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The lead imidazoquinoline compound was synthesized with a scheme developed by the Hubbell group [8] and modified with maleimide by EDC coupling to produce the polymerizable TLR-7/8 adjuvant.
Scheme 1. Synthesis of maleimide derivative by EDC coupling and polymer-TLR-7/8 conjugate using RAFT polymerization
RAFT Polymerization
Figure 2. Proposed mechanism of RAFT polymerization [9]
Purification conducted following standard dialysis protocol
Raw-Blue Assay
Upon confluency, 100,000 Raw-Blue cells seeded per well in 96-well plate
After cells attached to surface, 10 μg/mL, 50 μg/mL, and 100 μg/mL of pNIPAM, maleimide NIPAM conjugate polymer, and IMQ were added and incubated for 24 hours at 37 °C, 39 °C, and 40 °C
Immunogenicity was quantified via QUANTI-Blue detection of alkaline phosphatase, which corresponds to NF-κB activation
Cell Viability Assay
500,000 Raw-Blue cells seeded per well in two 24-well plates
Raw-Blue cells treated with 10 μg/mL, 50 μg/mL, and 100 μg/mL of IMQ and incubated for 24 hours at 37 °C and 40 °C
Cells harvested, washed with PBS, stained with 7-AAD nucleic acid dye, and analyzed by flow cytometry to estimate cell death
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