Doxorubicin-Conjugated Iron Oxide Nanoparticles Synthesized by Laser Pyrolysis: In Vitro Study on Human Breast Cancer Cells

Even today, breast cancer remains a global public problem, with a high mortality rate among women. Nanoparticle (NP) based systems are developed to enhance drug delivery, reducing the toxic effect of medicine molecules. By using iron oxide nanoparticles for cancer treatment, several advantages were highlighted: the ability to target specific locations derived from their magnetic properties and reduced side effects. The aim of this study was to examine on breast cancer cell line the anticancer potential of γ-Fe2O3 NPs loaded with doxorubicin (DOX) and stabilized with carboxymethylcellulose sodium (CMCNa). The γ-Fe2O3 NPs were synthesized by laser pyrolysis technique and their nanometric size and crystallinity were confirmed by X-ray diffraction and transmission electron microscopy. The loading efficiency was estimated by using absorption and fluorescence spectroscopy. The DOX conjugated//CMCNa coated γ-Fe2O3 NPs proved through the biological studies to have a good anticancer effect through the inhibition of tumoral cell proliferation, disruption of the cellular membrane, induction of cell death and reduced effects on normal breast cells. Our data showed that DOX cytotoxicity increases significantly when conjugated with ɣ-Fe2O3 and ɣ-Fe2O3_CMCNa, a 50% reduction of cancer cell viability was obtained with a concentration around 0.1 µg/mL.


Introduction
Breast cancer (BC) is the most common type of cancer in the world among women and it remains a challenge even today. For the next years, statistics show that the diagnosis will come out positive for 1 in 8 women, and it was demonstrated that with age, the incidence of BC increases, but after menopause, the frequency is lower [1,2]. In 2018, the World Health Organization estimated that 627,000 women died of breast cancer [3].
under the action of DOX-conjugated polymer-coated γ-Fe 2 O 3 NPs by 10-20% compared to free Dox. More recently, a significantly decrease of viability of human glioblastoma cells (GaMG line) was reported in the presence of magnetic γ-Fe 2 O 3 NPs conjugated with DOX in low concentration (10 nM) [29]. In contrast, in A549 lung cancer cells, the PEG-functionalized γ-Fe 2 O 3 NPs conjugated with DOX induced very high viability, possible due to the limited release of DOX. However, under alternating magnetic field (AMF), NPs exhibited excellent thermal effects thus favoring the release of DOX from γ-Fe 2 O 3 NPs and destruction of lung cancer cells [30].
The main purpose of this study is to demonstrate the efficiency of γ-Fe 2 O 3 loaded with DOX in breast cancer therapy. The IONPs were synthesized by laser pyrolysis technique using iron pentacarbonyl (Fe(CO) 5 ) vapors and synthetic air as iron and oxygen sources, respectively. NPs synthesized by laser pyrolysis come with major advantages. They present narrow distribution for particle size in comparison with other vapor condensation methods, extra small dimensions (a few nm), low degree of contamination with other elements and improved magnetic and structural properties due to the high temperatures used during synthesis in comparison with wet/chemical methods [31].
The nanopowders were analyzed by X-ray diffraction (XRD) and transmission electron microscopy (TEM).
The nanoparticles were stabilized with carboxymethylcellulose sodium salt (CMCNa) and loaded with DOX. The loading efficiency was estimated by using absorption and fluorescence spectroscopy. The conjugation of the drug with NPs was evidenced by FTIR spectroscopy. The DOX conjugated γ-Fe 2 O 3 NPs suspensions proved a good anticancer effect by the inhibition of tumoral cell proliferation, disruption of the cellular membrane, induction of cell death, and reduced effects on normal breast cells.

Synthesis of Iron Oxide Nanoparticles
The iron oxide nanoparticles were synthesized by laser pyrolysis technique ( Figure 1) using iron pentacarbonyl (Fe(CO) 5 ) (Merck, Darmstadt, Germany) and synthetic air as iron precursor and oxidizing agent. Ethylene (C 3 H 4 ) (Linde plc., Munich, Germany) was used as an energy transfer agent, also known as sensitizer. The principle of the method depends on the cross-flow configuration that ensures the resonance between the emission line of a CO 2 infrared laser and the absorption line of the gas sensitizer, subsequently heating the reacting gases through collision energy transfer [32].
Polymers 2020, 12, x FOR PEER REVIEW 3 of 18 (GaMG line) was reported in the presence of magnetic γ-Fe2O3 NPs conjugated with DOX in low concentration (10 nM) [29]. In contrast, in A549 lung cancer cells, the PEG-functionalized γ-Fe2O3 NPs conjugated with DOX induced very high viability, possible due to the limited release of DOX. However, under alternating magnetic field (AMF), NPs exhibited excellent thermal effects thus favoring the release of DOX from γ-Fe2O3 NPs and destruction of lung cancer cells [30].
The main purpose of this study is to demonstrate the efficiency of ɣ-Fe2O3 loaded with DOX in breast cancer therapy. The IONPs were synthesized by laser pyrolysis technique using iron pentacarbonyl (Fe(CO)5) vapors and synthetic air as iron and oxygen sources, respectively. NPs synthesized by laser pyrolysis come with major advantages. They present narrow distribution for particle size in comparison with other vapor condensation methods, extra small dimensions (a few nm), low degree of contamination with other elements and improved magnetic and structural properties due to the high temperatures used during synthesis in comparison with wet/chemical methods [31].
The nanopowders were analyzed by X-ray diffraction (XRD) and transmission electron microscopy (TEM).
The nanoparticles were stabilized with carboxymethylcellulose sodium salt (CMCNa) and loaded with DOX. The loading efficiency was estimated by using absorption and fluorescence spectroscopy. The conjugation of the drug with NPs was evidenced by FTIR spectroscopy. The DOX conjugated γ-Fe2O3 NPs suspensions proved a good anticancer effect by the inhibition of tumoral cell proliferation, disruption of the cellular membrane, induction of cell death, and reduced effects on normal breast cells.

Synthesis of Iron Oxide Nanoparticles
The iron oxide nanoparticles were synthesized by laser pyrolysis technique ( Figure 1) using iron pentacarbonyl (Fe(CO)5) (Merck, Darmstadt, Germany) and synthetic air as iron precursor and oxidizing agent. Ethylene (C3H4) (Linde plc., Munich, Germany) was used as an energy transfer agent, also known as sensitizer. The principle of the method depends on the cross-flow configuration that ensures the resonance between the emission line of a CO2 infrared laser and the absorption line of the gas sensitizer, subsequently heating the reacting gases through collision energy transfer [32]. Briefly, the focused continuous wave of CO2 laser radiation (70 W maximum output power, λ = 10.6 µm) is orthogonally intersected by the reactant flow. The combination between synthetic air (100 sccm) and Fe(CO)5 vapors carried by an ethylene flow(100 sccm) entered into the reaction chamber through the central inner tube. The confinement of both gas precursors to the flow axis, as well as of Briefly, the focused continuous wave of CO 2 laser radiation (70 W maximum output power, λ = 10.6 µm) is orthogonally intersected by the reactant flow. The combination between synthetic air Polymers 2020, 12, 2799 4 of 18 (100 sccm) and Fe(CO) 5 vapors carried by an ethylene flow(100 sccm) entered into the reaction chamber through the central inner tube. The confinement of both gas precursors to the flow axis, as well as of the newly nucleated nanoparticles, was attained by a coaxial argon (Ar) flow. The process was described in detail elsewhere [32]. The laser power and work pressure used were 55 W and 300 mbar, respectively, also presented in Table 1. The laser beam diameter was: ϕ = 2.0 mm. In order to stabilize and functionalize the nanoparticles, the following protocol was used (ensuring the concentrations of 1 mg/mL NPs, 0.6 mg/mL CMCNa and 0.08 mg/mL chemotherapeutic drug (DOX)): The corresponding quantities for γ-Fe 2 O 3 and CMCNa were mechanically mixed on an aluminum foil; at the same time, a glass vessel with the appropriate distilled water (pH = 5.5) load was placed in the ultrasound bath with the cooling system on. While in the ultrasound bath, a rotary mixer was introduced in the fluid and then the powders from the aluminium foil were slowly added. After all, the powder was introduced in the glass vessel, the mixer was removed, a cap was placed on the vessel, and it was left overnight in the ultrasound bath (59 kHz, 20 • C). The DOX powder was added at the end, and the final suspension was furthermore homogenised in the ultrasound bath for 1 h. The resulting suspensions γ-Fe 2 O 3 _CMCNa_DOX and γ-Fe 2 O 3 _DOX were centrifuged at 10 K rpm for 30 min, in order to eliminate the unanchored CMCNa and DOX. The solid deposit was then easily re-dispersed in distilled water maintaining the initial suspension volume using the same ultrasound bath for 30 min at 20 • C.
The resulted nanopowders were analyzed by the following methods: The phase composition and crystallinity were analized by X-ray diffraction (XRD) using a PANalytical X'Pert MPD theta-theta X-Ray diffraction apparatus with a Cu K α source (0.15418 nm); the morphology and structure were observed by transmission electron microscopy (TEM) and selected area electron diffraction (SAED) analysis, using a Philips CM 120ST (120 kV) Transmission Electron Microscope.

Spectroscopic Methods
The efficiency of DOX loading on γ-Fe 2 O 3 /γ-Fe 2 O 3 _CMCNa was quantified by measuring the absorbance values with a Lambda 950 UV-vis-NIR spectrophotometer (PerkinElmer, Inc., Waltham, MA, US) and 5 mm for DOX and 0.1 mm for γ-Fe 2 O 3 /γ-Fe 2 O 3 _CMCNa thickness optical quartz cuvettes (PerkinElmer, Inc., US). After solutions have been diluted from stock one, aiming to avoid saturation in the spectra, the absorption spectra were measured from 250 to 800 nm at room temperature (22-24 • C).
The efficiency of DOX loading was analysed also by laser induced fluorescence (LIF) home made set-up. The details of the experimental set-up were described elsewhere [24,25].
The laser excitation source consisted in the second harmonic generation (SHG) of a Nd:YAG laser (Continuum, US, Minilite II,) emitting at 532 nm, frequency 10 Hz. The laser pulse energies used in the experiments were smaller than 2.5 mJ to avoid a saturation effect of the processes and the DOX photodegradation. LIF signals were analyzed by a spectrograph (Acton Research, Acton, MA, US, model SpectraPro 2750) having a 150 tr/mm diffraction grating blazed at 500 nm. The detection was made by an iCCD (Princeton Instruments, Trenton, NJ, US, model PIMAX 1024 RB). The camera was triggered by a TTL laser-generated synchronizing pulse. The spectra were averaged on 1000 pulses.
The IR absorption study of the samples was performed by using a FTIR spectrometer (Thermo Fisher Scientific, Waltham, MA, US), model Nicolet iS50, resolution 4 cm −1 . The Dox solution and NPs suspensions were dried on the surface of optical grade KRS-5 plates and the resulting film spectra were recorded in the mid IR range between 4000 and 400 cm −1 . Live/Dead staining. The cells were seeded in 24 well plates at a density of 3 × 10 4 cells/mL and left to adhere overnight. After 24 and 48 h of treatment with NPs and DOX suspensions, the culture media was removed and replaced with a mix of calcein-AM and ethidium homodimer-1 solution following the manufacturer instructions of "LIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells" (L3224, Invitrogen, Carlsbad, CA). The live (labeled in green) and dead (labeled in red) cells were analyzed under an Olympus IX73 fluorescence microscope (Olympus, Tokyo, Japan) equipped with a Hamamatsu ORCA-03G camera (A3472-06, Hamamatsu, Japan). The images were acquired using fluorescein isothiocyanate (FITC) and tri-rhodamine-isothiocyanate (TRITC) filters and CellSens Dimension software (v1.11, Olympus). c.
Lactate dehydrogenase (LDH) assay. The activity of LDH released in cell culture media as a result of cell membrane permeabilization was measured using the "Cytotoxicity Detection Kit (LDH)" (cat. no. 11644793001, Roche, Basel, Switzerland) in a 96 well plate. After exposure for 24 and 48 h to IONPs and DOX suspensions, 50 µL culture media of treated and control cells were incubated with 50 µL mix reaction (catalyst and dye solution) from the kit for 15 min at room temperature, in the dark. The absorbance of the samples was read at 490 nm using a microplate reader. In vitro NO assay. The level of NO released in culture media was measured to estimate the inflammatory potential of the suspensions on tumor and normal breast cells using Griess method.
After the exposure of MCF7 and MCF-12A cells to 6.25 and 12.5 µg/mL NPs and, respectively, 50 and 100 ng/mL DOX for 24 and 48 h, 80 µL of culture media were mixed with 80 µL Griess reagent. The absorbance of the samples was measured at 540 nm. The NO concentration of the samples was determined using a NaNO 2 standard curve (0-100 µM) and expressed as percentages relative to control.

Statistical Analysis
The results were calculated as an average of three replicates and represented relative to control (untreated cells). Statistical significance was calculated using Student's test and a p-value < 0.05 was considered significant. Figure 2A shows the TEM micrograph for sample γ-Fe 2 O 3 , and it can be observed that the nanoparticles present spherical shape and a tendency for agglomeration in small ramified chain-like aggregates. By a lognormal fit, two maxima for particle diameter distribution were found, d 1m = 2.5 and d 2m = 3.5 nm. SAED analysis, presented as insert (2C), highlights the spinel structure of γ-Fe 2 O 3 /Fe 3 O 4 . These results are in agreement with the X-ray diffraction analysis pattern. Figure 3 presents the XRD patterns for the sample γ-Fe 2 O 3 . The main crystalline phases detected were magnetite (Fe 3 O 4 ) and maghemite (γ-Fe 2 O 3 ). The rather broad peaks exhibited by the XRD patterns could be correlated with the decreased particle size, which was confirmed by further calculations. The mean crystallite dimension was 3.2 nm and was calculated with Scherrer equation based on FWHM (full width half maximum) for (440) peak, found at around 2θ = 63 degrees.

Characterization of Synthesized γ-Fe 2 O 3 NPs
Previous papers were focused on structural and magnetic characterization of iron oxide NPs synthesized by laser pyrolysis in appropriate experimental conditions [32,33]. The magnetic hysteresis loops measured at different temperatures show a superparamagnetic feature for the as synthesized iron oxide NPs at room temperature with magnetic saturation of 32 emu/g and with almost zero remanent magnetization and coercivity above 150 K [33]. Only the iron oxide NPs with around 6 nm mean particle size show a small hysteresis loop at room temperature where the magnetic saturation grows up to 50 emu/g and the coercivity is less than 13 KA/m [32]. Figure 2A shows the TEM micrograph for sample γ-Fe2O3, and it can be observed that the nanoparticles present spherical shape and a tendency for agglomeration in small ramified chain-like aggregates. By a lognormal fit, two maxima for particle diameter distribution were found, d1m = 2.5 and d2m = 3.5 nm. SAED analysis, presented as insert (2C), highlights the spinel structure of ɣ-Fe2O3/Fe3O4. These results are in agreement with the X-ray diffraction analysis pattern.  Figure 3 presents the XRD patterns for the sample γ-Fe2O3. The main crystalline phases detected were magnetite (Fe3O4) and maghemite (γ-Fe2O3). The rather broad peaks exhibited by the XRD patterns could be correlated with the decreased particle size, which was confirmed by further  Previous papers were focused on structural and magnetic characterization of iron oxide NPs synthesized by laser pyrolysis in appropriate experimental conditions [32,33]. The magnetic hysteresis loops measured at different temperatures show a superparamagnetic feature for the as synthesized iron oxide NPs at room temperature with magnetic saturation of 32 emu/g and with almost zero remanent magnetization and coercivity above 150 K [33]. Only the iron oxide NPs with around 6 nm mean particle size show a small hysteresis loop at room temperature where the magnetic saturation grows up to 50 emu/g and the coercivity is less than 13 KA/m [32].
A dry deposit from the γ-Fe2O3_CMCNa NPs stabilized suspension was also investigated with TEM and SAED. As it can be seen in Figure 4A, the NPs-chains maintained the original feature, and they seem to be surrounded by a conformal polymeric-like shell with 3-7 nm thickness. Moreover, the SAED patterns ( Figure 4B) resemble the original NPs (only γ-Fe2O3/Fe3O4 crystalline phase were revealed) which proves that after the suspension preparation, the iron oxide NPs keep their original crystallinity and suggests thus that the CMCNa stabilizing shell can preserve the crystalline magnetic phase of iron oxide (maghemite/magnetite). A dry deposit from the γ-Fe 2 O 3 _CMCNa NPs stabilized suspension was also investigated with TEM and SAED. As it can be seen in Figure 4A, the NPs-chains maintained the original feature, and they seem to be surrounded by a conformal polymeric-like shell with 3-7 nm thickness. Moreover, the SAED patterns ( Figure 4B) resemble the original NPs (only γ-Fe 2 O 3 /Fe 3 O 4 crystalline phase were revealed) which proves that after the suspension preparation, the iron oxide NPs keep their original crystallinity and suggests thus that the CMCNa stabilizing shell can preserve the crystalline magnetic phase of iron oxide (maghemite/magnetite).  The attachment of CMCNa chains to iron oxide nanoparticles surface can be explained by the electrostatic attraction between the positive protonated Fe-OH 2+ groups in slightly acidic aqueous medium and the negatively charged -CH2-COO-groups of the polyelectrolyte, reinforced also by multiple hydrogen bonds provided by hydroxyls from both oxidic surfaces and cellulose-derived chains.

Spectroscopic Analysis
The absorption spectrum for the γ-Fe2O3_DOX sample was compared to the absorption spectrum for a solution of DOX in distilled water at 0.02 mg/mL ( Figure 5). The γ-Fe2O3_DOX suspension was diluted 1:10 to avoid saturation of the absorption signal. In the inset of Figure 5, it is shown the spectrum of the DOX conjugated NPs suspension after background subtraction. The attachment of CMCNa chains to iron oxide nanoparticles surface can be explained by the electrostatic attraction between the positive protonated Fe-OH 2+ groups in slightly acidic aqueous medium and the negatively charged -CH 2 -COO-groups of the polyelectrolyte, reinforced also by multiple hydrogen bonds provided by hydroxyls from both oxidic surfaces and cellulose-derived chains.

Spectroscopic Analysis
The absorption spectrum for the γ-Fe 2 O 3 _DOX sample was compared to the absorption spectrum for a solution of DOX in distilled water at 0.02 mg/mL ( Figure 5). The γ-Fe 2 O 3 _DOX suspension was diluted 1:10 to avoid saturation of the absorption signal. In the inset of Figure 5, it is shown the spectrum of the DOX conjugated NPs suspension after background subtraction.
From the absorbance values corresponding to the absorption peak of Dox located at 500 nm for the suspension of the NPs functionalised with DOX and the corresponding absorbance of DOX at a known concentration of 0.02 mg/mL, we determined the amount of DOX loaded on the NPs. A concentration of 0.8 µg/mL in the 1:10 dilution of γ-Fe 2 O 3 _DOX suspension has been found, this corresponding to a loading efficiency of 10%. The samples containing CMCNa in the suspension were difficult to analyze by absorption spectroscopy, due to the absorption band of CMCNa which hinders the subtraction of the DOX absorption band intensity in order to estimate the loading efficiency.
This suspension containing CMCNa was analyzed by LIF. The emission spectra excited with 532 nm at 2.5 mJ for Dox solution (0.02 mg/mL), γ-Fe 2 O 3 _CMCNa and γ-Fe 2 O 3 _CMCNa_DOX suspensions are shown in Figure 6. The NPs samples used were 1:10 dilutions of the stock suspensions. From the absorbance values corresponding to the absorption peak of Dox located at 500 nm for the suspension of the NPs functionalised with DOX and the corresponding absorbance of DOX at a known concentration of 0.02 mg/mL, we determined the amount of DOX loaded on the NPs. A concentration of 0.8 µg/mL in the 1:10 dilution of γ-Fe2O3_DOX suspension has been found, this corresponding to a loading efficiency of 10%. The samples containing CMCNa in the suspension were difficult to analyze by absorption spectroscopy, due to the absorption band of CMCNa which hinders the subtraction of the DOX absorption band intensity in order to estimate the loading efficiency.
This suspension containing CMCNa was analyzed by LIF. The emission spectra excited with 532 nm at 2.5 mJ for Dox solution (0.02 mg/mL), γ-Fe2O3_CMCNa and γ-Fe2O3_CMCNa_DOX suspensions are shown in Figure 6. The NPs samples used were 1:10 dilutions of the stock suspensions.   From the absorbance values corresponding to the absorption peak of Dox located at 500 nm for the suspension of the NPs functionalised with DOX and the corresponding absorbance of DOX at a known concentration of 0.02 mg/mL, we determined the amount of DOX loaded on the NPs. A concentration of 0.8 µg/mL in the 1:10 dilution of γ-Fe2O3_DOX suspension has been found, this corresponding to a loading efficiency of 10%. The samples containing CMCNa in the suspension were difficult to analyze by absorption spectroscopy, due to the absorption band of CMCNa which hinders the subtraction of the DOX absorption band intensity in order to estimate the loading efficiency.
This suspension containing CMCNa was analyzed by LIF. The emission spectra excited with 532 nm at 2.5 mJ for Dox solution (0.02 mg/mL), γ-Fe2O3_CMCNa and γ-Fe2O3_CMCNa_DOX suspensions are shown in Figure 6. The NPs samples used were 1:10 dilutions of the stock suspensions.  From the fluorescence signals intensities at the maximum of the emission band of DOX placed at 600 nm, we can extract the amount of DOX loaded on the NPs. In this case, we estimated a value of approximately 8% from the initial DOX quantity used at mixture preparation.
In both, absorption and fluorescence spectra, it can be noticed besides the main monomer bands (495 and 596 nm), the ones for dimers (537 and 641 nm) which can be formed by aggregation [34].
Maybe, due to the fact that Dox forms aggregates, the amount of DOX loaded on the NPs is rather small. The adsorption of DOX on NPs is proved by the shift of the fluorescence emission peak to shorter wavelength (593 nm) for the conjugates γ-Fe 2 O 3 _CMCNa_DOX compared to simple Dox solutions (596 nm).
The samples were analysed also by FTIR spectroscopy to emphasize the conjugation of NPs with the drug, via IR bands of the specific functional groups. In Figure 7, the IR absorption spectra for DOX, γ-Fe 2 O 3 _DOX, γ-Fe 2 O 3 _CMCNa_DOX, γ-Fe 2 O 3 _CMCNa and γ-Fe 2 O 3 samples are shown. It can be noticed the Dox specific bands present in the spectra of conjugated NPs: C-C bending at 1413 cm −1 , C-O, C-H deformation vibrations at 1015 cm −1 and respectively 986 cm −1 . The adsorption of Dox on NPs is evidenced by the C=O stretching vibration shift from 1722 cm −1 to 1688 cm −1 and C-O-C stretching vibration slightly shift from 1286 cm −1 to 1251 cm −1 . The attachment of DOX to Np can occur via the interaction of -NH 2 and -OH groups of DOX with -OH groups of absorbed water on Np through hydrogen bonding [35]. In support of this, N-H vibrations present in Dox spectrum at 1616 cm −1 and 760 cm −1 are clearly diminished in conjugated samples spectra. On the other side, the 1601 cm −1 H-O-H bending vibration of adsorbed water present in the spectra of iron nanoparticles disappears in the spectra of γ-Fe2O3_ DOX.
In both, absorption and fluorescence spectra, it can be noticed besides the main monomer bands (495 and 596 nm), the ones for dimers (537 and 641 nm) which can be formed by aggregation [34].
Maybe, due to the fact that Dox forms aggregates, the amount of DOX loaded on the NPs is rather small. The adsorption of DOX on NPs is proved by the shift of the fluorescence emission peak to shorter wavelength (593 nm) for the conjugates γ-Fe2O3_CMCNa_DOX compared to simple Dox solutions (596 nm).
The samples were analysed also by FTIR spectroscopy to emphasize the conjugation of NPs with the drug, via IR bands of the specific functional groups. In Figure 7, the IR absorption spectra for DOX, γ-Fe2O3_DOX, γ-Fe2O3_CMCNa_DOX, γ-Fe2O3_CMCNa and γ-Fe2O3 samples are shown. It can be noticed the Dox specific bands present in the spectra of conjugated NPs: C-C bending at 1413 cm −1 , C-O, C-H deformation vibrations at 1015 cm −1 and respectively 986 cm −1 . The adsorption of Dox on NPs is evidenced by the C=O stretching vibration shift from 1722 cm −1 to 1688 cm −1 and C-O-C stretching vibration slightly shift from 1286 cm −1 to 1251 cm −1 . The attachment of DOX to Np can occur via the interaction of -NH2 and -OH groups of DOX with -OH groups of absorbed water on Np through hydrogen bonding [35]. In support of this, N-H vibrations present in Dox spectrum at 1616 cm −1 and 760 cm −1 are clearly diminished in conjugated samples spectra. On the other side, the 1601 cm −1 H-O-H bending vibration of adsorbed water present in the spectra of iron nanoparticles disappears in the spectra of γ-Fe2O3_ DOX. The presence of polyanionic CMCNa chains wrapped on the Nps can enhance the adsorption of DOX due to their amino group electrostatic attraction towards carboxy negative groups of CMCNa, doubled by the possible formation of some hydrogen bonds between the hydroxyls groups of polymer and those from DOX [36]. The presence of polyanionic CMCNa chains wrapped on the Nps can enhance the adsorption of DOX due to their amino group electrostatic attraction towards carboxy negative groups of CMCNa, doubled by the possible formation of some hydrogen bonds between the hydroxyls groups of polymer and those from DOX [36].

Anticancer Cell Efficiency
The cytotoxicity of the DOX, γ-Fe 2 O 3 , γ-Fe 2 O 3 _CMCNa, γ-Fe 2 O 3 _DOX and γ-Fe 2 O 3 _CMCNa_DOX suspensions to MCF7 and MCF-12A cells was estimated and confirmed after 24 and 48 h of exposure by three tests: MTT, Live/Dead, and LDH assays. Morphological alterations were also observed and presented by optical microscopy images. Figure 8 representing MTT evaluation shows that both γ-Fe 2 O 3 _DOX and γ-Fe 2 O 3 _CMCNa_DOX induced a significant decrease of cell viability at doses higher than 12.5 µg/mL γ-Fe 2 O 3 and respectively 100 ng/mL DOX most likely caused by a change in the cell metabolic activity or enzyme activity in the mitochondrial respiratory chain. The cytotoxicity was time-and dose-dependent for both human cell lines. A decrease by 50% of MCF7 cell viability was obtained after 48 h incubation with γ-Fe 2 O 3 _DOX (in a dose of 12.5 µg/mL γ-Fe 2 O 3 + 100 ng/mL DOX) and only by 42% with γ-Fe 2 O 3 _CMCNa_DOX compared to control (unexposed cells). For MCF-12A cells, the decline of cell viability was closely similar to the one for MCF7 cells registering a decrease of 56% for the same conditions. For free DOX, γ-Fe 2 O 3 or γ-Fe 2 O 3 _CMCNa, the results indicated no decrease of MCF7 and MCF-12A cell viability during the experiment period in a concentration range between 3.125 and 31.25 µg/mL γ-Fe 2 O 3 NPs and 25 and 250 ng/mL DOX, suggesting high biocompatibility for NPs but no cytotoxicity of DOX in this range of concentrations. Only a dose of 250 ng/mL free DOX caused a slight decrease of MCF7 cells viability by 22.6% compared to control cells. . The statistical significance of results was noted with asterisk (*) as follow: * p < 0.05 (weakly significant); ** p < 0.01 (moderately significant); *** p < 0.001 (highly significant).
According to literature, the half-maximal inhibitory concentration (IC 50 ) for DOX was obtained at a concentration between 0.3 and 4.6 µg/mL after 48 h exposure of different human breast cells [37]. By comparison, our data showed that cytotoxicity of DOX increases significantly when conjugated with γ-Fe 2 O 3 and γ-Fe 2 O 3 _CMCNa; a 50% reduction of cell viability was obtained with a concentration around 0.1 µg/mL.
The anticancer efficiency of nanosuspensions conjugated with DOX was also confirmed by microscopy images of cells (Figure 9, bright-field images). The morphological observations revealed significant alterations of MCF7 cells compared to control cells. While control cells showed intact cell membrane, the treated MCF7 cells were characterized by cell shrinkage, extensions of the cell membrane, presence of large vacuoles in the cytoplasm and apoptotic bodies (cell fragments) when exposed to 12.5 µg/mL γ-Fe 2 O 3 + 100 ng/mL DOX γ-Fe 2 O 3 _DOX and γ-Fe 2 O 3 _CMCNa_DOX, which might indicate the occurrence of apoptosis. However, we found that the morphological changes in MCF-12A normal breast cells were less severe. significant alterations of MCF7 cells compared to control cells. While control cells showed intact cell membrane, the treated MCF7 cells were characterized by cell shrinkage, extensions of the cell membrane, presence of large vacuoles in the cytoplasm and apoptotic bodies (cell fragments) when exposed to 12.5 µg/mL γ-Fe2O3 + 100 ng/mL DOX γ-Fe2O3_DOX and γ-Fe2O3_CMCNa_DOX, which might indicate the occurrence of apoptosis. However, we found that the morphological changes in MCF-12A normal breast cells were less severe. Consistent with the findings from the morphological examination, the Live/Dead assay results showed a clear decrease in the number of MCF7 live cells (green fluorescence) concomitant with an increase in the number of dead cells (red fluorescence) for both γ-Fe2O3_DOX and ɣ-Fe2O3_CMCNa_DOX compared to control cells. In contrast, the number of live non-tumoral breast cells was slightly higher than of tumoral cells after 48 h of exposure. In addition, the Live/Dead images might suggest besides apoptotic death, a decrease in MCF7 cell proliferation because fewer cells were noticed in the acquired images. The antiproliferative effect of DOX on cancer cells was already demonstrated but the exact mechanism that inhibits cell proliferation is not yet fully understood [38].
After treatment, the LDH released in culture media was also measured to estimate the cell membrane damage after 24 and 48 h. As shown in Figure 10, loss of cell membrane integrity was present only for MCF7 cells treated with 12.5 µg/mL γ-Fe2O3 + 100 ng/mL DOX γ-Fe2O3_DOX and γ-Fe2O3_CMCNa_DOX, where the level of LDH increased significantly with a maximum of 36% and 34%, respectively, after 48 h of exposure compared to control. However, no modifications of LDH level were observed for MCF-12A cells, which suggests that normal breast cells succeeded in keeping their membrane intact. This result was not previously reported and is an important finding, which reveals opportunities for new therapeutic approaches to tackle this disease. Consistent with the findings from the morphological examination, the Live/Dead assay results showed a clear decrease in the number of MCF7 live cells (green fluorescence) concomitant with an increase in the number of dead cells (red fluorescence) for both γ-Fe 2 O 3 _DOX and γ-Fe 2 O 3 _CMCNa_DOX compared to control cells. In contrast, the number of live non-tumoral breast cells was slightly higher than of tumoral cells after 48 h of exposure. In addition, the Live/Dead images might suggest besides apoptotic death, a decrease in MCF7 cell proliferation because fewer cells were noticed in the acquired images. The antiproliferative effect of DOX on cancer cells was already demonstrated but the exact mechanism that inhibits cell proliferation is not yet fully understood [38].
After treatment, the LDH released in culture media was also measured to estimate the cell membrane damage after 24 and 48 h. As shown in Figure 10, loss of cell membrane integrity was present only for MCF7 cells treated with 12.5 µg/mL γ-Fe 2 O 3 + 100 ng/mL DOX γ-Fe 2 O 3 _DOX and γ-Fe 2 O 3 _CMCNa_DOX, where the level of LDH increased significantly with a maximum of 36% and 34%, respectively, after 48 h of exposure compared to control. However, no modifications of LDH level were observed for MCF-12A cells, which suggests that normal breast cells succeeded in keeping their membrane intact. This result was not previously reported and is an important finding, which reveals opportunities for new therapeutic approaches to tackle this disease.
Membrane extension, cell shrinkage and apoptotic bodies are features of apoptosis while the loss of cell membrane integrity is a feature of necrosis. Thus, our results might suggest a possible combination of more types of cell death induced in MCF7 cells by DOX-nanoconjugates, which could increase the anticancer efficiency by targeting more cell pathways.
Previous studies showed that different doses of DOX activate different regulatory mechanisms to induce either apoptosis, necrosis or cell death through mitotic catastrophe [39,40]. In vitro, free DOX has been shown to induce apoptosis via the activation of caspases and disruption of mitochondrial membrane potential. For example, Nestal de Moraes et al. demonstrated that DOX inhibited cell viability and induced DNA fragmentation and activation of caspases−3, −7 and −9 in the breast cancer-derived cell lines MCF7 and MDA-MB-231 [41]. However, more recently, it was reported the absence of active caspase 3 and cleavage of PARP 1 in MCF7 cells treated with DOX for 48 h [42]. By combining DOX with IONPs, it was possible to increase the drug intracellular concentration by escaping the endosomal retention and thus overcome resistance [43] and even cross the blood-brain barrier [44].

Oxidative and Inflammatory Potential
The level of ROS was analyzed to evaluate the capacity of DOX-nanoconjugates to induce oxidative stress in tumor and normal cells. As seen in Figure 11, the treatment with nanoconjugates caused a time-dependent increase of intracellular ROS in both MCF7 and MCF-12A cells. The level of ROS was higher in tumoral cells compared with normal cells. Overproduction of ROS was recorded for both doses of DOX-nanoconjugates in MCF7 cells and only for the higher dose in MCF-12A cells. More importantly, the results showed a higher production of ROS in MCF7 exposed to γ-Fe 2 O 3 _DOX compared to γ-Fe 2 O 3 _CMCNa_DOX for the dose of 12.5 µg/mL γ-Fe 2 O 3 + 100 ng/mL DOX starting with 4 h. No significant increase of ROS level was observed for free γ-Fe 2 O 3 _DOX but only for γ-Fe 2 O 3 _CMCNa after 48 h. We think that the negatively charged CMCNa-coating of γ-Fe2O3 NPs might attract a large number of protein molecules on the surface of NPs that possibly hinder the DOX cytotoxicity. Cancer cells are known to acidify their environment and, consequently, serum proteins tend to have positive charges which interact with the functional carboxymethyl groups of CMCNa and surround the NPs forming a biocorona of proteins.
It is well known that the generation of free radicals is one of the mechanisms by which DOX induces its anticancer effect. DOX accumulates in mitochondria by specifically binding to the phospholipid cardiolipin which is found abundantly in the inner mitochondrial membrane. At this level, the membrane perturbation inhibits complex I and complex II, disrupting the electron transport chain leading to ROS production. Besides this, it was demonstrated that DOX can also generate ROS through other mechanisms. For example, DOX can directly interact with iron to form reactive anthracycline-iron complexes resulting in an iron cycling between Fe 3+ and Fe 2+ associated with ROS production including the high-toxic hydroxyl radical (OH•) by the Fenton and Haber-Weiss reactions, thus altering iron homeostasis [45]. The statistical significance of results was noted with asterisk (*) as follow: * p < 0.05 (weakly significant); ** p < 0.01 (moderately significant).
Previous studies reported that DOX also increases nitric oxide synthase (NOS) activity and expression and, by direct binding to this enzyme, leading to nitric oxide (NO) production [45,46].
In this study, the NO level produced in culture media of tumoral and normal breast cells seems to be more a result of γ-Fe 2 O 3 NPs exposure and less of DOX one. As seen in Figure 12, exposure to simple γ-Fe 2 O 3 and γ-Fe 2 O 3 _CMCNa in a dose of 12.5 µg/mL resulted in a statistically significant elevation of NO production starting with 24 h for normal cells and 48 h for tumoral cells suggesting a slight inflammatory potential of bare NPs. However, an increase of NO production (no more than 17% over control) was also detected for both cell lines treated with γ-Fe 2 O 3 _CMCNa_DOX at a dose of 12.5 µg/mL γ-Fe 2 O 3 + 100 ng/mL DOX but the level was similar to the one found in cells exposed only to free NPs. These results suggested that DOX had no contribution to NO production. Interestingly, exposure of cells to γ-Fe 2 O 3 _DOX did not cause any change in NO level at none of the concentrations, which made us to conclude that the combination with DOX might decrease the cytotoxicity of bare γ-Fe 2 O 3 .

Introduction
Breast cancer (BC) is the most common type of cancer in the world among women and it remains a challenge even today. For the next years, statistics show that the diagnosis will come out positive for 1 in 8 women, and it was demonstrated that with age, the incidence of BC increases, but after menopause, the frequency is lower [1,2]. In 2018, the World Health Organization estimated that 627,000 women died of breast cancer [3].
Conventional strategies, such as chemotherapy, radiotherapy and surgical resection, either performed individually or in combination, manifest limited therapeutic effects for cancer because these types of treatment are non-specific [4]. The most common class of drugs utilized in BC chemotherapy is anthracyclines, such as doxorubicin (DOX) [5] or epirubicin [6] administrated in early and advanced stages of breast cancer. Recently, there were identified two possible mechanisms of DOX anticancer effect. The first mechanism refers to the DOX property to intercalate into DNA

Conclusions
The present study demonstrated the efficiency of γ-Fe 2 O 3 NPs loaded with DOX on the decrease of viability of breast cancer cells. The NPs were synthesized by laser pyrolysis, and their nanometric size and crystallinity were confirmed by XRD and TEM analyses.
The NPs were stabilized with CMCNa and loaded with DOX. The loading efficiency was estimated by using absorption and fluorescence spectroscopy, and it was about 10%. The conjugation of NPs with the drug was evidenced by FTIR spectroscopy.
According to the biological results obtained in the current investigation, both utilized DOX-nanoconjugates present a good anticancer potential demonstrated through the inhibition of tumoral cell proliferation, disruption of the cellular membrane, induction of cell death, and reduced effects on normal breast cells. However, we also found that the coating of γ-Fe 2 O 3 with CMCNa might hinder the DOX-ROS production and thus its cytotoxicity in comparison to uncoated γ-Fe 2 O 3 . Comparing with previous works, this study presents not only the cytotoxic effect of NPs but also their oxidative and inflammatory potential. Further studies are needed to explore the cytotoxic potential of these nanoconjugates in vivo.