Imaging of Hsp70-positive tumors with cmHsp70.1 antibody-conjugated gold nanoparticles

Abstract

Real-time imaging of small tumors is still one of the challenges in cancer diagnosis, prognosis, and monitoring of clinical outcome. Targeting novel biomarkers that are selectively expressed on a large variety of different tumors but not normal cells has the potential to improve the imaging capacity of existing methods such as computed tomography. Herein, we present a novel technique using cmHsp70.1 monoclonal antibody-conjugated spherical gold nanoparticles for quantification of the targeted uptake of gold nanoparticles into membrane Hsp70-positive tumor cells. Upon binding, cmHsp70.1-conjugated gold nanoparticles but not nanoparticles coupled to an isotype-matched IgG1 antibody or empty nanoparticles are rapidly taken up by highly malignant Hsp70 membrane-positive mouse tumor cells. After 24 hours, the cmHsp70.1-conjugated gold nanoparticles are found to be enriched in the perinuclear region. Specificity for membrane Hsp70 was shown by using an Hsp70 knockout tumor cell system. Toxic side effects of the cmHsp70.1-conjugated nanoparticles are not observed at a concentration of 1–10 µg/mL. Experiments are ongoing to evaluate whether cmHsp70.1 antibody-conjugated gold nanoparticles are suitable for the detection of membrane-Hsp70-positive tumors in vivo.

Keywords:

• heat shock protein 70
• tumor biomarker
• theranostics
• multimodal CT
• multispectral CT
• k-edge

Supplementary materials

Materials and methods

Aggregation

For coupling round shaped gold nanoparticles with a diameter of 30 nm were used (Nanopartz, Loveland, CO, USA). After washing away nonbound antibodies, vacant binding sites on the gold nanoparticles were saturated by bovine serum albumin (Sigma-Aldrich, St Louis, MO, USA). Antibody-conjugated gold nanoparticles or only bovine serum albumin-blocked negative control Au-NPs were used for experiments directly after coupling. For characterization of the nanoparticles and checking for aggregation, particles were analyzed for their size by dynamic light scattering (Zetasizer NanoS; Malvern Instruments, Malvern, UK). Only nanoparticles with a single peak around 40 nm were used for experiments.

Toxicity assays

Short- and long-term toxicity of gold nanoparticles was measured by Annexin-V staining and colony forming assays to determine apoptosis and clonogenic cell survival. Briefly, cells were seeded in 12-well plates and grown overnight. After adherence, cells were incubated with gold nanoparticles for 24 hours, 48 hours, or 72 hours. For measuring short-term toxicity Annexin V staining assays were performed 24 hours after incubation of the cells with gold nanoparticles. After 24 hours, cells were trypsinized, washed with Annexin binding buffer (140 mM sodium chloride, 10 mM 2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethansulfonacid (HEPES) buffer, 2.5 mM CaCl), stained with Annexin-V–FLUOS (Hoffman-La Roche Ltd., Basel, Switzerland). Annexin V binding was measured on a FACS Calibur (BD Biosciences, San Jose, CA, USA). For measuring long-term toxicity, colony forming assays were performed. Cells were incubated with gold nanoparticles at a concentration of 1 µg/mL for 24 hours, 48 hours, or 72 hours. After removing the cell culture medium, cells were washed with phosphate buffered saline, fixed with methanol (−20°C) for 5 minutes, and stained with crystal violet. Following intensive washing with water, colonies were counted on a Bioreader 3000 (Biosys, Karben, Germany).

Figure S1 Aggregation assay.

Notes: The size and the aggregation of gold nanoparticles were measured by dynamic light scattering assays. Data from freshly prepared gold nanoparticles conjugated with cmHsp70.1 antibody are shown in red. Upon storage for 4 weeks at 4°C peaks are visible with a size above 100 nm (green). These data show an increased aggregation of the nanoparticles during storage. For all cell-based experiments only freshly prepared nanoparticles with a single peak in the hydrodynamic diameter of ~40 nm were used. The graph shows a representative measurement of cmHsp70.1-coated gold nanoparticles either freshly prepared (red) or after 4 weeks of storage at 4°C (green).

Figure S2 Annexin V assay.

Notes: Tumor cells were seeded in 12-well chamber slides and grown overnight. After an incubation of the cells with gold nanoparticles (concentration of 1 µg/mL) for 24 hours, cells were trypsinized and incubated with Annexin-V-FLUOS. Cells were analyzed on a FACS Calibur instrument. Annexin-V-positively stained cells were considered as apoptotic. As a positive control for apoptosis, cells were incubated with campthotecin (1 µg/mL, 24 hours) (CAM). No difference in cell viability was observed in cells incubated with the different gold nanoparticles either unconjugated (Au-NP) or conjugated (Au-IgG, Au-Hsp70), or cells that were not incubated with nanoparticles (w/o); (A) CT26, (B) 4T1 wt, (C) 4T1 Hsp70−/− (n=3). Although basal apoptosis was slightly higher in 4T1 wt and 4T1 Hsp70−/− cells compared to CT26 cells, the incubation with gold nanoparticles did not further increase apoptosis.

Abbreviations: w/o, without; FACS, fluorescent activated cell sorting.

Figure S3 Cell viability assay.

Notes: Cells were seeded in 12-well chamber slides, incubated for 24 hours, 48 hours, or 72 hours with gold nanoparticles (Au-NP, Au-IgG, Au-Hsp70) or left untreated (w/o). Adherent cell colonies were counted 24 hours, 48 hours, and 72 hours after incubation with the gold nanoparticles and then stained with crystal violet. Colonies were counted automatically, and colony counts after 24 hours were set to 1. Shown are mean values of three independent experiments obtained with CT26 cells. For each plate, at least 30 colonies were counted in triplicates.

Abbreviation: w/o, without.

Figure S4 Representative view of a Z-stack light microscopic analysis of a CT26 tumor cell after incubation with Au-Hsp70 gold nanoparticles for 24 hours to demonstrate intracellular localization of the Au-Hsp70.

Notes: Magnification of 100×/1.3 oil using an EC plan-Neofluar objective, 15 ms; 22 slices were taken of the cells from the top to the bottom. The size of a single black dot was <500 nm, the size of the aggregates of black dots was ~2–3 µm. The scale bar indicates 20 µm. The yellow line depicts the outer plasma membrane of the cell at top position.

Abbreviation: ms, millisecond.

Acknowledgments

This work was supported by the Wilhelm Sander-Stiftung (2012.078.1), EU-CELLEUROPE (315963); BMBF (Strahlenkompetenz, 02NUK007E; 02NUK031B; Innovative Therapies, 01GU0823; NSCLC, 16GW0030; m⁴ – Leading Edge Cluster, 16EX1021C) and the DFG Cluster of Excellence: Munich Centre for Advanced Photonics. The research leading to these results has received funding from the Deutsche Forschungsgemeinschaft (DFG) under Grant Agreement No. SFB 824/2; INST 95/980-1 FUGG; INST 411/37-1 FUGG irradiation devices and the Helmholtz Zentrum München (HMGU), CCG – Innate Immunity in Tumor Biology. The BMBF (Federal Ministry of Education and Research, Germany) cutting edge cluster m⁴ (Individualized Medicine) (MAK, PBN, EJR) are acknowledged for funding (BMBF M4 PM8 801EX1021D).

Author contributions

All authors contributed toward data analysis, drafting and critically revising the paper and agree to be accountable for all aspects of the work.

Disclosure

The authors declare no conflict of interest in this work.