Functionalized magnetic nanowires for chemical and magneto-mechanical induction of cancer cell death

Exploiting and combining different properties of nanomaterials is considered a potential route for next generation cancer therapies. Magnetic nanowires (NWs) have shown good biocompatibility and a high level of cellular internalization. We induced cancer cell death by combining the chemotherapeutic effect of doxorubicin (DOX)-functionalized iron NWs with the mechanical disturbance under a low frequency alternating magnetic field. (3-aminopropyl)triethoxysilane (APTES) and bovine serum albumin (BSA) were separately used for coating NWs allowing further functionalization with DOX. Internalization was assessed for both formulations by confocal reflection microscopy and inductively coupled plasma-mass spectrometry. From confocal analysis, BSA formulations demonstrated higher internalization and less agglomeration. The functionalized NWs generated a comparable cytotoxic effect in breast cancer cells in a DOX concentration-dependent manner, (~60% at the highest concentration tested) that was significantly different from the effect produced by free DOX and non-functionalized NWs formulations. A synergistic cytotoxic effect is obtained when a magnetic field (1 mT, 10 Hz) is applied to cells treated with DOX-functionalized BSA or APTES-coated NWs, (~70% at the highest concentration). In summary, a bimodal method for cancer cell destruction was developed by the conjugation of the magneto-mechanical properties of iron NWs with the effect of DOX producing better results than the individual effects.


Iron nanowires synthesis
A 99.99 % pure aluminum substrate (Goodfellow, London, UK) was cleaned with acetone, isopropanol and deionized water followed by an electropolishing process for evening its surface.
A two-step anodization of the polished aluminum was carried out with 0.3 M oxalic acid at 4°C applying a voltage of 40 V to a sealed cell containing the Al film applying constant stirring, which resulted in the growth of a porous anodic alumina template with hexagonally highly ordained nanopores with diameters from 30 to 40 nm. The first anodization process lasted for 24 h creating set of random pores whereas that the second one had a duration of 4 h, yielding pores with parallel orientation. Between the anodization processes, the cell was washed with deionized water and then filled with an aqueous solution of 0.4 M H 3 PO 4 and 0.2 M CrO 3 to remove the alumina layer at a temperature of 30 ºC and constant agitation. Dendrites were synthesized in order to connect the bottom of the pores with the Al for the electrodeposition of a Fe solution, allowing the flow of current. Fe NWs were grown into the alumina template by pulsed electrodeposition with current pulses limited to 60 mA employing a solution composed of 0.5 M FeSO 4 (7H 2 O), 0.5 M Na 2 SO 4 , 0.4 M HBO 3 and 0.1g/100 mL of ascorbic acid.

Coating of iron nanowires
Fe NWs were coated with APTES and BSA through the formation of covalent bonds between the coating agent and the iron oxide (Fe 2 O 3 ) interphase that surrounds the NWs. a) APTES coating of iron nanowires Fe NWs were coated with (3-aminopropyl)triethoxysilane (APTES) by adding 0.0946 g of APTES per each 100-300 mg of Fe to the NWs suspended in ethanol with a final volume of 5 mL in a falcon tube and sonicating for 1 h. Since basic catalysis is required for the reaction, 200 μL of milliQ water and 10 μL of NaOH 1 M were added, followed by a second 1 h sonication process. Finally, the NWs were washed 5 times with 1 mL absolute ethanol and stored at room conditions in an Eppendorf tube. For further functionalization steps, APTES coated NWs were washed 3 times with phosphate buffer (PB), 10 mM and pH 7.4, in sterile conditions. b) BSA coating of iron nanowires For bovine serum albumin (BSA) coating, NWs were suspended in PB, 10 mM and pH 7.4, to a final volume of 2.5 mL in a glass vial and added with 0.8 mg of BSA/mg of Fe, followed by 1.5 h of sonication. The BSA coated NWs (BSA-NWs) were washed 3 times with PB and stored at 4°C in sterile conditions. The amount of albumin attached to the NWs was determined from Bradford assay of the supernatant containing BSA that remained in solution after the coating process.
The morphology of BSA and APTES coated NWs was analyzed by transmission electron microscopy (TEM) (TEM: TecnaiBioTWIN; FEI Company). For electron imaging, fresh samples were prepared in which NWs were released from the alumina template and rinsed several times with ethanol with sonication periods of 10 seconds in between the washes followed by the respective coating methods for each sample.

Functionalization of iron nanowires a) Activation of coated nanowires
For the functionalization of the coated NWs, the free primary amino groups present on APTES and BSA were activated by the addition of 2-Iminothiolane (2-IT) at a concentration of 500 μmol/g of Fe that adds thiol moieties (sulfhydryl groups) for further attachment of the drug, which was previously modified for the addition of a sulfhydryl group. The activation of the coated NWs was evaluated by quantifying the content of the sulfhydryl groups via the addition of aldrithiol, a molecule composed of two pyridines joined by a disulfide bond. The free sulfhydryl of the activated NWs reacts with the disulfide bound of the aldrithiol, releasing a 2pyridinethione that was quantified (λmax = 343 nm, ε 343 nm = 8080 L mol -1 cm -1 ) with a UV/Vis and fluorescence spectrophotometer Synergy H4 microplate reader (BioTek) using 96-well plates as previously described. 47 The activation process was done in sterile conditions. b) Drug attachment to activated BSA and APTES-nanowires Activated BSA and APTES coated NWs were suspended in 1 mL of phosphate buffered saline (PBS) of pH 7.4. DOX derivative 1 mM in DMF was added to reach 80 μmol of DOX/g Fe. The sample was incubated overnight at 37 °C in slow oscillation to maintain the NWs in suspension.
Functionalized NWs were retained with a magnetic rack and washed 3 times with PBS solution.
From the functionalization supernatant, the covalently immobilized DOX onto thiolated NWs was indirectly determined by quantification of free DOX in solution (λmax = 495 nm) by UV/Vis spectrophotometry and comparing the result with the result obtained from a solution of free DOX at the same concentration. All functionalization processes were carried out under sterile conditions and the DOX solution used was filtered through a 0.22-μm strainer.

Functionalized iron nanowires internalization and drug release monitoring by confocal reflection microscopy
Two 4-well plates were added with cover slips and seeded with 25000 cells/well and incubated for 24 h to reach confluence in the mentioned conditions. Cells were treated with the NWs formulations and incubated for 24 h and 72 h at 37 ºC, 5% CO 2 . Cells were washed twice with PBS and fixed with 500 µL/well of the solution containing 4% of paraformaldehyde and 0.5% of triton 100x for 5 minutes at room conditions followed by a second incubation period of 10-15 minutes with a solution containing only 4% paraformaldehyde removing first the previous solution. Thereafter, the fixing media was removed and the cells incubated with 500 µL of DAPI 300 nM for 5 minutes covering the plates from light. Finally, cells were washed twice with PBS solution, dried at room conditions and put on microscope slides with Fluoroshield™ for observation with a TCS Leica SP5 confocal laser scanning microscope (CLSM) using a confocal reflection mode. b) Prussian blue staining assay 40000 cells/well were seeded in cover slips put in a 24 well and incubated for 24 h at 37 ºC, 5% of CO 2 to reach confluence. The incubated cells were treated separately with non-coated Fe NWs, APTES-NWs, APTES-NWs-PEG and BSA-NWs. Before the treatment, NWs were washed 3 times with PBS and suspended in fresh medium in sterile conditions. Each one of these samples of Fe NWs was added with an amount of 0.01 mg Fe/well in duplicates and incubated for 24 h more at 37 ºC and 5% CO 2 . After this second incubation period, the cells were washed with PBS three times, fixed with paraformaldehyde and 1 mL of 4% potassium ferrocyanide (Fe(CN) 6 4-) was added together with 1 mL of 4% HCl for the acid catalysis (all PanreacQuímica) and incubated for 30 min at room conditions. Thereafter, each well was washed 3 times with milliQ water and counter stained with Neutral Red 0.5% (2-5 min). Finally the cover slips were washed 3 times with milliQ water, taken out from the 24 wells plate and dried at room conditions for 1 h. Once dried, the cover slips containing the cells were put over microscope slides adding depex mounting media (Sigma-Aldrich) for further observation in bright field microscopy.  BSA-NWs-DOX after 24 h of incubation. Scale bar is 10 µm.