Targeting tumor cells and neovascularization using RGD-functionalized magnetoliposomes

Abstract

Purpose

Magnetoliposomes (MLs) have shown great potential as magnetic resonance imaging contrast agents and as delivery vehicles for cancer therapy. Targeting the MLs towards the tumor cells or neovascularization could ensure delivery of drugs at the tumor site. In this study, we evaluated the potential of MLs targeting the αvβ3 integrin overexpressed on tumor neovascularization and different tumor cell types, including glioma and ovarian cancer.

Methods

MLs functionalized with a Texas Red fluorophore (anionic MLs), and with the fluorophore and the cyclic Arginine-Glycine-Aspartate (cRGD; cRGD-MLs) targeting the αvβ3 integrin, were produced in-house. Swiss nude mice were subcutaneously injected with 10⁷ human ovarian cancer SKOV-3 cells. Tumors were allowed to grow for 3 weeks before injection of anionic or cRGD-MLs. Biodistribution of MLs was followed up with a 7T preclinical magnetic resonance imaging (MRI) scanner and fluorescence imaging (FLI) right after injection, 2h, 4h, 24h and 48h post injection. Ex vivo intratumoral ML uptake was confirmed using FLI, electron paramagnetic resonance spectroscopy (EPR) and histology at different time points post injection.

Results

In vivo, we visualized a higher uptake of cRGD-MLs in SKOV-3 xenografts compared to control, anionic MLs with both MRI and FLI. Highest ML uptake was seen after 4h using MRI, but only after 24h using FLI indicating the lower sensitivity of this technique. Furthermore, ex vivo EPR and FLI confirmed the highest tumoral ML uptake at 4 h. Last, a Perl’s stain supported the presence of our iron-based particles in SKOV-3 xenografts.

Conclusion

Uptake of cRGD-MLs can be visualized using both MRI and FLI, even though the latter was less sensitive due to lower depth penetration. Furthermore, our results indicate that cRGD-MLs can be used to target SKOV-3 xenograft in Swiss nude mice. Therefore, the further development of this particles into theranostics would be of interest.

Keywords:

• tumor targeting
• SKOV-3
• cRGD
• magnetoliposomes
• MRI
• FLI

Acknowledgments

The authors would like to thank Mrs. Ann Van Santvoort and Mr. Jens Wouters (Biomedical MRI/MoSAIC, KU Leuven, Belgium) for their technical support. This work was supported by the European Commission for the FP7 MC-ITN ‘BetaTrain’ (EU-FP7, 289932); the Horizon 2020 research and innovation programme (H2020‐NMP‐2014‐2015) for the ‘PANA’ project under grant agreement n° 686009; the Agency for Innovation by Science and Technology (IWT n° 140061, SBO NanoCoMIT) and The Research Foundation - Flanders (FWO) for project G.0A75.14 and G.0B28.14. Current adress for Dr Rita Sofia Garcia Ribeiro is In vivo Cellular and Molecular Imaging Lab (ICMI), Research Cluster Imaging and Physical Sciences (BEFY), Vrij Universiteit Brussel, Laarbeeklaan 103, B-1090 Jette, Belgium.

Ethics approval

All principles of laboratory animal care were followed according to the Belgian (Royal Decree of 29 May 2013), Flemish (Decision of the Flemish Government to adapt the Royal Decree of 29 May 2013, 17 February 2017) and European (Directive 2010/63/EU) regulations on the protection of animals used for scientific purposes. The animal experimental procedures were approved by the Ethics Committee of the KU Leuven (ECD number p259/2015).

Supplementary materials

Cyclic RGD peptide-labeled MLs synthesis

Small anionic MLs were produced as previously described [30]. In short, sonicated vesicles containing a mixture of 1,2-Dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC) and 1,2-Dimyristoyl-sn-glycero-3-phosphorylglycerol sodium salt (DMPG) (both from Avanti Polar Lipids, Alabaster, Alabama) (9:1) in 2-[Tris(hydroxymethyl)-methylamino]-ethanesulfonic acid (TES) buffer (5 mM, pH 7.0; Sigma-Aldrich, Overijse, Belgium) were mixed at a lipid/Fe3O4 weight ratio of 1:5 with a water compatible magnetic fluid at room temperature (RT). Subsequently the mixture was dialyzed for 3 days against TES buffer with regular buffer changes. Separation of the resulting MLs from the excess vesicles was performed by high-gradient magnetophoresis (HGM) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-maleimide (DSPE-PEG-MAL, 2-5% mol) (Avanti Polar Lipids, Alabaster, Alabama) was added to the anionic vesicles. The RGD peptide was linked to the MLs formulation via a sulfhydryl group at the cysteine residue of the DSPE-PEG-MAL by overnight incubation at a 10:1 molar ratio (Supplementary Scheme 1). A custom-made cyclic RGD peptide, labeled with fluorescein isothiocyanate (FITC), from Pepscan B.V (Lelystad, The Netherlands) was used. Additionally, a fluorescent lipid conjugate 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE)-Texas Red (Avanti Polar Lipidis, Alabaster, Alabama) (0.75% mol) was added to the outer lipid layer of the MLs. Unbound cyclic RGD peptide and DPPE-Texas Red were separated from the magnetic MLs fraction by HGM.

Characterization of cRGD-MLs

A JEOL 2100 transmission electron microscope (JEOL Ltd., Tokyo, Japan) working at 80 keV was used to image the nanostructures. Cryogen frozen samples were prepared in a FEI Vitrobot™ (Thermo Fischer Scientific) under the following parameters: Sample volume, 7.5 μl; Blot time, 3 s; Wait time, 1 s; Drain time, 0 s; Blot force, −3; Blot total, 1. Lacey carbon coated, 300 mesh, copper grids (Ted Pella, Redding, CA, USA) were used for the samples. Hydrodynamic size and surface charge studies were performed on a Horiba nanoPartica SZ-100 (Horiba Ltd., Kyoto, Japan) instrument directly in water solutions. Measurements were performed directly in water at 37 ºC in disposable cuvettes. Carbon electrodes were used for the zeta potential measurements. In both cases, results are shown as the average of four independent measurements ± standard deviation.

High-content image analysis for cell viability, mitochondrial ROS, and morphology

To investigate the effect of following the exposure period, cells were washed twice with phosphate-buffered saline (PBS, Gibco Life Technologies) and were immediately fixed with 4% PFA to prepare for actin staining. Next, cells were permeabilized for 10 mins with Triton X-100 (1%, Sigma-Aldrich) and blocked for 30 min with 10% serum-containing PBS, followed by incubation with Acti-stain 488 phalloidin (TebuBio, Belgium) at RT. For samples reserved for viability, samples were fixed with 4% paraformaldehyde (PFA) and incubated with LIVE/DEAD^® Fixable Green Dead Cell Stain Kit (Thermo Fisher Scientific) for 30 min at 37°C and 5% CO2. Samples reserved for mitochondrial ROS studies, live cells were incubated at 37°C and 5% CO2 for 30 min with MitoTracker Red CMXRos (Molecular Probes, Life Technologies Europe, Belgium). Subsequently, all cells were washed twice with PBS and counterstained with 1:500 Hoechst (Thermo Fisher Scientific) for 10 min in the dark at RT. All samples were kept in PBS, in the dark at 4°C, until analyzed with the InCell Analyzer 2000 (GE Healthcare Europe GmbH, Belgium). Data processing was performed using Investigator Tool 1.6.1 (GE Healthcare Europe GmbH) software on which the cells were segmented and fluorescence intensities were determined in individual cells.

Figure S1 Schematic representation of RGD functionalized magnetoliposomes synthesis procedure. The RGD peptide was linked via a sulfhydryl (-SH) group of the cysteine in the cyclic-RGD peptide that couples to the maleimide groups at the distal end of the DSPE-PEG-MAL group. The liposome suspension was incubated with cyclic RGD peptide at a molar ratio of 10:1 overnight.

Figure S2 Experimental outline of the in vivo imaging experiments. Two separate groups of animals were used for the fluorescence imaging (FLI) and Magnetic Ressonance Imaging (MRI) experiments.

Figure S3 Representation of high content image analysis for (A) cell viability, (B) membrane damage, (C–D) cell morphology (cell size and skewness) and (E–F) mitochondrial ROS formation and stress levels for BEAS-2B, GRX and SKOV-3 cells labeled with increasing concentrations of anionic (AN) and cRGD-MLs for 24 h via color-coded heat maps. Data are presented as mean ± SD vs control. Acquired data were analyzed using two-way ANOVA. No significance difference was found after incubating the cells with either anionic or RGD-MLs.

Author contributions

All authors contributed to data analysis, drafting and revising the articles, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.

Disclosure

Dr Rita Sofia Garcia Ribeiro reports grants from European Comission, Agency for Innovation by Science and Technology, and Flemish Wetenschap Onderzoek (FWO), during the conduct of the study. Miss Sarah Belderbos reports grants from KU Leuven, during the conduct of the study. Prof. Dr. Uwe Himmelreich reports grants from European Commission, Agency for Innovation by Science and Technology and Research Foundation Flanders, during the conduct of the study. The authors report no other conflicts of interest in this work.