VLA-4 antagonist peptide binds to MM cells, and inhibits their adhesion to fibronectin. (a) MM.1S, NCI-H929, U266 and IM9 cell lines all express VLA-4 subunits α₄- and β₁-integrins as determined by flow cytometry. Black columns are primary antibodies and gray columns are isotype controls. (b) Structure of VLA-4 antagonist peptide (VLA4-pep). (c) Cellular-binding assays were performed using FITC-labeled VLA4-pep and was detected by flow cytometry. Control experiments were done with FITC-labeled nonspecific peptide and the background binding was subtracted for each data point. VLA4-pep binds to U266, NCI-H929 and IM9 cell lines with apparent K_d of ∼250 nM. (d) VLA4-pep inhibits adhesion of MM cell lines to fibronectin-coated plates. BSA-coated plates were used as controls, and no adhesion of MM cells was observed. No inhibition of adhesion was observed in the control experiments done with nonspecific peptide (results not shown). All experiments were done in triplicates and data represents means (±s.d.).

Synthesis and characterization of VLA-4 targeting, Dox-conjugated multifunctional nanoparticles (NP_{Dox/VLA4−pep}). (a) Schematic illustration of the multifaceted synthetic steps for peptide conjugation to DSPE-PEG2000-NH₂ using solid support. (b) Schematic illustration of Dox conjugation to DPPE-GA. (c) Illustration of multifunctional micellar nanoparticles that incorporate VLA4-pep and Dox. (d) Dynamic light-scattering analysis of nanoparticles. VLA-4 targeting, Dox conjugated (NP_{Dox/VLA4−pep}), only Dox conjugated (NP_Dox), only VLA4-pep conjugated (NP_VLA4−pep), Dox and nonspecific peptide conjugated (NP_Dox/ns), nonspecific-peptide conjugated (NP_ns) and bare nanoparticles (NP_bare) all gave an average size distribution of ∼20 nm. (e) Drug release profile of Dox from the nanoparticles in pH=5.5 and pH=7.4. Rate of hydrolysis was quantified via HPLC, taking measurements at pre-determined time intervals and observing the absorbance at wavelength of 477 nm. Data shown are from a representative experiment.

Cellular uptake studies. (a) Rhodamine-labeled nanoparticles with varying valency of VLA4-pep conjugates (n=0–40/nanoparticle) were prepared and incubated with NCI-H929 cells at 37 °C for the indicated time points. N=20 peptide conjugates per nanoparticle triggered the most efficient uptake as determined by flow cytometry. (b) In a separate experiment, control experiments with NP_ns, and competition experiment with excess free VLA4-pep (2 mM) was performed to determine receptor-mediated specificity of nanoparticle uptake. Data represent means (±s.d.) of triplicate experiments. (c) Internalization of VLA-4-targeting nanoparticles was confirmed with a Nikon A1R confocal microscope using a × 40 oil lens. Image acquisition was performed by Nikon Elements Ar software.

NP_{Dox/VLA4−pep} induces cytotoxicity against MM cells. NCI-H929 MM cells were cultured in the presence of equivalent Dox concentrations of NP_{Dox/VLA4−pep}(▪), NP_Dox (▴), or free Dox (•) for 48 and 72 h. In all cases, cell viability was assessed by cell counting kit-8 (CCK-8), and data represent means (±s.d.) of triplicate cultures. Control experiments performed with NP_Dox/ns showed similar results to NP_Dox. NP_bare and NP_VLA4−pep did not show any cytotoxic effects at the concentrations tested (results not shown).

NP_{Dox/VLA4−pep} induces DNA DSBs and apoptosis in MM cells. NCI-H929 cells were treated with 250 nM Dox equivalents of NP_{Dox/VLA4−pep} or free Dox for 0–48 h. (a) Phosphorylation of DNA damage response protein H2AX at Ser¹³⁹ was assayed by western blotting. (b) Respective H2AX foci formation was assayed by immunocytochemistry (right). Representative images are shown. Apoptosis was assessed by flow cytometry following Annexin V-FITC staining (c), and by western blotting for PARP cleavage, and caspase-8 and caspase-9 activation (d). For flow cytometric analysis, data represent means (±s.d.) of triplicate experiments. For western blotting, representative images are shown.

NP_{Dox/VLA4−pep} inhibits adhesion of MM cells to fibronectin and overcomes CAM-DR. (a) Calcein-labeled NCI-H929 MM cells were allowed to adhere to fibronectin-coated plates alone, or with increasing concentrations of NP_VLA4−pep. Nonadherent cells were removed by washing with PBS, and adherent cells were quantitated in a fluorescence multi-well plate reader. Data represents means (±s.d.) of triplicate experiments. *P<0.05, **P<0.01 when compared with control. (b) NCI-H929 cells were allowed to adhere to fibronectin- or BSA-coated plates for 1 h, and then treated with equivalent Dox concentrations of NP_{Dox/VLA4−pep}, or free Dox for 72 h. Cell viability was assessed by cell counting kit-8 (CCK-8), and data represent means (± s.d.) of triplicate cultures. (c) An alternate illustration of data presented in (b).

In vivo characterization of NP_{Dox/VLA4−pep} in a xenograft model of MM. Tumor bearing SCID mice were injected, intravenously, with free Dox, NP_{Dox/VLA4−pep}, or NP_Dox at a dose of 6 mg/kg Dox equivalents on days 1, 3 and 5. (a) Tumor growth inhibition was detected by caliper measurements. All mice in free Dox group were killed on day 7 because of high systemic toxicity (weight loss >15%). NP_{Dox/VLA4−pep}, was significantly more efficacious than NP_Dox with *P<0.05. Data shown are means (±s.e.) of n=6–8 per treatment group. (b) Percentage of body weight of the animals as a measure of systemic toxicity. Free Dox group dramatically lost weight (>15%) and demonstrated moribundity by day 7. Therefore, mice in this group were killed on day 7. Only ∼10% weight loss was observed with NP_{Dox/VLA4−pep} or NP_Dox during the 2-week study period. (c) Ex-vivo mechanistic analysis of tumors for apoptosis. Three additional mice from each group were dissected on day 5 and tumors were stained for activated caspase-3. Representative images of tumor cross-sections that were captured using a Nikon Eclipse TS100 microscope at × 20 magnification are shown. (d) Tissue biodistribution of Dox following treatment. Three mice from each group were treated, intravenously, with 10 mg/kg of Dox equivalent drugs. Mice were killed 24 h after treatment and tissues were analyzed for Dox accumulation. Data shown are means (±s.e.). ^*P<0.05, ^**P<0.01 when compared with free Dox group. (e, f) Complete blood count and organ weights as a measure of systemic toxicity. Three additional mice from each group were dissected on day 5, and complete blood count (white blood cell, red blood cell and thrombocyte) was performed. (e) Weights of excised heart, kidney, spleen and liver are shown. (f) Data represent means (±s.e.). ^*P<0.05, when compared with free Dox group.