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Figure 3. Demonstration of thermoresponsive nature of pNIPAM
(a) At temperatures below its LCST, pNIPAM molecules remain soluble in aqueous solution.
(b) pNIPAM undergoes liquid-liquid phase separation at its cloud point, resulting in a turbid solution.
Table 1. Polymer characterization data
Polymer molecular weight (Mn), percent conversion, degree of polymerization (DP), and percent by weight adjuvant were determined by NMR. (a) Ratios of starting materials used in each polymerization, M = TLR-7/8 adjuvant, N = N-isopropylacrylamide (in excess), CTA = 2-(((ethylthio)carbonothioyl)thio)propanoic acid or pNIPAM (macro-CTA), I = Azobisisobutyronitrile
Figure 4. Characterization of polymer-TLR-7/8 conjugates
(a) Polymers (1 mg/mL in H2O) exhibited LCSTs above physiological temperature via UV-Vis transmittance at 600 nm.
(b) Polymer nanoparticles (100 μg/mL in H2O) observed by DLS at 35 °C, 37 °C, 39 °C, 41 °C, 43 °C, and 45 °C. For the maleimide NIPAM polymer, particle size decreased as temperature increased, which was the opposite trend observed for the control, pNIPAM.
Figure 5. Raw-Blue cells were treated with pNIPAM (negative control), the maleimide NIPAM polymer, and IMQ (positive control) and incubated for 24 hours at 37 °C, 39 °C, or 40 °C. Data are represented as mean ± SD (n=3) and normalized relative to pNIPAM (10 μg/mL) and IMQ (50 μg/mL). Overall, the TLR-7/8 polymer conjugate showed decreased immunogenicity above physiological temperature.
Figure 6. Flow cytometry histograms of Raw-Blue cells stained with 7-AAD dye treated with (a) PBS (negative control) and (b) IMQ (100 μg/mL) and incubated for 24 hours at 37 °C and 40 °C. Live/dead assays indicate that hyperthermia has minimal impact on cell viability, confirming that conjugating a TLR-7/8 adjuvant to thermoresponsive pNIPAM is responsible for the reduced immunogenicity observed at high temperatures.
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