biodistribution and pharmacokinetic evaluation of choline - bound gold nanoparticles in a human prostate tumor xenogra model

Purpose: Gold nanoparticles (GNPs) have attracted signi cant attention in the treatment of cancer due to their potential as novel radiation enhancers, particularly when functionalized with various targeting ligands. e aim of this study was to assess the biodistribution and pharmacokinetic characteristics of a novel choline-bound GNP (choline-GNP) stabilized with polyethelenimine (PEI). Methods: Choline bound to 27 nm diameter GNPs was characterized using transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). Toxicity of choline-GNPs was examined on DU-145 prostate cancer cells using an MTT assay. Using balb/c mice bearing ank DU-145 prostate tumors, choline-GNPs bio-distribution was measured using inductively coupled mass spectroscopy (ICP-MS). Blood, heart, lung, liver, spleen, brain, kidney and tumor gold content were examined at multiple time points over a 24-hour period a er tail vein injection. Results: An MTT assay using DU-145 prostate cancer cells yielded a 95% cell viability 72 hours a er choline-GNP administration. e tumor GNP area under the concentrationtime curve during the rst 4 hours (AUC0-4) was 2.2 μg/ml h, representing 13% of the circulating blood GNP concentration over the same time period. e maximum intratumor GNP concentration observed was 1.4% of the injected dose per gram of tumor tissue (%ID/g) one hour post injection. Conclusions: GNPs functionalized with choline demonstrates a viable future nanoparticle platform with increased intra-tumor uptake as compared to unconjugated GNPs. Decreased intra-hepatic accumulation appears to be the reason for the improved systemic bioavailability. e next logical translational investigation will incorporate external beam radiation with the observed maximum intra-tumor uptake. ORIGINAL RESEARCH © 2013 CIM Clin Invest Med • Vol 36, no 3, June 2013 E133 Correspondence to: Wilson Roa MD, FRCPC Department of Radiation Oncology Cross Cancer Institute11560 University Avenue Edmonton, Alberta, CANADA, T6G 1Z2 email: [email protected] Manuscript submitted 25th September, 2011 Manuscript accepted 15th May, 2013 Clin Invest Med 2013; 36 (3): E133-E142. e application of gold nanoparticles (GNPs) to enhance radiation therapy has evolved into a promising frontier in cancer research [1-7]. Roeske et al. demonstrated that materials with high atomic numbers, such as gold, when coupled with low energy x-rays, can provide relatively large increases in dose enhancement factors (DEF) [4]. Following the pioneering work by Herold et al. with gold microspheres, Hainfeld et al. performed pre-clinical in vivo experiments to establish proof-ofprinciple that intravenously-administered GNPs can accumulate within tumors in su cient enough quantity to impart external beam radiation enhancement [7,8]. Recent in vivo experimental work further illustrates that GNPs are capable of providing radiation enhancement in the setting of radiation resistant squamous cell carcinoma cells, yielding enhanced cytotoxicity [2]. Radiation enhancement has been shown to involve mechanisms beyond the predicted local DEF. Roa et al. demonstrated that GNPs enhance the e ects of radiotherapy on prostate cancer cells through the increased accumulation of cells in the radiation sensitive G2/M phase of the cell cycle [9]. GNPs can be bound to various ligands through a robust amine or thiol covalent bond, conferring unique functional capabilities, in order to improve tumor targeting [9-12]. Choline is a quaternary amine that is ubiquitously distributed in cells, mostly in the form of phospholipids [13]. In vitro and in vivo magnetic resonance spectroscopy studies have shown an over-expression of choline kinase and a signi cant increase in choline in prostate cancer cells. Although the liver and kidney are major sites for choline oxidation, the bladder has only negligible activity, which allows for enhanced prostate imaging sensitivity [14-16]. ese favorable features have garnered signi cant clinical interest in the applicability of radiolabelled choline as a potential positron emission tomography (PET) tracer in the imaging of prostate malignancy [17-20]. Polyethylenimine (PEI) has been extensively studied as a vehicle for non-viral gene delivery and therapy. PEI is widely utilized as a polymer-based gene carrier secondary to e cient polyplex (complex of nucleic acid and polymer) transfection, as demonstrated both in vitro and in vivo [21]. e positivelycharged PEI polymer is thought to interact with the anionic cell membrane proteoglycans resulting in improved cellular uptake [22]. PEI retains a substantial bu er capacity over a wide pH range and it has been hypothesized that this simple molecular property is associated with the e ciency by which the complex multistage process of transfection takes place [22]. e “proton sponge” nature of PEI is thought to provide bu ering within the endosomes, improving PEI bound GNP stability a er cellular internalization [23-27]. Furthermore, the steric hindrance imparted by PEI surface functionalization imparts added colloidal stability for the gold in solution [27]. For these reasons, its incorporation into a nanoparticle platform may provide enhanced systemic bioavailability and intratumor uptake a er intravenous administration. In this study, GNPs, with a diameter of 27 nm, were covalently functionalized with both choline and PEI (Figure 1). In addition to the potential enhancement of GNP cell uptake, PEI provides additional stability of our construct in solution. e objective of this study was to assess the biodistribution pattern of choline-bound GNPs (choline-GNPs) using a human prostate cancer xenogra mouse model. Materials and Methods Materials and reagents All chemicals used in the experiment were obtained from Sigma–Aldrich (WI, USA) or Acros Organics (NJ, USA), unless otherwise stated. io-choline synthesis e procedure for the preparation of 2-mercaptoethyl-N,N,Ntrimethylammonium chloride (mta or thiocholine) was adapted from a previously established protocol [28]: 0.5 g (2.52 mmol) of acetylthiocholine chloride was dissolved in 2 ml (1 M) of HCl and re uxed at 110°C under an inert atmosphere for 16 hours. Acetic acid, HCl and deionized water were removed via vacuum distillation leaving behind a white solid. Recrystallization with methanol produced a yield of 83.0%. in layer chromatography (2:1 CHCl3: MeOH) results demonstrated a retention factor = 0.23. 1H NMR (CD3OD, 300 MHz): 3.50, 3.11 and 2.91 ppm. Seed solution preparation HAuCl4 (0.5 ml of 1% HAuCl4 solution) was added to 50 ml deionized water. e solution was heated to re ux and 0.5 ml (0.02 g/ml) of branched PEI (MW: 25000 Da) was added, followed by 1.0 ml of 1% sodium citrate solution. e solution was then boiled for 5 minutes and allowed to cool to room temperature. e resulting colloidal gold solution seen in Figure 2 was used as the seed for the subsequent synthesis of 27.3 nm gold nanoparticles. Synthesis of PEI stabilized choline bound GNPs HAuCl4 (0.5 ml of 1% HAuCl4 solution) in 50 mL deionized water was added to 2.0 ml of the seed GNP solution produced in the previous step. Sodium citrate (0.15 ml of 1% sodium citrate solution) and 0.5 ml (0.03 M) of hydroquinone were Razzak et al. Prostate Cancer Targeted GNPs © 2013 CIM Clin Invest Med • Vol 36, no 3, June 2013 E134 added and stirred continuously at 750 rpm for 20 min. iocholine (0.20 ml; 0.2 M) was added and the resulting solution was stirred continuously for an additional 20 minutes, creating the nal PEI stabilized choline bound GNP (choline-GNP) end product. Choline-GNP was stored at 40C. e mean diameter of synthesized choline-GNPs was achieved by measuring 100 nanoparticles using TEM ( JEOL-2100, Japan). X-ray photoelectron spectroscopy (XPS) Room-temperature XPS experiments were performed at Alberta Center for Surface and Engineering Science (ACSES) using a Kratos Axis spectrometer with monochromatized Al Kα (hυ = 1486.71 eV). e spectrometer was calibrated by the binding energy (84.0 eV) of Au 4f7/2 with reference to Fermi Razzak et al. Prostate Cancer Targeted GNPs © 2013 CIM Clin Invest Med • Vol 36, no 3, June 2013 E135 FIGURE 1. PEI stabilizes choline-GNPs via covalent conjugation. level. e pressure of analysis chamber during experiments was greater than 5×10-10 Torr. A hemispherical electron-energy analyzer working at the pass energy of 20 eV was used to collect core-level spectra while survey spectrum within a range of binding energies from 0 to 1100 eV was collected at analyzer pass energy of 160 eV. Charge e ects were corrected by using C 1s peak at 284.8 eV. A Shirley background was applied to subtract the inelastic background of core-level peaks. Non-linear optimization using the Marquardt Algorithm (Casa XPS) was used to determine the peak model parameters such as peak positions, widths and intensities. e model to describe XPS core-level lines for curve tting was a product of Gaussian and Lorentzian functions. Fourier transform in ared spectroscopy (FTIR) Transmission spectra for the gold nanoparticles and its cholineGNP complex were obtained by forming transparent KBr pellets containing the choline-GNPs centrifuged in the FTIR spectrometer (Nicolet 8700, Nicoletermo). e KBr mixtures were placed on a vacuum line overnight before pellet formation, and then the pellets were again placed on a vacuum line before use. e transmission spectra were obtained, a er purging in dry air, and were background corrected using a reference “blank” KBr pellet. FTIR spectra were recorded in the range of 400–4000 cm−1 at a resolution of 2 cm-1. DU-145 prostate cancer cell culture Human prostate carcinoma cell line DU-145 was used in all the experiments (American Type Culture Collection, VA, USA). Cells were maintained in Dulbecco’s modi ed Eagle’s medium supplemented with 10% FBS, 20 mM D-glucose, 100 UI/ml penicillin G and 100 μg/ml streptomycin in a humidied incubator with 5% CO2 in the air at 37oC