Fig. 1. Synthesis and characterization of UAN. (A) UAN monomer is synthesized by covalently linking 2-HEMA, PEG, and glycerol propoxylate at 2:1:1 M ratio. (B) NTA-UAN is formed by cross-linking UAN monomers with AIBN and conjugation of NTA to PEG. NTA-UAN forms nanoparticles when suspended in aqueous solution. (C) TEM images of UAN nanoparticles after staining with uranyl acetate. Scale bar is shown. (D) The function and specificity of Ni-NTA on UAN nanoparticles were confirmed by their binding to a His- peptide column. (EeF) The release kinetics of hydrophobic dyes and drugs from UAN were inferred by measuring the rate of escape through dialysis tubes without (E) or with (F) UAN. The fit of the first-order kinetics to the data is shown as a line. (G) The delay in the rate of escape (s_UAN-s) of the payloads (HPTS, FITC, celastrol, CPT) is plotted against their respective solubility values in water on a semi-log plot.

Fig. 2. LFA-1 I domains engineered for high affinity for targeting ICAM-1. (A) Structural diagrams of low (inactive) and high affinity (active) conformations of LFA-1 I domains. Structurally conserved regions between the two states are in light gray, while the regions that differ more than 1 Angstrom in their Ca positions are in dark gray (inactive) and in medium gray (active). The metal ions in the metal ion-dependent adhesion site are shown as spheres. (B) The structure of the I domain in complex with ICAM-1 domain 1 (D1). Allosteric activation sites found in our previous study [38] are displayed in spheres along the peptide backbone. The metal ion and three oxygen atoms of water molecules are depicted as spheres, while the residues that coordinate to the metal ion are drawn with the sticks. (A) and (B) are adapted from Fig. 5 of Xuebo et al [51]. (C) Schematic of ICAM-1 domains. Domain 1 binds to LFA-1 I domain. The mAbs LB2 and R6.5 bind domain 1 and domain 2, respectively. (D) Surface plasmon resonance data for binding of I domains (100 mM of WT (wild-type) and F292A and 1 mM of F265S, F292G, and F265S/F292G) to ICAM-1 immobilized (adapted from Fig. 2 of Jin et al [38]). Compared to the wild-type, each mutation of I domain (F292A, F265S, F292G, F265S/F292G) increases the binding affinity of I domain to ICAM-1 in a step-wise manner. The numbers in parentheses denote the equilibrium dissociation constants, KD.

Fig. 3. Affinity- and expression-dependent delivery of I domain-UAN(FITC) to ICAM-1 expressing cells. (A) UAN nanoparticles encapsulating FITC and conjugated to different I domains were delivered to HeLa and MDA-MB-231 cells. (B) Immunofluorescence flow cytometry measurement of the delivery into HeLa of UAN(FITC) with no I domain, WT, or I domain variants (n=3). (C) The levels of ICAM-1 expression in HeLa, MDA-MB-231, and KTC-1 cells were measured by mAb R6.5 (open histogram) or with secondary antibody alone as a control (filled histogram). (D) Immunofluorescence flow cytometry measurements of F265S/F292G-UAN(FITC) binding to HeLa, MDA-MB-231, and KTC-1 cells.

Fig. 4. Affinity dependent cytotoxicity by I domain-UAN(celastrol or CPT). I domain-UAN nanoparticles encapsulating celastrol (A, B) or CPT (C) were delivered to HeLa cells. The cells treated with celastrol were stained with crystal violet for better visualization. Cell viability was then quantified by measuring the amount of crystal violet after cell lysis and shown as bar graphs (DeF), normalized to the levels with WT-UAN (n=3). (G) The percentage of viable cells after HeLa cells treated with UAN(celastrol or CPT) was plotted as a function of I domain affinity to ICAM-1 (n=8). Values were normalized to that of the wild-type. The significance of the differences in cell viability due to I domain-UAN was analyzed by one-way ANOVA and Tukey’s HSD post hoc test statistics.

Fig. 5. Comparison of cytotoxicity due to free celastrol, UAN(celastrol) without targeting, and F265S/F292G-UAN(celastrol). (A) Cytotoxicity of HeLa due to the treatment with celastrol, UAN(celastrol), or F265S/F292G-UAN(celastrol) was analyzed. HeLa cells were stained with a crystal violet for better visualization (images were taken 28 h after the treatment) and subsequently lysed for quantification of cell viability. Each condition contained 100 mM of celastrol. (B) The measured cell viability was shown in bar graphs, normalized to that of ‘No I domain Control’ (n=3).

Fig. 6. Antibody-mediated targeting of ICAM-1. (A) In order to demonstrate the versatility of UAN platform, UAN nanoparticles were encapsulated with DPA or CPT, while the surface of UAN was coated with four different combinations of protein A and mAb R6.5. HeLa cells were imaged 2 h after incubation with UAN(DPA) and 28 h with UAN(CPT). (B) The bar graphs were normalized to that of no protein A and no R6.5 control (‘-/-’, n=3).

Fig. 7. Minimal binding of I domain-UAN to cells with basal expression of ICAM-1. (A) Immunofluorescence flow cytometry measurement of mAb LB2 binding to HMEC-1 and THP-1 cells with (black open) or without (gray open) LPS treatment, or without labeling (filled histograms) to show the induction of ICAM-1 expression level. (B, C) Immunofluorescence flow cytometry measurement of F265S/F292G-UAN(FITC) (black open) binding to HMEC-1 and THP-1 cells with or without LPS treatment. UAN(FITC) without the I domain (gray open histograms) was used to measure non-specific binding to cells. Cells without labeling is shown in filled histograms. (D) Immunofluorescence flow cytometry results expressed as a bar graph.