Transcutaneous immunization using lipid colloidal systems and microneedles
Background: Needle-free immunization is a novel vaccination approach and provides many advantages over traditional vaccination. The present study focused on transcutaneous immunization (TCI) which is a promising approach due to the abundance of antigen presenting cells (APCs), residing in the skin. However the main obstacle for TCI is the delivery of the vaccine through the stratum corneum (SC) to the APCs in the deeper skin layers. Many approaches have been used to enhance skin penetration and in this study lipid colloidal systems and microneedles (MCNs) were utilized as a synergistic approach for vaccine delivery through the skin. A preliminary study investigated the ability of various lipid carriers including liposomes, transfersomes, ethosomes and cubosomes to enhance TCI. Subsequently, the effect of an optimal lipid colloidal formulation in combination with MCNs on TCI was examined in vitro and in vivo. Methods: Liposomes and transfersomes were prepared by the film-hydration method and ethosomes prepared by the modified reverse phase method. These vesicle systems were made of L-α-phosphatidylcholine. Cubosomes were made of phytantriol and prepared using the lipid precursor method. All formulations were loaded with a constant amount of peptide antigen and the adjuvants monophosphoryl lipid A (MPL) and Quil A (QA) and were characterized in terms of size, zeta potential, deformability, morphology, entrapment efficiency, interfacial behavior and immune reactivity in vitro. Permeation of the vaccines formulations (with or without MCN pretreatment) through stillborn piglet skin was investigated in vitro under occlusive conditions using Franz diffusion cells. Permeation and skin retention of peptide was visualized using confocal laser scanning microscopy (CLSM), optical coherence tomography (OCT) and two photon microscopy (2PM). The immune response to topically delivered peptides in vivo was investigated in C57BL/6 mice by measuring the expansion of antigen-specific CD4+ and CD8+ T cells, antigen-specific recall responses and the production of IFN-γ. Results: All formulations contained negatively charged particles of similar size (134–200 nm). Incorporation of peptide antigen into the formulations was variable ranging from 20 to 80%. Addition of QA to the formulations destabilised the monolayers, reduced peptide loading and increased particle deformability. The most deformable system was cubosomes and they were also an effective vaccine delivery system to BMDCs in vitro. Although there was no detectable permeation of the peptide through the intact skin, cubosomes and ethosomes showed superior skin retention compared with liposomes and transfersomes. Imaging studies revealed greater peptide penetration into skin via the intercellular pathway when vaccine was formulated in cubosomes. With the other systems peptide was mostly located in the vicinity of the hair follicles and within the hair shaft. From the preliminary results, cubosomes were identified as a potential candidate formulation to be used in combination with MCNs to improve TCI. The utilization of MCNs significantly improved vaccine permeation and penetration. A combined approach using cubosomes with MCNs increased skin retention but not skin permeation. This was likely due to partitioning between the hydrophilic epidermis and lipid particles. Vaccine antigen appeared to be preferentially taken up by a subpopulation of skin APC known as dermal dendritic cells. Interestingly delivery of vaccine using MCNs in conjunction with cubosomes appeared to stimulate a CD8+ T cell biased immune response. Conclusion: A combined approach of using cubosomes and MCNs improved vaccine uptake into skin. It appears that by transiently disrupting the SC with MCN, vaccine was targeted to a different subset of APCs than that it would have encountered had the vaccine been applied to intact skin and that a qualitatively different immune response is produced.
Advisor: Hook, Sarah; Rades, Thomas
Degree Name: Doctor of Philosophy
Degree Discipline: School of Pharmacy
Publisher: University of Otago
Keywords: skin vaccination; microneedles; Optical Coherence Tomograph; two-photon microscopy; lipid-based colloidal systems
Research Type: Thesis