Grafting aptamers onto gold nanostars increases in vitro efficacy in a wide range of cancer cell types

Abstract

We report the design of a nanoconstruct that can function as a cell-type independent agent by targeting the ubiquitous protein nucleolin. Gold nanostars (AuNS) loaded with high densities of nucleolin-specific DNA aptamer AS1411 (Apt-AuNS) produced anticancer effects in a panel of 12 cancer lines containing four representative subcategories. We found that the nanoconstructs could be internalized by cancer cells and trafficked to perinuclear regions. Apt-AuNS resulted in downregulation of antiapoptotic Bcl-2 mRNA expression by ca. 200% compared to cells without the nanoconstructs. The caspase 3/7 activity (apoptosis) and cell death in cancer cells treated with Apt-AuNS increased by 1.5 times and by ca. 17%, respectively, compared to cells treated with free AS1411 at over 10 times the concentration. Moreover, light-triggered release of aptamer from the AuNS further enhanced the in vitro efficacy of the nanoconstructs in the cancer line panel with a 2-fold increase in caspase activity and a 40% decrease in cell viability compared to treatment with Apt-AuNS only. In contrast, treatments of the nanoconstructs with or without light-triggered release on a panel of normal cell lines had no adverse effects.

Figure 1

Nucleolin expression in non-nuclear extracts of cancer and normal cells. (A) Full-length (106 kDa) and the proteolysis product (98 kDa) of nucleolin appear in the non-nuclear lysates of all cancer cells. (B) MCF-10A cells show minimal expression of nucleolin, while fibroblast cells show nucleolin expression comparable to cancer cells.

Figure 2

Gold nanostars (AuNS) as a drug delivery platform. (A) TEM image of multibranched AuNS. (B) Higher magnification TEM image of a representative AuNS shape, whose average hydrodynamic diameter is ca. 40 nm. (C) Scaled-up production of Apt-AuNS indicating that up to 5 L of Apt-AuNS can be synthesized in an hour.

Figure 3

Uptake of Apt-AuNS in 12-cancer cell line panel and 3-normal cell line panel. Cy5-labeled Apt-AuNS (red) in the cytoplasm and near DAPI-stained nuclei (blue) in (A) cancer cells and (B) normal cells suggest that Apt-AuNS can be internalized. Cells are arranged in order of increased doubling time. All images were collected at the same magnification. Confocal images are 30 μm × 30 μm. Cancer cell panel: HCT-116 (colon), HT-1080 (connective tissue), A-549 (lung), HeLa (cervix), MCF-7 (breast), U-87 (brain), DU-145 (prostate), MDA-MB-231 (breast), SK-MEL-2 (skin), SKOV-3 (ovary), PANC-1 (pancreas), A-498 (kidney). Normal cell panel: MCF-10A (epithelial mammary), WI-38 (lung fibroblast), HS-27 (skin fibroblast).

Figure 4

Apt-AuNS uptake and expression of nucleolin in plasma membrane and cytoplasmic in cancer and normal cells. Higher Au content and expression of non-nuclear nucleolin was found in cancer and fibroblast cell lines compared to mammary epithelial cells.

Figure 5

Downregulation of antiapoptotic Bcl-2 gene in cancer cells after treatment with Apt-AuNS. A 400% reduction of Bcl-2 mRNA expression was observed in PANC-1 cancer cells after treatment with 0.3 nM Apt-AuNS (33 nM AS1411) compared to untreated cancer cells. Gene expression was reduced further (200% to 1500%) in cancer cells after a single treatment with Apt-AuNS + hν. Only seven out of 12 cancer cells (HCT-116, HT-1080, HeLa, MDA-MB-231, PANC-1, SK-MEL-2, and U-87) treated with 450 nM free AS1411 decreased by minimal amounts. No change of Bcl-2 mRNA levels was found in normal cells. p-values were determined using a one-way ANOVA test. (*) and (**) indicate p < 0.05 and p < 0.1, respectively.

Figure 6

Elevation of caspase 3/7 activity in cancer cells after treatment with Apt-AuNS and Apt-AuNS + hν. Higher increases in caspase activities (up to 6-fold) are observed in all cancer cells treated with Apt-AuNS + hν. Up to a 4-fold increase of caspase 3/7 activities after treatment with Apt-AuNS indicates that large populations of cancer cells entered apoptosis. Negligible increase in caspase 3/7 activities in cancer cells from treatment with 450 nM AS1411 (less than 1.5-fold). Notably, there are no significant adverse effects observed in normal cells after exposure to laser irradiation. p-values were determined using one-way ANOVA test. Lines over bars indicate groups that are not significantly different.

Figure 7

Light-triggered release of Apt from AuNS increases the percentage cell death. Cell viability analysis showed a 70% decrease of cell viability after aptamer release. Approximately 25% decrease in cell viability after treated with Apt-AuNS. Cancer cells treated with 450 nM of free AS1411 showed reduced effects on cell death (less than 10%) compared to those treated with 0.3 nM Apt-AuNS (33 nM AS1411) and three times lower than that from light-triggered treatment. No significant cell death was observed in any of the normal cell lines. p-values were determined using a one-way ANOVA test. Lines over bars indicate groups that are not significantly different. (*) and (**) indicate p < 0.05 and p < 0.1, respectively.