Cellular effects of paclitaxel-loaded iron oxide nanoparticles on breast cancer using different 2D and 3D cell culture models

Stephan Lugert1 Department of Otorhinolaryngology, Head and Neck Surgery, Section of Experimental Oncology and Nanomedicine (SEON), Else Kröner-Fresenius-Stiftung-Professorship, Universitätsklinikum Erlangen, Erlangen, Germany, [email protected];2 Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany

Harald Unterweger1 Department of Otorhinolaryngology, Head and Neck Surgery, Section of Experimental Oncology and Nanomedicine (SEON), Else Kröner-Fresenius-Stiftung-Professorship, Universitätsklinikum Erlangen, Erlangen, Germany, [email protected]

Marina Mühlberger1 Department of Otorhinolaryngology, Head and Neck Surgery, Section of Experimental Oncology and Nanomedicine (SEON), Else Kröner-Fresenius-Stiftung-Professorship, Universitätsklinikum Erlangen, Erlangen, Germany, [email protected]

Christina Janko1 Department of Otorhinolaryngology, Head and Neck Surgery, Section of Experimental Oncology and Nanomedicine (SEON), Else Kröner-Fresenius-Stiftung-Professorship, Universitätsklinikum Erlangen, Erlangen, Germany, [email protected]

Sebastian Draack3 Institut für Elektrische Messtechnik und Grundlagen der Elektrotechnik, TU Braunschweig, Braunschweig, Germany

Frank Ludwig3 Institut für Elektrische Messtechnik und Grundlagen der Elektrotechnik, TU Braunschweig, Braunschweig, Germany

Dietmar Eberbeck4 Physikalisch-Technische Bundesanstalt Braunschweig und Berlin, Berlin, Germany

Christoph Alexiou1 Department of Otorhinolaryngology, Head and Neck Surgery, Section of Experimental Oncology and Nanomedicine (SEON), Else Kröner-Fresenius-Stiftung-Professorship, Universitätsklinikum Erlangen, Erlangen, Germany, [email protected]

Ralf P Friedrich1 Department of Otorhinolaryngology, Head and Neck Surgery, Section of Experimental Oncology and Nanomedicine (SEON), Else Kröner-Fresenius-Stiftung-Professorship, Universitätsklinikum Erlangen, Erlangen, Germany, [email protected]Correspondence[email protected]

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Abstract

Background

Magnetic drug targeting (MDT) is an effective alternative for common drug applications, which reduces the systemic drug load and maximizes the effect of, eg, chemotherapeutics at the site of interest. After the conjugation of a magnetic carrier to a chemotherapeutic agent, the intra-arterial injection into a tumor-afferent artery in the presence of an external magnetic field ensures the accumulation of the drug within the tumor tissue.

Materials and methods

In this study, we used superparamagnetic iron oxide nanoparticles (SPIONs) coated with lauric acid and human serum albumin as carriers for paclitaxel (SPION^LA-HSA-Ptx). To investigate whether this particle system is suitable for a potential treatment of cancer, we investigated its physicochemical properties by dynamic light scattering, ζ potential measurements, isoelectric point titration, infrared spectroscopy, drug release quantification, and magnetic susceptibility measurements. The cytotoxic effects were evaluated using extensive toxicological methods using flow cytometry, IncuCyte^® live-cell imaging, and growth experiments on different human breast cancer cell lines in two- and three-dimensional cell cultures.

Conclusion

The data showed that next to their high magnetization capability, SPION^LA-HSA-Ptx have similar cytostatic effects on human breast cancer cells as pure paclitaxel, suggesting their usage for future MDT-based cancer therapy.

Keywords:

Supplementary materials

Figure S1 Effect of different solvents on the size of SPION^LA-HSA-Ptx and SPION^LA-HSA.

Notes: SPION^LA-HSA-Ptx and SPION^LA-HSA were diluted in (A, B) H₂O, (C, D) RPMI, (E, F) RPMI + 10% FBS, (G, H) DMEM, and (I, J) DMEM + 10% FBS. Z-average and PDI were analyzed on day 1 after preparation and after 7 days of storage at 4°C by DLS, and the results are depicted in representative graphs. The strong increase in the PDI in samples containing 10% FBS is obviously the result of the additional FBS signal at 7.3 nm.

Abbreviations: DLS, dynamic light scattering; DMEM, Dulbecco’s Modified Eagle’s Medium; PDI, polydispersity index; Ptx, paclitaxel; SPION, superparamagnetic iron oxide nanoparticles; SPION^LA-HSA, lauric acid- and human serum albumin-coated SPIONs; SPION^LA-HSA-Ptx, SPION^LA-HSA functionalized with paclitaxel; RPMI, Roswell Park Memorial Institute; Z-average, intensity-weighted harmonic mean size.

Figure S1 Effect of different solvents on the size of SPIONLA-HSA-Ptx and SPIONLA-HSA.Notes: SPIONLA-HSA-Ptx and SPIONLA-HSA were diluted in (A, B) H2O, (C, D) RPMI, (E, F) RPMI + 10% FBS, (G, H) DMEM, and (I, J) DMEM + 10% FBS. Z-average and PDI were analyzed on day 1 after preparation and after 7 days of storage at 4°C by DLS, and the results are depicted in representative graphs. The strong increase in the PDI in samples containing 10% FBS is obviously the result of the additional FBS signal at 7.3 nm.Abbreviations: DLS, dynamic light scattering; DMEM, Dulbecco’s Modified Eagle’s Medium; PDI, polydispersity index; Ptx, paclitaxel; SPION, superparamagnetic iron oxide nanoparticles; SPIONLA-HSA, lauric acid- and human serum albumin-coated SPIONs; SPIONLA-HSA-Ptx, SPIONLA-HSA functionalized with paclitaxel; RPMI, Roswell Park Memorial Institute; Z-average, intensity-weighted harmonic mean size.

Figure S2 Viability of breast cancer cells 24 hours after Ptx treatment.

Notes: (A) BT-474, (B) MCF-7, (C) MDA-MB-231, and (D) T-47D cells were incubated for 24 hours with increasing amounts of free Ptx, SPION^LA-HSA-Ptx, and SPION^LA-HSA and analyzed by multiparameter flow cytometry. Viability was determined by AxV–FITC and PI staining (first column), yielding the percentage of viable (Ax− PI−), apoptotic (Ax+ PI−), and necrotic (PI+) cells. The status of the mitochondrial membrane potential was analyzed by DiIC1(5) staining and distinguished cells with intact (DiIC1(5) positive) and depolarized (DiIC1(5) negative) membranes (middle column). DNA degradation and cell cycle were determined by PIT staining and showed the amount of degraded DNA, diploid DNA (G1 phase), and double-diploid DNA (synthesis/G2 phase) (last column). Positive controls contain 2% DMSO, and negative controls represent the corresponding amount of solvent instead of drug or ferrofluid. Data are expressed as the mean ± SD (n=4 with technical triplicates). Statistical significance of viability, intact membrane potential, and diploid DNA content between control and samples are indicated with *P<0.01, **P<0.001, and ***P<0.0001, and were calculated via Student’s t-test analysis.

Abbreviations: AxV, Annexin A5; DiIC1(5), 1,1′,3,3,3′,3′-hexamethylindodicarbocyanine iodide; DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate; MMP, mitochondrial membrane potential; PI, propidium iodide; PIT, propidium iodide–Triton X-100; Ptx, paclitaxel; SPION, superparamagnetic iron oxide nanoparticles; SPION^LA-HSA, lauric acid- and human serum albumin-coated SPIONs; SPION^LA-HSA-Ptx, SPION^LA-HSA functionalized with paclitaxel.

Figure S2 Viability of breast cancer cells 24 hours after Ptx treatment.Notes: (A) BT-474, (B) MCF-7, (C) MDA-MB-231, and (D) T-47D cells were incubated for 24 hours with increasing amounts of free Ptx, SPIONLA-HSA-Ptx, and SPIONLA-HSA and analyzed by multiparameter flow cytometry. Viability was determined by AxV–FITC and PI staining (first column), yielding the percentage of viable (Ax− PI−), apoptotic (Ax+ PI−), and necrotic (PI+) cells. The status of the mitochondrial membrane potential was analyzed by DiIC1(5) staining and distinguished cells with intact (DiIC1(5) positive) and depolarized (DiIC1(5) negative) membranes (middle column). DNA degradation and cell cycle were determined by PIT staining and showed the amount of degraded DNA, diploid DNA (G1 phase), and double-diploid DNA (synthesis/G2 phase) (last column). Positive controls contain 2% DMSO, and negative controls represent the corresponding amount of solvent instead of drug or ferrofluid. Data are expressed as the mean ± SD (n=4 with technical triplicates). Statistical significance of viability, intact membrane potential, and diploid DNA content between control and samples are indicated with *P<0.01, **P<0.001, and ***P<0.0001, and were calculated via Student’s t-test analysis.Abbreviations: AxV, Annexin A5; DiIC1(5), 1,1′,3,3,3′,3′-hexamethylindodicarbocyanine iodide; DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate; MMP, mitochondrial membrane potential; PI, propidium iodide; PIT, propidium iodide–Triton X-100; Ptx, paclitaxel; SPION, superparamagnetic iron oxide nanoparticles; SPIONLA-HSA, lauric acid- and human serum albumin-coated SPIONs; SPIONLA-HSA-Ptx, SPIONLA-HSA functionalized with paclitaxel.

Acknowledgments

The present work was performed in fulfillment of the requirements for obtaining the degree “Dr. med.” (Stephan Lugert). This study was supported by the Deutsche Forsc-hungsgemeinschaft (SPP1681: Grant Numbers: AL 552/5-3, VI 892/1-1, and WI 4230/1-2), by the Emerging Fields Initiative BIG-THERA of the Friedrich-Alexander-Universität Erlangen–Nürnberg (FAU) and the Forschungsstiftung Medizin am UK-Erlangen. The authors also acknowledge the support by Deutsche Forschungsgemeinschaft and FAU within the funding program of Open Access Publishing. Eveline Schreiber, Bianca Weigel, and Julia Band are gratefully acknowledged for their expert technical help in the chemistry and cell culture laboratory.

Disclosure

The authors report no conflicts of interest in this work.