Targeted polyethylene glycol gold nanoparticles for the treatment of pancreatic cancer: from synthesis to proof-of-concept in vitro studies

Abstract

The main objective of this study was to optimize and characterize a drug delivery carrier for doxorubicin, intended to be intravenously administered, capable of improving the therapeutic index of the chemotherapeutic agent itself, and aimed at the treatment of pancreatic cancer. In light of this goal, we report a robust one-step method for the synthesis of dicarboxylic acid-terminated polyethylene glycol (PEG)-gold nanoparticles (AuNPs) and doxorubicin-loaded PEG-AuNPs, and their further antibody targeting (anti-Kv11.1 polyclonal antibody [pAb]). In in vitro proof-of-concept studies, we evaluated the influence of the nanocarrier and of the active targeting functionality on the anti-tumor efficacy of doxorubicin, with respect to its half-maximal effective concentration (EC₅₀) and drug-triggered changes in the cell cycle. Our results demonstrated that the therapeutic efficacy of doxorubicin was positively influenced not only by the active targeting exploited through anti-Kv11.1-pAb but also by the drug coupling with a nanometer-sized delivery system, which indeed resulted in a 30-fold decrease of doxorubicin EC₅₀, cell cycle blockage, and drug localization in the cell nuclei. The cell internalization pathway was strongly influenced by the active targeting of the Kv11.1 subunit of the human Ether-à-go-go related gene 1 (hERG1) channel aberrantly expressed on the membrane of pancreatic cancer cells. Targeted PEG-AuNPs were translocated into the lysosomes and were associated to an increased lysosomal function in PANC-1 cells. Additionally, doxorubicin release into an aqueous environment was almost negligible after 7 days, suggesting that drug release from PEG-AuNPs was triggered by enzymatic activity. Although preliminary, data gathered from this study have considerable potential in the application of safe-by-design nano-enabled drug-delivery systems (ie, nanomedicines) for the treatment of pancreatic cancer, a disease with a poor prognosis and one of the main current burdens of today’s health care bill of industrialized countries.

Keywords:

• PEGylated gold nanoparticles
• doxorubicin
• potassium channel targeting
• hERG1
• pancreatic cancer
• antibody-drug conjugate

Supplementary materials

Table S1 Effects of PEG-AuNPs on the cell cycle of PANC-1 cells after 24 hours of exposure

Table S2 Zeta potential values for PEG-AuNPs dispersed in PBS at pH 7

Figure S1 Dynamic light scattering (DLS) of PEG-AuNPs.

Abbreviations: PEG-AuNPs, polyethylene glycol-gold nanoparticles; AuPEG, PEG-coated AuNPs.

Figure S2 Schematic representation of PEGylated nanostructured surface.

Notes: (A) Gold surface modification with cysteamine by SAM process; (B) AuPEG_1 immobilization via amide bond formed between activated COOH groups (using EDC/NHS) decorating the nanoparticles’ surface and the amine group of cysteamine.

Abbreviations: PEG, polyethylene glycol; SAM, self-assembled monolayer; NHS, N-hydroxysuccinimide; EDC, N′-ethylcarbodiimidehydrochloride; AuNPs, gold nano-particles; AuPEG, PEG-coated AuNPs.

Figure S3 Changes in cell number when PANC-1 cells were exposed to AuPEG_3 or AuPEG_4 at concentration equal to their LC₅₀ (corresponding to 1.3×10⁻⁷ M for AuPEG_3 and 8.5×10⁻⁸ M for AuPEG_4) for 3, 6, and 24 hours.

Notes: Changes were detected by high-content screening and analysis (HCSA) based on nuclei counterstain. Data are shown as average ± standard error of the mean (n_replicates=3; n_tests=2), and are normalized to the respective negative controls (NT).

Abbreviations: LC_50, half-maximal lethal concentration; AuPEG, PEG-coated AuNPs; PEG, polyethylene glycol; AuNPs, gold nanoparticles; h, hours.

Figure S4 XPS analysis of cysteamine gold surface before (CE-Au) and after (PEG-CE-Au) immobilization of pegylated gold nanoparticles.

Abbreviations: XPS, X-ray photoelectron spectroscopy; PEG, polyethylene glycol.

Figure S5 XRD spectra of AuPEG_1.

Notes: Diffraction peaks were detected at 2θ=22.68°, 38.41°, 43.78°, 44.86°, 64.87°, and 77.74° which correspond to (110), (111), (200), (200), (220), and (311) lattice plane, respectively.

Abbreviations: XRD, X-ray diffraction; AuPEG, PEG-coated AuNPs; PEG, polyethylene glycol; AuNPs, gold nanoparticles.

Figure S6 Representative SEM image of AuPEG_1 deposited on a planar gold surface.

Abbreviations: SEM, scanning electron microscopy; AuPEG, PEG-coated AuNPs; PEG, polyethylene glycol; AuNPs, gold nanoparticles.

Figure S7 NTA of PEG-AuNPs dispersed in (A, B) PBS, (C, D) FBS, or (E, F) DMEM supplemented with 10% FBS.

Notes: Measurements were carried out (A, C, E) immediately after dispersion (t=0 hour) or (B, D, F) following 24 hours of incubation at 37°C (t=24 hours). Graphs show the average particle concentration for sizes ranging from 0 to 700 nm. Data of control experiments (ie, dispersing media without PEG-AuNPs) were subtracted and curves were then normalized from 0 to 1.

Abbreviations: NTA, nanoparticle tracking analysis; PEG-AuNPs, polyethylene glycol-gold nanoparticles; PBS, phosphate-buffered solution; FBS, fetal bovine serum; DMEM, Dulbecco’s Modified Eagle’s Medium; AuPEG, PEG-coated AuNPs; h, hours.

Figure S8 NTA of PEG-AuNPs dispersed in (A, B) PBS, (C, D) FBS, or (E, F) DMEM supplemented with 10% FBS.

Notes: Measurements were carried out (A, C, E) immediately after dispersion (t=0 hour) or (B, D, F) following 24 hours of incubation at 37°C (t=24 hours). Graphs show the average particle concentration ± standard deviation for sizes ranging from 0 to 700 nm. NTA measurements for dispersing media (ie, PBS, FBS, or DMEM supplemented with 10% FBS) without PEG-AuNPs are also reported in each graph, showing that these did not interfere with the analysis of PEG-AuNPs dispersions. (D) Inset shows differences between NTA graphs of FBS without PEG-AuNPs (in gray) and of AuPEG_1 (in black). (A–F) Differences in the maximal concentration of samples are associated with the dilution tested and do not influence the outcome of the experiment.

Abbreviations: NTA, nanoparticle tracking analysis; PEG-AuNPs, polyethylene glycol-gold nanoparticles; PBS, phosphate-buffered solution; FBS, fetal bovine serum; DMEM, Dulbecco’s Modified Eagle’s Medium; AuPEG, PEG-coated AuNPs; h, hours.

Figure S9 Determination of LC₅₀ values.

Notes: (A–D) Percentage (%) of live cells as quantified by flow cytometry after exposing PANC-1 cells to various concentrations of PEG-AuNPs (ranging from 0 to 2.5×10⁻⁴ M) for 24 hours. Dose–response curves are fitted to extrapolate the LC₅₀ value of PEG-AuNPs. Scale bars are not comparable among the graphs. (E–H) LC₅₀ values of PEG-AuNPs as extrapolated from dose–response curves. LC₅₀ values are reported as mean ± 95% confidence interval.

Abbreviations: PEG-AuNPs, polyethylene glycol-gold nanoparticles; LC₅₀, half-maximal lethal concentration; AuPEG, PEG-coated AuNPs.

Figure S10 UV–vis absorption spectra of DOX at increasing known concentrations.

Note: A calibration curve was extrapolated from such measurements and the amount of DOX molecules grafted onto PEG-AuNPs surface was estimated based on this curve.

Abbreviations: DOX, doxorubicin; PEG-AuNPs, polyethylene glycol-gold nanoparticles.

Figure S11 Percentage of live cells as quantified by flow cytometry after exposing PANC-1 cells to COOH-terminated PEG (67.1 μg/mL) for 24 hours.

Notes: Values for the negative (untreated cells [NT]) and positive (cells exposed to 70% acetone for 30 minutes, PT) control are also shown for comparison. The symbol (***) indicates significant changes (P<0.001) as compared to the negative control. Column statistics followed by a one-sample t-test analysis was carried out (n_replicates=2) (Prism, Graph-Pad Software Inc., La Jolla, CA, USA). A P-value <0.05 was considered statistically significant.

Abbreviation: PEG, polyethylene glycol.

Figure S12 Representative confocal micrographs of PANC-1 cells cultured in the presence of inhibitors of 1) the cellular energy-dependent mechanisms of uptake (NaN₃) for 3 hours; 2) RME pathways (4°C for 4 hours); 3) clathrin-dependent endocytosis (Pitstop^® 2) for 6 hours; 4) calveolin-dependent endocytosis (Filipin III) for 30 minutes; or 5) the protein transport from the endoplasmic reticulum to the Golgi apparatus (Brefeldin A) for 6 hours.

Notes: A negative control (−), consisting of PANC-1 cells exposed to supplemented cell medium, was included in the experimental design. Cells were stained with rhodamine phalloidin (F-actin, in red) and Hoechst 33342 (nuclei, in blue). No significant changes in the organization of the cytoskeleton could be detected. Scale bars: 20 μm (63× objective lens).

Abbreviation: RME, receptor-mediated endocytosis.

Figure S13 Untreated PANC-1 cytotoxicity response to inhibitors.

Notes: Changes in cell count in the presence of inhibitors of 1) the cellular energy-dependent mechanisms of uptake (NaN₃) for 3 hours; 2) RME pathways (4°C for 4 hours); 3) clathrin-dependent endocytosis (Pitstop^® 2) for 6 hours; 4) calveolin-dependent endocytosis (Filipin III) for 30 minutes; or 5) the protein transport from the endoplasmic reticulum to the Golgi apparatus (Brefeldin A) for 6 hours. A negative control (−), consisting of PANC-1 cells exposed to supplemented cell medium, was included in the experimental design. Cells nuclei were stained with Hoechst 33342 and counted by HCSA. No significant change in cell count could be detected, as confirmed by statistical analysis (two-way ANOVA followed by a Bonferroni post-test) (Prism, Graph-Pad Software Inc.). A P-value <0.05 was considered statistically significant. Data are reported as average ± standard error of the mean (n_replicates=3; n_tests=3) and are normalized on the (−) control.

Abbreviations: RME, receptor-mediated endocytosis; HCSA, high-content screening and analysis; ANOVA, analysis of variance.

Figure S14 Percentage of DOX released over time from AuPEG_3 into aqueous PBS at pH 4.2 (black squares) and at pH 7.2 (red dots).

Notes: The percentage of DOX released by AuPEG_3 incubated in DMEM supplemented with 10% FBS was also monitored. Data are reported as average ± standard deviation. Release curves were fitted.

Abbreviations: PEG, polyethylene glycol; PBS, phosphate-buffered solution; FBS, fetal bovine serum; DMEM, Dulbecco’s Modified Eagle’s Medium; DOX, doxorubicin; AuNPs, gold nanoparticles; AuPEG, PEG-coated AuNPs.

Acknowledgments

We would like to thank Dr C Méthivier (LRS-UPMC-Paris) for technical assistance and discussion about XPS analysis. This work was partially supported by the EU FP7 MUL-TIFUN project (contract # 262943), QualityNano project Transnational Access (TCD-TAF-344 and TCD-TAF-456), and Science Foundation Ireland under the AMBER centre research (grant #SFI/12/RC/2278).

Disclosure

The authors report no conflicts of interest in this work.