Sulfated lactosyl archaeol (SLA; 6′-sulfate-β-D-Galp-(1,4)-β-D-Glcp-(1,1)-archaeol) was synthesized, as described previously [20 ]. Cy5.5-OVA conjugate was purchased from Nanocs (New York, NY, USA), while CellVueTM NIR815 was purchased from Thermofisher Scientific (Waltham, MA, USA).
Thin-film hydration archaeosomes were prepared, as previously described [10 (link)]. In brief, SLA lipid that was dissolved in chloroform/methanol underwent solvent removal under N2 gas with mild heating to form a thin lipid film. Dried lipids were then hydrated in Milli-Q water for more than 12 h to form a uniform lipid suspension. Next, sonication was applied at 40 °C in an ultrasonic water bath (Fisher Scientific, Ottawa, ON, Canada) for up to an hour until the desired particle size (~100 nm) was obtained. After that, 10 × PBS (Millipore Sigma Canada, Oakville, Ontario) was added and archaeosomes stored at 2–8 °C until used. Note that we have previously demonstrated that archaeosomes stored under similar conditions were stable up to 6 months of testing, which was well beyond the storage time of ~1 month in the current study [21 (link)]. On the day of immunization, antigen solution (ovalbumin; OVA; type VI, Sigma-Aldrich, St. Louis, MO, USA) was added to the pre-formed archaeosomes to reach a desired final concentration.
Thin-film hydration archaeosomes were prepared, as previously described [10 (link)]. In brief, SLA lipid that was dissolved in chloroform/methanol underwent solvent removal under N2 gas with mild heating to form a thin lipid film. Dried lipids were then hydrated in Milli-Q water for more than 12 h to form a uniform lipid suspension. Next, sonication was applied at 40 °C in an ultrasonic water bath (Fisher Scientific, Ottawa, ON, Canada) for up to an hour until the desired particle size (~100 nm) was obtained. After that, 10 × PBS (Millipore Sigma Canada, Oakville, Ontario) was added and archaeosomes stored at 2–8 °C until used. Note that we have previously demonstrated that archaeosomes stored under similar conditions were stable up to 6 months of testing, which was well beyond the storage time of ~1 month in the current study [21 (link)]. On the day of immunization, antigen solution (ovalbumin; OVA; type VI, Sigma-Aldrich, St. Louis, MO, USA) was added to the pre-formed archaeosomes to reach a desired final concentration.