Fig. 2.

Quantitative screening of albumin/AlbiCpG nanocomplexes for LN delivery. a Structures, radiolabeling, and formulations of CpG derivatives for PET-based screening. Shown in the middle is the molecular structure of NMEB used for radiolabeling of AlbiCpG. Bioconjugations were conducted using thiol on Cp, maleimide on NMEB (for AlbiCpG), or NOTA (for CpG and IFA(CpG)), as well as maleimide and alkyne on bifunctional PEG20K and azide on NOTA for PEG–CpG. b Upper: representative coronal (coro) PET images showing FVB mice at 6 h post s.c. injection (dose: 4.4–5.5 Mbq) of CpG derivatives at tail base. Nanocomplexes of albumin with four MEB–CpG derivatives, respectively, efficiently delivered CpG to LNs, relative to free CpG, PEG–CpG, and IFA(CpG). Lower: representative transverse (trans) PET images showing LN delivery of MH₂C at 6 h post injection. White arrow heads mark IN and AX LNs. c Amounts of CpG derivatives in IN and AX LNs quantified from three-dimensional (3D)-reconstructed, decay-corrected PET images (n = 4–8). The IFA(CpG) signal in LNs was too low to visualize for quantification. d Biodistribution of tested compounds in resected organs measured by γ counting 3 days post injection (n = 4). (Liver/gal: liver with gallbladder; intest.: intestine). e An AFM image of HSA/AlbiCpG nanocomplexes (premixed AlbiCpG: HSA = 1:1). Scale bar: 200 nm. f, g In C57BL/6 mice, AlbiCpG s.c. injected at the tail base ameliorated the systemic toxicity of CpG (n = 4) as shown by lower IL-6 and IL-12p40 titers in blood (f) (dose: 5 nmol CpG equivalents) and ameliorated splenomegaly (g) on day 6 (dose: 5 nmol CpG equivalents on day 0 and day 3). Scale bar for spleen: 1 cm. Wt: weight. Data show mean ± s.e.m. of two–three independent experiments. ***p < 0.001, **p < 0.01, *p < 0.05, ns: not significant (p > 0.05) by one-way ANOVA with Bonferroni post test. Asterisks in c indicate statistically significant differences between the corresponding compounds with MH₂C