Fig 1.
Schematic structure and synthesis method.
In step 1, α-D-Mannopyranosylphenyl isothiocyanate was conjugated to -NH2 group of the dendrimer, using a ratio of 50:1 mannose to dendrimer. In step 2, Cy7 NHS ester was conjugated to -NH2 group of the dendrimer, using a ratio of 20:1 Cy7 to dendrimer. In step 3, Suc-HYNIC-Tfa was conjugated to -NH2 group of the dendrimer, using a ratio of 10:1 Suc-HYNIC-Tfa to dendrimer.
Fig 2.
(A) PAMAM G5 dendrimers, (B) PAMAM G5 dendrimers-mannose, (C) PAMAM G5 dendrimers-manose-Cy7 and (D) PAMAM G5 dendrimers-manose-Cy7-HYNIC-Tfa.
Fig 3.
RP-HPLC analysis and stability.
(A) RP-HPLC analysis of PAMAM G5 dendrimer-mannose-Cy7-HYNIC-Tfa UV (λ = 214) detector, the retention time was 9.10 min. (B) RP-HPLC analysis of radiolabeled 99mTc-HYNIC-dendrimers-mannose-Cy7, the retention time was 9.20 min. (C) Stability of 99mTc-HYNIC-dendrimers-mannose-Cy7 versus histidine and PBS. 100% was the total activity of the compound determined at time 0, n = 3 for each point on the graph.
Fig 4.
Flow cytometry analysis of BMDMs and differentiation.
(A) BMDMs were first gated on FSC and SSC to remove debris and conjugates. Mature BMDMs were defined as F4/80+ subpopulations with the purity displayed as a percentage. (B) After treatment with LPS + IFNγ or IL-4 for 24 hours, BMDMs show different morphology.
Fig 5.
BMDMs were treated with LPS and IFN-γ or IL-4 for 24 hours. The polarization of BMDMs was confirmed by quantitative RT-PCR. (A) iNOS, (B) MRC1, and (C) Arg1 expression levels were normalized by β-actin (mean ± SD). A comparison in each group was performed by one-way ANOVA followed by Tukey-Kramer test (***p<0.0001).
Fig 6.
Cell uptake of 99mTc-HYNIC-dendrimer-mannose-Cy7.
BMDMs untreated and treated with LPS + IFNγ and IL-4 were incubated with 99mTc-HYNIC-dendrimer-mannose-Cy7. Cellular uptake was analyzed 1 to 6 hours after incubation. Data are expressed as % dose (mean ± SD). A comparison in each group was performed by two-way ANOVA followed by the Bonferroni test (***p<0.001, **p<0.01 and ns = p>0.05).
Fig 7.
Cell uptake of 99mTc-HYNIC-dendrimer-mannose-Cy7 with block.
(A) BMDMs uptake of 99mTc-HYNIC-dendrimer-mannose-Cy7, cells treated with different concentrations of D-mannose. (B) BMDMs uptake of 99mTc-HYNIC-dendrimer-mannose-Cy7, cells treated with different concentrations of D-glucose. Data are expressed as % dose (mean ± SD). A comparison in each group was performed by two-way ANOVA followed by the Bonferroni test (*p < 0.005). Ctr (control), Man (mannose), Glc (glucose).