Ionic gelation technique was employed for the preparation of TMC nanoparticles. A 2 mg/mL solution of TMC in 10 mM HEPES buffer was treated with 1 mL Tripolyphosphate (TPP) aqueous solution (1.7 mg/mL) drop by drop while continuous stirring. TPP, the crosslinking agent, induces ionic complexation resulting in the formation of an opalescent dispersion indicating the formation of nanoparticles. The nanoparticles were harvested by centrifugation at 15,000 rpm for 10 min on a 10 µL glycerol bed. The particles were stored at −20°C until further use. Particles were resuspended in HEPES buffer pH 7.4 and after a brief sonication, the size, polydispersity index, and zeta potential were measured by using Nanosizer ZS (Malvern Instruments, Malvern, UK). Particles were observed and analyzed by SEM and TEM imaging. For imaging sample preparation the particles were processed as described earlier by Malik et al.31 (link)
Fifty micrograms of aqueous soluble TMC nanoparticles were subcutaneously injected individually to 6–8-week old female Balb/c mice (n=8) with or without CpG ODN (20 µg/mice). A booster dose was given 2 weeks post-immunization and mice sera were collected 28 days after the immunization. The pooled sera were stored at −80°C until further use.
Fifty micrograms of aqueous soluble TMC nanoparticles were subcutaneously injected individually to 6–8-week old female Balb/c mice (n=8) with or without CpG ODN (20 µg/mice). A booster dose was given 2 weeks post-immunization and mice sera were collected 28 days after the immunization. The pooled sera were stored at −80°C until further use.
