a combined chemical and magneto-mechanical...
TRANSCRIPT
A Combined Chemical and Magneto-Mechanical Induction of Cancer Cell Death
by the Use of Functionalized Magnetic Iron Nanowires
Thesis by
Aldo Isaac Martínez Banderas
In Partial Fulfillment of the Requirements
For the Degree of
Master of Science in Bioscience
King Abdullah University of Science and Technology
Thuwal, Kingdom of Saudi Arabia
© April 11th, 2016
Aldo Isaac Martínez Banderas
All Rights Reserved
2
EXAMINATION COMMITTEE APPROVALS FORM
The thesis of Aldo Isaac Martínez Banderas is approved by the examination committee.
Committee Chairperson: Dr. Jürgen Kosel Committee Co-Chair: Dr. Timothy Ravasi Committee Members: Dr. Jasmeen Merzaban, Dr. Hossein Fariborzi
3
ABSTRACT
A Combined Chemical and Magneto-Mechanical Induction of Cancer Cell Death
by the Use of Functionalized Magnetic Iron Nanowires
Aldo Isaac Martínez Banderas
Cancer prevails as one of the most devastating diseases being at the top of death
causes for adults despite continuous development and innovation in cancer
therapy. Nanotechnology may be used to achieve therapeutic dosing, establish
sustained-release drug profiles, and increase the half-life of drugs. In this context,
magnetic nanowires (NWs) have shown a good biocompatibility and cellular
internalization with a low cytotoxic effect. In this thesis, I induced cancer cell
death by combining the chemotherapeutic effect of iron NWs functionalized with
Doxorubicin (DOX) with mechanical disturbance under a low frequency
alternating magnetic field. Two different agents, APTES and BSA, were
separately used for coating NWs permitting further functionalization with DOX.
Internalization was qualitatively and quantitatively assessed for both
formulations by confocal reflection microscopy and inductively coupled plasma-
mass spectrometry. From confocal reflection analysis, BSA formulations
demonstrate to have a higher internalization degree and a broader distribution
4
within the cells in comparison to APTES formulations. Both groups of
functionalized NWs generated a comparable cytotoxic effect in MDA-MB-231
breast cancer cells in a DOX concentration-dependent manner, (~60% at the
highest concentration tested) that was significantly different from the effect
produced by the free DOX (~95% at the same concentration) and non-
functionalized NWs formulations (~10% at the same NWs concentration). A
synergistic cytotoxic effect is obtained when a low frequency magnetic field (1
mT, 10 Hz) is applied to cells treated with the two formulations that is again
comparable (~70% at the highest concentration). Furthermore, the cytotoxic effect
of both groups of coated NWs without the drug increased notoriously when the
field is applied (~25% at the highest concentration tested). Here, a novel bimodal
method for cancer cell destruction was developed by the conjugation of the
magneto-mechanical properties of the iron NWs coupled with the chemotoxic
effect of an anticancer drug. Moreover, it was demonstrated that iron nanowires
possess an outstanding biocompatibility and showed high efficacy as drug
delivery agents coupled to a high degree of cell internalization. Finally, the
proposed method benefits from the low power fields applied during treatment.
This poses much less safety risks and allows using cheaper and simpler
equipment.
5
ACKNOWLEDGEMENTS
I want to express my gratitude to Prof. Jürgen Kosel and Prof. Timothy Ravasi for their support and advice during the realization of this project. Thank you both for the opportunity of being part of your research groups and for enriching my academic experience on having transmitted to me some of your expertise and knowledge.
Prof. Jürgen Kosel, thank you so much for the confidence placed in me and for encouraging me to always go further.
I want to extend my gratitude to the Madrid Institute for Advanced Studies (IMDEA) Nanoscience where part of this project was performed under the guidance of Prof. Aitziber L. Cortajarena and Dr. Antonio Aires. Thank you both for the nice opportunity of making an internship in your group.
This work is dedicated to the King Abdullah University of Science and Technology for the postgraduate education received which is an important pillar in my academic career. Thank you KAUST for giving me the opportunity to experience this amazing multicultural environment.
I dedicate this thesis to my relatives, especially to my parents and my sister, Oriana, whom have always encouraged me to pursuing my goals and taught me to never surrender in to the adversity. Thank you for making of me who I am now.
I would like to thank Mafe, Efrain and Michael for their support, patience and the time dedicated to me since I initiated my research experience at KAUST. I hope we will continue being friends even across borders and oceans. Also, many thanks to Nouf and the people from the Integrative Systems Biology group for being such a nice and supportive team.
My gratitude also goes to all my friends including: the Kaustians; Liliana, Pablo, Arturo, Gil, Natalia, Amoudi, Vlad, Aziz, Maddie, Maha, Allison, Juan, Nastia, Adrian, Ohood and many others with whom I have shared this nice experience; My lifetime friends; Gonzo, Boludo, Lecona, Sure, Maris, Yeya, Dany, Aranza, Xavier, Isak, Zara, Mario, Anuar, Josesito, Camilo, Alex, Deny, Jorge and Erick. All of you (and many others that are not mentioned here) are an important part in my life and my achievements. Thank you all.
Especially, my heartfelt gratitude is extended to my girlfriend, Anna, for the incredible moments together during this time and for making me realize that there is no unreachable goal.
6
STATEMENT OF COLLABORATION
This master’s project was done in collaboration between the Madrid Institute for Advanced Studies (IMDEA), Nanoscience and Nanobiotechnology Unit associated to Centro Nacional de Biotecnología (CNB-CSIC) in Madrid, Spain and the King Abdullah University of Science and Technology in Thuwal, Saudi Arabia. Prof Aitziber Lopez Cortajarena from IMDEA and Prof. Jürgen Kosel from KAUST supervised the project. Prof. Timothy Ravasi from KAUST acted as co-supervisor.
Because of the interdisciplinary nature of this project, the experiments were performed by a group of people of different expertise.
The in-vitro alternating magnetic field generator was provided and set up for the conditions required by Dr. Francisco Terán at IMDEA. Dr. Antonio Aires provided the doxorubicin derivative employed for the functionalization of the iron nanowires.
Dr. Antonio Aires and Aldo Isaac Martínez Banderas performed the cell culture, the iron nanowires coating and functionalization, the drug release assay, the Prussian blue assay, the in vitro cell viability assays, the inductively coupled plasma mass spectrometry studies and prepared the samples for confocal light reflection microscopy imaging. Confocal reflection microscopy images were taken by Sylvia Gutiérrez Erlandsson at the Confocal Microscopy Service CNB-CSIC associated to IMDEA.
Iron Nanowires were synthesized by Aldo Isaac Martínez Banderas and José Efraín Pérez Rodríguez at KAUST. The preparation of samples for transmission electron microscopy imaging, scanning electron microscopy imaging and energy-dispersive X-Ray spectroscopy coupled to scanning transmission electron microscopy was done by José Efraín Pérez Rodríguez, Nouf Alsharif and Aldo Isaac Martínez Banderas. Images from transmission electron microscopy, scanning electron microscopy were taken by José Efraín Pérez Rodríguez, Nouf Alsharif, Aldo Isaac Martínez Banderas and Manuel Roldan Rodriguez at the Imaging and Characterization Core Lab at KAUST.
Analysis and images from the energy-dispersive X-Ray spectroscopy coupled to scanning transmission electron microscopy were done by Manuel Roldan Rodriguez at the Imaging and Characterization Core Lab at KAUST.
7
TABLE OF CONTENTS
Page
TITLE PAGE ............................................................................................................ 1
EXAMINATION COMMITTEE APPROVALS FORM ...................................... 2
ABSTRACT .............................................................................................................. 3
ACKNOWLEDGEMENTS ..................................................................................... 5
STATEMENT OF COLLABORATION ................................................................ 6
TABLE OF CONTENTS ......................................................................................... 7
LIST OF ABBREVIATIONS ................................................................................... 10
LIST OF FIGURES ................................................................................................... 12
LIST OF TABLES ..................................................................................................... 14
1. Introduction ......................................................................................................... 15
1.1 Magnetic nanomaterials for cancer treatment………………………… . 15
1.2 Magnetic nanowires.…………………………………………………….. . 17
1.3 Nanowires internalization…………………………………………… ...... 20
1.4 Coating and functionalization of magnetic nanomaterials…..………… 22
1.4.1 Coating of nanomaterials ……………………………………….. .... 22
1.4.2 Functionalization of magnetic nanomaterials with an anticancer drug ............................................................................................................... 24
1.4.3 Strategies for functionalization of nanomaterials………….….. .. 25
1.5 Magneto-mechanical effects of nanomaterials…………….…………… 25
1.6 Summary……………………………………………………….. ................. 27
1.7 Objective/Motivation .................................................................................. 29
1.8 Thesis outline ................................................................................................ 30
8
2. Materials and Methods....................................................................................... 33
2.1 Chemical Reagents ....................................................................................... 33
2.2 Alternating magnetic field generator ........................................................ 33
2.3 Cell culture .................................................................................................... 34
2.4 Drug derivative synthesis ........................................................................... 35
2.5 Iron nanowires synthesis ............................................................................ 35
2.6 Iron nanowires characterization ................................................................ 37
2.7 Coating of iron nanowires .......................................................................... 38
2.7.1 APTES coating of iron nanowires .................................................... 39
2.7.2 BSA coating of iron nanowires ......................................................... 39
2.7.3 Characterization of nanowires´ coating .......................................... 40
2.7.4 PEG coating of iron nanowires ........................................................ 40
2.8 Functionalization of iron nanowires ......................................................... 40
2.8.1 Activation of coated nanowires ....................................................... 40
2.8.2 Drug attachment to activated BSA- and APTES-nanowires ........ 41
2.9 Drug release of functionalized iron nanowires ....................................... 42
2.10 Iron nanowires quantification .................................................................. 43
2.10.1 Prussian blue staining assay ......................................................... 43
2.10.2 Inductively coupled plasma mass spectrometry ....................... 44
2.11 Functionalized iron nanowires internalization and drug release monitoring by confocal reflection microscopy ................................................ 46
2.12 In vitro cell viability assay in the presence of functionalized iron nanowires with/without low frequency alternating magnetic field .............. 47
2.13 Statistical Analysis ..................................................................................... 48
3. Results ................................................................................................................... 49
3.1 Iron nanowires characterization ................................................................ 49
9
3.2 Coating of iron nanowires .......................................................................... 50
3.3 Colloidal stability and internalization of coated iron nanowires by Prussian blue staining assay .................................................................................................. 52
3.4 Functionalization of iron nanowires ......................................................... 55
3.5 Drug-release quantification ........................................................................ 56
3.6 Inductively coupled plasma mass spectrometry for iron nanowires quantification ........................................................................................................... 58
3.7 Functionalized iron nanowires internalization and drug release monitoring by Confocal Reflection Microscopy ...................................................................... 59
3.8 Bimodal strategy for cancer cell death induction with functionalized iron nanowires .................................................................................................................. 63
4. Discussion and Conclusions .............................................................................. 66
4.1 Discussion ..................................................................................................... 66
4.2 Conclusions ................................................................................................... 73
REFERENCES…………………………………………………………………… .. 75
PUBLICATIONS..……………………………………………………………… ... 87
10
LIST OF ABBREVIATIONS
2-IT 2- iminothiolane AMF alternating magnetic field Al2O3 aluminium oxide APTES (3-aminopropyl)triethoxysilane APTES-NWs nanowires coated with (3-aminopropyl)triethoxysilane APTES-PEG-NWs nanowires functionalized with (3-
aminopropyl)triethoxysilane and polyethylene glycol APTES-NWs-DOX nanowires coated with (3-aminopropyl)triethoxysilane and
functionalized with doxorubicin BSA bovine serum albumina BSA-NWs nanowires coated with bovine serum albumina BSA-NWs-DOX nanowires coated with bovine serum albumina and
functionalized with doxorubicin CLSM confocal laser scanning microscope CrO3 chromium trioxide DMEM dulbecco’s modified eagle’s medium DNA deoxyribonucleic acid DOX doxorubicin EDX energy-dispersive X-Ray spectroscopy Fe iron FexOy iron oxide Fe2O3 iron(III) oxide FeSO4(7H2O) iron(II) sulfate heptahydrate Fe(CN)63- potassium ferricyanide Fe(CN)64- potassium ferrocyanide FBS fetal bovine serum H magnetic field HBO3 boric acid H3PO4 phosphoric acid Hz Hertz IC50 inhibitory concentration 50 ICP-MS inductively coupled plasma mass spectrometry J Joule MDA-MB-231 breast cancer cell MNP(s) magnetic nanoparticle(s) NaOH sodium hidroxide Na2SO4 sodium sulfate
11
Ni niquel NP(s) nanoparticle(s) NW(s) nanowire(s) PB phosphate buffer PBS phosphate buffer saline PEG polyethylene glycol PEG-SH thiol-polyethylene glycol SEM scanning electron microscopy STEM scanning transmission electron microscopy T Tesla TEM transmission electron microscopy
12
LIST OF FIGURES
Figure 1. Functionalized magnetic nanoparticle ................................................ 16
Figure 2. Superparamagnetic beads used for cell separation ........................... 17
Figure 3. Schematic representation of the force generated by a single NW when an AMF is applied ................................................................................................... 18
Figure 4. Transport of neurons by Ni NWs by and external magnetic field .. 19
Figure 5. TEM images of Ni NWs internalized at 24 and 72h .......................... 21
Figure 6. HRTEM image of a Fe NW showing its iron oxide outside layer ... 22
Figure 7. Scheme of functionalization of MNP ................................................... 23
Figure 8. Combined chemotherapeutic and hyperthermia treatment of an induced subcutaneous xenograft in the backside of a nude mice with functionalized MNP ............................................................................................... 27
Figure 9. Schematic of the bimodal strategy for cancer cell death induction with functionalized Fe NWs ........................................................................................... 28
Figure 10. In vitro alternating magnetic field generator .................................... 34
Figure 11. Synthesis of Fe NWs by a two-step anodization process ............... 37
Figure 12. General scheme of Fe NWs coating and functionalization ............ 42
Figure 13. Characterization of Fe NWs ................................................................ 50
Figure 14. TEM micrographs of APTES-NWs and BSA-NWs .......................... 52
Figure 15. Internalization and distribution of Fe NWs coated with different agents into MDA-MB-231 cells 24 h post incubation using Prussian blue staining for iron oxide detection .......................................................................................... 53
Figure 16. Release kinetics of DOX from APTES-NWs-DOX and BSA-NWs-DOX .................................................................................................................................... 57
Figure 17. Confocal reflection microscopy images from MDA-MB-231 cells incubated with APTES-NWs-DOX after 24 h and after 72 h and incubated with BSA-NWs-DOX after 24 h and after 72 h ............................................................. 60
13
Figure 18. Z-stack of a MDA-MB-231 cell treated with APTES-NWs-DOX and BSA-NWs-DOX after 72 h of incubation ............................................................. 61
Figure 19. Morphology and distribution of functionalized NWs in MDA-MB-231 cells ............................................................................................................................ 62
Figure 20. Viability of MDA-MB-231 cells incubated with different formulations and with or without application of a low frequency AMF ............................... 65
14
LIST OF TABLES
Table 1. Calculated Fe mass for the different NW-formulation stock solutions according to the selected concentrations of doxorubicin..................................... 45
Table 2. Correlation between the calculated and quantified concentration of Fe in the different NW-formulation stock solutions…………………………………... 58
15
Chapter 1
Introduction
1.1 Magnetic nanomaterials for cancer treatment
Regardless of the continuous development and innovation in cancer therapy,
cancer prevails at the top of death causes for adults as one of the most
devastating diseases.1,2 Current cancer treatment options such as surgery,
radiation and chemotherapy are highly aggressive to the organism by their
invasiveness and side effects and additionally, the chemical agents are affected
by the development of the multidrug resistance phenotype in cancer cells.3 Most
pharmaceutical preparations have their primary targets within the cell where
they may be transported; therefore selective subcellular delivery may increase
the therapeutic efficiency and simultaneously overcome secondary effects. In this
regard, nanotechnology may be used to achieve therapeutic dosing, establish
sustained-release drug profiles,4 and increase the half-life of drugs avoiding
efflux or degradation.5
Nanomaterials possess novel structural, optical and electromagnetic
properties and their pharmacokinetic parameters may be altered according to
size, shape, and surface functionalization.4 Their vast surface area provides them
with the possibility of surface modifications for further conjugation of large
amounts of therapeutic molecules such as targeting agents and anticancer drugs
16
as shown in Figure 1.3,6,7
Figure 1. Functionalized magnetic nanoparticle. Adapted from Latorre et al., 20148
In addition to their drug loading capability, the potential of
electromagnetic nanomaterials as a therapeutic agent arises from the intrinsic
properties of the magnetic core combined with the biomedical properties
generated by different surface coatings.9 These surface modifications alter the
particokinetics and toxicity in addition to surface attachment for biomolecule
binding through covalent linkages.10 The electromagnetic properties of the core
allow remote manipulation of nanomaterials through the application of an
electromagnetic field. It has been observed that magnetic nanomaterials can be
trapped, concentrated, 11-14 or used in cell separation.15-17
17
Figure 2. Superparamagnetic beads used for cell separation. Adapted from Wang et al., 2004.15
Moreover, the influence of alternating fields can induce heat 18 or rotate
the nanostructures.19,20Nowadays, many kinds of nanomaterials and nanodevices
have been developed and designed being some of the most commonly utilized
quantum dots, magnetic nanoparticles (MNPs), gold NPs, carbon nanotubes,
polymers, dendrimers, liposomes and magnetic nanowires (NWs).7 Most of the
research done lately have focused in MNPs and their enhancement as a
therapeutic option for cancer.
1.2 Magnetic nanowires
NWs are anisotropic colloidal objects with submicronic diameters and lengths in
the range of 1 to 100 μm grown by electrochemical deposition in nanoporous
templates.21,22
18
Recently, it has been reported that magnetic NWs offer potential
advantages over nanobeads because of their higher magnetic moments per
volume and larger surface area to volume ratios.23,24 Their magnetic moments
enable them to generate large forces and torques,24 while large aspect ratios
provide ferromagnetic NWs with large remanent magnetizations, and hence can
be used in low-field environments where the superparamagnetic beads do not
perform at all.21
Figure 3. Schematic representation of the force generated by a single NW when an AMF is applied. Adapted from Contreras et al., 2014.25
In biomedical applications, iron (Fe) and niquel (Ni) are the magnetic
materials usually used as NWs. Several studies support the efficiency and utility
of Ni NWs in diverse applications such as cell separation, manipulation, and
purification,16,17,22-24,26-28 as well as in cargoes delivering including biological
entities.29
19
Figure 4. Transport of neurons by Ni NWs by and external magnetic field. Adapted from Choi et al., 2007. 17
Furthermore, they have been utilized as therapeutic agents for
hyperthermia30 and cell inflammation induction31 in cultures of human
embryonic cells. Although a large amount of evidence place Ni as a good
candidate material, an important genotoxicity and cytotoxicity effects have also
been reported in Ni-containing dust particles.32 Fe NWs have shown a good
biocompatibility and low cytotoxic effect even at high concentrations with long
incubation periods.33 A cross-comparison of Fe and Ni NWs among studies34,35
implies that Fe NWs have a lower impact in cell viability than Ni NWs at a given
concentration. However, Fe NWs tend to aggregate more, since they have a
higher remanence magnetization value.36
20
1.3 Nanowires internalization
Once NWs are in close proximity to the cellular membrane, the uptake
mechanism is triggered that in turn determines the intracellular fate of the
nanostructures.34 Endocytosis has been accepted as the most common passive
mechanism for MNP uptake by different cellular types (HeLa, fibroblast, and
dendritic cells).37 It has been demonstrated that at least three types of endocytosis
occur for NPs: clathrin-mediated endocytosis, caveolin-mediated endocytosis,
and clathrin- and caveolin-independent endocytosis.38 NWs internalization by
cells has been documented over the past years for both Ni and Fe describing
different ways of entry. Regarding Ni NWs, it has been reported that the
internalization takes place through the activation of the integrin-mediated
phagocytosis pathway,24 a process in which several integrin membrane receptors
link the actin cytoskeleton to extracellular components, driving the cell
membrane to engulf the foreign particle for internalization and final degradation
as recently assessed for the Ni NWs (Figure 5) that, after being internalized by
this pathway, are dissolved in lysosomes.39,40
21
Figure 5. TEM images of Ni NWs internalized at 24 and 72h. Modified from Perez et al., 2015.40
Furthermore, it has been described that Fe NWs, individually and in
clusters, exhibit a fast but continuous internalization process and two main
mechanisms for the uptake were proposed depending on the NW size, including
non-specific pynocitosis for short NWs and perforation of the pouter plasma
membrane for longer NWs.34 Likewise, after 24 hours of incubation with
polymeric NWs made of iron oxide particles, it was seen that most of them were
located in the cytosol, with a small fraction located in late endosomal/lysosomal
compartments, where, due to the lower pH, they were probably broken in
smaller pieces for further degradation.22 Moreover, uptake and intracellular
location of nanostructures have been shown to depend on several factors such as
the NW aspect ratio, cell line, NW surface charge, etc.41 Thus, more experiments
are necessary to reach a definite conclusion concerning the portals of entry of the
wires into the cells.22
22
1.4 Coating and functionalization of magnetic nanomaterials
1.4.1 Coating of nanomaterials
Regardless of the synthesis method or particle morphology, metal particles will
be oxidized when exposed to air or oxygen. At room temperature, zero covalent
Fe nanoparticles exposed to air will always be covered by a thin interphase of
oxide42 and it has been well studied that iron oxide have great bicompatibility,43
therefore the low toxicity of Fe NWs may be related to the surface iron oxide
(Figure 6). This FexOy-Fe structure also has potential applications, for instance
the ionic or covalent attachment of bioactive molecules or coating reagents for
enhancing the colloidal properties and biocompatibility.21,34,44,45
Figure 6. HRTEM image of a Fe NW showing its iron oxide outside layer. Adapted from Song et al., 201034
Different coatings have been employed in recent years for improving
biocompatibility, colloidal stability and the further functionalization process of
nanomaterials including polymers such as poly(ethylene glycol) (PEG),46,47 and
23
dimercaptosuccinic acid.48 Interestingly, galliumnitride NWs have been coated
with 3-Aminopropyltriethoxysilane (APTES), one of the most explored coupling
agents in the functionalization of semiconductor materials that has the ability to
covalently attach to oxidized or hydroxylated surfaces and works as an initial
step for the immobilization of biomolecules.49-52 Nevertheless, due to their
superior biocompatibility and hydrophilicity, proteins or polypeptides are
considered the most promising molecules as biocompatible coating agents for
biomedical applications.53 In this manner, bovine serum albumin (BSA) has been
covalently immobilized in MNP allowing further functionalization of an
anticancer-drug and improving their colloidal properties under physiological
conditions as well as cellular uptake of the MNP resulting in the increase of the
chemotherapeutic effect (Figure 7).54
Figure 7. Scheme of functionalization of MNP. Modified from Aires et al., 2015
24
1.4.2 Functionalization of magnetic nanomaterials with an anticancer drug
As mentioned above, coating agents allow or enhance the further attachment of
biomolecules such as drugs providing nanomaterials with the capability to act as
a drug delivery system.
Currently, several anticancer chemotherapeutic agents including
doxorubicin, methotrexate and gemcitabine have been formulated with
nanodevices.8,54-59 Doxorubicin (DOX) is an anthracyline drug widely used in the
treatment of several cancers including breast, lung, gastric, ovarian, thyroid, non-
Hodgkin’s and Hodgkin’s lymphoma, multiple myeloma, sarcoma, and pediatric
cancers.54,60,61 There are two proposed mechanisms by which DOX acts in the
cancer cell; by intercalation into DNA and disruption of topoisomerase-II-
mediated DNA repair and the generation of free radicals and their damage to
cellular membranes, DNA and proteins.62 However, to reduce its systemic
toxicity and side effects, this drug is constantly under investigation to be used in
a drug carrier system that can be activated. (e.g. by heat or pH-sensitive
liposomes).63-67 An advantage of DOX is its strong visible absorption and
fluorescence emission that makes it easy to monitor during the different steps of
the functionalization strategy and has shown great efficacy in both solid and
liquid tumors, but the emergence of drug resistance and several side effects such
as heart muscle damage are important limitations for successful cancer
treatment.68
25
1.4.3 Strategies for functionalization of nanomaterials
To integrate chemotherapeutics into the nanomaterials, strategies such as
covalent binding with cleavable linkages have been explored and ideally these
chemotherapeutic agents are inactivated till they are release inside the cell.69 In
this regard, different linkers sensitive to certain intracellular triggering stimulus
such as pH3,70 and the presence of some enzymes71-73, or external stimuli such as
temperature74,75 have been employed to connect and release drugs from magnetic
nanoparticles in a controlled manner.
It has been assessed that covalent bonds sensible to acid hydrolysis are a
suitable and an efficient option for attaching the drug to the magnetic
nanoparticles due to the pH difference existing between the extra and
intracellular compartments 8, commonly considering the pH of blood (7.4) and
endosomes (5.5–6.4).3,76,77 Recently, specialized functionalizing linkers have been
designed for releasing the drug without any chemical modification only when
the specific release-condition is fulfilled and have demonstrated efficiency as
cancer therapeutic agents with the extra advantage of allowing quantifying the
drug immobilized.54
1.5 Magneto-mechanical effects of nanomaterials
Studies of the alterations in cellular features due to magneto mechanical effects
from alternating magnetic fields (AMFs) have been done before. A remarkable
26
decrease of viability of dendritic cells previously loaded with MNPs was
observed after 30 min of exposure to an hyperthermia-like AMF (16 mT, 260
kHz), without raising the temperature of the cell culture. Clear morphological
changes including cell membrane leakage and cell shrinkage after magnetic field
application were observed and adjudicated as the main reason for the cell
death.37
Recently, colon cancer cells were incubated with Ni NWs and exposed to
low frequency AMF (0.5 mT and 1 Hz or 1 kHz) for 10 or 30 minutes which
exerted a force on the Ni NWs, triggering a mechanical disturbance to the cells
and therefore inducing cell death in a non-chemotoxic way.25
What is more, Hilger et al., 2015 have exploited two properties of the
magnetic nanomaterials, magnetic hyperthermia and chemotherapy, combining
them for tumor cell killing. As a result, a synergic effect was produced, translated
in a strong enhancement of the cytotoxicity of the functionalized MNPs in vitro
and in vivo.55
27
Figure 8. Combined chemotherapeutic and hyperthermia treatment of an induced subcutaneous xenograft in the backside of a nude mice with functionalized MNP. Modified from Kossatz et al.,
201555
1.6 Summary
In this thesis, I introduce a new bimodal strategy for cancer cell death induction
by combining the chemotherapeutic effect of DOX functionalized Fe NWs with
the mechanical disturbance exerted by them when a low frequency AMF is
applied as shown in Figure 9. The effectiveness of this approach was evaluated
through the decrease of the viability of breast cancer cells. Fe NWs were
functionalized with DOX through a pH-sensitive covalent bond to permit the
selective drug release after internalization of NWs and therefore trigger a
chemotoxic effect. A low frequency AMF induces a force over the NWs that are
interacting (internalized or attached to the plasmatic membrane) with the seeded
cells and therefore triggers cell death in non-chemotoxic way (Figure 9).
28
Figure 9. Schematic of the bimodal strategy for cancer cell death induction with functionalized Fe NWs. DOX (D) functionalized NWs are internalized by phagocytosis in endosomes where the lower pH induce the release of DOX that will diffuse across the cytoplasm reaching its main
target in the nucleus where it triggers the chemotherapeutical effect. At the same time, a magneto mechanical effect is produced by the NWs when a low frequency AMF is applied generating a
synergic induction of cancer cell death.
Additionally, two different agents, APTES and BSA were separately used
for coating Fe NWs permitting further functionalization with DOX. Both NWs
formulations were evaluated and compared against each other. NWs
internalization was qualitatively and quantitatively assessed for both
formulations by confocal reflection microscopy images and inductively coupled
plasma mass spectrometry (ICP-MS) for Fe measurement, respectively.
29
1.7 Objective/Motivation
To this day, cancer prevails at the top of death causes for adults regardless the
huge effort in developing new options for treatment. Furthermore, most of this
cancer treatment approaches, such as surgery; radiation and chemotherapy are
highly aggressive to the organism by their invasiveness and side effects.
Additionally, most of the pharmaceutical preparations have their primary targets
within the cell; therefore selective subcellular delivery may increase the
therapeutic efficiency and simultaneously overcome secondary effects, goals in
which nanotechnology could be an important contributor.
Recently, many studies have proposed magnetic NWs as a new
nanomaterial that offers potential advantages over nanobeads mainly because of
their morphology and magnetic properties. NWs have shown a good
biocompatibility and cellular internalization with a low cytotoxic effect, and have
been used in diverse applications such as cell separation, manipulation, and
purification as well as in cargoes delivering and hyperthermia. For biomedical
applications, Fe and Ni are the magnetic materials usually used as NWs.
In this thesis, we aim to use Fe NWs as a device for bimodal cancer cell
death induction by exploiting their capability of being functionalized with an
anticancer drug combined with their electromagnetic properties, specifically the
mechanical cell disturbance exerted by them when a low frequency
30
electromagnetic field is applied. The effectiveness of the proposed model will be
evaluated in MDA-MB-231, breast cancer, cells.
Different coating reagents will be evaluated for enhancing the colloidal
properties, biocompatibility and internalization degree of Fe NWs as well as the
efficiency of functionalization with an anticancer drug and further chemotoxic
effect, comparing the performance of the different formulations at each aspect.
Fe NWs will be functionalized with a derivative of DOX trough a pH-
sensitive covalent bond to permit the selective drug release after internalization
of NWs in breast cancer cells and therefore trigger a chemotoxic effect.
1.8 Thesis outline
This thesis is structured in a manner where the order of the chapters helps the
reader to understand the main idea about what has been done along this
research project. The first chapter contains an introduction covering topics such
as the involvement of nanotechnology in cancer treatments describing some of
the main nanomaterials employed and studied by now. Emphasis is made on
magnetic NWs, their characteristics, properties and applications. Some of the
main research that has been done with magnetic NWs is presented focusing in
the NW properties that where exploited during this project. Importantly, a
summary of the thesis project is presented in this section stating the bimodal
strategy for cancer cell death induction introduced in this thesis project.
31
Furthermore, the motivation/objective of this thesis project is stated at the end of
this section.
Chapter two describes the materials and method employed in this
research project starting from NW synthesis, characterization and
functionalization with an anticancer drug. Internalization studies employing
different techniques and Fe quantification techniques are also described followed
by in-vitro cell viability assays in breast cancer cells in the presence and absence
of an AMF.
Chapter three includes a profound description of the results obtained in
each of the experiments performed presenting the main charts, images and
graphs.
Chapter four is directly linked to chapter three, and meticulously
discusses the results obtained during this project at each step. The efficiency of
the combination of the magneto-mechanical effect of the Fe NW in the presence
of a low frequency AMF coupled to the chemotherapeutical effect of DOX linked
to the NWs for cancer cell induction is discussed. Furthermore, a comparison is
made between the two coating agents employed in this study regarding the
internalization degree, biocompatibility, functionalization efficiency and both
ways of cancer cell death induction in combination and separated. Finally, a
32
conclusions section is presented formed by various statements of the
achievements of the thesis project.
33
Chapter 2
Materials and Methods
2.1 Chemical Reagents
APTES, BSA and DOX were purchased from Sigma-Aldrich. Polyethylene glycol-
Thiol (Mw = 2000 Dalton, PEG-SH) was from Creative PEGWorks (USA). Double
distilled water was used in all experiments.
2.2 Alternating magnetic field generator
The in vitro AMF generator employed in this study (Figure 10) is a home-made
air-cooled ferrite core with a C-shape and a gap of 16 mm, coiled with Litz wires.
The AMF generator is part of an LCR resonant circuit allowing to independently
adjusting the frequency and intensity. The core gap allows placing NUNC(TM) 4
well dishes (inner well diameter is 10 mm) in an AMF. The AMF direction is
perpendicular to the wells, and its intensity gradient is about 10% from the
center of the well to the external border. Before the in vitro studies, we checked
that the AMF generator does not heat up the cell media under the conditions
employed in this study. The applied AMF was 1 mT and 10 Hz.
34
Figure 10. In vitro alternating magnetic field generator (A) and four- well plate placed in the 16 mm gap of the C-shape magnetic coil (B).
2.3 Cell culture
MDA-MB231 cell line was purchased from American Type Culture Collections
(Manassas, VA, USA). MDA-MB231 cell line was grown as monolayer in
Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal
bovine serum (FBS), 2 mM L-glutamine, 0.25 μg/mL fungizone, 100/mL units of
penicillin and 100 µg/mL of streptomycin. All reagents were purchased from
GIBCO. Cell lines were maintained in an incubator at 37 ºC in a humidified
atmosphere of 95% air and 5% CO2.
35
2.4 Drug derivative synthesis
A DOX derivative [(5-Maleimidovaleroyl) hydrazone of Doxorubicin] was
synthesized as previously described,78 using as precursor 5-aminovaleric acid
instead of 6-aminocaproic acid, to functionalize the coated NWs with DOX.
1H NMR (400 MHz, MeOD, δ): 7.92 (bd, lH), 7.81 (t, lH), 7.55 (d, lH), 6.57 (m,
2H), 5.51 (m, lH), 5.07 (m, lH), 4.54 (d, lH), 4.25 (m, lH), 4.06 (s, 3H), 3.61-2.7 (m,
5H), 2.55-2.26 (m, 4H), 2.20-1.90 (m, 3H), 1.62-1.25 (m, 10 H); HRMS (ESI) m/z:
[M + H]+ calcd for C36H41N4O13, 727.7226; found, 727.3628.
2.5 Iron nanowires synthesis
The Fe NWs were fabricated by chemical electrodeposition into nanoporus alu-
mina membranes as explained in previous studies.25,35,79-81. A 99.99 % pure
aluminum substrate (Goodfellow, London, UK) was cleaned with acetone,
isopropanol and deionized water followed by an electropolishing process for
even its surface. As represented in Figure 10, a two-step anodization of the
polished aluminum was carried out with 0.3 M oxalic acid at 4°C applying a
voltage of 40V to a sealed cell containing the Al film applying constant stirring
which resulted in the growth of a porous anodic alumina (Al2O3) template with
hexagonally highly ordained nanopores with diameters from 30 to 40 nm. The
first anodization process last for 24h creating set of random pores, (Figure 10A1
and 10A2) whereas that the second one has duration of 4h yielding pores with
36
parallel orientation (Figure 10C1 and 10C2). Between the anodization processes,
the cell was washed with deionized water and then filled with an aqueous
solution of 0.4 M phosphoric acid (H3PO4) and 0.2 M chromium trioxide (CrO3)
to remove the alumina layer at a temperature of 30 ᵒC and constant agitation
(Figure 10B1 and 10B2). Dendrites are 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 (Figure 10D1 and 10D2). As represented in Figure 10E1, Fe NWs
were grown into the alumina template by pulsed electrodeposition to deposit the
Fe with current pulses limited to 60 mA employing an solution composed by 0.5
M iron(II) sulfate heptahydrate (FeSO4(7H2O)), 0.5 M sodium sulfate (Na2SO4),
0.4 M boric acid (HBO3) and 0.1g/100 mL of ascorbic acid. The NWs’ length of
~7 µm was controlled by the deposition time (1.5 h). Thereafter, the template
containing the NWs (Figure 10E2) was dissolved with 1 M sodium hydroxide
(NaOH) in an Eppendorf tube for for 20 min and the alumina membrane was
removed. The NaOH solution was changed every hour for four times with the
help of a magnetic rack (DynaMag™-2; Life Technologies, Carlsbad, CA, USA).35
Finally, the NWs were collected with the magnetic rack and rinsed thoroughly
with ethanol for 10 to 15 times with 10-second sonication steps in between and
leaving the released NW´s suspended in 1 mL of absolute ethanol.
37
Figure 11. Synthesis of Fe NWs by a two-step anodization process. A. First anodization of the Al disk (1) and SEM image of the non-ordered nanopores formed (2). B Etching of the alumina layer revealing the ordered domains on the Al substrate (1) and SEM image (2). C Second anodization
process obtaining highly ordered pores with narrow diameter size distribution (1) and SEM image (2). D. Formation of dendrites through reduction of the alumina layer (1) and image
magnification (2) for allowing the flow of current in the next step. E. Fe NWs formation after pulsed chemical electrodeposition (1) and Al disk showing the deposition area (2).
2.6 Iron nanowires characterization
The morphology, length and diameter of the Fe NWs were investigated by
scanning electron microscopy (SEM) and transmission electron microscopy
(TEM) (SEM: Quanta 3D; FEI Company, Hillsboro, OR, USA; and TEM: Tecnai
BioTWIN; FEI Company). Energy-dispersive X-Ray spectroscopy (EDX) was
used for determining the chemical composition of NWs (scaning TEM (STEM)
Tecnai BioTWIN; FEI Company). Images were analyzed using ImageJ software
38
as previously described.25 For TEM imaging, fresh samples were prepared in
which NWs were released from the alumina template and rinsed several times
with ethanol with sonication periods of ten seconds. For SEM imaging sonication
steps were skipped to prevent fragmentation of the NWs. Likewise, the NWs
were quantified using the methodology described by Contreras et al., 2015 in
which the amount of pores in determined area were quantified from SEM
images, and extrapolated to the total deposition area based on the assumption
that every pore contains a NW. The assumed correlation is that 225 NWs are
contained in 2.06E-8 cm2. The deposition area was determined using ImageJ
software from where the number of NWs was obtained for further Fe mass
calculation. The calculated mass of one Fe NW of a length of ~7 µm and a radius
of 17 nm is 4.88 x 10-11 mg. This calculated Fe mass was used for the preparation
of the NW stock solutions for the further experiments of coating and
functionalization.
2.7 Coating of iron nanowires
Fe NWs were coated with different agents through the formation of covalent
bonds between the coating agent and the iron oxide (Fe2O3) interphase that
surrounds the NWs, which is formed after releasing the NWs from the alumina
template as explained in Figure 11.
39
2.7.1 APTES coating of iron nanowires
Fe NWs were coated with (3-aminopropyl)triethoxysilane (APTES) (Figure 11A1)
by adding 0.0946g 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 sonicated for 1h. Since
basic catalysis is required for the reaction, 200 μL of miliQ water and 10 μL of
NaOH 1M were added, followed by a second 1h sonication process. Finally, the
NWs were washed five times with 1 mL absolute ethanol and stored at room
conditions in an Eppendorf tube. For further functionalization steps, APTES
coated NWs (APTES-NWs) were washed three times with phosphate buffer (PB)
10 mM and pH 7.0, in sterile conditions.
2.7.2 BSA coating of iron nanowires
For bovine serum albumin (BSA) coating (Figure 11A2), 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 of sonication. The BSA coated NWs
(BSA-NWs) were washed three 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
coating process.
40
2.7.3 Characterization of nanowires´ coating
The morphology of BSA and APTES coated NWs was analyzed by TEM (TEM:
Tecnai BioTWIN; 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 ten seconds in between the
washes followed by the respective coating methods for each sample.
2.7.4 PEG coating of iron nanowires
NWs were also coated with thiol-poliethilen glycol (PEG-SH) (50 μmol PEG/g
Fe) (Figure 11C). In this case, NWs have to be first coated with APTES and
further activation with 2-Iminothiolane (2-IT) (Figure 11B) and aldrithiol (Figure
11C) as previously described,54 to finally react with PEG-SH (2 kD) giving
APTES-PEG-NWs.
2.8 Functionalization of iron nanowires
2.8.1 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-IT at a
concentration of 500 μmol/g of Fe that adds thiol moieties (sulfhydryl groups)
(Figure 11B) for further attachment of the drug, which was modified in a
different way for the addition of a sulfhydryl group as previously described.8
The activation of the coated NWs was evaluated by quantifying the content of
41
sulfhydryl groups by the addition of aldrithiol, a molecule composed of two
pyridines joined by a disulfide bond (Figure 11C). The free sulfhydryl of the
activated NWs reacts with the disulfide bound of the aldrithiol, releasing a 2-
pyridinethione that was quantified (λmax = 343 nm, ε343 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.54 The activation
process was done in sterile conditions.
2.8.2 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 dimethylformamide
was added to reach 80 μmol of DOX/g Fe (Figure 11D). The sample was
incubated overnight at 37°C in slow oscillation to maintain the particles in
suspension. Both formulations of functionalized NWs (APTES-NWs-DOX and
BSA-NWs-DOX) were retained with the 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 compared to the result given from 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.
42
Figure 12. General scheme of Fe NWs coating and functionalization. (A1) Fe NWs were coated with APTES, APTES-NWs or (A2) BSA, BSA-NWs (B) Introduction of free thiol groups by the
addition of 2-IT in APTES-NWs and BSA-NWs, both represented by the presence of free amino groups. C. Evaluation of the thiol groups introduction efficiency and coating of APTES-NWs with
PEG, APTES-PEG-NWs. D. Functionalization of APTES-NWs and BSA-NWs with a DOX derivative generating the formulations APTES-NWs-DOX and BSA-NWs-DOX.
2.9 Drug release of functionalized iron nanowires
The release of DOX from the functionalized NWs is based on the pH sensibility
of the drug derivative linker. The amount of DOX released was quantified by
measuring the fluorescence of DOX released in solution (λexc = 495 nm, λem =
520–750 nm) at regular time intervals after passing 0.5 mg of functionalized NWs
from PBS solution of pH 7.4 to acetate buffer solution of pH 5.0 at 37 ºC. The
values were compared to the ones from a reference sample of functionalized
43
NWs suspended in PBS solution of pH 7.4. The same process was done for
APTES and BSA coated NWs.
2.10 Iron nanowires quantification
In order to quantify the amount of NWs contained in the NW-formulation stock
solutions that are added to the seeded cells and also the NWs that interact (get
internalized or attached to the membrane) with the incubated cells, Prussian blue
staining and inductively coupled plasma mass spectrometry (ICP-MS) for Fe
quantification were performed.
2.10.1 Prussian blue staining assay
The presence and distribution of Fe NWs in MDA-MB-231 cells was assessed by
Prussian blue staining assay, comparing different coating agents. The oxidation
state of Fe in the oxide interphase surrounding the NWs was also determined in
this assay. 40000 cells/well were seeded in cover slips put in a 24 well and
incubated for 24h at 37 ºC, 5% of CO2 to reach confluence. The incubated cells
were treated separately with 0.05 mg Fe/mL of non-coated Fe NWs, APTES-
NWs, APTES-NWs-PEG and BSA-NWs and compared to a control group of cell
without treatment. Before the treatment, NWs were washed 3 times with PBS
solution 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 24h more at 37 ºC and 5% CO2. After this second incubation
44
period, the cells were washed with PBS three times, fixed with
paraformaldehyde and added with 1 mL of 4% potassium ferrocyanide
(Fe(CN)64-; containing Fe2+) or potassium ferricyanide (Fe(CN)63-, containing
Fe3+) together with 1 mL of 4% HCl for the acid catalysis (all Panreac Química)
and incubated for 30 min at room conditions. Thereafter, each well was washed 3
times with miliQ water and counter stained with Neutral Red 0.5% (2-5 min).
Finally, the cover slips were washed 3 times with miliQ water, taken out from
the 24 well plate and dried at room conditions for 1h. Once dried, the cover slips
containing the cells were putted over microscope slides adding depex mounting
media (Sigma-Aldrich) for further observation in bright field microscopy.
2.10.2 Inductively coupled plasma mass spectrometry
The quantification of Fe NWs in the NW-formulation stock solutions was
performed by ICP-MS directly from an aliquot of each formulation before being
added to the cells. Each DOX-NW stock solution was prepared from selected
fixed concentrations of DOX (1 and 3 µM), which in conjunction with the DOX
immobilization percentage obtained from the UV/Vis spectrophotometry studies
(65% of immobilization for APTES-NWs; ~50 μmol DOX/g Fe and 32% for BSA-
NWs; ~25μmol DOXO/g Fe), indicated the amount of NWs (mg of Fe) that have
to be added to each solution as shown in Table1. 30% more was considered for
covering possible loss during the coating and functionalization process.
45
Table 1. Calculated Fe mass for the different NW-formulation stock solutions according to the selected concentrations of doxorubicin
NW-formulation stock solution
DOX (µM) mg of Fe (30% more added)
DOX_1 0.5
DOX_2 1
NW-APTES_1
0.02275 NW-APTES_2
0.06825
NW-APTES-DOX_1 1 0.02275 NW-APTES-DOX_2 3 0.06825
NW-BSA_1 0.032 NW-BSA_2 0.096
NW-BSA-DOX_1 1 0.032 NW-BSA-DOX_2 3 0.096
The results obtained from the ICP measurements of the stock solutions
were directly used for performing the quantification of Fe NWs in MDA-MB-231
cells, comparing different coating agents. 40000 cells/well were seeded and
incubated for 24 h to reach confluence in the mentioned conditions. The cells
were treated with NWs formulations 26 µg Fe/mL, 1.3 µM of DOX for APTES-
NWs-DOX and 28 µg Fe/mL, 0.73 µM of DOX for BSA-NWs-DOX (values
obtained from the quantification of the NW-stock solution formulation) and
incubated for 24 h at 37 ºC, 5% CO2. The cells were washed twice with PBS and
incubated for 10-15 min with 500 µL of Tripsin at 37 ºC. Unattached cells of each
treated group were collected in Eppendorf tubes and counted by bright field
microscopy in a Neubauer chamber. The Eppendorf tubes were centrifuged at
10000 rpm for 20 min and the supernatant discarded carefully. 300 µL of 37%
HCl was added to the cell pellet and the resultant suspension was sonicated for
46
30 minutes at 40 ºC. Finally, 2700 mL of bi-distilled water were added. The Fe
concentration was determined by measuring the sample using an ICP-MS,
NexION 300XX (Perkin Elmer) (n = 4).
2.11 Functionalized iron nanowires internalization and drug release monitoring
by confocal reflection microscopy
The internalization of the different Fe NWs formulations was assessed using
confocal reflection microscopy. Two 4-well plates were added with cover slips,
seeded with 25000 cells/well and incubated for 24 h to reach confluence in the
mentioned conditions. Cells were treated with the NWs formulations (28 µg
Fe/mL and 0.73 µM of DOX for BSA-NWs-DOX, 26 µg Fe/mL and 1.3 µM of
DOX for APTES-NWs-DOX) and incubated for 24 h and 72 h at 37 ºC, 5% CO2.
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 5min 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 min 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.
47
Intracellular focal plane independent images as well as intracellular z-stacks
were taken from samples incubated 24 and 72 h with NWs formulations.
2.12 In vitro cell viability assay in the presence of functionalized iron nanowires
with/without low frequency alternating magnetic field
To assess cell death induction, cells were cultured on a 4-well plate at a density
of 2.5 x 104 cells per well in 500 µl of DMEM containing 10% FBS. After 24 h, the
growth medium was removed and the cells were incubated for 24 h at 37 ºC in
the presence of different concentrations of free DOX (0.5 and 1 µM), APTES-NWs
(8.7 and 26 µg Fe/mL), APTES-NWs-DOX (8.7 µg Fe/mL, 0.44 µM of DOX and
26 µg Fe/mL 1.3 µM of DOX), BSA-NWs (9.3 and 28 µg Fe/mL) and BSA-NWs-
DOX (9.3 µg Fe/mL, 0.25 µM of DOX and 28 µg Fe/mL, 0.73 µM of DOX). The
NWs’ concentration tested in this experiment was found by ICP-MS from the
stock NWs’ formulations, as previously described. As controls, non-treated cells
and empty wells were used. After incubation, cells were washed three times with
PBS and then maintained in 0.5 mL of DMEM containing 10% FBS at 37ºC and
5% CO2 incubator. Then, the magnetic field was applied for 10 minutes
maintaining a temperature of 37°C. A homemade equipment described before
was used for the generation of an electromagnetic field with a strength
(amplitude) of 1 mT and a frequency (ν) of 10 Hz. After 72 h of postincubation,
the medium was replaced with of DMEM containing 10% FBS and 10% of
Resazurin dye (1 mg/ml PBS). Cells were maintained at 37ºC and 5% CO2
48
incubator for 6 h and then, a Synergy H4 microplate reader was used to
determine the amount of Resazurin by measuring the absorbance of the reaction
mixture (excitation 540 nm, emission 590 nm). 600 µl of 10% of Resazurin dye
was added to empty wells as a negative control. The viability of the cells was
expressed as the percentage of absorption of treated cells in comparison with
control cells (without NWs). All experiments were carried out in two sets of
quadruplicates, one set with the AMF and one without it.
2.13 Statistical Analysis
All the data obtained from the in vitro cell viability assay were plotted and
statistically analyzed using the software package GraphPad Prism version 5.0 for
Windows. All samples were compared using a one-way ANOVA and Bonferroni
post-hoc test (*P < 0.05, **P < 0.01, and ***P < 0.001). Only significant differences
among the samples are indicated in the charts.
49
Chapter 3
Results
3.1 Iron nanowires characterization
Fe NWs were fabricated by electrodeposition into alumina membranes produced
by a two-step anodization process as explained in Chapter 2. NWs were released
from the alumina and their morphology (Figure 12A and 12B) and chemical
composition (Figure 12C and 12D) were analyzed showing a core of solid Fe
surrounded by an iron oxide (Fe2O3) interphase. The Fe NWs presented an
average length of 6.4+/-1.3 µm and a diameter of 30 to 40 nm (Figure 12A and
12B).
50
Figure 13. Characterization of Fe NWs. A. SEM image of Fe NWs on top of a silicon wafer
substrate, the inset corresponds to the NWs length distribution. Scale bar = 10 µm. B. TEM image of a single Fe NW. Scale bar = 50 nm. C. Scanning TEM image of a single Fe NW indicating its Fe2O3 surrounding layer (1) and core (2). Scale bar = 50 nm. D. Point EDX spectra showing the
composition analysis of fabricated Fe NWs at its surrounding layer (1) and core (2).
3.2 Coating of iron nanowires
The Fe NWs were functionalized with three different biocompatible coatings
(APTES, BSA and APTES-PEG) to improve their stability under physiological
conditions (Figure 11 in Chapter 2).
APTES and BSA were covalently attached to the NWs by the reaction with
the iron oxide interphase (Fig. 11A1 and 11A2). Both coating agents were added
51
in excess to the Fe NWs samples as described in Chapter 2. A Bradford assay of
the supernatant after coating process indicated an immobilization of 410 µg
BSA/mg de Fe. The presence of the BSA coating on the NWs’ surface was also
verified by TEM imaging that showed a homogenous layer of protein around the
NW that is absent in non-coated NWs (Figure 13A and 13B).
These two coating reagents provide the NW with free primary amino
groups that were used for the introduction of free thiol groups by the addition of
2-IT generating thiol moieties (Figure 11B). This process was evaluated by the
quantification of the 2-pyridinethione released from the reaction between the free
thiol groups and alditriol, added in excess, as exemplified in Figure 11C
The addition of alditriol also serves as an activation step, in which
disulfide bonds onto the APTES-NWs were introduced. The functionalization of
APTES-NWs with PEG was achieved by the formation of disulfide bonds
between the reactive thiol of the thiol-PEG and the activated sulfhydryl groups
of the modified APTES-NWs (Figure 11C). The process led to NWs bearing
approximately 50 µmol of PEG /g Fe.
The evaluation of the thiol moeites showed that ~65 μmol of 2-
pyridinethione/g Fe were immobilized in APTES-NWs and 32 μmol of 2-
pyridinethione/g Fe for BSA-NWs, showing an efficient activation for further
functionalization.
52
Figure 14. TEM micrographs of APTES-NWs (A) and BSA-NWs (B). The scale bars correspond to 50 nm.
3.3 Colloidal stability and internalization of coated iron nanowires by Prussian
blue staining assay
In order to determine which coating improved the NWs colloidal stability and
the cellular internalization in MDA-MB-231 breast cancer cells, a Prussian blue
staining assay for iron oxide detection was performed. To this end, MDA-MB-231
breast cancer cells were incubated with the three different coated Fe NWs at 0.05
mg Fe/mL for 24 h at 37ºC, followed by several washes with PBS and slides
where prepared for bright field imaging as explained in Chapter 2 (Figure 14).
Furthermore, by the use of two types of cyanide iron salts, we were able to
determine the oxidation state of the Fe contained in the iron oxide interphase
(Figure 14E).
53
Figure 15. Internalization and distribution of Fe NWs coated with different agents into MDA-MB-231 cells 24 h post incubation using Prussian blue staining for iron oxide detection. (A) Fe NWs without coating. (B) APTES-NWs. (C) BSA-NWs. (D) APTES-PEG-NWs. Samples A to D where
added with potassium ferrocyanide (Fe(CN)64-; containing Fe2+). (E) Fe NWs without coating added with potassium ferricyanide (Fe(CN)63-, containing Fe3+). Scale bar = 10 µm.
54
From images in Figures 14A, 14B, 14C and 14D, blue stained NWs are
present in all the samples treated with potassium ferrocyanide (Fe(CN)64-) while
the sample treated with potassium ferricyanide (Fe(CN)63-) remain the same or
showed an slightly brown tonality (Figure 4E). In this manner, it was assessed
that Fe3+ is contained in the oxide interphase surrounding the NWs. Explained
more in detail, in the one hand, the Fe(CN)64- solution contains Fe2+ and will
react with Fe3+when present in the NWs outside layer producing a dark blue
precipitate called Prussian Blue as observed in Figures 14A, 14B, 14C and 14D
and is represented by the next reaction.
K+(aq) + Fe3+(aq) + [Fe(CN)6]4-(aq) <==> KFe[Fe(CN)6](s)
Contrastingly, when Fe2+ is present in the outside layer this reaction won’t
occur presenting no appreciable change or slightly brown coloration in the
coloration of the NWs.
On the other hand, Fe(CN)63- containing Fe3+ will give a dark blue
coloration if Fe2+ is present and although this precipitate is known as Turnbull's
blue, it is identical with Prussian blue which is represented in the next reaction.
K+(aq) + Fe2+(aq) + [Fe(CN)6]3-(aq) <==> KFe[Fe(CN)6](s)
In this case, if Fe3+ is present in the iron oxide interphase of the nanowires
an slightly brown coloration or no change is observed as obtained in Figure 14E.
55
In this manner the presence of Fe3+ in the outside layer or interphase is
assessed by two different chemical reactions.
Interestingly, a difference in the morphology, size and distribution of NW
agglomerates can be observed between the different coatings (Figures 14B, 14C
and 14D), when compared to the non-coated NWs (Figure 14A). Thereby, the
three coating agents reduced the size of the agglomerates and increased the
homogeneous distribution of the NWs across the sample. The effect is more
pronounced for APTES-NWs and BSA-NWs (Figure 14B and 14C) than for
APTES-PEG-NWs (Figure 14D). Regarding the internalization, we found BSA
and APTES coating agents increased the internalization of the NWs, while
APTES-PEG-NWs seemed to remain outside the cells attached to the plasmatic
membrane.
3.4 Functionalization of iron nanowires
Based on the previous results, BSA-NWs and APTES-NWs were selected
for further functionalization with DOX to evaluate their potential on cancer cell
death induction by combining the chemotherapeutic effect with the magneto-
mechanical one. The coated Fe NWs were functionalized with DOX through a
pH-sensitive covalent bond to permit the selective drug release after
internalization of the NWs. As stated in Chapter 1, covalent bonds sensible to
acid hydrolysis are a suitable and efficient option for attaching drugs to magnetic
56
nanoparticles due to the pH difference existing between the extra (pH 7.4) and
intracellular compartments (pH 5.5–6.4 in endosomes).3,8,77,82 The
functionalization was achieved first introducing free thiol groups on the coated
NWs by the reaction between 2-IT and the amine groups of the coated NWs as
previously explained. Then, DOX was immobilized on the activated NWs by the
reaction of the free thiols and the maleimide group of the DOX derivative (Figure
11D) and the success of the functionalization was evaluated
spectrophotometrically. In this manner, 65% of immobilization was quantified
for APTES-NWs, which is approximately 50 μmol DOX/g Fe and 32% for BSA-
NWs that counts for ~25μmol DOXO/g Fe. It was noted that almost double the
amount of DOX was immobilized in APTES-NWs.
3.5 Drug-release quantification
Before performing in vitro cytotoxicity experiments, I studied the release of DOX
from the functionalized NWs through the pH-sensitive covalent bond that allows
the selective release of the unmodified drug in intracellular conditions. DOX was
released from the two groups of DOX functionalized NWs after transfer them
from PBS pH 7.4 to acetate buffer pH 5.0 (Figure 15) in a time dependent manner.
Fluorescence spectrophotometric measurements showed that 80% of DOX was
released from APTES-NWs-DOX and BSA-NWs-DOX after 4 h, and reached a
total release after 10 hours as shown in the release kinetics (Figure 15). In
contrast, no appreciable release was observed in the control experiments where
57
the functionalized NWs remained in PBS solution, which confirms the stability of
the nanoformulations.
Figure 16. Release kinetics of DOX from APTES-NWs-DOX and BSA-NWs-DOX with (pH 7.4, empty circles, crosses and solid line; pH 5.0, black squares, empty triangles and dashed line).
Both formulations of functionalized NWs release 80% of the drug at 4h and present a complete release after 8 h of incubation in acetate buffer pH 5. Functionalized NWs in PBS solution pH 7.4
were used as control.
58
3.6 Inductively coupled plasma mass spectrometry for iron nanowires
quantification
The concentration of Fe was quantified directly from aliquots of each one of the
NW-formulation stock solutions by ICP-MS as previously explained in Chapter
2. The values obtained for each stock solution are presented in Table 2 which also
includes the calculated concentrations derived from the values presented in
Table 1 contained in Chapter 2. From the ICP values obtained and the
immobilization percentage, the concentration of DOX can be recalculated.
Table 2. Correlation between the calculated and quantified concentration of Fe in the different NW-formulation stock solutions.
NW-stock solution
DOX Fe concentration
calculated (µg Fe/mL)
Fe concentration ICP values
(µg Fe/mL) DOX (µM ) adjusted
DOX_1 0.5 0.5 DOX_2 1 1
NW-APTES 2
32.5 8.7 NW-APTES 3
97.5 26
NW-APTES-D2
1 32.5 8.7 0.44
NW-APTES-D3
3 97.5 26 1.3
NW-BSA 2 45.7 9.3 NW-BSA 3 138.6 28
NW-BSA-D2 1 45.7 9.3 0.25 NW-BSA-D3 3 138.6 28 0.73
In order to quantify the amount of Fe NWs that interact with the seeded
breast cancer cells, ICP-MS measurements were also carried out on cells
59
incubated with NWs for 24h, washed several times and lysed. The results
showed no significant difference in the total amount of Fe from the cells
incubated with APTES-NWs (31±5 pg Fe/cell) and BSA-NWs (26±3 pg Fe/cell).
These results represent a total internalization of 19% and 15% for APTES-NWs
and BSA-NWs when compared with the values obtained from the stock
solutions.
3.7 Functionalized iron nanowires internalization and drug release monitoring
by Confocal Reflection Microscopy
The NWs internalization and the selective intracellular drug release in breast
cancer cells were evaluated through confocal microscopy studies. Images of
intracellular focal planes were acquired after 24 and 72 h of incubation with
APTES-NWs-DOX (Figures 16A and 16B) and BSA-NWs-DOX (Figures 16C and
16D). The red fluorescence of DOX permits its intracellular imaging. From
Figures 16B-I and 16D-I, the emergence of red fluorescence derived from the
presence of DOX after 72h (Figure 16B and 16D) is notorious when compared
with 24h (Figs. 16A-I and 16C-1). At 72h the DOX fluorescence signal overlaps
with the nucleus stained with DAPI (panel III), as shown in Figures 16B and 16D.
In all the conditions, functionalized NWs were observed as white structures, due
to their capability of reflecting light in the reflection mode used to acquire the
images (Figure 16, panels II).
60
Figure 17. Confocal reflection microscopy images from MDA-MB-231 cells incubated with APTES-NWs-DOX after 24 h (A) and after 72 h (B) and incubated with BSA-NWs-DOX after 24 h
(C) and after 72 h (D). Four different channels: I. Red fluorescence of DOX, II. Light reflected from the Fe NWs, III. DAPI nuclear staining, and IV. DIC. M. shows the merged image. Scale bars
= 10 µm.
61
Furthermore, intracellular Z-stacks were acquired to substantiate and
compare the uptake and internalization of both NW formulations (Fig.17A and
17B). In all the conditions NWs were observed at the same focal plane as the
nuclear staining, confirming their intracellular localization.
Figure 18. Z-stack of a MDA-MB-231 cell treated with (A) APTES-NWs-DOX and (B) BSA-NWs-DOX after 72 h of incubation. Scale bar is 10 µm.
62
A different distribution of the NWs clusters is observed for both
formulations. APTES-NWs-DOX (Figure 18A) were less distributed within the
field, when compared to BSA-NWs-DOX (Figure 18B) that showed a broad
distribution across the cell appearing in different focal planes of the Z-stack
(Figure 17B). Morphological and quantity differences are also appreciated in both
formulations. BSA-NWs-DOX agglomerated in less compact clusters and
presented a needle-like shape while more compact clusters with a round shape
were produced by APTES-NWs-DOX. This observation is perceived in greater
detail in Figure 18A and 18B that show broader field images of an intracellular
focal plane denoted by the presence of the nucleus. Additionally, a larger
amount of BSA-NWs-DOX is observed in Figure 18A, indicating a higher degree
of internalization when compared to APTES-NWs-DOX (Figure 18B).
Figure 19. Morphology and distribution of functionalized NWs in MDA-MB-231 cells. (A) Cells treated with APTES-NWs-DOX after 24 h of incubation. (B) Cells treated with BSA- NWs-DOX
after 24 h of incubation. Scale bars are 25 µm.
63
3.8 Bimodal strategy for cancer cell death induction with functionalized iron
nanowires
The ability of BSA-NWs-DOX and APTES-NWs-DOX to induce cancer cell death
by combining the selective drug release with the mechanical disturbance upon
the application of a low frequency AMF was examined on MDA-MB 231 cells by
Alamar Blue (in vitro cell viability) assay (Figure 19). For standardizing this test,
drug concentration was equalized for both groups of NW-formulation stock
solutions employing the selected concentrations (1 and 3 µM) of DOX, as shown
in Table 1 and Table 2. A correlation between the calculated and quantified
concentration of Fe (by ICP-MS) in the NW-formulation stock solutions is
represented in the Table 2 from which the DOX concentration value was
adjusted. It can be noted that due to the difference immobilization percentage of
both groups of formulations, BSA-NWs-DOX_1 and BSA-NWs-DOX_2 contain
approximately the half of DOX content of APTES-NWs-DOX_1 and APTES-
NWs-DOX_2.
A first important observation is that applying a low frequency AMF of 1
mT and 10 Hz to cells without NWs did not affect the cell viability. Similarly, the
cytotoxic effect produced by the free drug was not affected by the AMF at the
two DOX concentrations tested (0.5 and 1 µM) (Figure 19).
Fe NWs coated with APTES or BSA did not reduce the cell viability. On
the other hand, a significant cytotoxic effect emerged when the AMF was applied
64
to cells that were incubated with NWs. A decrease of ~14% and 23% in cell
viability was observed for APTES-NWs_1 (8.7 µg of Fe/mL) and APTES-NWs_2
(26 µg of Fe/mL). A higher cytotoxic effect, showing a decrease of ~21% and 28%
in cell viability, was generated by BSA-NWs_1 (9.3 µg of Fe/mL) and BSA-
NWs_2 (28 µg of Fe/mL) for similar amounts of Fe (Figure 9).
In the case of DOX-functionalized NWs, the cytotoxic effect due to the
DOX release showed a decrease of approximately 32% and 54% in cell viability
for APTES-NWs-DOX_1 (8.7 µg of Fe/mL, 0.44 µM DOX) and APTES-NWs-
DOX_2 (26 µg of Fe/mL, 1.3 µM DOX), respectively. For the BSA-coated NWs a
decrease of 31% and 58% in cell viability was determined for BSA-NWs_1-DOX
(9.3 µg of Fe/mL, 0.25 µM DOX) and BSA-NWs_2 (28 µg of Fe/mL, 0.73 µM
DOX), respectively (Figure 19).
Finally, an additive effect of the cytotoxicity was observed when an AMF
was applied to cells treated with both formulations of DOX-functionalized NWs.
An additional decrease of ~10% in cell viability was observed for APTES-NWs-
DOX_1 (8.7 µg of Fe/mL, 0.44 µM DOX) and APTES-NWs-DOX_2 (26 µg of
Fe/mL, 1.3 µM DOX), and an additional decrease of ~15% and 8% in cell
viability, was observed by BSA-NWs_1-DOX (9.3 µg of Fe/mL, 0.25 µM DOX)
and BSA-NWs-DOX_2 (28 µg of Fe/mL, 0.73 µM DOX). These experiments were
performed using similar amounts of Fe but less amounts of DOX in the case of
BSA-NWs-DOX. The synergic cytotoxic effect was statistically equal for both
65
formulations, leading to a final decrease in cell viability of ~45% and 69% for
APTES-NWs-DOX, and ~48% and 73% for BSA-NWs-DOX (Figure 19).
Figure 20. Viability of MDA-MB-231 cells incubated with different formulations and with or
without application of a low frequency AMF. APTES-NWs: 8.7 µg Fe/mL (APTES-NWs_1), 26 µg Fe/mL (APTES-NWs_2). APTES-NWs-DOX: 8.7 µg Fe/mL and 0.44 µM of DOX (APTES-NWs-
DOX_1), 26 µg Fe/mL and 1.3 µM of DOX (APTES-NWs-DOX_2). BSA-NWs: 9.3 µg Fe/mL (BSA-NWs_1), 28 µg Fe/mL (BSA-NWs_2). BSA-NWs-DOX: 9.3 µg Fe/mL and 0.25 µM of DOX (BSA-
NWs-DOX_1), 28 µg Fe/mL and 0.73 µM of DOX (BSA-NWs-DOX_2). Free DOX: 0.5 µM (DOX_1) and 1 µM (DOX_2). (*p < 0.05, **p < 0.01, and ***p < 0.001).
66
Chapter 4
Discussion and Conclusions
4.1 Discussion
Fe NWs were successfully coated with to different agents, APTES that is an
aminosile and BSA, a 583 aminoacid protein. Both agents were covalently
attached to the Fe2O3 interphase that surrounds the Fe0 core of the NWs and is
formed immediately after being released form the aluminum-oxide alumina
template and get in contact with O2 from the air. From the TEM image in figure
12B, the presence of this interphase can be distinguished, being a layer of ~8nm
observed at both sides of the Fe NWs which slightly differs from the dimensions
described by Song et al, 2009.34 Moreover, a TEM image of a nanowire coated
with BSA in Figure 13B showed an amorphous and non-uniform layer of
surrounding the Fe NW.
Both coatings provided Fe NWs with free amino groups, necessary for the
functionalization process that were further activated with 2-IT. This activation
process provided the NWs with thiol moieties that were necessary for the
attachment of the drug derivative.
The evaluation of the activation process showed that ~65 and ~32 μmol of
2-pyridinethione/g Fe were immobilized for APTES-NWs and BSA-NWs,
respectively. This number correlates with the amount of drug that can be
67
potentially immobilized. From these values, it was already noted that BSA-NWs
were provided with approximately half of the thiol moieties needed for the
functionalization of the anticancer drug. Furthermore, this difference in the
activation could be addressed to the fewer amount of free amino groups
available for activation provided on the BSA coating, probably related to the
bigger size of BSA, as a protein, and also due to the conformational changes that
it may suffer during the functionalization process where amino groups could be
hidden inside of the protein.
From the Prussian blue staining assay (Figure 14) and the point EDX
(Figure 12C and 12D), the composition of the interphase surrounding the Fe
NWs was assessed, demonstrating the presence of an Fe oxide layer in which
Fe3+ but not Fe2+ was observed, in contrast to the findings by Song et al. 2009 in
which they suggest an FeO layer.28 Furthermore, APTES-NWs and BSA-NWs
showed a higher degree of internalization when compared to non-coated NWs
while APTES-PEG-NWs appeared not to be internalized at all, as described by
Sharma et al, 2015.83 The colloidal properties of the NWs were improved by the
coating process due to the fact that smaller clusters of NWs were observed for
APTES-NWs, BSA-NWs and APTES-NWs-PEG together with a broader
distribution across the field when compared to the non-coated NWs. A higher
internalization level can be noted as an insight of the higher biocompatibility of
BSA over APTES (Figure 14).
68
Based on their better colloidal properties and outstanding
biocompatibility, BSA-NWs and APTES-NWs were selected for further
functionalization with DOX. Functionalized NWs are the product of the
attachment of the DOX derivative to the activated NW. The design and synthesis
of the DOX derivative is reported by Latorre et al, 2014 in which DOX is added to
a designed liker that allows its attachment to the activated NWs.8 The
immobilization of DOX was a successful process that directly represents the
values obtained from the thiol moieties quantification acquiring ~50 and ~25
μmol of DOX/g Fe for APTES-NWs-DOX and BSA-NWs-DOX, respectively,
which in turn represent the 65% and 32% of the drug derivative added. This
suggests a ratio 2:1 in the amount of drug attached in both formulations. The
design of the linker in the DOX derivative allows the drug release through the
acid hydrolysis of the amide bond, that directly attaches the drug to the linker
and permit the release of the DOX without any modification.8,54,55 Amide bonds
are sensible to acid hydrolysis and therefore, a decrease in the pH of the media
will induce the release of DOX without any modification in one step. In this
manner, DOX was released from the functionalized NWs when passed to an
acidic environment having a complete release after 8h for both formulations
similarly to what has been observed for MNP.8,54,55 No significant difference in
the release kinetics of both formulations was perceived in Figure 15.
69
From the Fe quantification in both of the Fe NWs stock solutions, the ICP-
MS output revealed a loss of NWs up to 62% and 71% for APTES-NWs and BSA-
NWs, respectively during the functionalization process. Additionally, no
significant difference in the quantified Fe from the seeded cells after 24h of
incubation with the two different formulations presenting 31±5 pg Fe/cell for
APTES-NWs and 26±3 pg Fe/cell for BSA-NWs. It is worth to mention that these
values refer not only to internalized NWs, but also to the NWs embedded or
stuck in the extracellular structures surrounding the plasmatic membrane.
The NWs internalization and the selective intracellular drug release in
breast cancer cells were assessed through confocal microscopy studies. The
fluorescent properties of DOX allowed its intracellular imaging for tracking its
movement inside of the breast cancer cells (Figure 16). After 24h of incubation no
signal from DOX was perceived for both formulations in contrast with the
intense signal observed in the images of cells after 72h of incubation with
APTES-NWs-DOX and BSA-NWs-DOX. These results indicate the efficient
release and nuclear localization of the chemotherapeutic agent. Z-stacks from
cells incubated 72 h with both formulations confirmed the internalization of both
formulations (Figure 17). Both formulations of NWs appeared as clusters located
mainly around the nucleus and were present in different focal planes of the Z-
stacks. A higher level of detail in the distribution and morphology of the NW
clusters across a broader field at the nuclear focal plane can be appreciated in
70
Figure 18. BSA-NWs-DOX were more distributed across the field and also
agglomerated in less compact clusters and presented a needle-like shape.
Moreover, a higher degree of internalization was observed for BSA-NWs-DOX
denoted by the larger amount of functionalized NWs observed at the nucleus
focal plane. Finally, the potential of BSA-NWs-DOX and APTES-NWs-DOX on
cancer cell death induction by combining the chemotherapeutic with the
magneto-mechanical effect was evaluated in MDA-MB-231 cells (Figure 19). Both
formulations of functionalized Fe NWs generated a comparable cytotoxic effect
in MDA-MB-231 cells that was significantly different from the effect produced by
the free DOXO and non-functionalized NWs formulations. For both of the
conditions stablished for the in-vitro cell viability assay, which are the presence
and absence of the low frequency AMF (1 mT and 10 Hz), non-treated cells were
used as a negative control were no change of the cell viability was observed at
the conditions tested. In comparison with the reported inhibitory concentration
50 (IC50) of 25 nM for DOX in MDA-MB-231 cells84, high drug concentrations (0.5
and 1 µM) were used to assess an appreciable decrease of the cell viability as free
drug, effect that was taken as a comparison point of cell viability decrease and
that was also not affected when the low frequency AMF was applied.
A very low affection of the cell viability was caused by Fe NWs coated
with APTES or BSA, although a slightly higher cytotoxic effect was produced by
BSA-NWs. Contrastingly, a significant cytotoxic effect emerged when the AMF
71
was applied to cells that were incubated with both formulations of coated NWs
that was higher in the cells incubated with BSA-NW at the two concentrations
tested.
Theoretically, higher amounts of Fe mass were added for BSA
formulations due to the lower immobilization percentage being the apparent
reason of this, nevertheless ICP-mass assays for Fe quantification substantiate
that practically the same amount of Fe was added for both NWs formulations
Thus, this slightly difference can be attributed to a higher internalization level,
broader distribution of the BSA-NWs insinuated from the Prussian blue images
and then corroborated in the confocal reflection images. In general, these results
confirm the high degree of biocompatibility of Fe NWs and the possibility to kill
cancer cells by magneto-mechanical stimulation. They also suggest that BSA is a
more efficient coating agent with respect to killing of cancer cells by this method.
It is important to mention again that the magneto-mechanical effect is carried out
by the active contribution of both, internalized and embedded NWs.
As described in Chapter 3, the cytotoxic effect due to the DOX release
from DOX-functionalized NWs showed a decrease of approximately 32% and
54% in cell viability for APTES-NWs-DOX_1 and APTES-NWs-DOX_2,
respectively. For the BSA-coated NWs a decrease of 31% and 58% in cell viability
was determined for BSA-NWs-DOX _1 and BSA-NWs-DOX_2, respectively.
These results show the efficacy of the selective intracellular drug release and the
72
cytotoxic effect of the DOX. Moreover, they imply that Fe NWs are highly
effective as drug carriers. Although the BSA-NWs carry less amount of DOX than
the APTES-NWs, the cell viability reduction is about the same. This indicates a
higher efficacy of the BSA-NWs, which might be related to a more efficient
internalization as found in the confocal microscopy study.
As a final point, an additive reduction of the cell viability was observed
when the AMF was applied to the cells treated with both formulations of DOX-
functionalized NWs.
An additional decrease of ~10% in cell viability was observed for APTES-
NWs-DOX_1 and APTES-NWs-DOX_2, and an additional decrease of ~15% and
8% in cell viability was observed by BSA-NWs_1-DOX and BSA-NWs-DOX_2.
As mentioned previously in Chapter 3, these experiments were performed
using similar amounts of Fe but less amounts of DOX in the case of BSA-NWs-
DOX and the synergic cytotoxic effect was statistically equal for both
formulations, leading to a final decrease in cell viability of ~45% and 69% for
APTES-NWs-DOX, and ~48% and 73% for BSA-NWs-DOX. Taking into account
that BSA-NWs-DOX had 1.8 times less amount of DOX/Fe a significant increase
in the effective cytotoxicity was detected for the BSA-NWs-DOX, which we
attribute to their higher stability and cellular internalization.
73
In this regard, confocal reflection images from cells treated with both NWs
formulations in Figure 18 clearly exhibit a higher degree of internalization for the
BSA-coated functionalized NWs reflected by the larger amount of NWs present.
This higher internalization level coupled to the broader distribution within the
cell and the morphological differences observed such as clustering in smaller and
less compact agglomerates of the BSA formulations supports the difference in the
cytotoxicity in both chemical and magneto-mechanical ways, separately and
when combined.
4.2 Conclusions
In this thesis, a novel method for bimodal cancer cell destruction was developed
by combining the intrinsic magneto-mechanical properties of Fe NWs coupled to
the chemotherapeutic effect performed by an anticancer drug attached to the
NWs. The Fe NWs and the two different coating agents employed, APTES and
BSA, demonstrated to be highly biocompatible. The coatings also proved to be
efficient for the further pH sensitive functionalization of NWs with a
chemotherapeutic agent. The NWs were readily internalized and turned out to
be very efficient carriers for drug delivery. Due to their magnetic properties, such
remotely controllable drug carriers could have advantages over other methods.
The BSA-NWs formulations displayed a higher internalization degree and a
broader distribution within the cells in addition to bunch in smaller and less
compact clusters, making it a more efficient candidate for the induction of cell
74
death. Furthermore, the functionalized NWs generated a large cytotoxic effect in
MDA-MB-231 cells. The combination of the chemotoxic and magneto-mechanical
treatment modes was found to have synergistic effects, making the proposed
method an attractive option for new cancer therapies. Compared to
hyperthermia-based methods, the magneto-mechanical treatment mode requires
only low field strengths and low frequencies. This is not only relevant from a
safety point of view, but it also reduces the power consumption by several orders
of magnitude, making it very efficient in terms of costs and technical
implementation.
75
REFERENCES
1 Torre, L. A. et al. Global Cancer Statistics, 2012. Ca-a Cancer Journal for
Clinicians 65, 87-108, (2015).
2 Thakor, A. S. & Gambhir, S. S. Nanooncology: The Future of Cancer
Diagnosis and Therapy. Ca-a Cancer Journal for Clinicians 63, 395-418,
(2013).
3 Banerjee, S. S. & Chen, D. H. Multifunctional pH-sensitive magnetic
nanoparticles for simultaneous imaging, sensing and targeted intracellular
anticancer drug delivery. Nanotechnology 19, 8, (2008).
4 Faraji, A. H. & Wipf, P. Nanoparticles in cellular drug delivery. Bioorganic
& Medicinal Chemistry 17, 2950-2962, (2009).
5 Swai, H. et al. Nanomedicine for respiratory diseases. Wiley
Interdisciplinary Reviews-Nanomedicine and Nanobiotechnology 1, 255-263,
(2009).
6 Bhabra, G. et al. Nanoparticles can cause DNA damage across a cellular
barrier. Nature Nanotechnology 4, 876-883, (2009).
7 Sajja, H. K. et al. Development of Multifunctional Nanoparticles for
Targeted Drug Delivery and Non-invasive Imaging of Therapeutic Effect.
Current drug discovery technologies 6, 43-51, (2009).
8 Latorre, A., Couleaud, P., Aires, A., Cortajarena, A. L. & Somoza, A.
Multifunctionalization of magnetic nanoparticles for controlled drug
76
release: A general approach. European Journal of Medicinal Chemistry 82,
355-362, (2014).
9 Kim, J. E., Shin, J. Y. & Cho, M. H. Magnetic nanoparticles: an update of
application for drug delivery and possible toxic effects. Archives of
Toxicology 86, 685-700, (2012).
10 Wang, L. A., Neoh, K. G., Kang, E. T. & Shuter, B. Multifunctional
polyglycerol-grafted Fe3O4@SiO2 nanoparticles for targeting ovarian
cancer cells. Biomaterials 32, 2166-2173, (2011).
11 Gooneratne, C. P., Yassine, O., Giouroudi, I. & Kosel, J. Selective
Manipulation of Superparamagnetic Beads by a Magnetic Microchip. IEEE
Transactions on Magnetics 49, 3418-3421, (2013).
12 Gooneratne, C. P., Giouroudi, I., Liang, C. & Kosel, J. A giant
magnetoresistance ring-sensor based microsystem for magnetic bead
manipulation and detection. Journal of Applied Physics 109, 3, (2011).
13 Li, F. & Kosel, J. An efficient biosensor made of an electromagnetic trap
and a magneto-resistive sensor. Biosensors & Bioelectronics 59, 145-150,
(2014).
14 Li, F. & Kosel, J. A Magnetic Method to Concentrate and Trap Biological
Targets. IEEE Transactions on Magnetics 48, 2854-2856, (2012).
77
15 Wang, D. S., He, J. B., Rosenzweig, N. & Rosenzweig, Z.
Superparamagnetic Fe2O3 Beads-CdSe/ZnS quantum dots core-shell
nanocomposite particles for cell separation. Nano Letters 4, 409-413, (2004).
16 Gao, N., Wang, H. J. & Yang, E. H. An experimental study on
ferromagnetic nickel nanowires functionalized with antibodies for cell
separation. Nanotechnology 21, 8, (2010).
17 Choi, D. et al. Transport of living cells with magnetically assembled
nanowires. Biomedical Microdevices 9, 143-148, (2007).
18 Wei-Syuan, L., Hong-Ming, L., Hsiang-Hsin, C., Yeu-Kuang, H. & Yuh-
Jing, C. Shape Effects of Iron Nanowires on Hyperthermia Treatment.
Journal of Nanomaterials 2013, (2013).
19 Keshoju, K., Xing, H. & Sun, L. Magnetic field driven nanowire rotation in
suspension. Applied Physics Letters 91, 3, (2007).
20 Gunther, A., Bender, P., Tschope, A. & Birringer, R. Rotational diffusion of
magnetic nickel nanorods in colloidal dispersions. Journal of Physics-
Condensed Matter 23, 14, (2011).
21 Reich, D. H. et al. Biological applications of multifunctional magnetic
nanowires (invited). Journal of Applied Physics 93, 7275-7280, (2003).
22 Malak, S. et al. Interactions between magnetic nanowires and living cells:
Uptake, toxicity and degradation. Abstracts of Papers of the American
Chemical Society 243, 1, (2012).
78
23 Hultgren, A., Tanase, M., Chen, C. S., Meyer, G. J. & Reich, D. H. Cell
manipulation using magnetic nanowires. Journal of Applied Physics 93,
7554-7556, (2003).
24 Hultgren, A. et al. Optimization of yield in magnetic cell separations using
nickel nanowires of different lengths. Biotechnology Progress 21, 509-515,
(2005).
25 Contreras, M. F., Sougrat, R., Zaher, A., Ravasi, T. & Kosel, J. Non-
chemotoxic induction of cancer cell death using magnetic nanowires.
International Journal of Nanomedicine 10, 2141-2153, (2015).
26 Hultgren, A., Tanase, M., Chen, C. S. & Reich, D. H. High-yield cell
separations using magnetic nanowires. IEEE Transactions on Magnetics 40,
2988-2990, (2004).
27 Ning, G. et al. Antibody-functionalized magnetic nanowires for cell
purification. Proceedings of the SPIE - The International Society for Optical
Engineering 7318, (2009).
28 Yi, Z. & Hansong, Z. Rotational maneuver of ferromagnetic nanowires for
cell manipulation. IEEE Transactions on Nanobioscience 8, 226-236, (2009).
29 Li, Z., Petit, T., Peyer, K. E. & Nelson, B. J. Targeted cargo delivery using a
rotating nickel nanowire. Nanomedicine: Nanotechnology, Biology and
Medicine 8, 1074-1080, (2012).
79
30 Choi, D. S. et al. Hyperthermia with magnetic nanowires for inactivating
living cells. Journal of Nanoscience and Nanotechnology 8, 2323-2327, (2008).
31 Choi, D. S. et al. Magnetically driven spinning nanowires as effective
materials for eradicating living cells. Journal of Applied Physics 111, 3,
(2012).
32 Kasprzak, K. S., Sunderman, F. W. & Salnikow, K. Nickel carcinogenesis.
Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis
533, 67-97, (2003).
33 Wang, C. B. & Zhang, W. X. Synthesizing nanoscale iron particles for
rapid and complete dechlorination of TCE and PCBs. Environmental
Science & Technology 31, 2154-2156, (1997).
34 Meng-Meng, S. et al. Cytotoxicity and cellular uptake of iron nanowires.
Biomaterials 31, 1509-1517, (2010).
35 Perez, J. E., Contreras, M. F., Vilanova, E., Ravasi, T. & Kosel, J.
Cytotoxicity and Effects on Cell Viability of Nickel Nanowires.
International Conference on Biological, Medical and Chemical Engineering
(BMCE), 178-184, (2013).
36 Ferre, R., Ounadjela, K., George, J. M., Piraux, L. & Dubois, S.
Magnetization processes in nickel and cobalt electrodeposited nanowires.
Physical Review B 56, 14066-14075, (1997).
80
37 Marcos-Campos, I. et al. Cell death induced by the application of
alternating magnetic fields to nanoparticle-loaded dendritic cells.
Nanotechnology 22, 13, (2011).
38 Conner, S. D. & Schmid, S. L. Regulated portals of entry into the cell.
Nature 421, 37-44, (2003).
39 Dupuy, A. G. & Caron, E. Integrin-dependent phagocytosis - spreading
from microadhesion to new concepts. Journal of Cell Science 121, 1773-1783,
(2008).
40 Perez, J. E. et al. Cytotoxicity and intracellular dissolution of nickel
nanowires. Nanotoxicology, 1-10, (2015).
41 Zhang, L. W. & Monteiro-Riviere, N. A. Mechanisms of Quantum Dot
Nanoparticle Cellular Uptake. Toxicological Sciences 110, 138-155, (2009).
42 Wang, C. M. et al. Void formation during early stages of passivation:
Initial oxidation of iron nanoparticles at room temperature. Journal of
Applied Physics 98, 7, (2005).
43 Pisanic, T. R., Blackwell, J. D., Shubayev, V. I., Finones, R. R. & Jin, S.
Nanotoxicity of iron oxide nanoparticle internalization in growing
neurons. Biomaterials 28, 2572-2581, (2007).
44 Gao, J. H., Gu, H. W. & Xu, B. Multifunctional Magnetic Nanoparticles:
Design, Synthesis, and Biomedical Applications. Accounts of Chemical
Research 42, 1097-1107, (2009).
81
45 McCarthy, J. R. & Weissleder, R. Multifunctional magnetic nanoparticles
for targeted imaging and therapy. Advanced Drug Delivery Reviews 60,
1241-1251, (2008).
46 Herve, K. et al. The development of stable aqueous suspensions of
PEGylated SPIONs for biomedical applications. Nanotechnology 19, 7,
(2008).
47 Park, J. S., Kim, J. M., Kim, H. Y., Yi, S. & Perepezko, J. H. Oxidation
resistance coatings of Mo−Si−B alloys via a pack cementation process.
Met. Mater. Int. 14, 1-7, (2008).
48 Salas, G. et al. Controlled synthesis of uniform magnetite nanocrystals
with high-quality properties for biomedical applications. Journal of
Materials Chemistry 22, 21065-21075, (2012).
49 Kang, B. S. et al. Electrical detection of immobilized proteins with ungated
AlGaN/GaN high-electron-mobility transistors. Applied Physics Letters 87,
3, (2005).
50 Baur, B. et al. Chemical functionalization of GaN and AlN surfaces. Applied
Physics Letters 87, 3, (2005).
51 Arranz, A. et al. Influence of surface hydroxylation on 3-
aminopropyltriethoxysilane growth mode during chemical
functionalization of GaN surfaces: An angle-resolved X-ray photoelectron
spectroscopy study. Langmuir 24, 8667-8671, (2008).
82
52 Williams, E. H. et al. Solution-based functionalization of gallium nitride
nanowires for protein sensor development. Surface Science 627, 23-28,
(2014).
53 Elzoghby, A. O., Samy, W. M. & Elgindy, N. A. Albumin-based
nanoparticles as potential controlled release drug delivery systems. Journal
of Controlled Release 157, 168-182, (2012).
54 Aires, A. et al. BSA-coated magnetic nanoparticles for improved
therapeutic properties. Journal of Materials Chemistry B 3, 6239-6247, (2015).
55 Kossatz, S. et al. Efficient treatment of breast cancer xenografts with
multifunctionalized iron oxide nanoparticles combining magnetic
hyperthermia and anti-cancer drug delivery. Breast Cancer Research 17,
(2015).
56 Chorny, M. et al. Targeting stents with local delivery of paclitaxel-loaded
magnetic nanoparticles using uniform fields. Proceedings of the National
Academy of Sciences of the United States of America 107, 8346-8351, (2010).
57 Mu-Yi, H. et al. Magnetic-nanoparticle-modified paclitaxel for targeted
therapy for prostate cancer. Biomaterials 31, 7355-7363, (2010).
58 Kohler, N. et al. Methotrexate-immobilized poly(ethylene glycol) magnetic
nanoparticles for MR imaging and drug delivery. Small 2, 785-792, (2006).
59 Pouponneau, P., Leroux, J. C., Soulez, G., Gaboury, L. & Martel, S. Co-
encapsulation of magnetic nanoparticles and doxorubicin into
83
biodegradable microcarriers for deep tissue targeting by vascular MRI
navigation. Biomaterials 32, 3481-3486, (2011).
60 Arcamone, F. et al. Adriamycin, 14-hydroxydaunomycin, a new antitumor
antibiotic from S. peucetius var. caesius (Reprinted from Biotechnology
and Bioengineering, vol 11, pg 1101-1110, 1969). Biotechnology and
Bioengineering 67, 704-713, (2000).
61 Cortes-Funes, H. & Coronado, C. Role of anthracyclines in the era of
targeted therapy. Cardiovascular Toxicology 7, 56-60, (2007).
62 Gewirtz, D. A. A critical evaluation of the mechanisms of action proposed
for the antitumor effects of the anthracycline antibiotics Adriamycin and
daunorubicin. Biochemical Pharmacology 57, 727-741, (1999).
63 Oliveira, H. et al. Magnetic field triggered drug release from
polymersomes for cancer therapeutics. Abstracts of Papers of the American
Chemical Society 246, 1, (2013).
64 Anirudhan, T. S. & Sandeep, S. Synthesis, characterization, cellular uptake
and cytotoxicity of a multifunctional magnetic nanocomposite for the
targeted delivery and controlled release of doxorubicin to cancer cells.
Journal of Materials Chemistry 22, 12888-12899, (2012).
65 Kaaki, K. et al. Magnetic Nanocarriers of Doxorubicin Coated with
Poly(ethylene glycol) and Folic Acid: Relation between Coating Structure,
84
Surface Properties, Colloidal Stability, and Cancer Cell Targeting.
Langmuir 28, 1496-1505, (2012).
66 Li, L. et al. Mild hyperthermia triggered doxorubicin release from
optimized stealth thermosensitive liposomes improves intratumoral drug
delivery and efficacy. Journal of Controlled Release 168, 142-150, (2013).
67 Yarmolenko, P. S. et al. Comparative effects of thermosensitive
doxorubicin-containing liposomes and hyperthermia in human and
murine tumours. International Journal of Hyperthermia 26, 485-498, (2010).
68 Thorn, C. F. et al. Doxorubicin pathways: pharmacodynamics and adverse
effects. Pharmacogenetics and Genomics 21, 440-446, (2011).
69 Brzozowska, M. & Krysinski, P. Synthesis and functionalization of
magnetic nanoparticles with covalently bound electroactive compound
doxorubicin. Electrochimica Acta 54, 5065-5070, (2009).
70 Fang, C. et al. Fabrication of magnetic nanoparticles with controllable drug
loading and release through a simple assembly approach. Journal of
Controlled Release 162, 233-241, (2012).
71 Wang, H. W. et al. Magnetic-Fe/Fe3O4-nanoparticle-bound SN38 as
carboxylesterase-cleavable prodrug for the delivery to tumors within
monocytes/macrophages. Beilstein Journal of Nanotechnology 3, 444-455,
(2012).
85
72 Cengelli, F. et al. Surface-Functionalized Ultrasmall Superparamagnetic
Nanoparticles as Magnetic Delivery Vectors for Camptothecin.
Chemmedchem 4, 988-997, (2009).
73 Lee, G. Y. et al. Theranostic Nanoparticles with Controlled Release of
Gemcitabine for Targeted Therapy and MRI of Pancreatic Cancer. Acs
Nano 7, 2078-2089, (2013).
74 Chiper, M. et al. Colloidal stability and thermo-responsive properties of
iron oxide nanoparticles coated with polymers: advantages of Pluronic (R)
F68-PEG mixture. Nanotechnology 24, 11, (2013).
75 Dionigi, C. et al. Regulating the thermal response of PNIPAM hydrogels
by controlling the adsorption of magnetite nanoparticles. Applied Physics a-
Materials Science & Processing 114, 585-590, (2014).
76 Miao, G. et al. Multilayer nanoparticles with a magnetite core and a
polycation inner shell as pH-responsive carriers for drug delivery.
Nanoscale 2, 434-441, (2010).
77 Pack, D. W., Hoffman, A. S., Pun, S. & Stayton, P. S. Design and
development of polymers for gene delivery. Nature Reviews Drug Discovery
4, 581-593, (2005).
78 Willner, D. et al. (6-maleimidocaproyl)hydrazone of doxorubicin - A new
derivative for the preparation of immunoconjugates of doxorubicin.
Bioconjugate Chemistry 4, 521-527, (1993).
86
79 Masuda, H. & Fukuda, K. Ordered metal nanohole arrays made by a 2-
step replication of honeycomb structures of anodic alumina. Science 268,
1466-1468, (1995).
80 Nielsch, K., Muller, F., Li, A. P. & Gosele, U. Uniform nickel deposition
into ordered alumina pores by pulsed electrodeposition. Advanced
Materials 12, 582-586, (2000).
81 Pirota, K. R., Navas, D., Hernandez-Velez, M., Nielsch, K. & Vazquez, M.
Novel magnetic materials prepared by electrodeposition techniques:
arrays of nanowires and multi-layered microwires. Journal of Alloys and
Compounds 369, 18-26, (2004).
82 Guo, M. et al. Multilayer nanoparticles with a magnetite core and a
polycation inner shell as pH-responsive carriers for drug delivery.
Nanoscale 2, 434-411, (2010).
83 Sharma, A. et al. Inducing cells to disperse nickel nanowires via integrin-
mediated responses. Nanotechnology 26, 12, (2015).
84 Smith, L. et al. The analysis of doxorubicin resistance in human breast
cancer cells using antibody microarrays. Molecular Cancer Therapeutics 5,
2115-2120, (2006).
87
PUBLICATIONS
From this master thesis project the publication cited bellow was generated.
Martínez-Banderas, A.I., Aires, A., Perez, J.E., Alsharif, N., Teran, FJ., Cadenas
J.F., Kosel, J. & Cortajarena, A.L. Functionalized magnetic nanowires for
chemical and magneto-mechanical induction of cancer cell death. Submitted to
Scientific Reports, (2016).