Sequestering survivin to functionalized nanoparticles: a strategy to enhance apoptosis in cancer cells

Abstract

Survivin belongs to the family of inhibitor of apoptosis proteins (IAP) and is present in most cancers while being below detection limits in most terminally differentiated adult tissues, making it an attractive protein to target for diagnostic and, potentially, therapeutic roles. Sub-100 nm poly(propargyl acrylate) (PA) particles were surface modified through the copper-catalyzed azide/alkyne cycloaddition of an azide-terminated survivin ligand derivative (azTM) originally proposed by Abbott Laboratories and speculated to bind directly to survivin (protein) at its dimer interface. Using affinity pull-down studies, it was determined that the PA/azTM nanoparticles selectively bind survivin and the particles can enhance apoptotic cell death in glioblastoma cell lines and other survivin over-expressing cell lines such as A549 and MCF7 relative to cells incubated with the original Abbott-derived small molecule inhibitor.

Fig. 1

Reaction scheme for synthesis of survivin ligands, TM and azTM

Fig. 2

(a) Structures of survivin ligands, Abbott8 and Abbott17 (targeting molecule TM) proposed by Abbott Labs and an azide modified targeting molecule (azTM). (b) Schematic of poly(propargyl acrylate) (PA) particles surface modified with an azide-terminated survivin ligand (azTM) through an aqueous-phase “click” transformation sequestering anti-apoptotic protein survivin.

Fig. 3

Preliminary cell viability data in glioblastoma cells, U251MG and U118MG, using MTT assay. Untreated cells were used as the control (ctl). Cells were treated with the small molecule ligands at the indicated concentrations for 24 hours. Cell viability is presented as a percentage of viable cells in the total population. Significant difference from control value was indicated by *p<0.05. (a) TM and (b) azTM treatment of the glioblastomas.

Fig. 4

Increased apoptosis in glioblastoma cells, U251MG and U118MG, with azTM small molecule ligand when compared to TM. Untreated cells were used as the control (ctl). Cells were treated with TM or azTM ligand at indicated concentrations for 24 hours. (a) In situ Wright staining for detection of morphological features of apoptosis. Bar diagram shows percent apoptosis based on Wright staining for (b) TM and (c) azTM treatments. Cell death is presented as percentage of apoptosis in total population. Significant difference from control value was indicated by *p < 0.05 or **p < 0.01.

Fig. 5

Purification of survivin and affinity pull-down of survivin by surface-functionalized particles. (a) SDS-PAGE of purified recombinant survivin (ca. 0.8 μg); (b) Poly(propargyl acrylate) (PA) particles were surface modified with azTM (cf. Figure 2) and azPEG. Control particles lacked the presence of ligand. Both sets of particles were incubated with survivin or BSA protein. The supernatant was removed and the particles were washed with buffer. Proteins retained on the particles were eluted. The supernatant (S), wash (W) and elution (E) were separated on a 15 % polyacrylamide gel and stained with Coomassie blue. (c) Percentage of bound proteins on functionalized and control particles (data taken from SDS-PAGE in Part (b) as well as two other pull-down experiments).

Fig. 6

(a) Survivin conjugate azTM disrupted the activity of survivin to promote Caspase mediated apoptosis. Both glioblastoma cell lines were treated with survivin conjugate azTM as a free small molecule or attached to the surface of nanoparticles (1.0 μM ligand concentration) prior to extraction of protein samples. Protein samples were resolved by 4 – 20 % SDS-PAGE and Western blotting was performed using the primary IgG antibodies against survivin, caspase-3, and β-actin. Quantification of expression of survivin and caspase-3 after treatment with free ligand (azTM) and modified particles (PA/ azTM) in (b) U251MG or (c) U118MG cells. Significant difference from control value was indicated by *p<0.1, **p<0.01 and #p<0.001.

Fig. 7

Cell viability (MTS assay) of A549 cells treated with small molecule azTM and PA/ azTM particles with two different grafting densities (gd), 0.58 and 1.91 azTM/nm², at varying concentrations for 48 hours. Unmodified and azPEG modified particles were used as additional controls. Cell viability is presented as percentage of viable cells compared to the control.

Fig. 8

Viability of A549 cells with PA/ azTM particles of varying ligand surface density. The cells were treated with azTM and PA/ azTM of varying different grafting densities (gd) of 0.58, 0.90, 1.71, 1.77 and 1.91 azTM/nm² and at varying concentrations for 48 hours. Cell viability is presented as percentage of viable cells compared to the control and the MTS assay was employed.

Fig. 9

Imaging of A549 cells treated with 15 μM PA/ azTM/ azSQ for 24 hours and fluorescence images were taken under RFP filter. All scale bars represent 100 μm.

Fig. 10

Fluorescence microscope images of A549 cells after incubation with 25 μM of azTM, PA/ azTM particles with two different grafting densities, PA/ azTM/ azPEG particles and PA particles. Cells were treated with an apoptotic/necrotic/healthy cells detection kit to differentiate the status of the cells. All scale bars represent 200 μm.