Abstract
Developing a technology that enables oral vaccines to work efficiently remains a considerable effort since a number of difficulties must be addressed. The key objective being to ensure the safe passage through the harsh conditions within the gastrointestinal tract, promoting delivery that induces enhanced immune response. In the particular case of hepatitis B, the oral formulation in the nanostructured silica SBA-15 is a viable approach. As a result of its porous structure, low toxicity and structural stability, SBA-15 is capable to protect and release the hepatitis B surface antigen (HBsAg), used in the vaccination scheme, at the desired destination. Furthermore, when compared to the currently used injection based delivery method, better or similar antibody response has been observed. However, information about the organisation of the antigen protein remains unknown. For instance, HBsAg is too large to enter the 10 nm ordered mesopores of SBA-15 and has a tendency to agglomerate when protected by the delivery system. Here we report on the pH dependence of HBsAg aggregation in saline solution investigated using small angle X-rays scattering that resulted in an optimisation of the encapsulation conditions. Additionally, X-ray microscopy combined with neutron and X-ray tomography provided full 3D information of the HBsAg clustering (i.e. agglomeration) inside the SBA-15 macropores. This method enables the visualisation of the organisation of the antigen in the interior of the delivery system, where agglomerated HBsAg coexists with its immunological effective uniformly distributed counterpart. This new approach, to be taken into account while preparing the formulation, can greatly help in the understanding of clinical studies and advance new formulations.
Highlights
The nanostructured mesoporous silica SBA-15, formed by ~20 μm particles with hexagonal ordered mesopores with a diameter of 10 nm and macropores larger than 50 nm, is especially interesting[6,7,8,9,10,11]
This study demonstrates the potential of combining neutron and X-ray imaging techniques with X-ray microscopy to visualise the distribution of the protein antigen hepatitis B surface antigen (HBsAg) used for hepatitis B vaccination in the oral delivery vehicle SBA-15
Ab initio modelling of the small angle X-ray scattering (SAXS) data of HBsAg protein in solution resulted in a half sphere with a diameter of 32 nm, confirming that the antigen is too large to enter the 10 nm mesopores of SBA-15 and must be protected in the macropores
Summary
The nanostructured mesoporous silica SBA-15, formed by ~20 μm particles with hexagonal ordered mesopores with a diameter of 10 nm and macropores larger than 50 nm, is especially interesting[6,7,8,9,10,11]. Recent in vivo mice studies have reported that SBA-15 induces better or similar immunological response compared to the traditional injection based vaccination method, as after immunisation a higher level of antibodies and immune cells are observed[7]. Another important consideration is the fact that the macrophages can recognize size and www.nature.com/scientificreports/. Using X-ray and neutron tomography, we verified that for different HBsAg to SBA-15 mass ratio, the antigen and its buffer were observed together, but non-uniformly distributed These measurements demonstrated that HBsAg agglomerates the least upon encapsulation in the mass ratio 1:40, making this the optimal condition for oral delivery. An important application of this approach during the formulation process is the ability to control the distribution of the protein in the delivery vehicle as well as to clarify if the efficiency of oral vaccines is directly related to the morphology of the carrier
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