Article pubs.acs.org/bc
Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX
Gastrin-Releasing Peptide Receptor- and Prostate-Specific Membrane Antigen-Specific Ultrasmall Gold Nanoparticles for Characterization and Diagnosis of Prostate Carcinoma via Fluorescence Imaging Marc Pretze,*,† Andreas Hien,‡ Matthias Rad̈ le,‡ Ralf Schirrmacher,§ Carmen Wan̈ gler,∥ and Björn Wan̈ gler*,† †
Molecular Imaging & Radiochemistry, Department of Clinical Radiology and Nuclear Medicine and ∥Biomedical Chemistry, Medical Faculty Mannheim of Heidelberg University, Mannheim 68167, Germany ‡ Institute of Process Control and Innovative Energy Conversion, Mannheim University of Applied Sciences, Mannheim 68163, Germany § Oncologic Imaging, Department of Oncology, University of Alberta, Edmonton 6820, Alberta, Canada S Supporting Information *
ABSTRACT: Gold nanoparticles (AuNPs) have widely been used for 70 years in cancer treatment, but only in the last 15 years has the focus been on specific AuNPs with homogeneous size and shape for various areas in science. They constitute a perfect platform for multifunctionalization and therefore enable the enhancement of target affinity. Here we report on the development of tumor specific AuNPs as diagnostic tools intended for the detection of prostate cancer via fluorescence imaging and positron emission tomography (PET). The AuNPs were further evaluated in vitro and in vivo and exhibited favorable diagnostic properties concerning tumor cell uptake, biodistribution, clearance, and tumor retention.
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multimodal imaging techniques26 as well as for theranostic purposes.27−31 Many approaches are based on a phenomenon typically known as enhanced permeability and retention (EPR) due to passive extravasation of nanoparticles across the perforated vasculature of tumors.32 In this work, we report on the functionalization of targetspecific AuNPs for the diagnosis of different prostate cancers via near-infrared (NIR) fluorescence imaging. We show the straightforward synthesis and characterization of modified AuNPs with target-specific peptides (Bombesin (7−14) (BBN(7−14))33,34 and the Lys-urea-Glu motif (LUG)35) binding the gastrin-releasing peptide receptor (GRPR) and the prostate-specific membrane antigen (PSMA), respectively. The different AuNPs were tested for stability, avidity, and cell association, as well as fluorescence imaging in vitro. Furthermore, their biodistribution, tumor enrichment, and imaging in vivo were evaluated.
INTRODUCTION Gold nanoparticles (AuNPs) have been used for nearly 70 years for the therapy1−3 and diagnosis4 of different cancers. Early on, those colloidal solutions were unspecific and consisted of AuNPs of heterogeneous size distribution, restricting their use to interstitial brachytherapeutic applications.5 The development of methods for the synthesis of monodisperse AuNPs6 followed by surface-modification for enhanced stability and homogenization of AuNPs7−9 paved the way for further functionalization.10 The high affinity of sulfur toward gold surfaces and the formation of stable and covalent Au−S bonds11 enables a fast and facile functionalization of AuNPs with thiol-modified (bio)molecules. Although cytotoxic effects are known for citrate-capped AuNPs,12−14 polyethylene glycol-containing (PEG)ylated AuNPs exhibit toxic effects only at high concentrations of >3 g/kg.4,15,16 Furthermore, the PEGylation of the AuNPs leads to a higher bioavailability because the in vivo formation of a protein corona around the AuNP is hindered.17,18 Therefore, more target-specific AuNPs could be developed and labeled with small molecules,19 antibodies,20 peptides,21 natural products,22,23 radionuclides,24 and magnetic resonance imaging (MRI) relevant metals.25 Thus, AuNPs represent a perfect platform for multimerization of targetspecific effectors at their surface and modification for © XXXX American Chemical Society
Received: January 25, 2018 Revised: March 12, 2018 Published: March 15, 2018 A
DOI: 10.1021/acs.bioconjchem.8b00067 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry
Figure 1. Synthetic pathway for differently functionalized NIR-AuNPs 4, 7, 8, and NODAGA-AuNPs 10, 11, and 12.
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(50 nm) has no impact on the specific tumor-targeting properties of the nanoparticle,38,39 because the big nanoparticles exhibit slower kinetics and therefore the biodistribution is strongly influenced by the nanoparticle itself and not by the tumor vector. The herein reported ultrasmall AuNP (80% confluency using 0.05% trypsin/EDTA solution. In contrast to PC3 and A431 cells, LNCaP cells were grown longer in well-plates before using them in in vitro assays (two days instead of one) and washed more carefully, since they grow less adherent. Bradford assays were performed after uptake, internalization, and avidity experiments. For cell uptake studies, Tris-Mn buffer was used (see SI for detailed recipe). Cells were seeded onto a 24 well plate 2−3 days prior to the experiment to obtain ∼1.6 × 106 cells per well. Cells were incubated with [68Ga]AuNPs (4.3 MBq/well) for a maximum of 5 h at 37 °C. Afterward, cells were washed carefully 3× with PBS and lysed 2× with 1 M NaOH for 10 min at 37 °C. The NaOH fractions were collected and measured in a gamma counter (2470 WIZARD2, PerkinElmer). Internalization Assay. Cells were seeded onto a 12 well plate 2−3 days prior to the experiment to reach ∼2.4 × 106 cells per well. Cells were incubated with [68Ga]AuNPs (4.4 MBq/well) for 4−5 h at 37 °C with and without blocking substance (60 μg BBN(7−14)/well (M = 940 g/mol) or 24 μg EG-LUG (EG = ethylene glycol) 14/well (M = 464 g/mol), ∼1000× excess each). After incubation, the cells were washed 3× with PBS, incubated 2× with glycine buffer (pH 2.8) for 5 min at 37 °C and the supernatant fraction was collected for gamma counting. Finally, the cells were lysed 2× with 1 M NaOH and the NaOH fractions were collected for gamma counting. For the different well sizes the recommended working volumes were used for incubation (2 mL for 12 well plate, 1 mL for 24 well plate). All cell experiments were performed in triplicate. Fluorescence Microscopy. Cells were seeded onto coverslips for 2 days, then washed with PBS and incubated for 24 h at 37 °C in 5% CO2 with the respective media containing different amounts of AuNPs (1−100 μg/mL). Afterward, the cells were washed with PBS and incubated with CellMask Orange-solution (1× working solution) for 15 min at 37 °C. Cells were fixed with 1:1 medium:4% formaldehyde in PBS for 2 min at ambient temperature and then with 4% formaldehyde in PBS for 15 min at ambient temperature. Cells were then washed 3× with PBS and coverslips were prepared onto an object plate with Sytox Green-solution (8.3 μM, 10 μL). Fluorescence microscopy was performed on a Leica TCS SP8 confocal microscope with lasers at λ = 488, 552, and 638 nm. Overlays of microscopies were generated using FIJI software (v1.50e). Avidity Studies. The avidities of the GRPR-specific or PSMA-specific AuNPs were determined similar to Fischer et al.53 In brief, cells were seeded on 12- or 24-well plates for 2 days. 125Iod-Tyr4-Bombesin (125I-BBN) (∼370 kBq, ∼81 GBq/ μmol) was purchased from PerkinElmer and 177Lu-PSMA-617 (∼972 MBq, ∼53.7 GBq/μmol) was obtained from University Medical Center Mainz. 125I-BBN (0.06 nM, 106.7 pg/mL) was added together with the GRPR-specific AuNP derivatives in medium in different concentrations (0.01−60 μg/mL). 177LuPSMA-617 (1 nM, 1.21 ng/mL) was added together with the PSMA-specific AuNP derivatives in medium in different concentrations (0.01−60 μg/mL). Cells were incubated with OptiMEM for 1 h at 37 °C, washed 3× with PBS and lysed 2× with 1 M NaOH. The NaOH fractions were collected and measured in the gamma counter. IC50 values were determined using Origin 8.1 software.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.8b00067. Synthesis data, electron microscopy (EM), dynamic light scattering (DLS), thermogravimetric analysis (TGA) measurements, confocal fluorescence microscopies, fluorescence imaging, NMR spectroscopy measurements, and ex vivo and in vivo data (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. Phone: +49 621 383 6045. *E-mail:
[email protected]. Phone: +49 621 383 5594. ORCID
Marc Pretze: 0000-0002-6432-5694 Ralf Schirrmacher: 0000-0002-7098-3036 Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Research Campus M2OLIE funded by the German Federal Ministry of Education and Research (BMBF, Funding Code 13GW0091B and 13GW0091E) and the “Zentrales Innovationsprogramm Mittelstand des Bundesministeriums für Wirtschaft und Energie” (AiF Project GmbH, Funding Code KF2035759AK3 and KF3086202AK3). We would like to thank Tobias Timmermann for measuring NMR spectra and Dr. Uwe Seibold for measuring MALDI-MS spectra and Dr. Mareike Roscher for fruitful discussion concerning the animal experiments. We like to thank Dr. Karsten Richter from the German Cancer Research Center (DKFZ) for measuring the EM. We also want to thank Prof. Dr. Wolfgang Schubert for using the TGA and Prof. Dr. Thorsten Röder for using the DLS at Mannheim University of Applied Sciences. We would like to thank Ms. G
DOI: 10.1021/acs.bioconjchem.8b00067 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Anne-Maria Suhr and Ms. Stephanie Riester for their technical assistance in the in vitro and in vivo experiments.
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ABBREVIATIONS AuNP, gold nanoparticle; CT, computer tomography; TEM, transmission electron microscopy; DLS, dynamic light scattering; TGA, thermogravimetric analysis; NMR, nuclear magnetic resonance spectroscopy; LUG, Lys-urea-Glu motif; BBN, bombesin; PSMA, prostate-specific membrane antigen
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DOI: 10.1021/acs.bioconjchem.8b00067 Bioconjugate Chem. XXXX, XXX, XXX−XXX
