over the past decades, consuming a considerable proportion of the health care budget. Allergen-specific subcutaneous immunotherapy represents the only intervention treating the underlying causes of type I allergies, but still suffers from unwanted side effects and low compliance. There is an urgent need for novel approaches improving safety and efficacy of this therapy. In the present study we investigated carbohydrate-mediated targeting of allergens to dermal antigen-presenting cells and its influence on immunogenicity and allergenicity. Mannan, high (40kDa) and low (6kDa) molecular weight dextran, and maltodextrin were covalently attached to ovalbumin and papain via mild carbohydrate oxidation resulting in neoglycocomplexes of various sizes. In particular, mannan-conjugates were efficiently taken up by dendritic cells in vivo leading to elevated humoral immune responses against the protein moiety and a shift from IgE to IgG. Beyond providing an adjuvant effect, papain glycocomplexes also proved to mask B-cell epitopes, thus rendering the allergen derivative hypoallergenic. The present data demonstrate that carbohydrate-modified allergens combine targeting of antigen presenting cells with hypoallergenicity, offering the potential for low dose allergen-specific immunotherapy while concomitantly reducing the risk of side effects.

Preparation and validation of a skin model for the evaluation of intradermal powder injection devices.

A very promising novel needle-free application method is epidermal powder immunisation, a method delivering particulate vaccines into the viable epidermis of human skin where a dense network of immunocompetent cells resides. These antigen-presenting cells (Langerhans cells) are able to recognise antigens, process them and present them to naïve T-cells and induce effective immune responses. Powder injection devices are being developed, and their evaluation is essential before applying them on live animals and individuals. An appropriate skin model will accelerate the development of such injection devices. Different films made from gelatin, silicon and agar were prepared and investigated as skin model candidates for the evaluation of powder injection devices. The mechanical properties of the skin model candidates were measured with an indentation method using a texture analyser, and the results were compared to the properties of human skin and pig skin. The indentation behaviour of the model films and the biological skin samples suggest that gelatin films plasticised with glycerol are very well suitable for a skin model. The mechanical properties of gelatin based films can be tailored by changing the glycerol content in the film making it even possible to simulate human skin with different mechanical properties as the mechanical properties depend on the individual, age, sex and site of injection. The stability of the gelatin films was also investigated under long-term storage. In addition, confocal laser scanning microscopy was used as a novel tool to determine the depths and size of fluorescently labelled particles in the gelatin model.

Devices for intradermal vaccination.

New insights in vaccine development, the need for safe, economic and efficient vaccine administration and the increasing mechanistic knowledge of immune responses induced by targeting the intradermal layers of the skin have all driven the engineering of devices for intradermal vaccination. In this review we highlight different delivery devices that make the epidermal and dermal layers of the skin accessible for vaccine administration. Depending on the device the desired vaccine can be applied either as a liquid formulation or as solid, powdered vaccine particles. The process of intradermal injection employs micron-sized needles that are inserted 1.5mm perpendicularly into the skin, and which inject approximately 100-200µl of a liquid vaccine formulation into the dermal skin layers. Tattoo devices, on the other hand, can be used to deliver liquid vaccine formulations into the dermal layer of the skin by the use of oscillating needles. Microneedle arrays are made of vaccine-coated solid microneedles or biodegradable microneedles. These are inserted into the dermal layers of the skin where either the vaccine coating is dissolved, or the microneedle itself dissolves in place. Jet-injectors operate by generating a high pressured stream, which flushes the liquid vaccine formulation into the deeper skin layers. Delivery devices using liquid vaccine formulations are advantageous, as established vaccine formulations can be used as provided without the need for reformulation. However, approaches that deliver vaccines in a solid form may also prove to be promising. One such method is the ballistic approach, in which solid vaccine particles or vaccine-coated gold particles are accelerated towards the skin by needle-free devices, so that the particles are deposited in the epidermal and dermal layers of the skin. These various delivery devices are explored in this review with regard to their delivery mechanism and ease of handling, their efficacy in clinical trials and their suitability for practical use.

Recombinant spider silk particles for controlled delivery of protein drugs.

The engineered and recombinant spider silk protein eADF4(C16) has been shown to be a promising biomaterial for the use as drug delivery system. In previous studies, eADF4(C16) particles were loaded with low molecular weight drugs exhibiting a positive net-charge and sufficient hydrophobicity. Here, we demonstrate that also macromolecular drugs like proteins can be loaded on eADF4(C16) particles. Using lysozyme as a model protein, remarkably high loading of up to 30% [w/w] was feasible and high loading efficiencies of almost 100% were obtained. Furthermore, using confocal laser scanning microscopy, it is demonstrated that fluorescently labeled lysozyme is not only adsorbed to the negatively charged particles' surface, but also diffusing into the matrix of eADF4(C16) particles. The release of lysozyme is shown to be dependent on the ionic strength and pH of the release medium. To improve the long-term stability of eADF4(C16) containing dispersions, lyophilization is shown as a suitable tool. Disaccharides (sucrose, trehalose) and mannitol served as stabilizers to prevent aggregation and/or particle degradation during freeze-drying. The slowly biodegradable eADF4(C16) particles are a promising new particulate drug carrier system for the delivery of susceptible drugs like therapeutic proteins.

Immunostimulatory lipid implants containing Quil-A and DC-cholesterol.

Biocompatible lipid implants which promote the sustained release of antigen have potential as novel vaccine delivery systems for subunit antigen as they may reduce or remove the requirement for multiple administrations. Of particular interest are sustained release systems that release antigen incorporated into particles. Previous work has demonstrated that lipid implants prepared from phosphatidylcholine, cholesterol, the adjuvant Quil-A, and ovalbumin as the model antigen could stimulate an immune response equivalent to that induced by a prime and boost with a comparable injectable vaccine. However, entrapment of antigen into particles released from the implant was low. Therefore the aim of this study was to firstly determine if the inclusion of a cationic derivative of cholesterol, DC-cholesterol, into the implants increased antigen entrapment and immunogenicity, and secondly, if a cationic implant could induce at least a comparable immune response as compared to a prime and boost with an injectable vaccine. The inclusion of DC-cholesterol had only a minor effect on antigen entrapment into particles released from the implants and the implants did not stimulate cellular responses as effectively as the comparable injectable vaccine or the unmodified implant containing Quil-A and cholesterol, although the vaccine did induce stronger responses than either soluble protein alone, or protein co-delivered in alum.

In vivo activity of cationic immune stimulating complexes (PLUSCOMs).

A particulate vaccine delivery system consisting of cationic ISCOM derivatives (PLUSCOMs) was compared to classic anionic ISCOMs with regard to antigen attachment and ability to elicit in vivo T cell responses against a model protein antigen (ovalbumin [OVA]). ISCOMs did not incorporate hydrophilic OVA whilst OVA readily adsorbed onto PLUSCOMs with increasing adsorption at higher protein concentrations. The zeta-potential of PLUSCOMs significantly decreased with increasing protein load, suggesting neutralization of the cationic charge upon absorption of the anionic OVA. Antigen-specific CD8 T cell responses were demonstrated in mice vaccinated with either PLUSCOMs or ISCOMs. Ex vivo restimulation of harvested T cells demonstrated that cells isolated from PLUSCOM and ISCOM vaccinated mice responded to the secondary OVA challenge more efficiently than mice vaccinated with OVA in solution. Restimulated cells from the mice vaccinated with particulate vaccines produced significantly more INF-gamma. Therefore PLUSCOMs are as effective as classic ISCOMs in inducing antigen-specific CD8 T cell responses and have advantages with regard to the incorporation of purified anionic antigens.

Immunostimulatory biodegradable implants containing the adjuvant Quil-A--Part I: Physicochemical characterisation.

Sustained-release vaccines offer the potential to reduce, or obviate, the need for repeated dosing of vaccines. In this study, we report the development and characterisation of sustained-release lipid implants that release immunogenic, self-assembling colloidal particles. Lipid implants consisting of cholesterol (CHOL), phosphatidylcholine (PC), the adjuvant Quil-A (QA) and the model antigen ovalbumin (OVA) were formulated and investigated using a variety of techniques. Transmission electron microscopy was utilised to demonstrate the release of colloidal structures from these implants over time. The nature of the colloidal particles varied depending on the ratio of QA:CHOL:PC. The release of the model antigen as well as its incorporation into the colloidal particles was investigated using a fluorescent tag covalently coupled to OVA and quantified using fluorospectrophotometry. The antigen release was modified by the incorporation of excess CHOL into the formulation and was not only dependent on the ratio of QA:CHOL:PC but also on the nature of the model antigen. Alteration of the hydrophobicity of the model antigen resulted in an increased incorporation into the colloidal structures. Surface changes of the implants were analysed using scanning electron microscopy. The implant formulations investigated in this study show a potential for the delivery of subunit vaccines.

Immunostimulatory biodegradable implants containing the adjuvant Quil-A--Part II: In vivo evaluation.

Sustained-release formulations have drawn the attention of formulation scientists working in the area of vaccine research because these systems may reduce the need for booster immunisations. This would be of great advantage especially for the administration of subunit vaccines. The aim of this study was to illustrate the performance of liposome-forming, sustained-release lipid implants containing 2% of the adjuvant Quil-A (QA) (w/w of total lipids) and ovalbumin (OVA) as a model antigen, in an in vivo study using C57Bl/6 mice. QA/OVA-containing lipid implants were administered subcutaneously and stimulated a similar magnitude of immune response when compared with an immediate-release formulation that contained an equivalent amount of adjuvant and antigen but was administered twice. The novel implant system presented here combines the advantages of both sustained release and particulate delivery in one formulation.

On the preparation, microscopic investigation and application of ISCOMs.

ISCOM matrices constitute colloidal structures formed from Quillaja saponins, cholesterol and phospholipid. Addition of protein antigens to these matrices leads to the formation of ISCOMs. In this review we report on microscopic investigations of ISCOM matrices and ISCOMs as well as related colloidal structures, such as helices, worm-like micelles, ring-like micelles, and lamellae structures. We briefly outline the immunologic basis for the use of ISCOMs as vaccine delivery systems, and describe the various methods to form ISCOMs. Negative staining transmission electron micrographs of all colloidal structures are presented and described. On the basis of our microscopic investigations, different formation mechanisms of ISCOMS are discussed.

Cationic cage-like complexes formed by DC-cholesterol, Quil-A, and phospholipid.

This study describes the formation of cationic, cage-like complexes which have a structure similar to classic anionic ISCOMs. In order to prepare these complexes cholesterol, a major component of classic ISCOM formulations, was substituted with a cationic derivative, 3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholesterol (DC-CHOL). Colloidal dispersions with varying compositions of DC-CHOL, phosphatidylcholine, and Quil-A, which is a mixture of anionic triterpene saponins, were prepared by the lipid-film hydration method and characterised by transmission electron microscopy and laser Doppler electrophoresis. The colloidal structures obtained are presented in pseudo-ternary phase diagrams with two buffer systems as the pseudo-component. It was found that the formation of cationic, cage-like particles is highly depending on the formulation buffer. With TRIS buffered saline (TBS) pH 7.4, cage-like particles formed at compositions with high proportions of DC-CHOL and had a strongly positive zeta-potential. These could be purified by differential centrifugation. With phosphate buffered saline pH 7.4, the formation of cage-like particles was much reduced. It was shown that the formation of cage-like particles with a positive charge depended on suitable concentrations of TRIS in the hydration buffer.

Immuno-stimulating complexes prepared by ethanol injection.

This study describes the formulation of immuno-stimulating complexes (ISCOMs) utilising the ethanol injection technique. Cholesterol and phosphatidylcholine were dissolved in ethanol and the resulting solution was rapidly injected into a stirred, aqueous solution of the triterpene-saponin mixture Quil-A. The reversed experiment was also carried out by adding the aqueous Quil-A solution to a solution of cholesterol/phosphatidylcholine dissolved in ethanol. This was done by either rapid injection or dropwise addition of the aqueous Quil-A solution. The colloidal dispersions obtained by ethanol injection and reversed addition were compared with formulations obtained by the dialysis and lipid-film hydration methods. In a further experiment, the preparation of ISCOMs from liposomes as precursor structures was investigated. Transmission electron microscopy was used to analyse the resulting colloidal dispersions. Samples were also compared with respect to homogeneity of obtained particle species. The ethanol injection technique led to formation of ISCOMs in high numbers within 2 h post formulation. The reversed rapid injection resulted in a similar colloidal dispersion, differing from the former mainly due to the presence of some helical micellar structures. The reversed, dropwise addition led to the formation of helices as the predominant colloidal structure. Of the three previously established methods, only dialysis led to the formation of ISCOMs within 48 h. The lipid-film hydration method and the approach using liposomes as precursor structures did not produce ISCOMs under the conditions and within the time periods investigated. However, it is known that dispersions prepared by the hydration method equilibrate towards ISCOMs after longer storage. Ethanol injection and reversed rapid injection are simple, cost-effective and quick methods to produce ISCOMs.