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Effective cancer theranostics with polymer encapsulated superparamagnetic nanoparticles: Combined effects of magnetic hyperthermia and controlled drug release Nanasaheb D. Thorat, Raghvendra A Bohara, Mohamed Radzi Noor, Dinesh Dhamecha, Tewfik Soulimane, and Syed A. M. Tofail ACS Biomater. Sci. Eng., Just Accepted Manuscript • Publication Date (Web): 28 Nov 2016 Downloaded from http://pubs.acs.org on November 28, 2016

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ACS Biomaterials Science & Engineering

Effective cancer theranostics with polymer encapsulated superparamagnetic nanoparticles: Combined effects of magnetic hyperthermia and controlled drug release Nanasaheb D. Thorat†‡*, Raghvendra A Bohara§*, Mohamed Radzi Noor‡⊥, Dinesh Dhamechaџ Tewfik Soulimane‡⊥ and Syed A.M. Tofail†‡ †

Department of Physics, University of Limerick, Limerick, Ireland Bernal Institute, University of Limerick, Limerick, Ireland § Research and Innovations for Comprehensive Health Care (RICH), Dr. D. Y. Patil Hospital and Research Centre, D. Y. Patil University, Kolhapur, India џ Dr. Prabhakar Kore Basic Science Research Center, KLE University, Nehru Nagar, Belagavi-590010, Karnataka, India ⊥ Department of Chemical Sciences, University of Limerick, Limerick, Ireland ‡

Corresponding Author Nanasaheb D. Thorat, Department of Physics, University of Limerick, Limerick, Ireland ‡ Bernal Institute, University of Limerick, Limerick, Ireland [email protected], * These authors equally contributed to the manuscript

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Abstract: A combination of chemotherapy with non-conventional nanoparticle based physical destruction therapy has been proposed clinically to reduce the prospect of evolution of drug resistance in cancer. Superparamagnetic nanoparticles have been actively used for synergetic cancer therapy including magnetic fluid hyperthermia (MFH) guided by magnetic resonance imaging (MRI). To explore this direction of potential applications in cancer therapy, we have functionalized superparamagnetic La0.7Sr0.3MnO3 nanoparticles (SPMNPs) with an oleic acidpolyethylene glycol (PEG) polymeric micelle (PM) structure, and loaded it with anticancer cancer drug doxorubicin (DOX) in a high loading capacity (~60.45 %) for in vitro delivery into cancer cells. The micellar structure provided good colloidal stability and biocompatibility. Upon drug loading, the cancer cell death rate of 89 % was comparable to free DOX (75 %) for 24 h, and that the counterstrategy of DOX conjugated SPMNPs-induced hyperthermia resulted the cancer cell extinction up to 80 % under in vitro conditions within 30 min. In addition, the preliminary effect of protein corona formation on in vitro drug release and delivery was studied. Finally, in vivo bio distribution of micellar SPMNPs is observed in mice model for 50 mg kg-1 dose of SPMNPs. Taken together, polymeric micelle SPMNPs reported here can serve as a promising candidate for effective multimodal cancer theranostics such as in the combined chemotherapy–hyperthermia cancer therapy.

Keywords: LSMO; magnetic nanoparticles; polyethylene glycol; drug delivery; anticancer drug; in vivo biocompatibility;


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1. Introduction New class of magnetic nanosystem such as superparamagnetic La0.7Sr0.3MnO3 nanoparticles (SPMNPs) and other ferrite based superparamagnetic nanoparticles have been considered as a potential mediator in novel cancer theranostics that synergistically combine chemotherapy and diagnostic image guided therapy such as magnetic hyperthermia.1,2 This is due to the fact that SPMNPs can act both as a carrier for target specific pharmaceutics and a controlled heat source through the use of magnetic hysteresis and superparamagnetic behavior. The successful therapeutic implementation of SPMNPs is contingent on the intrinsic magnetization and their biophysical properties such as biocompatibility, superficial functionality, dispersion stability and specific absorption rate (SAR) at both physiological and tumor conditions.3 These attributes can be obtained through the functionalization of the outer surface of SPMNPs using biomolecules and chemical compounds.4,5 Magnetic fluid hyperthermia (MFH) is a non-invasive and externally controlled therapy. However, MFH alone is deficient therapy in oncology for complete tumor suppression and, thus, is often used complementarily to chemotherapy.6,7 When compared to synergetic cancer therapy over magnetic nanoparticles (MNPs), SPMNPs can achieve a much higher rate of cancer-cell destruction, both in vitro and in vivo, under the action of a non-invasive low-frequency magnetic field applied for a short duration.8–11 SPMNPs with a Curie temperature (Tc) of 42-50 °C (~ 315-325 K) is an ultimate MFH mediator for in vivo clinical trials and strontium doped lanthanum manganite (LSMO) such as La0.7Sr0.3MnO3, compound fulfills above conditions.3,12 LSMO is a known multiferroic half-metal and exhibits extraordinary magnetic properties such as colossal magnetoresistance (CMR), which makes the material attractive for spintronic devices. 3 ACS Paragon Plus Environment


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Superparamagnetic LSMO NPs generate efficient heat under relatively low strength and frequency magnetic field.13–15 These magnetic properties combined with its excellent MRI contrast due to T2 relaxivity can be utilized for the purpose of theranostics e.g. image guided cancer diagnostics and therapy. In this context, biocompatible superparamagnetic LSMO NPs encapsulated in polyethyleneglycol (PEG) micelle have been successfully synthesized with the resulting improvement of SAR, colloidal stability and biocompatibility for MFH and MRI applications.16 Recently, liposomes, polymeric NPs and SPMNPs are investigated for cancer tumorspecific drug delivery by various researchers. SPMNPs based drug delivery has distinct advantages over other analogous structures mentioned above in that its fundamental superparamagnetism contributes a control over magnetization, allowing for a regulated movement of SPMNPs under an external alternating current (AC) magnetic field. Therefore, SPMNPs-based therapeutic cargos can be distributed to tumor locations by applying a low strength non-invasive magnetic field. More significantly, MRI technique can be used to quantify the localized MNP-accompanying drug that may help in formative site-specific (sub)optimal dosing. Combined with the high size flexibility of MNPs, SPMNPs have clear advantages compared to other structures.17 In the present investigation, we report the development and optimization of magneticpolymeric micelle structure for simultaneous thermo-chemotherapy through local heat generation and delivery of chemotherapeutic agents. Polymeric micelle structures on SPMNPs were formed by encapsulating SPMNPs in PEG polymer through oleic acid (OA), as PEG capping has been found to be highly advantageous in making biocompatible, colloidally stable SPMNPs due to their protein-resistant and highly hydrophilic nature. Chemotherapeutic anticancer drug doxorubicin (DOX) was then encapsulated into the micelles. The colloidal stability, toxicity, hemocompatibility of polymeric micelle (PM)-


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SPMNPs (PM@SPMNPs) have already been investigated by us to confirm the usefulness of intravenous injection (i.v.). Here, the consequence of hyperthermia produced by these magnetic-polymeric core/shell nanostructures on cancer cells is evaluated in vitro. The in-vitro efficacy of the developed PM@SPMNPs as mediators of synergetic cancer theranostics is evaluated for both MFH and chemotherapeutic drug delivery. The results admit a noteworthy intensification (> 80%) in the cancer cell killing with the synergetic therapy that combines magnetic hyperthermia and controlled drug release.

2. Results 2.1. Formation of DOX conjugated nanoparticle-micelle structure The formation of polymeric micelle through oleic acid (OA)-PEG on SPMNPs surface is schematically illustrated in Fig. 1. The OA molecular fragments are chemically absorbed on the surface of SPMNPs. Their hydrophobic methyl-segment are oriented towards the outside, hence OA-SPMNPs can be dispersed in apolar solvents. The conjugation of the OA-SPMNPs with PEG in solution results in an interdigitated hydrophobic interaction between the hydrophobic segment of PEG with the OA moieties. At the same time, the hydrophilic terminal carboxylic-acid blocks oriented outwards confer a negative charge and water solubility property to [email protected] Changes in the surface charge on the NPs due to the application of functional coatings are measured using zeta potential measurements. The pH dependent zeta potential values of SPMNPs, PM@SPMNPs and DOX- PM@SPMNPs dispersions in water are shown in Fig. S1a. The isoelectric point of SPMNPs, PM@SPMNPs and DOX- PM@SPMNPs is 3, 5 and 3.5, respectively. At pH 7.4, a negative zeta potential of -20.04 mV was observed for SPMNPs, while the subsequent surface charge alteration with PEG and DOX loading changed the zeta potential to -25 and -15 mV, respectively. 5 ACS Paragon Plus Environment


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To evaluate the PM size and the aggregation behavior of bare and drug loaded PM@SPMNPs, DLS measurements were performed in water. The relative hydrodynamic distribution of non-coated SPMNPs in water is ranging from ~ 10-110 nm16 (Fig. S1b). After PM formation, the aggregation was reduced to between 20 and 60 nm. Formation of PM structures and DOX conjugation on SPMNPs was further analyzed by Fourier transferinfrared spectroscopy (FTIR) and shown in the Fig.S2. The FTIR spectrum for the bare SPMNPs shows a distinct peak at 600 cm-1. Upon polymeric micelle formation, major peaks appeared at 960, 1354 and 1102 cm−1. The synergy between DOX molecules and PM@SPMNPs was investigated by fluorescence measurements in water at pH 7 (Fig. 2a). Other physical characterizations of SPMNPs and PM@SPMNPs including X-ray diffraction (XRD), thermogravimetric analysis (TGA) and particle size distribution by transmission electron microscopy (TEM) was reported in our recent publication.16

2.2. Drug release in physiological and in vitro conditions By studying DOX release at pH 5 and 7.4 (Fig. 2b) in water, a pH-dependence release profile from DOX-PM@SPMNPs over time could be observed. The effect of protein corona on drug release was also studied, where the corona formation on DOX- PM@SPMNPs was performed by referring to previous reports as outlined in the experimental methods section. About 46 % of DOX was released from DOX- PM@SPMNPs after 24 h, which is comparable to the ~42 % drug release with human serum protein-conjugated DOXPM@SPMNPs (HSP-DOX- PM@SPMNPs) after the same length of time at pH 5. At pH 7.4, ~14 % and 11 % drug release was observed from DOX-PM@SPMNPs and HSP-DOXPM@SPMNPs, respectively. In parallel, using confocal laser scanning microscopy (CLSM), the subcellular (in vitro) DOX release was qualitatively visualized. The Fig.2c spectacles the CLSM images of MCF7 cells when incubated with only DOX, DOX-PM@SPMNPs and


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HSP-DOX-PM@SPMNPs for 4 h, where it is clear that there is an internalization of DOXPM@SPMNPs with the subsequent DOX release into MCF7 cells.

2.3. Synergetic cancer therapy The AC magnetic field-dependent temperature profile and SAR of PM@SPMNPs in water are shown in Fig. S3a and physical mechanism behind the magnetic heating and improvement in specific absorption rate (SAR) is discussed in our previous review article.4 Initially we tested anticancer efficacy of DOX and DOX-PM@SPMNPs on MCF7 cells. Cancer cell killing efficacy of DOX- PM@SPMNPs were assessed through a MTT and MTS assay. The Fig. 3a shows cell viability data of MCF7 cells treated with various formulations, the cell viability decreased obviously as the DOX concentration increased. From this study the IC50 values of DOX-PM@SPMNPs were ~25 µg mL-1 determined for MCF7 cells. At the highest investigated DOX-PM@SPMNPs concentration i.e. 1.0 mg mL-1 (~60 µg mL-1 DOX), the viabilities of MCF7 cells further dropped to just 10.11 % however, 25.02 % cell viability observed for only DOX. To obtain a quantitative view of the effect of DOX and DOX-PM@SPMNPs, cell proliferation was appraised by MTS colorimetric biochemical assay. The data shows (Fig. 3b) that both DMSO and PM@SPMNPs negative controls did not affect cell growth of MCF7 cells, DOX-PM@SPMNPs and HSP-DOX-PM@SPMNPs exhibited a gradual dose-reliant cell proliferation inhibitory effect. During the in vitro magnetic hyperthermia tests, the temperature of the cell suspension with nano-formulation was rigorously sustained at 43-44 °C and was ensured to not overcome the hyperthermia threshold of 45-46 °C. The in vitro hyperthermia study (Fig. 4) reveals that the cancer cells incubation with PM@SPMNPs without magnetic field or the presence of magnetic field alone did not cause a cogent decrease of cell viability. This signifies that the exposure of PM@SPMNPs or magnetic field alone does not influence



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cancer cell viability. Results also revealed that the hyperthermia generated by water bath heating of MCF7 cells at 43-44 °C for 60 min and the SPMNPs based MFH for 60 min decreased cell viability to 72 ± 3% and 40 ± 2 %, respectively, showing a comparable effect. DOX-PM@SPMNPs incubation for 4 h decreased cell viability of MCF7 cells to 76 ± 2 %. Based on the release profile (Fig. 2b), after 4 h ∼18 % of the loaded DOX on PM@SPMPNs was released, accessible DOX concentration of ~10.8 µg mL−1 is calculated to affect the cells viability. MCF7 cells exposed to a drug delivery and MFH synergetic treatment by DOX– PM@SPMNPs exhibited a reduced cell viability (10 ± 1 %) than the single modality treatments i.e. only drug delivery with DOX– PM@SPMNPs (72 ± 3 %) as reflected in Fig.4. As shown in Fig. S4, PM@SPMNPs swiftly introverted the MCF7 cell viability after an elevated exposure of AMF. Only PM@SPMNPs based MFH therapy can kill ~80 % MCF7 cells. Important markers of complete cell death (apoptosis) by synergetic cancer therapy, can be qualitatively determined by CLSM using propodeum iodide (PI), 4',6diamidino-2-phenylindole (DAPI) and Fluorescein Diacetate (FDA) dyes. The nucleuspenetrating PI dye cannot internalize into of living cells through the intact membrane, whereas early apoptotic or necrosis cells can internalize PI. In the context of this study, we used DAPI for nucleus staining of live and dead, PI for dead cells only and FDA for live cell nucleus staining (Fig. 5).

2.4. In vivo toxicity and biodistribution study The in vitro toxicity and hemocompatibility of PM@SPMNPs was investigated in our recent report.16 The in vitro study is performed on different cell lines including non-cancer (L929) and cancer (MCF7) and results demonstrated PM@SPMNPs are biocompatible at various tested concentrations. Hemocompatibility study also revealed that PM@SPMNPs did not cause hemolysis of RBCs up to 24 h. In the present study, we extended the previous in vitro


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study16 to analyze the in vivo toxicity of PM@SPMNPs. Potential acute toxicity and biodistribution in major organs (heart, liver, kidney, spleen, brain and lungs) of rat postintravenous injection was assessed by dissection and compared with the negative control. An essential feature of NPs is their biocompatibility; hence, primary organs of rat post administration of PM@SPMNPs were tested via the pathological assay (H & E staining). In Fig. 6 it can be seen that no observable morphological changes were seen in these organs with respect to control, which clearly ruled out the presence of acute injury induced by the injection of PM@SPMNPs. Histology of the heart, brain, liver is not altered with any abnormalities (e.g. clogged central veins), lungs (e.g. profusion accompanied by interstitial hemorrhage) and kidneys (e.g. peritubular profusion and hyaline droplet change in the tubule).19

3. Discussions The formation of PEGylated micelle structure on SPMNPs and DOX conjugation was determined using zeta potential and DLS techniques (Fig S1a, b). As DOX was loaded onto PM@SPMNPs through EDC-NHS coupling chemistry, the shift in zeta potential values can be associated to the existence of charge on PEG polymeric chain and DOX molecules. Carboxyl group of PEG molecules can provide a higher electrostatic hindrance and improves colloidal stability of NPs. Protonated positively-charged primary amine in DOX can bind negatively charged PM@SPMNPs and once again shifting in zeta potential was observed. The zeta potential analysis of DOX-PM@SPMNPs shows a clear shift in isoelectric point from -5 to -3.4 mV. The upsurge in zeta potential values of DOX- PM@SPMNPs are attributed to the cationic nature of DOX molecules.20 The particle size distribution of the PM@SPMNPs in water is observed ~20 - 60 nm (Fig. S1b). This implies that thin polymeric micelle structures have been formed on SPMNPs



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with a narrow size distribution. After DOX loading the size distribution remained more or less the same (30-60). DLS study implies that the PEG micelle formation provide a steric hindrance amongst the SPMNPs particles in a highly acidic or basic environment, thus helping to decrease the agglomeration rate of the SPMNPs under these conditions. This implies that the degree of coverage or the thickness of the PEG layer on the OA-SPMNPs surface is sufficient in stabilizing the particles even in strongly acidic or basic conditions. Spherical polymeric micelle-SPMNPs with size of 15-20 nm range visualized by TEM are shown in recent publication.16 The FTIR spectrum for the bare SPMNPs shows distinct peak at 600 cm-1 corresponding to a metal-oxygen bonding as would be expected in a La-Sr-Mn-oxide. After polymeric micelle formation, major peaks appeared at 1354 and 1102 cm−1, suggesting the asymmetric and symmetric stretching of C–O–C, and the out-of-plane bending of the CH group in the polymeric chains at 960 cm−1.21 Moreover, PEG itself exhibits a number of noticeable peaks between 600-900 cm−1. The magnetic core of the La-Sr-Mn (i.e. bare SPMNPs) and OA molecules did not produce any FTIR signal in this range. The stretching vibration mode of the carbonyl group in the DOX-conjugated PM@SPMNPs (Fig. S2c) is represented at 1720 cm-1. The prominent vibration IR peaks of DOX molecules appeared at 1280, 1210, 1016 and 1000 cm-1 , which suggest DOX conjugation [email protected] Apart from IR spectroscopy, the DOX on PM@SPMNPs can also be quantified on the basis of its distinctive absorbance at 485 nm as well as fluorescence at 560 and 595 nm. The fluorescence intensity of DOX-conjugated PM@SPMNPs is gradually decreased with respect to control DOX solution. By using a linear plot, the concentration on DOX on PM@SPMNPs surface can be calculated from fluorescence spectra. A 60.45 % DOX loading has been achieved with 1:10 DOX to PM@APMNPs (w/w) ratio. To perform the pH-dependent drug release profile of DOX- PM@SPMNPs and HSP-DOX-PM@SPMNPs in a physiological


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tumor-tissue environment, their respective release profiles were investigated at different pH values at 37 °C. The physiological pH of blood is 7.4, whereas cancerous cells (cellular compartments) have pH < 5.0.23 The release rate of DOX is gradually advanced at cancerous cells pH compared to physiological pH of blood for DOX- PM@SPMNPs. The drug release profile slightly affected after HSP binding on DOX- PM@SPMNPs. However, the DOX proclamation observed at the pH of 7.4 is as little as 15 % and 11 % after 24 h for DOXPM@SPMNPs and HSP-DOX-PM@SPMNPs, respectively. This observation is highly appealing for oncology, as the low pH in tumors reasonably can act as a specific stimulatory signal for drug release at the target site.24 Finally, the overall DOX release from DOXPM@SPMNPs formulation was superior at tumor cells pH and inferior at the normal physiological pH. It can be presumed that this effect was caused by the amide bond cleavage at acidic pH, as DOX was indeed covalently grafted through amide bonds. However, around 47.8 % of DOX was released at pH 5.0 from DOX-PM@SPMNPs formulation, which simulated the pH value of tumors microenvironment. Almost ~4 % decrease in drug release was observed with HSP-DOX-PM@SPMNPs composition and this implies that the HSP binding on DOX-PM@SPMNPs composition did not induced remarkable effect on drug release. However, this is a preliminary study and further detailed studies are required to understand the effect of HSP binding on drug release from nanocomposites under physiological conditions. In the unbound (free) DOX-treated batch, a large portion of the drug was located evenly within the medium as observed during experiments. Most of the free DOX was accumulated in the cellular cytoplasm, although some DOX was also internalized into nuclei of cells after 4 h (Fig. 3c). However, in the DOX-PM@SPMNPs batch, initial 2 h of incubation found a lower amount of DOX in the cytoplasm (not shown here) and the DOX internalization increased after 4 h. Similar to the previous report 20, our data here suggest that



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the release of DOX from the PM@SPMNPs was lengthened in the acidic endosomes. Given the intrinsic red fluorescence of DOX, MCF7 cells incubated with DOX-PM@SPMNPs showed a bright fluorescence demonstrating that once internalized, these particles exhibit an efficient intracellular delivery into the nuclei of tumor cells. The results indicated that DOX-PM@SPMNPs could release DOX more specifically and act as a ‘smart’ controlled release system in cancerous tumor’s acidic environment, and thus encouraging for targeted thermo-chemotherapy with a concomitant reduction of its systemic in vivo toxicity. Therefore, polymeric micelle formed on SPMNPs not only improved the stability of SPMNPs, but also encapsulated chemotherapeutic drug and further released in an acidic environment. The similar trend of DOX release was observed for HSPDOX-PM@SPMNPs composite (Fig. 2c(D)). Uptake of DOX-PM@SPMNPs complexes could be attributed to endocytosis. Recently Peng et. al. reported a detailed cellular uptake (mainly endocytosis) mechanism of chitosan grafted NPs into MCF7 cells.25 The study demonstrated a variety of forms of endocytosis to be involved in the cellular uptake of chitosan NPs formulation. In our study herein, endocytosis is also the main means of DOXPM@SPMNPs entry into cells. As a proof-of-concept for the potential of PM@SPMNPs in a combined chemotherapy-hyperthermia cancer treatment, the effect of DOX-PM@SPMNP-prompted MFH together with DOX delivery was determined as a function of cell viability. As evaluated in earlier section (section 3.2), the incubation of PM@SPMNPs with MCF7 cells did not result in a significant cytotoxicity at various concentrations. We initially tested the DOX, DOX-PM@SPMNPs and HSP-DOX-PM@SPMNPs toxicity in concentrationdependent manner for 24 h, with the DOX concentration on PM@SPMNPs being semiquantified through fluorescence spectroscopy. The observed toxicity is endorsed by PEG-


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mediated internalization of DOX-PM@SPMNPs that occurred 2 h after DOX is co-localized in the subcellular cytoplasmic compartment and started its cancer cell killing function.27,28 To observe a synergetic magneto-chemotherapy effect, the parameters for hyperthermal dosage and DOX-PM@SPMNPs incubation time with cells were adjusted such that the cell death caused by the individual treatments would not mask the effect of combined application. Based on the initial colloidal stability, biocompatibility and hyperthermia properties of PM@SPMNP, its in vitro magnetic hyperthermia effect on MCF7 (breast cancer) cells was analyzed. There was a steady increase in the number of apoptotic cells over the post-hyperthermia time (Fig. 5), presumably due to the programmed and controlled nature of apoptosis. In the meantime, the apoptosis cells steadily developed from the premature apoptoic stage to late stage i.e. necrosis.29 Although, PI-marked MCF7 cells are not observable prior to MFH (Fig. 5A), cell death after 30 and 60 min of synergetic treatment was reflected by a large number of PI-marked cells (Fig. 5B and C; imaging were performed after 24 h of MFH therapy). The PI staining correlated well negatively with the FDA staining, which was performed before the application of MFH to confirm healthy cell morphology. After 60 min of synergetic treatment, cells formed vesicle-like, intact membrane-lacking structures (i.e. apoptotic bodies).30 The confocal microscopy observations also enabled the monitoring of cell apoptosis at early stage (plasma membrane disruption) last stage (nucleus fragmentation) and at necrotic stage (overall cell shrinkage). Cancer cell death can be significantly enhanced within a short interval during MFH by having an efficient thermal prescription induced by PM@SPMNPs after AMF application. To attain such an optimal dose required in a clinical implementation for synergistic cancer therapy, high SAR responsive PM@SPMNPs were synthesized. Our previous works on LSMO demonstrated that surface grafted (colloidally stable) particles are capable of generating efficient thermal dose within a precisely controlled time duration.35,36 This



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strategy was further exploited to develop stable, dispersible, cyto-hemocompatible PM@SPMNPs (i.e. PEG capped La0.7Sr0.3MnO3 MNPs) with six fold improved SAR compared to their pristine counterpart. The enhancement of SAR and stimulated drug delivery resulted in an efficient internalization of drug by efficient thermal dose and the consequent increased cancer cell deaths.37,38 Nevertheless, the results presented herein show that multimodality i.e. DOX delivery (chemotherapy) with MFH (magneto-thermotherapy) using PM@SPMNPs is more effective than using single modality. As it is known that certain NPs are highly effective but also toxic to patients, we performed a preliminary histopathological examination of exposed mice organs focusing on tissue damage, inflammation or lesions (Fig. 6). With multiple possible routes of PM@SPMNPs administration, we selected the intravenous route (i.v.) based on a similar previous report.39

Our in vivo study showed no indication of extreme injuriousness or

toxicity. Solid signs of mortality, which is up to 50 mg kg-1 dose of PM@SPMNPs (via i.v. routes), led to the proposition of a safety limit of PM@SPMNPs at a dosage of 50 mg kg-1. At this dosages, as shown in Fig. 6 no unusual appearances were noted, although the liver and spleen seemed dark on the borders suggestive of local effects. The blackish nature of liver could be caused by a substantial intracellular uptake of the NPs aided by the rich blood supply to this organ and its permeability.19 In fact, many animal model studies have detailed an accumulation of MNPs preferentially in these two organs, including a report where about 55 % of iron was localized in the liver after 6 h following injection.40 It is, therefore, safe to conclude that PM@SPMNPs exhibit a good biocompatibility, at least in mice. We also extended the in vivo study and calculated La, Sr and Mn (as elements) concentration in blood and liver. Determining the fate of i.v. administered PM@SPMNPs and the concentration of its constituent metals in blood and other organs is necessary for identifying their exact toxicity level and to fulfil conditions for future applications such as


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MRI. We estimated La, Sr and Mn concentration up to 4 h after administration by following previously-published methods.19 In blood, the levels of all three elements slowly reduced after introduction into the animals, despite the fact there was an initial increase in the liver, in agreement with results of Reihaneh et.al.19

4. Conclusion DOX-conjugated PM@SPMNPs nanocomposites possess a great therapeutic efficacy by exhibiting a superior hyperthermic response in AMF and DOX release in cancer cell to effectively eliminate the target cancer cells through mild apoptosis and necrosis-mediated cell death. Furthermore, the localized antitumor effect of the proposed DOX-PM@SPMNPs has considerably increased due to the drug sensitization by trivial hyperthermia application. In conclusion, polymeric micelle formation on SPMNPs conjugate anticancer drug and effectively act as theranostics agent on cancer. Under optimized conditions, MFH of DOXPM@SPMNPs shows cancer cell killing up to 90 % over apoptosis and late necrosis pathway. Preliminary in vivo bio distribution study shows no harmful effects of this formulation on different organs. Finally, an integrative cancer theranostics which combines chemotherapy and MFH using this PM@SPMNPs formulation would provide a feasible and modest strategy in oncology in the future.

4. Experimental 4.1. Materials All chemicals, reagents and cell culture media used were sourced from Sigma-Aldrich Chemical Co. MCF7 cells were purchased from National Centre for Cell Sciences (NCCS), Pune, India. Whole human serum of a healthy male was obtained from D. Y. Patil Hospital Kolhapur, India and used within a week. MTT assay kit was provided from EZ-Cytox, South Korea. 4.2. Synthesis of PM@SPMNPs The superparamagnetic La0.7Sr0.3MnO3 (SPMNPs) were prepared by a method described earlier. Polymer micelle structure on SPMNPs were prepared by a two-step method and detailed in our recent publication.16 4.3. Physical characterization



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Fourier-transform infrared spectra (FTIR) were acquired on a Perkin-Elmer spectrometer (Model No.783, USA) in the range 400 to 4000 cm-1. Zeta potential measurements were carried on PSS-NICOMP-380 ZLS (USA) particle sizing system and the average values are reported from three successive replications. MNPs were inductively heated for hyperthermia application using an induction heating unit (Easy Heat 8310, Ambrell; UK) in accordance with the protocol we previously reported.16 4.4. Loading and release of anticancer drug (Doxorubicin) Initially 1 mg DOX is dissolved in 1 mL DD water, to this solution EDC (5 mg,) in 1 mL of H2O, NHS (10 mg,) was added drop wise and solution kept at 4 °C overnight on magnetic stirrer. Further the prepared solution is added into 10 mg PM@SPMNPs solution made in 10 mL water and final solution is kept for more 24 h on stirring at 4 OC. The DOX encapsulated particles were separated magnetically, washed thoroughly with ultrapure H2O. The doxorubicin loaded on PM@SPMNPs were quantified by UV absorbance (Cary 60 UV-Vis spectrophotometer, Agilent Technologies, Inc., USA) and Fluorescence (Fluorolog-3 spectrofluorometer) measurements for this DOX-PM@SPMNPs (100 µg mL-1) were dispersed in DI water. The quantification of DOX and other DOX release experiments were performed in accordance with protocol reported recently.32 4.5. Human serum protein (HSP) binding to PM@SPMNPs To load HSP on DOX-PM@SPMNPs, 50 mg DOX-PM@SPMNPs were incubated in 10 wt./v.% HS (total volume 100 mL) for 24 h at 37 °C and then solution was centrifuged at 10,000 rpm for 10 min to recover HSP-bound DOX-PM@SPMNPs (HSP-PM@SPMNPs) samples as pellet. The HSP-PM@SPMNPs samples were lyophilized using the Labconco Freeze Dry the lyophilized HSP-PM@SPMNPs samples were stored at 4 °C for further experiments. 4.6. In vitro drug release and Cancer cell killing In vitro DOX release in MCF7 cells (1×105 cells/mL) was performed in µ-Dish (35 mm tall) glass bottom obtained from ibidi Germany and observed under LEICATCS SP5 confocal microscope (Leica Microsystems, Germany). The cancer cell killing experiments are performed in accordance with our previous protocol.32 4.7. Cell proliferation Cell proliferation of MCF7 cells accompanying with SPMNPs, DOX-PM@SPMNPs and HSP-DOX-PM@SPMNPs was inspected by MTS assay. Briefly, MCF7 cells (1×105 cells/mL in each well) were grown into a 96-well plate. The cells were then incubated with different concentrations of PM@SPMNPs, DOX-PM@SPMNPs and HSP-DOXPM@SPMNPs for 24 h, washed once with PBS, resuspended in 1 mL new cell media having 20 µL MTS solution. The overall cell-NPs suspension was incubated for 3 h and finally absorbance at 492 nm was noted using a microplate reader. The percentage of cell proliferation was estimated by equating the absorbance of NPs treated cells against the untreated cells. 4.8. Animal model In vivo (animal experiments) study were conducted by prior approval from the institutional animal ethics committee of KLE University, Belagavi and in accordance with guidelines mentioned by Ministry of Environment and Forest, Govt. of India. Briefly, female Wistar rats of 180-200 g were selected for the study. Animals were provided by the central animal facility from the institute and acclimatized under natural light/dark conditions for two weeks at temperature of 25 ± 2 °C, RH 50% - 60 % before experiments. All animals were divided into Group I and II each containing six animals. Group I is control group, treated with normal saline and group II treated with PM@SPMNPS (50 mg/kg) intravenously through tail vein with prior calculation of dose as per body weight. After experimentation animals were sacrificed by 16 ACS Paragon Plus Environment

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cervical dislocation and tissues (Hearts, Lungs, Liver, Kidney, spleen and brain) from all experimental animal groups were stored in 10 % buffered formalin. Collected tissues were embedded in paraffin blocks and sliced into 5 µm in thickness, mounted on slides, stained with hematoxylin and eosin and examined under light microscope (Olympus BX-50 Corporation Tokyo, Japan) to get histopathological images. 4.9. Statistical data analysis Zeta potential, DLS, fluorescence, cellular toxicity and cell proliferation experiments were performed in triplicates and the results are expressed herein as mean ± standard error (SD) of the mean. All biological experiments are also replicated three times and the statistical significance of the data was examined by an unpaired, two tailed Student t-test, where a difference between datasets at p < 0.05 was considered statistically significant.

Acknowledgement The financial support from the Irish Research Council under the Government of Ireland Postdoctoral Fellowship-2015, Grant No. GOIPD/2015/320, is gratefully acknowledged. Supporting Information: zeta potential, DLS, FTIR magnetic hyperthermia and in vitro hyperthermia study of SPMNPs, PM@SPMNPs and DOX-PM@SPMNPs References (1) Quinto, C. A.; Mohindra, P.; Tong, S.; Bao, G. Multifunctional Superparamagnetic Iron Oxide Nanoparticles for Combined Chemotherapy and Hyperthermia Cancer Treatment. Nanoscale 2015, 7 (29), 12728–12736. (2) Patra, S.; Roy, E.; Karfa, P.; Kumar, S.; Madhuri, R.; Sharma, P. K. Dual-Responsive Polymer Coated Superparamagnetic Nanoparticle for Targeted Drug Delivery and Hyperthermia Treatment. ACS Appl. Mater. Interfaces 2015, 7 (17), 9235–9246. (3) McNamara, K.; Tofail, S. A. M. Nanosystems: The Use of Nanoalloys, Metallic, Bimetallic, and Magnetic Nanoparticles in Biomedical Applications. Phys. Chem. Chem. Phys. 2015, 17 (42), 27981–27995. (4) Bohara, R. A.; Thorat, N. D.; Pawar, S. H. Role of Functionalization: Strategies to Explore Potential Nano-Bio Applications of Magnetic Nanoparticles. RSC Adv. 2016, 6 (50), 43989–44012. (5) Ediriwickrema, A.; Saltzman, W. M. Nanotherapy for Cancer: Targeting and Multifunctionality in the Future of Cancer Therapies. ACS Biomater. Sci. Eng. 2015, 1 (2), 64–78. (6) Misra, S. K.; Ghoshal, G.; Gartia, M. R.; Wu, Z.; De, A. K.; Ye, M.; Bromfield, C. R.; Williams, E. M.; Singh, K.; Tangella, K. V.; et al. Trimodal Therapy: Combining Hyperthermia with Repurposed Bexarotene and Ultrasound for Treating Liver Cancer. ACS Nano 2015, 9 (11), 10695–10718. (7) Guardia, P.; Riedinger, A.; Nitti, S.; Pugliese, G.; Marras, S.; Genovese, A.; Materia, M. E.; Lefevre, C.; Manna, L.; Pellegrino, T. One Pot Synthesis of Monodisperse Water Soluble Iron Oxide Nanocrystals with High Values of the Specific Absorption Rate. J. Mater. Chem. B 2014, 2 (28), 4426. (8) Hergt, R.; Dutz, S. Magnetic Particle Hyperthermia—biophysical Limitations of a Visionary Tumour Therapy. J. Magn. Magn. Mater. 2007, 311 (1), 187–192. (9) Peng, E.; Ding, J.; Xue, J. M. Concentration-Dependent Magnetic Hyperthermic Response of Manganese Ferrite-Loaded Ultrasmall Graphene Oxide Nanocomposites. New J. Chem. 2014, 38 (6), 2312. 17 ACS Paragon Plus Environment


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Fig. 1


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Fig. 1. Schematic illustration showing the overall mechanism of anticancer drug (DOX) conjugation into polymer encapsulated SPMNPs and the concept of cancer cell killing through synergetic therapy using an alternating magnetic field (AMF).

Fig.2

Fig. 2. Doxorubicin (DOX) loading on PM@SPMNPs and release in water. (a) Fluorescence spectra 21 ACS Paragon Plus Environment


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of DOX and DOX-PM@SPMNPs (DOX: Ex. 480 nm, Em. 590 nm.). The equivalent DOX concentration is used as positive control while only PM@SPMNPs are used as negative control. (b) DOX release from DOX- PM@SPMNPs and HSP-DOX- PM@SPMNPs in water at different pH values. (c) In vitro DOX delivery in to MCF7 cells. Cells were incubated with optimum DOX-PM@SPMNPs and HSP-DOX- PM@SPMNPs for 4 h and stained with DAPI for confocal imaging. DOX-PM@SPMNPs and HSP-DOX- PM@SPMNPs are internalized into MCF7 cells DOX is accumulated into cytoplasm, observed from fluorescence imaging.

Fig.3.

Fig.

3

Cancer cell killing efficacy of DOX-PM@SPMNPs HSP-DOX- PM@SPMNPs assessed through a MTT (a) and cell proliferation(b) assay.


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Fig. 4



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Fig. 4 In vitro magneto-chemotherapy on cancer cells by PM@SPMNPs, DOXPM@SPMNPs and HSP-DOX-PM@SPMNPs. Values are expressed as mean ± SD, n = 3.

Fig. 5


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Fig. 5 Confocal microscopy images of untreated and treated MCF7 cells by MFH generated with DOX-PM@SPMNPs for 30 (B) and 60 (C) min, and stained with DAPI, PI and FDA after 24 h incubation (scale 20 µm). The background fluorescence due to DOX was removed by using LSM imaging software.

Fig. 6



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Fig. 6. (a) Photomicrograph of HE stained sections of organs including heart, liver, lung, spleen, kidney and brain from the control group and experimental groups subjected to 50 mg kg Dex-1 PM@SPMNPs via i.v. route. The concentrations of lanthanum (La), strontium (Sr) and manganese (Mn) were estimated by atomic absorption spectrometry (AAS) in blood (b) and liver (c) after the administration of PM@SPMNPs.


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Superparamagnetic nanoparticles (SPMNPs) has been considered as a potential mediator in novel cancer theranostics that synergistically combine chemotherapy and diagnostic image guided therapy such as magnetic hyperthermia. Graphic for manuscript 591x238mm (150 x 150 DPI)

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