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. 2022 Oct 26;14(11):2291.
doi: 10.3390/pharmaceutics14112291.

Evaluation of Adjuvant Activity and Bio-Distribution of Archaeosomes Prepared Using Microfluidic Technology

Yimei Jia  1 , Gerard Agbayani  1 , Vandana Chandan  1 , Umar Iqbal  1 , Renu Dudani  1 , Hui Qian  2 , Zygmunt Jakubek  3 , Kenneth Chan  1 , Blair Harrison  1 , Lise Deschatelets  1 , Bassel Akache  1 , Michael J McCluskie  1
Affiliations

Evaluation of Adjuvant Activity and Bio-Distribution of Archaeosomes Prepared Using Microfluidic Technology

Yimei Jia et al. Pharmaceutics. .

Abstract

Archaeosomes, composed of sulfated lactosyl archaeol (SLA) glycolipids, have been proven to be an effective vaccine adjuvant in multiple preclinical models of infectious disease or cancer. They have classically been prepared using a thin-film hydration method with an average particle size of 100-200 nm. In this study, we developed methods to generate SLA archaeosomes at different sizes, i.e., 30 nm and 100 nm, via microfluidic mixing technology and evaluated their physicochemical characteristics, as well as adjuvant activity and in vivo biodistribution in mice. Archaeosomes, prepared using thin-film and microfluidic mixing techniques, had similar nanostructures and physicochemical characteristics, with both appearing stable during the course of this study when stored at 4 °C or 37 °C. They also demonstrated similar adjuvant activity when admixed with ovalbumin antigen and used to immunize mice, generating equivalent antigen-specific immune responses. Archaeosomes, labeled with CellVueTM NIR815, had an equivalent biodistribution with both sizes, namely the highest signal at the injection site at 24 h post injection, followed by liver, spleen and inguinal lymph node. The presence of SLA archaeosomes of either size helped to retain OVA antigen (OVA-Cy5.5) longer at the injection site than unadjuvanted OVA. Overall, archaeosomes of two sizes (30 nm and 100 nm) prepared using microfluidic mixing maintained similar physicochemical properties, adjuvant activity and biodistribution of antigen, in comparison to those compared by the conventional thin film hydration method. This suggests that microfluidics based approaches could be applied to generate consistently sized archaeosomes for use as a vaccine adjuvant.

Keywords: archaeosome; bio-distribution; glycolipid; liposome; microfluidics; sulfated lactosyl archaeol; vaccine adjuvant.

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Conflict of interest statement

Yimei Jia, Bassel Akache, Lakshmi Krishnan and Michael McCluskie are inventors on a SLA archaeosome-related patent application. All other authors have no competing/conflicts of interest.

Figures

Figure 1
Figure 1
TEM images of SLA-30 and SLA-100 SLA archaeosomes. Nanoparticle morphology was visualized using transmission electron microscopy for SLA-100 (left) and SLA30 (middle) and those prepared using thin-film hydration (right). Two types of vesicles were observed: One displayed a bilayer membrane with hollow aqueous core component (yellow arrow), the other displayed as solid particles with a filled inner compartment (white arrow).
Figure 2
Figure 2
Stability of archaeosomes, 100 nm (A); 30 nm (B), prepared using microfluidic mixing that were incubated at 4 °C and 37 °C for 3 weeks. All data were measured in the absence of antigen and represent the mean ± SD of three individual measurements.
Figure 3
Figure 3
OVA-specific antibody titers in mice. Mice were immunized twice with OVA alone or adjuvanted with SLA archaeosomes at days 0 and 21 by IM. On Day 20 (A) and 28 (B) (7 days post 2nd immunization) antibody response was assessed by ELISA on individual serum samples. Grouped data is presented as geometric mean + 95% Confidence Interval (n = 4–5 per group). **** p < 0.0001.
Figure 4
Figure 4
In vivo cytolytic activity in mice. Mice were immunized with OVA antigen alone or adjuvanted with SLA archaeosomes. Target cells (CFSE-labeled splenocytes from naïve mice pulsed with OVA specific CD8 epitope) were transferred to immunized mice on Day 27. On Day 28 (7 days post 2nd immunization), splenocytes were collected and the levels of the target cells measured by flow cytometry. Grouped data is presented as Geometric mean + SD (n = 5 per group). ** p < 0.01, **** p < 0.0001.
Figure 5
Figure 5
OVA- specific T cells as determined by ELISpot. Splenocytes of mice immunized with OVA antigen alone or formulated with SLA archaeosomes were collected on Day 28 (7 days post 2nd immunization). Cells were stimulated in vitro with OVA peptide SIINFEKL and IFN-γ secretion measured by ELISpot assay. Grouped data is presented as geometric mean ± SD (n = 4–5 per group). **** p < 0.0001.
Figure 6
Figure 6
In vivo biodistribution analysis by IVIS imaging of C57BL/6 mice after intramuscular injection of SLA-30 (A,C) and SLA-100 (B,D), respectively, at a 24 h time point. (A,B); whole body images of dorsal (top) and ventral (bottom) of archaeosomes at 24 h postinjection; (C,D) the averaged fluorescent signals obtained for SLA-30 (C) and SLA-100 (D), respectively, in major organs at 24 h post injection. Data is presented as Mean ± SD, n = 4/group.
Figure 7
Figure 7
The whole body in vivo biodistribution analysis by IVIS imaging of a representative C57BL/6 mouse on dorsal surface after intramuscular injection of OVA-cy5.5 in the presence of archaeosomes of 30 nm and 100 nm, respectively, and in the absence of archaeosome at predetermined time points.
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Grants and funding

This research was funded by Human Health Therapeutics Research Center, National Research Council Canada.

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