Sarah Hook

Preparation of phytantriol cubosomes by solvent precursor dilution for the delivery of protein vaccines

Different delivery strategies to improve the immunogenicity of peptide/protein-based vaccines are currently under investigation. In this study, the preparation and physicochemical characterisation of cubosomes, a novel lipid-based particulate system currently being explored for vaccine delivery, was investigated. Cubosomes were prepared from a liquid precursor mixture containing phytantriol or glycerylmonooleate (GMO), F127 for particle stabilisation, and a hydrotrope (ethanol or polyethylene glycol (PEG(200)) or propylene glycol (PG)). Several liquid precursors were prepared, and the effect of varying the concentrations of F127 and the hydrotrope on cubosome formation was investigated. Formulations were prepared by fragmentation for comparison. The model protein ovalbumin (Ova) was also entrapped within selected formulations. Submicron-sized particles (180-300 nm) were formed spontaneously upon dilution of the liquid precursors, circumventing the need for the preformed cubic phase used in traditional fragmentation-based methods. The nanostructure of the phytantriol dispersions was determined to be cubic phase using SAXS whilst GMO dispersions had a reverse hexagonal nanostructure coexisting with cubic phase. The greatest entrapment of Ova was within phytantriol cubosomes prepared from liquid precursors. Release of Ova from the various formulations was sustained; however, release was significantly faster and the extent of release was greater from fragmented dispersions compared to liquid precursor formulations. Taken together, these results suggest that phytantriol cubosomes can be prepared using liquid precursors and that it is a suitable alternative to GMO. Furthermore, the high entrapment and the slow release of Ova in vitro highlight the potential of phytantriol cubosomes prepared using liquid precursors as a novel vaccine delivery system.

Liquid Crystalline Systems of Phytantriol and Glyceryl Monooleate Containing a Hydrophilic Protein: Characterisation, Swelling and Release Kinetics

Swelling and phase behaviour of phytantriol and glyceryl monooleate (GMO) matrices with varying water loadings were investigated. Release of a model protein, FITC-Ova was subsequently examined. Polarised light microscopy and small angle X-ray scattering analysis showed that the addition of FITC-Ova only altered the liquid crystalline structure of phytantriol matrices at low water loadings, and that postrelease study, the phase structure of matrices at both low and high loading reflected that of the binary system. Addition of FITC-Ova to GMO matrices also altered the liquid crystalline structure when compared to the respective binary system at low but not at high loading. All samples analysed after the release study had transformed to the reverse hexagonal phase (H(II)). Swelling studies revealed a faster and more extensive swelling of GMO when compared to phytantriol. Release of FITC-Ova from phytantriol matrices was faster and occurred to a greater extent most likely due to the conversion of GMO matrices into the H(II) phase. No effect on release as a function of initial water content was observed for either lipid. We have confirmed that phytantriol based liquid crystalline matrices can sustain the release of a hydrophilic protein, suggesting its suitability as a potential sustained antigen-delivery system.

Bicontinuous cubic liquid crystals as sustained delivery systems for peptides and proteins.

Importance of the field: Self-assembling lipid-based liquid crystalline systems are a broad and active area of research. Of these mesophases, the cubic phase with its highly twisted bilayer and two non-intersecting water channels has been investigated extensively for drug delivery. The cubic phase has been shown to accommodate and control the release of drugs with varying physicochemical properties. Also, the lipids used to prepare these delivery systems are generally cheap, safe and biodegradable, making these systems highly attractive. Early research investigating the potential of cubic phases as delivery systems showed that several peptides or proteins entrapped within these gel-based systems showed retarded release. Furthermore, entrapment within the cubic phase protected the selected peptide or protein from chemical and physical degradation with its native confirmation and bioactivity retained. Areas covered in this review: In this review, the literature pertaining to the delivery of various bioactives from cubic liquid crystalline phases is examined, with a particular focus on peptides and proteins. The scope and limitations of the cubic phases in this respect and the future of cubic liquid crystalline systems as sustained delivery systems are highlighted. What the reader will gain: The reader will be able to gain an understanding of the properties of the bicontinuous cubic phase and how its structural attributes make these systems desirable for sustained delivery of bioactives, in particular peptides and proteins, but also how these same structural properties have hindered progress towards clinical applications. Current strategies to overcome these issues will also be discussed. Take home message: The bicontinuous cubic phase offers great potential in the field of peptide and protein delivery, but limited research in this area precludes definite conclusions to its future in this respect.

Cubosomes containing the adjuvants imiquimod and monophosphoryl lipid A stimulate robust cellular and humoral immune responses.

New generation vaccines increasingly utilize highly purified peptides and proteins as the target antigen, however these are often poorly immunogenic. One of the most promising strategies for improving immunogenicity of such subunit vaccines is through incorporation into particulate carriers. Here we report the preparation, physicochemical characterization and in vivo immunological activity of cubosomes, a novel lipid-based nanostructured particulate carrier, modified to include the Toll-like receptor agonists monophosphoryl lipid A and imiquimod. The immunological activity of cubosome formulations was compared to that of liposome and alum formulations. Sustained release of the model antigen ovalbumin (Ova) was observed in vitro and in vivo from cubosomes. Cubosomes+adjuvants induced robust CD8⁺ and CD4⁺ T cell proliferation and interferon-γ production, as well as the production of Ova-specific antibodies. Cubosomes+adjuvants were more efficient at generating Ova-specific cellular responses and were equally as effective in generating humoral responses when compared to liposomes+adjuvants and alum. Overall, the results show that cubosomes have the potential to act as effective sustained release vaccine delivery systems.

Comparative study of liposomes, transfersomes, ethosomes and cubosomes for transcutaneous immunisation: characterisation and in vitro skin penetration.

Objectives  Lipid colloidal vaccines, including liposomes, transfersomes, ethosomes and cubosomes, were formulated, characterised and investigated for their ability to enhance penetration of a peptide vaccine through stillborn piglet skin in vitro.

Mannosylated liposomes as antigen delivery vehicles for targeting to dendritic cells

The immune stimulating ability of mannosylated liposomes containing FITC‐ovalbumin as a model antigen and displaying either a branched tri‐mannose or a mono‐mannose ligand on the liposome surface was investigated in human monocyte‐derived dendritic cells (MoDCs) and murine bone‐marrow‐derived dendritic cells (BMDCs). Uptake of liposomes, dendritic cell activation and proliferation of CD8+ T cells from OT‐I transgenic mice were determined by flow cytometry. Uptake of liposomes displaying the tri‐mannose ligand was enhanced in human MoDCs compared with both non‐mannosylated liposomes and liposomes displaying mono‐mannose ligands. However, this increased uptake did not result in an increase in expression of CD80 or CD86 on the surface of the MoDCs. In contrast, neither tri‐mannose‐ nor mono‐mannose‐containing liposomes were taken up by murine BMDCs to a greater extent than non‐mannose‐containing liposomes. The expression of CD86 and CD40 on the surface of BMDCs was not increased after exposure to mannosylated lipo‐somes and BMDCs incubated with mannosylated liposomes were not able to stimulate proliferation of CD8+ T cells to any greater extent than BMDCs incubated with non‐mannosylated liposomes. These findings suggest that while mannose‐containing ligands can enhance the uptake of antigen‐containing liposomes by some dendritic cells, important differences in the affinity of carbohydrate‐binding receptors for mannose‐containing ligands do exist between species. In addition, the increase in uptake of antigen by dendritic cells using mannosylated liposomes does not necessarily result in enhanced dendritic cell activation.

Oral insulin delivery using nanoparticles based on microemulsions with different structure-types : Optimisation and in vivo evaluation

The purpose of this study was to optimise entrapment of insulin in poly(alkylcyanoacrylate) nanoparticles prepared from microemulsions with different microstructure containing isopropyl myristate, caprylocaproyl macrogolglycerides, polyglyceryl oleate and insulin solution and to investigate the in vitro release and bioactivity of insulin in nanoparticles dispersed in the microemulsion templates. Entrapment efficiency and release of insulin were studied using a reverse-phase HPLC assay. Morphology of the nanoparticles was examined with scanning electron microscopy. Bioactivity of insulin was studied using a streptozotocin-diabetic rat model. Nanoparticles were spherical with 200-400 nm in size without significant difference between different microemulsion templates, types and amounts of monomer. Entrapment efficiency increased significantly with increasing monomer concentration but decreased with increasing aqueous fraction in the microemulsion template. Insulin loading however, showed an opposite trend. In vitro release profiles of insulin from the nanoparticles dispersed in the microemulsion templates were controlled by the monomer concentration only. In vivo, a consistent and significant hypoglycemic effect over controls was found for up to 36 h depending on the type of monomer. No significant serum insulin levels were detectable. This study showed that the strategy of delivering insulin orally, entrapped in nanoparticles and dispersed in a biocompatible microemulsion is promising and highlights the importance of optimisation studies in combination with in vivo experiments.

Activation of the NLRP3 inflammasome is not a feature of all particulate vaccine adjuvants.

Particulate vaccine formulations, designed to improve the delivery of antigens to antigen‐presenting cells (APCs) and to stimulate an immune response, have been shown to activate the NLRP3 inflammasome. This leads to the processing and secretion of interleukin (IL)‐1β, which supports the recruitment of pro‐inflammatory immune cells into the tissue and can therefore be beneficial for vaccine potency. Recent work suggested that this may be a common mechanism of action for all particulate formulations. The aim of this study was to investigate whether the activation of the NLRP3 inflammasome was common to many delivery systems. We prepared polymer‐based chitosan nanoparticles (CNPs), lipid‐based cubosomes, a water in oil emulsion of incomplete Freunds adjuvant (IFA) and alum formulations and examined inflammasome activation in vitro using murine bone‐marrow‐derived dendritic cells and human peripheral blood mononuclear cells and in vivo in mice. The formulations differed in their morphology, size and zeta‐potential. Only the positively charged particles (CNPs and alum) were able to activate the inflammasome and increase the secretion of IL‐1β. A decrease in the activation of the inflammasome with these particulates was observed when cathepsin B‐mediated effects were blocked, implying a role of lysosomal rupture in the activation process. These findings demonstrate a role for the surface charge of particulates in the activation of the NLRP3 inflammasome, which should be considered when designing a novel vaccine formulation.

In vitro and in vivo investigation of thermosensitive chitosan hydrogels containing silica nanoparticles for vaccine delivery.

In this work silica nanoparticles (SNP) containing the model antigen ovalbumin (OVA) were incorporated into a thermosensitive chitosan hydrogel, and the resulting formulation investigated for its potential to act as a particulate sustained release vaccine delivery system. OVA-loaded SNP and chitosan hydrogels containing OVA-loaded SNP were prepared and characterised in vitro, and examined for their ability to elicit OVA-specific immune responses in vivo. Optimised SNP were found to be approximately 300nm in size with a moderate level of heterogeneity, a highly negative zeta potential, and an entrapment efficiency of approximately 7%. A porous particulate structure was indicated both by electron microscopy and a rapid release of fluorescently-labelled OVA (FITC-OVA) from SNP. Following successful incorporation of SNP into chitosan hydrogels, the release of both soluble and SNP-associated antigen from gel systems was quantified. Approximately 16% of total protein was released in a particulate form over a 14-day period, while approximately 35% was released as soluble antigen. Gel-based systems containing SNP-associated or soluble antigen in the presence or absence of the adjuvant Quil A (QA) demonstrated an ability to stimulate both cell mediated and humoral immunity in vivo. Chitosan gels containing OVA-loaded SNP and the adjuvant QA showed a significantly greater ability to induce CD4(+) T cell proliferation than chitosan gel containing soluble OVA and QA, indicating the future promise for such a system.

Protein delivery using nanoparticles based on microemulsions with different structure-types

Poly(alkylcyanoacrylate) nanoparticles based on microemulsions with different structure-types and containing insulin as a model protein were prepared and characterised in this study. A phase diagram of the pseudoternary system isopropyl myristate, caprylocaproyl macrogolglycerides, polyglycerol oleate and water was established. All compounds used in this study were pharmaceutically acceptable and biocompatible. The area in the phase diagram containing optically isotropic, monophasic systems was designated as the microemulsion region. Systems within this region were identified as water-in-oil (w/o), bicontinuous and oil-in-water (o/w) microemulsions with viscosity, conductivity, differential scanning calorimetry and self-diffusion NMR. The size distributions of the resulting nanoparticles prepared by interfacial polymerisation from selected microemulsions using ethyl (2) cyanoacrylate and butyl (2) cyanoacrylate were unimodal but template- and monomer-dependent and ranged from 160 to 400 nm. Entrapment and release of insulin were also studied. Entrapment ranged from 11.5 to 20.9% and a near zero-order release was observed after an initial burst. Release of insulin was monitored for 6h. Insulin-loaded nanoparticles were 320-350 nm in size. The microemulsion-structure was retained during the polymerisation process as determined by NMR. This study showed that these microemulsions with flexible formulation possibilities for the solubilisation of peptides and proteins depending on their microstructure could serve well as a platform for designing encapsulation processes for oral delivery of insulin.