Combinatorial delivery of superparamagnetic iron

Materials Science and Engineering C 75 (2017) 1128–1143

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Combinatorial delivery of superparamagnetic iron oxide nanoparticles (γFe2O3) and doxorubicin using folate conjugated redox sensitive multiblock polymeric nanocarriers for enhancing the chemotherapeutic efficacy in cancer cells Chetan Nehate, M.R. Aji Alex, Arun Kumar, Veena Koul ⁎ Centre for Biomedical Engineering, Indian Institute of Technology Delhi, New Delhi 110016, India Biomedical Engineering Unit, All India Institute of Medical Sciences, New Delhi 110029, India

a r t i c l e

i n f o

Article history: Received 19 October 2016 Received in revised form 2 January 2017 Accepted 2 March 2017 Available online 06 March 2017 Keywords: Hyperthermia Chemotherapy Folate Glutathione sensitive nanoparticles

a b s t r a c t Redox sensitive, folate conjugated multiblock polymeric system of (-PLGA-PEG-PLGA-urethane-ss-) demonstrated self-assembly into stable nanoplatforms. The polymeric nanocarriers were encapsulated with doxorubicin and highly crystalline γFe2O3 superparamagnetic iron oxide nanoparticles (SPIONs), for co˗delivery of the same to cancer cells, with average particle size of ~170 nm and zeta potential of ~−33 mV. Furthermore, the designed formulation was evaluated for protein adsorption, hemo˗cytocompatibility and stability. Glutathione (GSH) induced redox sensitivity of the nanocarriers was depicted by ~4.47 fold increase in drug release in the presence of 10 mM GSH. In vitro cellular uptake studies of the designed nanocarriers showed synergistic cytotoxic effect in folate overexpressing cells (HeLa and MDA-MB-231), after subjecting the cells to radio frequency (RF) induced hyperthermia (~43 °C). Negligible effect of the combinatorial therapy was observed in normal cells (L929). The developed polymeric system depicted facile synthesis, reproducibility and potential for achieving combinatorial and targeted delivery of drug and SPIONs to cancer cells. This combinatorial approach can help in achieving better therapeutic effect with minimal side effects of chemotherapy. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Tremendous efforts have been carried out in past decades for developing targeted delivery systems to combat cancer, but still it remains a major threat to mankind due to its non-resectability in 40% of tumors. The ability of radio frequency (RF) assisted hyperthermia therapy has got renewing interest for scientists, as it is non-hazardous and nonionizing. RF waves have high penetration power and deeply rooted cancer can be well treated by RF therapy. Mostly RF assisted therapy utilizes nano-heating agents such as gold nanoparticles and iron oxide nanoparticles [1,2]. Gold nanoparticles have deleterious cytotoxic effects than iron oxide nanoparticles [3]. Iron oxide nanoparticles have already been approved by FDA as magnetic resonance imaging (MRI) contrast agents [4]. Moreover they have excellent in vivo biodegradability, and the iron ions released after degradation can be assimilated by body's own tightly regulated physiological mechanism [5,6]. Hyperthermia is emerging an attractive therapeutic modality for cancer treatment as local therapy. During hyperthermia treatment, temperature of tumor tissue is maintained at 42–46 °C to destroy the tumor cells. As this ⁎ Corresponding author. E-mail address: [email protected] (V. Koul).

http://dx.doi.org/10.1016/j.msec.2017.03.024 0928-4931/© 2017 Elsevier B.V. All rights reserved.

modality has less side effects in comparison to chemo- and radiotherapy, it acts as promising tool for combinatorial treatment with other modalities to treat cancer. Different clinical trials have already demonstrated the usefulness of such combinations [7,8]. Doxorubicin (DOX) plays a pivotal role in chemotherapy because of its effective cancer killing potential, but it suffers from major drawbacks such as P-glycoprotein mediated multidrug resistance and cardiotoxicity [9]. Recent research is being focused on co˗delivery of other non-toxic therapeutic agents along with chemotherapeutics in order to achieve better pharmacological effect. For this purpose, a suitable delivery vehicle which can dispose the loaded therapeutic agents specifically to the tumor mass is necessary. The designed nanocarriers should also effectively overcome various systemic and cellular barriers to reach the target site. This can be accomplished by tailoring hydrophobic/hydrophilic ratio, providing stealth nature and conjugating targeting ligands etc. Cancer cells exhibit high redox potential environment with 100–1000 times higher level of reducing GSH tripeptide in their cytoplasm and nuclei (1–10 mmol) in comparison to blood and extracellular milieu (2–20 μM) [10,11]. This higher level of GSH is attributed to anticancer drug resistance of cells, mediated due to GSH-assisted phase II detoxification mechanism [12]. Moreover it is stated that tumor tissues are highly hypoxic and with at least 4 fold higher GSH level than normal

C. Nehate et al. / Materials Science and Engineering C 75 (2017) 1128–1143

tissue [13]. Hence, designing a nanosystem with redox sensitive functionalities can selectively release encapsulated drug in cancer cells by triggered release mechanism in the high redox potential environment of cancer cells [14]. There are reports where combinatorial approach for hyperthermia and chemotherapy with magnetoliposomes containing anticancer drug and iron oxide nanoparticles showed favourable results on cultured cells [15,16]. The size of these magnetoliposomes was N200 nm which limits their application for in vivo. Moreover nanoparticles with size N200 nm can be rapidly cleared from the circulation by the reticuloendothelial system (RES). Similar combinatorial approach was reported where DOX and iron oxide nanoparticles were loaded into polyethyleneimine (PEI, 25,000 Da) shell [17], but there are several studies demonstrating non-biodegradable nature and toxicity of PEI [18,19]. The authors of our research group have previously synthesized multiblock polymers based on PCL˗PEG˗PCL for drug delivery application and successfully demonstrated their reproducibility [20]. In the present investigation, we have synthesized PLGA based stealth redox

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sensitive multiblock copolymer (-PLGA-PEG-PLGA-urethane-ss-) and encapsulated SPIONs and DOX in the nanocarriers of same, for targeted tumor therapy. Multiple disulfide linkages in the polymer chain can be cleaved in 10 mmol GSH, resulting into degradation of polymeric nanoparticles and release of encapsulated cargos to tumor environment (Scheme 1). The cellular uptake of nanoparticles was evaluated in folate receptor overexpressing and non-expressing cell lines. In vitro cytotoxicity studies were conducted to ascertain the potential of developed nanosystem for combination therapy of RF induced hyperthermia and drug delivery. 2. Materials and methods Poly(ethylene glycol) (PEG, Mn ~200 Da), lactide, glycolide, tin(II) 2ethylhexanoate [Sn(oct)2], 2-hydroxyethyl disulfide, hexamethylene diisocyanate (HMDI), dibutyltin dilaurate, (DBTL), sodium oleate, Iron(III) chloride hexahydrate (FeCl3·6H2O) N,N′-dicyclohexyl carbodiimide (DCC), N-hydroxysuccinimide (NHS), Potassium hexacyanoferrate(II) trihydrate (K4[Fe(CN)6]·3H2O), glutaraldehyde,

Scheme 1. Schematic representation of GSH responsive folate-(PLGA-PEG-PLGA-urethane-ss-)j multiblock polymeric nanoparticles for combined delivery of DOX and SPIONs to cancer cells.

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reduced glutathione (GSH), bovine serum albumin (BSA) and 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St Louis, MO, USA) and used without further purification. A dialysis membrane of 3.5 kDa was obtained from spectrum labs. Fetal bovine serum (FBS), penicillin–streptomycin solution and Dulbecco's modified Eagle's medium (DMEM), Leibovitz's L-15 medium, trypsin EDTA solution, SlowFade® Diamond Antifade

Mountant with DAPI were purchased from Thermofisher scientific (NY, USA). Tetrahydrofuran (THF), methanol, dimethyl sulfoxide (DMSO), dichloromethane (DCM), hydrochloric acid (HCL), and triton X-100 were obtained from Merck Millipore (Mumbai, India) and were used as received without further purification. Amicon, stirred ultrafiltration cell was obtained from Merck Millipore (Bellerica, M.A., USA) Doxorubicin was obtained as a gift sample from Ranbaxy Laboratories Ltd.,

Scheme 2. Reaction scheme for synthesis of Folate-(PLGA-PEG-PLGA-urethane-ss-)j multiblock copolymer.

C. Nehate et al. / Materials Science and Engineering C 75 (2017) 1128–1143 Table 1 Composition of nanoparticles with their notation. Nanoparticles notation

Composition

SPIONs mb NPs mbSP NPs mbD NPs mbSPD NPs

Oleic acid coated superparamagnetic nanoparticles. Multiblock copolymer NPs. SPIONs loaded multiblock copolymer NPs. DOX loaded multiblock copolymer NPs. SPIONs and DOX loaded multiblock copolymer NPs.

New Delhi, India. Ultrapure water with 18 MΩ cm resistivity was obtained from Milli-Q system (Merck Millipore, Billerica, MA, USA). 2.1. Synthesis of multiblock copolymers based on PLGA-PEG-PLGA Synthesis of multiblock copolymer is two step reaction as depicted in Scheme 2. In the first step, the PLGA-PEG-PLGA triblock copolymer was synthesized by ring opening polymerization of lactide and glycolide using PEG as initiator. In the second step, polymerization of PLGA-PEG-PLGA was carried out with 2-hydroxyethyl disulfide and hexamethylene diisocyanate to form multiblock copolymer with urethane linkages. Further, folic acid was conjugated with terminal hydroxyl groups of the multiblock copolymer via DCC-NHS coupling. 2.1.1. Synthesis of the PLGA-PEG-PLGA triblock copolymer Polyethylene glycol (Mn ~ 200 Da, 2 g, 10 mmol) was dissolved in 10 mL of dry toluene. Lactide (2.77 equivalents, 4 g, 27.7 mmol), glycolide (3.44 equivalents, 4 g, 34.4 mmol) were added in solution and catalyst, tin-2-ethylhexanaote (5 mol% of the initiator, 161.9 μL) was added to reaction medium in the Schlenk flask, the reaction was continued under a N2 atmosphere for 24 h at 125 °C. After 24 h, crude product was concentrated over rotavapor and obtained crude product was precipitated in diethyl ether thrice to obtain pure product. The final product was dried under high vacuum and characterized by 1H NMR (400 MHz, Brukers, U.S.A.) and gel permeation chromatography (GPC, Waters, U.S.A.) with refractive index detector (Waters 2414) using THF based styragel HR4 (MW 5000–600,000 Da) and styragel HR3 (MW, 500–30,000 Da) in series. THF was used as mobile phase with 1 mL/min flow rate. Column heater temperature was kept at 40 °C. Molecular weight calibration curve was plotted using series of polystyrene standards. Yield = 92%.

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round bottom flask containing activated folic acid and stirred at 30 °C for 24 h in the presence of N2. After 24 h, polymer was dialyzed against distilled water for 24 h to remove unreacted folic acid and the product was lyophilized. The yellow colored polymer was characterized by FTIR (Perkin- Elmer, USA), and CHNS analyzer (Vario EL III Element Analyzer, Elementar Analysensysteme GmbH, Germany). 2.2. Synthesis of oleic acid coated superparamagnetic nanoparticles Monodisperse hydrophobic SPIONs with high crystallinity and saturation magnetization were synthesized by thermal decomposition method developed by Park et al. [21]. 2.2.1. Synthesis of iron-oleate complex The iron-oleate complex was prepared by reacting sodium oleate with ferric chloride. In a typical reaction, 4 g of iron chloride (FeCl3·6H2O, 24.66 mmol) and 13.518 g of sodium oleate (44.40 mmol) was dissolved in solvent mixture comprising of 30 mL ethanol, 20 mL distilled water and 50 mL hexane. The solution was allowed to heat at 75 °C and refluxed for 4 h. After completion of 4 h, the upper organic layer containing the iron-oleate complex was washed thrice with 50 mL distilled water by using separatory funnel. The hexane was evaporated on rotavapor resulting in dark brown waxy solid of iron-oleate complex (yield 72%). 2.2.2. Synthesis of SPIONs The monodispersed and highly crystalline, γFe2O3 (maghemite) SPIONs with average particle size of 9.2 ± 0.69 nm were prepared. In typical synthesis, 12 g (13.33 mmol) of the iron-oleate complex and 1.9 g of oleic acid (6.72 mmol) were mixed with 115 g of 1octadecene at room temperature. The reaction mixture was heated with a constant heating rate of 3 °C min− 1 up to 320 °C, and kept at this temperature for 30 min till the solution became brownish black. The resulting solution containing the nanoparticles was then cooled to 80 °C and purged with air for 6 h. The mixture was again heated to 320 °C with heating rate of 3 °C min− 1 and kept at this temperature for 1 h, then solution was allowed to cool to room temperature. SPIONs were precipitated in ethanol and were collected by high strength magnet (0.2 T). The process of precipitation was repeated thrice to remove all 1-octadecene. The remaining SPIONs in 1-octadecene were stored at 4 °C for further use. 2.3. Nanoparticles (NPs) preparation and characterization

2.1.2. Synthesis of multiblock copolymer based on PLGA-PEG-PLGA (mbPLGA) Triblock copolymer PLGA-PEG-PLGA (2 g, 2.01 mmol), 2hydroxyethyl disulfide (30% of the triblock copolymer, 74 μL, 0.60 mmol), hexamethylene diisocyanate (382.26 μL, 2.37 mmol), and catalyst, dibutyltin dilaurate (~ 78 μL, 0.13 mmol) were added in 10 mL of dry THF in Schlenk flask and the reaction was carried out for 24 h at 80 °C under the presence of N2. After 24 h, THF was evaporated on rotavapor and crude polymer was obtained. The crude polymer was further dissolved in DCM and precipitated three times in diethyl ether. The product was isolated, vacuum dried and characterized by 1H NMR and GPC. Yield = 88%. 2.1.3. Conjugation of folic acid to multiblock copolymer (mbPLGA-FA) Folic acid was conjugated to multiblock copolymer in two steps. In the first step, γ-carboxylic group of folic acid was activated by DCCNHS chemistry. In a typical process, folic acid (99.05 mg, 0.22 mmol, 1.2 equivalents of the copolymer) was dissolved in dry dimethyl sulfoxide (10 mL) in 50 mL Schlenk flask, followed by addition of DCC (55.56 mg, 0.26 mmol) and NHS (30.95 mg, 0.26 mmol) and the reaction was stirred at 30 °C for 24 h under a N2 atmosphere. The reaction byproduct, dicyclohexyl urea was removed by filtration. In the second step, the multiblock copolymer (1 g, 0.187 mmol) was added into

Four types of NPs were prepared for cell culture studies by nanoprecipitation method. The composition of each NPs is given in Table 1. DOX was deprotonated according to earlier reported method with little modification [22]. For preparation of DOX loaded polymeric NPs (mbD NPs), 10 mg of polymer and 1 mg of DOX was dissolved in 1 mL of DMSO. This solution was added dropwise to 10 mL of milli-Q water with continuous stirring for 20 min. In preparation of DOX and SPIONs loaded NPs (mbSPD NPs), 200 μL of SPIONs suspension (15 mg/mL in THF) was mixed with 1 mL of DMSO containing 1 mg of DOX and 10 mg of polymer. This solution was further added dropwise to 10 mL of water with continuous stirring. SPIONs loaded NPs (mbSP NPs) were prepared in similar way keeping concentration of SPIONs in organic phase same as used for mbSPD NPs preparation. Blank NPs (mb NPs) were also prepared using similar method without adding DOX or SPIONs in organic phase. The unloaded SPIONs and DOX were removed by Amicon, stirred ultrafiltration cell (Millipore, U.S.A.) with polyether sulfone membrane (300 kDa). The purified NPs were lyophilized with 10% trehalose as cryoprotectant. DOX loading in NPs was determined using HPLC method. Waters HPLC system with PDA detector and empower 3 software. The column symmetry shield RP18 with pore size 5 μm and 4.6 × 150 mm dimension was used for separation (Waters, Milford, MA, USA). The main

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column was protected by guard column of same chemistry 5 μm pore size and 3.9 × 20 mm dimension (Waters, Milford, MA, USA). Mobile phase was comprised of 0.05 M sodium acetate buffer (pH 4.0)-acetonitrile (70:30) was delivered at flow rate of 1 mL/min. The column compartment temperature was kept at 37 °C. The column elute was monitored at 481 nm with a band width of 16 nm. Quantification of DOX was done using the standard calibration curve with concentration range 0.1–25 μg/mL of DOX (Supporting information Fig. S9). The retention time of DOX was found to be 3.72 min (Supporting information Fig. S10). Loading of SPIONs in NPs was determined as per their iron (Fe) content, using flame ionization detection (atomic absorption spectrophotometer, AAS, Thermo Scientific, M Series GE 650268 v1.30, USA) at wavelength of 248.3 nm. Air acetylene flame at 1.1 L/min was used. Chamber preconditioning and nebulizer uptake was kept for 4 s. The total iron concentration in NPs was determined using standard curve made with different concentrations of FeCl3·6H2O. Prior to the AAS measurements, a weighed sample of freeze-dried NPs were digested with 5 mL of 6 N hydrochloric acid for 6 h. The analysis was done with Solar-11.02 software. Loading and encapsulation efficiency of DOX and SPIONs was calculated using Eqs. (1, 1.1) and (2, 2.1) respectively. Loading efficiency of DOX ¼

ðamt:of DOX loaded in NPsÞ 100 ðamt:of NPsÞ

Encapsulation efficiency of DOX ðamt:of DOX loaded in NPsÞ ¼ 100 ðamt:of DOX taken for loading in NPsÞ Loading efficiency of SPIONs ¼

ð1Þ

ð1:1Þ

ðamt:of SPIONs loaded in NPsÞ 100 ðamt:of NPsÞ ð2Þ

2.5.1. Hemolysis One milliliter of blood was centrifuged at 1500 rpm for 10 min to obtain RBCs pellet. The pellet was redispersed in PBS, washed and centrifuged thrice to remove any adsorbed proteins. Stock solution was prepared by diluting 50 μL of RBCs with 10 mL of PBS. NPs in PBS (100 μL) were incubated with RBC stock solution for 1 h at 120 rpm, so that final concentration is in the range of 10–80 μg of Fe/mL and 2.5–21 μg of DOX/mL. After 1 h, supernatant was collected by centrifugation at 1500 rpm and analysed for released haemoglobin using UV– visible spectrophotometer at 540 nm. The percent hemolysis was determined using PBS as negative control and 1% triton X-100 as positive control. Blank was used with same concentration of NPs in PBS 7.4. Percent hemolysis was calculated using following Eq. (3), Hemolysis% ¼

Sample540 nm −negative control540 nm positive control540 nm −negative control540

100 nm

ð3Þ

2.5.2. Coagulation study The mbSPD NPs, suspended in 100 μL of PBS were incubated in 900 μL of blood for 1 h, so that final concentration is in the range of 10–80 μg of Fe/mL and 2.5–21 μg of DOX/mL. Platelet poor plasma was obtained from centrifugation of blood at 4000 rpm for 10 min and incubated at 37 °C. Tissue factor was added to platelet poor plasma followed by an excess of calcium chloride solution (25 mmol) and prothrombin time (PT) was determined. Activated partial thromboplastin time (aPTT), was determined by adding micronized silica and cephalin in platelet poor plasma. Mixture was mixed properly and calcium chloride solution (25 mmol) was added. The PT and aPTT were determined based on the fibrin clot formation time using automated coagulation analyzer (Diagnostica Stago, Germany). 2.6. Colloidal stability studies of NPs

Encapsulation efficiency of SPIONs ðamt:of SPIONs loaded in NPsÞ 100 ¼ ðamt:of SPIONs taken for loading in NPsÞ

ð2:1Þ

The size of NPs were measured by using dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern Instruments Ltd., U.K.), highresolution transmission electron microscope (HRTEM; Technai G2, 200 kV, FEI, U.S.A.), Scanning electron microscope (SEM; Zeiss EVO 50, Carl Zeiss Microscopy GmbH, Germany), Atomic force microscopy (AFM; Nanoscope Multimode AFM, Digital Instruments, U.S.A.), CHNS analysis, Alternating gradient magnetometer (AGM, Micromag 2900, from Princeton Measurement Corp.), XRD (Rigaku D/max 2500 diffractometer, Japan) and X-ray photoelectron spectroscopy (XPS, SPECS, Germany). 2.4. Drug release studies The DOX release study was carried out in phosphate buffer saline pH 7.4 in the presence and absence of 10 mmol and 20 μM GSH to simulate the reducing conditions present in cytoplasm of cancer cells and blood respectively [11,23]. mbSP NPs containing 1 mg of DOX were dialyzed against 10 mL of PBS pH 7.4 with and without 10 mmol and 20 μM GSH at 37 °C at 120 rpm. The 1 mL of buffer was taken out at different time intervals and fresh 1 mL PBS was added to the dialysis medium to maintain sink condition. The collected samples were analysed by HPLC method. 2.5. Blood compatibility Blood compatibility of NPs was evaluated using hemolysis and coagulation study. Blood was obtained from blood bank of All India Institute of Medical Sciences (AIIMS), New Delhi, India.

The colloidal stability of NPs (mbSPD NPs) was determined in different media such as water, PBS 7.4, DMEM, DMEM supplemented with 10% FBS, PBS 7.4 with 10 mmol GSH and PBS 7.4 with 20 μM GSH for 6 h at 37 °C. Briefly 0.1 mL of 5 mg/mL solution of NPs was diluted with 3 mL of respective medium at 37 °C. The particle size and PDI of NPs was monitored at different time intervals using DLS. The GSH effect on disulfide linkages of NPs was evaluated by using DLS. 2.7. Protein adsorption study Protein adsorption on NPs surface was determined by using Biuret assay. In typical experiment, 5 mg of NPs were incubated with 4% of bovine serum albumin (BSA) solution in PBS 7.4 (10 mL) in incubator shaker over the period of 24 h at 100 rpm. The suspension was centrifuged at 25000 rpm for 30 min. Supernatant (1 mL) was taken and biuret reagent (4 mL) was added with further incubation at 37 °C for 30 min at 100 rpm. The unabsorbed protein in supernatant was analysed using UV–vis spectrophotometer (Biotek PowerWave XS2) at 540 nm. A standard calibration curve of known concentrations of BSA (0.5 to 2.5 mg/mL) was prepared for biuret test. 2.8. NPs-cells interactions NPs cell interactions were visualized by light microscopy using Prussian blue imaging method and fluorescence microscopy. L929, HeLa and MDA-MB-231 cells were seeded on sterile cover slips in 24 well plate at density of 3 × 104 in cell culture medium supplemented with 10% FBS and 1% penicillin-streptomycin as an antibiotic (DMEM was used for L929 and HeLa cells while Leibovitz's L-15 was used for MDA-MB-231 cells) in 5% CO2 atmosphere at 37 °C for 24 h. After 24 h, mbSPD NPs at concentration of 15 μg of Fe/mL or 4 μg of DOX/mL were incubated in


wells for next 12 h. In light microscopy experiment, Prussian blue staining technique was used to identify NPs-cells interactions. In typical process, after 12 h incubation of NPs, cells were washed with PBS three times and fixed with 3% glutaraldehyde for 10 min, washed and incubated with equal volume of 2% hydrochloric acid and 2% K4[Fe(CN)6]·3H2O for next 20 min. Cells were washed with water and counterstained with 0.5% nuclear red fast red for 2 min and washed. Coverslips were removed, air-dried, and images were taken at 40× in an Olympus IX73 microscope under bright light illumination. While for fluorescence imaging, nuclei of cells were stained with SlowFade® diamond antifade mountant with DAPI and DOX fluorescence was visualized under similar exposure time for all cell lines. Control experiment was performed for non-targeted mbSPD NPs while folic acid competition assay was performed by incubating cells with 1 mmol free folic acid for 1 h prior to addition of mbPSD NPs and fluorescence images were taken after 12 h incubation.

2.9. Radiofrequency (RF) induced hyperthermia 2.9.1. Capacitively coupled RF heating instrument and conditions for experiment As shown in Fig. 1, we used capacitive 13.56 MHz heating system (COMDEL, USA). The instrument comprised of Teflon platform in midplane between two insulated copper electrodes with spacing of 7.5 cm. The tuner was adjusted so that capacitance and impedance will be 113 and 194 respectively. System is comprised of match pro impedance matching network (MATCH PRO CPMX 2500, CODEL INC) which is further connected to RF power generator amplifier (CX1250S, fan cooled RF generator, COMDEL) through N-type cable. This matches the impedance between amplifier signals to the water cooled, solenoid antenna. The impedance was matched so that reflected RF waves were at minimum level by adjusting standing wave ratio (SWR) near to 1 during each RF exposure. The system has adjustable power output ranging from 10 W to 600 W and operated at fixed frequency of 13.56 MHz. All the RF assisted hyperthermia experiments were conducted at 50 W of RF generator power. For each RF exposure to samples the amplifier setting was kept at 50 W for 10 min. During RF exposure, unwanted heating due to elemental composition of culture medium was avoided by replacing media with 40% FBS in Milli Q water.

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2.9.2. Heating profile of NPs under selected RF conditions Heating ability of mbSPD NPs was evaluated under 50 W for 15 min. Specific absorption rate (SAR) is important parameter for hyperthermia treatment which provides a measure of rate at which energy is absorbed and converted into heat per unit time and mass of SPIONs. NPs suspension (0.5 mg/mL) was kept in 24 well plate between the electrodes and RF was applied, temperature change was determined by digital probe thermometer for every 1 min interval. Specific absorption rate was determined by following Eq. (4), SAR ¼

CVs dT m dt

ð4Þ

where, C is specific heat capacity of water (Cwater = 4.185 J/g °C), Vs is sample volume, m is mass of SPIONs, ðdT Þ is initial slope of time dependt dent temperature curve. 2.9.3. Anti-cancer efficacy of NPs under optimum RF conditions The cytotoxic effect of NPs, single or combined effect of hyperthermia and chemotherapy was evaluated on different cell lines such as L929, HeLa, and MDA-MB-231 for 24 h. Cytotoxic evaluation was done by using MTT assay which is a colorimetric test based on the formation of purple colored formazan crystals into viable cells. After reaching 90% confuency, cells were trypsinized and seeded at density of 104 cells/well with cell culture media supplemented with 10% FBS and 1% penicillinstreptomycin as an antibiotic (DMEM was used for L929 and HeLa cells while Leibovitz's L-15 was used for MDA-MB-231 cells) in flat bottom 96 well plate and incubated in CO2 incubator at 37 °C for 24 h. After 24 h, for evaluating the combined effect of hyperthermia and chemotherapy, mbSPD NPs were added to cells so that final concentration of NPs is in the range of 10–80 μg of Fe/mL and 2.5–21 μg of DOX/mL and incubated for 12 h. While for analysing hyperthermia and chemotherapeutic effect separately, mbSP NPs and mbD NPs were incubated with similar concentrations as described for combined effect. The biocompatibility of mb NPs and mbSP NPs without RF exposure was also analysed on same cell lines. After 12 h incubation, samples with media were removed from each well and further washed with PBS 7.4 thrice. After washing, 100 μL of special media containing 40% FBS in milli Q water was added in each well. The plate was kept in RF chamber and RF with set power of 50 W was applied for 10 min. All experiments were performed with SWR near to 1, so that reflected power was at minimum level. The plate was carefully removed and 40% FBS in milli Q water was replaced by fresh culture media and incubated for next 12 h. After 12 h, 10 μL of MTT solution (5 mg/mL in PBS 7.4) was added in each well and incubated for 4 h. The media with MTT solution was removed and 200 μL of DMSO was added to dissolve the formazan crystals. The plate was further incubated for 15 min at 37 °C. The absorbance readings were recorded using microplate spectrophotometer (PowerWave XS2, BioTek Instruments, USA) at 540 nm. Cell viability was calculated relative to negative control (PBS 7.4) and a positive control (1% Triton X-100) using the following Eq. (5), Cell viability ð%Þ ¼

sample540 nm −positive control540 nm negative control540 nm −positive control540

100 nm

ð5Þ

Fig. 1. (A) Prototype capacitive radiofrequency heating device. The metal chamber containing high-voltage matching circuits to produce radiofrequency fields. (B) Capacitive radiofrequency heating system where radiofrequency frequency is applied between two copper electrodes. (C) A 13.56 MHz signal is applied across Teflon coated layer electrodes which produces a high-voltage radiofrequency field (|E | = 15 kV m−1 at 600 W of radiofrequency generator power) over a variable air gap.

Cellular compatibility of this special media under RF exposure was checked with above mentioned cell lines at 50 W for 10 min, keeping negative control as cell culture media and positive control as 1% triton X-100. RF exposure was not provided for both control samples. Cell viability was calculated as per Eq. (5). The combined effect of hyperthermia and chemotherapy was evaluated using Valeriote's method [24]. Considering A, B and (A + B) as percent cell viability for different treatments, A (hyperthermia), B (chemotherapy), A + B (combined therapy of hyperthermia and chemotherapy). The combined effects were defined as follows: synergistic,

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Table 2 CHNS analysis of mb NPs and folic acid conjugated mb NPs using elemental analyzera. Elemental composition (%) Polymer

C

H

N

S

mbPLGA mbPLGA-FA

46.00 ± 1.32 48.95 ± 1.67

5.21 ± 1.45 6.44 ± 0.73

2.91 ± 1.53 3.21 ± 1.24

1.88 ± 1.19 1.91 ± 0.94

a

Mean ± SD, n = 3.

(A + B) b (A) × (B) / 100; additive, (A + B) = (A) × (B) / 100; subadditive, (A) × (B) / 100 b (A + B) b (A), if (A) b (B), interference, (A) b (A + B) b (B), if (A) b (B), antagonistic, (B) b (A + B), if (A) b (B). 2.10. Statistical analysis All in vitro data are expressed as mean ± standard deviation (SD). Statistical analysis was performed with graph pad prism software (Version 5, Graph pad software, U.S.A.) using one-way analysis of variance (one-way ANOVA) with Bonferroni multiple comparison test. Statistical significance was considered at p value b 0.05.

Fig. 3. XPS wide scan spectra of mb NPs, (A) after and (B) before folic acid conjugation. Stronger nitrogen 1s peak (400–402 eV) in folic acid conjugated nanoparticles than non-conjugated nanoparticles confirmed the successful folic acid conjugation.

3.1.1. Synthesis of multiblock copolymer based on PLGA-PEG-PLGA In the first step, synthesis of triblock copolymer PLGA-PEG-PLGA was carried out by ring opening polymerization of lactide and glycolide with PEG 200 as initiator and tin(II) octoate as catalyst. The successful synthesis of triblock copolymer was confirmed by 1H NMR, 13C NMR and GPC (Supporting information, Figs. S1, S2, S7). According to the 1H NMR spectrum, the presence of characteristic peaks at δ 1.50–1.84 and δ 4.80 corresponding to lactide methyl and methylene protons while peak at δ 5.22 corresponding to glycolide methylene units confirmed the synthesis of the triblock polymer. The PEG units in triblock copolymer were confirmed with multiplet broad peaks at δ 3.64 and δ 4.27 due to methylene units of PEG. The molecular weight was determined using 1H NMR and GPC. 1 H NMR (300 MHz, CDCl3, δ (ppm)): 1.50–1.84 (m, \\OCHCH3OCO\\), 3.64–3.74 (m, \\OCH2CH2O\\, PEG unit), 4.27 (m,

\\OCH2CH2O\\), 4.80 (m, \\OCHCH3OCO\\), 5.22 (m, HO\\CH2\\ OCO\\), Mn = 1409. 13 C NMR (75 MHz, CDCl3, δ (ppm)): 16.80 (\\OCHCH3OCO\\), 60.94 (HO\\CH2\\OCO\\), 64.70 (\\OCH2CH2O\\, PEG unit), 66.84 (\\OCH2CH2O\\, PEG unit), 70.68 (\\OCHCH3OCO\\), 167.11 (\\OCHCH3OCO\\), 169.56 (HO\\CH2\\OCO\\). GPC: Mn (GPC) = 994 Da, Mw (GPC) = 1452 Da, molecular weight distribution (MWD) = 1.46. FTIR: 3512, 2951, 1748, 1392 cm−1. In the second step, multiblock copolymer was synthesized by using triblock copolymer PLGA-PEG-PLGA, 2-hydroxyethyl disulfide and hexamethylene diisocyanate at molar ratio 1:0.29:1.18. The reaction was carried out in the presence of dibutyltin dilaurate at 80 °C in dry THF for 24 h. The multiblock copolymer was characterized by 1H NMR, 13 C NMR, FTIR and GPC. 1H NMR confirmed the synthesis of the multiblock copolymer (Supporting information, Fig. S2). Multiplet peaks at δ 1.35, δ 1.63 and δ 3.16 depicts the methylene units of hexamethylene diisocyanate in the multiblock copolymer backbone which forms urethane linkages in polymer chain. Triplet peaks at δ 2.92 are due to methylene groups attached to disulfide groups in 2hydroxyethyl disulfide, while triplet peaks at δ 3.16 is due to methylene groups of 2-hydroxyethyl disulfide. Thus 1H NMR confirmed the incorporation of disulfide linkages into the polymeric backbone. All the

Fig. 2. FTIR spectra of synthesized polymers (A) Triblock copolymer PLGA-PEG-PLGA, (B) Folic acid conjugated multiblock copolymer based on PLGA-PEG-PLGA (mbPLGA).

Fig. 4. Over lay of X-ray diffraction pattern for (A) mbSPD NPs, (B) mb NPs, DOX and SPIONs as physical mixture, (C) SPIONs, (D) mb NPs, (E) DOX.

3. Result and discussion 3.1. Synthesis and characterization of multiblock copolymers The synthesis of folate conjugated random multiblock copolymer based on PLGA-PEG-PLGA triblock copolymer is shown in Scheme 2.


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Fig. 5. (A) DLS of SPIONs and mbSPD NPs (n = 3), (B) TEM size of SPIONs (n = 200), (C) zeta potential of mbSPD NPs (n = 3).

other peaks corresponding to lactide, glycolide and polyethylene glycol were retained in the 1H NMR spectrum. The molecular weight of polymer was determined by 1H NMR and GPC (Supporting information, Figs. S3, S8). The polymer has low molecular weight distribution as 1.26. Thus the triblock copolymer PLGA-PEG-PLGA was repeated with intermediate disulfide and urethane linkages in polymer backbone. From 1H NMR spectrum it was found that 2-hydroxy ethyl disulfide has repeated thrice, thus each polymer chain has ~3 disulfide linkages. As shown in Table 2, appearance of sulfur in CHNS analysis of multiblock copolymer also confirmed the presence of disulfide linkages in polymer backbone. Further, in FTIR spectra, N\\H stretching vibrations was observed at 3337 cm−1 while urethane linkages in polymer backbone was confirmed by\\NH\\CO\\vibration band at 1531 cm−1. The vibration band at 1751 cm−1 confirmed the presence of ester linkages in the polymer chain as shown in Fig. 2. The synthesized multiblock copolymer can be self-assembled in NPs, after adding to aqueous phase, due to its hydrophilic-lipophilic balance in polymer chain. 1 H NMR (300 MHz, CDCl3, δ (ppm)): 1.35 (m, \\OCNHCH2 CH2CH2CH2CH2CH2NHCO\\), 1.46 (m, \\OCHCH3CO\\), 1.63 (m, \\OCNHCH2CH2CH2CH2CH2CH2NHCO\\), 2.92 (m, \\OCH2CH2\\SS \\CH2CH2OCO\\), 3.16 (m, \\OCNHCH2CH2CH2CH2CH2CH2NHCO\\ and\\OCH2CH2\\SS\\CH2CH2OCO), 3.69 (m, OCH2CH2O\\, PEG Unit), 4.31 (m, \\OCH2CH2O\\, PEG Unit) 4.68–4.91 (m, \\OCH2CO\\) 5.20–5.25 (m,\\OCHCH3CO\\). Mn = 8114 Da. 13 C NMR (75 MHz, CDCl3, δ (ppm)): 16.70 (\\OCHCH3CO\\), 25.55 (\\OCNHCH2CH2CH2CH2CH2CH2NHCO\\), 29.61 (\\OCH2CH2\\SS \\CH2CH2OCO\\), 37.75 (\\OCH2CH2\\SS\\CH2CH2OCO\\), 40.79 (\\OCNHCH2CH2CH2CH2CH2CH2NHCO\\), 60.42 (\\OCH2CH2\\SS

\\CH2CH2OCO\\), (\\OCH2CO\\), 67.56 (OCH2CH2O\\, PEG Unit) 70.51 (\\OCHCH3CO\\), 156.30 (\\OCNHCH2CH2CH2CH2CH2CH2 NHCO\\), 169.28 (\\OCH2CO\\), 175.45 (\\OCHCH3CO\\). GPC: Mn = 5339 Da, Mw = 6758 Da, MWD, 1.26. FTIR: 3337, 2937, 1751, 1531, 1393 cm−1. 3.2. Folic acid conjugation to multiblock copolymer Folic acid has been used as a targeting ligand for nanoparticle uptake in cancer cell lines. This ligand has been immensely explored as a targeting ligand for folate over expressed cell lines and tumors [25,26]. DCC-NHS coupling was used to conjugate folic acid to the terminal hydroxyl group of polymer backbone. γ-Carboxylic acid group of folic acid was activated by DCC-NHS and the activated folic acid was then reacted with terminal hydroxyl groups of the multiblock copolymer. The successful conjugation of folic acid to polymer was determined by different methods. As shown in Table 2, CHNS analysis showed the nitrogen content of folic acid conjugated polymer was increased by 1.11 fold. The increase in nitrogen content was also corroborated with XPS scan which showed increase in nitrogen signal intensity with folic acid conjugated NPs than non-conjugated NPs as depicted in Fig. 3. Folic acid non-conjugated NPs showed weak nitrogen signal (N1s) at a binding energy of 401 eV, whereas after folic acid conjugation, the nitrogen signal intensity was increased, indicating an increased nitrogen content on the surface of the NPs. This increased nitrogen signal intensity can be attributed to nitrogen from the folic acid molecules. 1H NMR of folic acid conjugated multiblock copolymer (Supporting information, Fig. S5) depicts characteristic peaks for folic acid, while UV visible spectroscopy

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Fig. 6. HRTEM images for (A and B) SPIONs, (C) mbD NPs (D) mbSPD NPs (E) Selected area of diffraction for SPIONs.

(Supporting information, Fig. S6) showed blue shift in absorption spectrum which confirms folic acid conjugation. Thus CHNS analysis, XPS and UV visible spectroscopy confirmed the successful conjugation of folic acid to polymer chain. 1 H NMR (300 MHz, CDCl3, δ (ppm)): 1.35 (m, \\OCNHCH2CH2CH2 CH2CH2CH2NHCO\\), 1.59 (m, \\OCHCH3CO\\). 1.73 (m, \\OCNHCH2CH2CH2CH2CH2CH2NHCO\\), 2.95 (m, \\OCH2CH2 \\SS\\CH2CH2OCO\\), 3.33 (m, \\OCNHCH2CH2CH2CH2CH2CH2 NHCO\\ and \\OCH2CH2\\SS\\CH2CH2OCO), 3.60 (m, OCH2CH2O\\, PEG Unit), 4.47 (m, \\OCH2CH2O\\, PEG Unit), 4.77–4.90 (m, \\OCH2CO\\) and (OCOCH2CH2CH(NH\\)COOH, Folic acid unit),

5.13–5.23 (m, \\OCHCH3CO\\), 2.18 (OCOCH2CH2CH(NH\\)COOH), 10.57 (s, OCOCH2CH2CH(NH\\)COOH), 6.63 (Ar\\CH), 7.48 (Ar\\CH). 3.3. Formulation and characterization of NPs 3.3.1. X-ray powder diffraction (XRD) studies XRD pattern was recorded on Rigaku D/max 2500 diffractometer (Rigaku, Japan) using Cu Kα radiation at scanning rate of 5°/min in the 2θ range 20–75° in continuous mode. XRD analysis of SPIONs reveals the γ-maghemite (γ-Fe2O3) structure showing broad peaks at dspacing values of (111), (220), (311), (400), (511) and (440) planes of


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Fig. 7. Characterization of NPs using, (A) SEM of mbSPD NPs, (B) AFM of SPIONs, (C) AFM of mbSPD NPs.

face-centered cubic (fcc) lattice of iron oxide. The lattice constant α = 0.82, matches to the reported standard data (JCPDS file 39-1346). The crystallite size of SPIONs was calculated using Scherrer equation, D = 0.9λ / βcosθ from the lower full-width-at-half maximum (FWHM) of (3 1 1) diffraction reflection, where D is average crystallite size, λ is x ray wavelength used (nm), β is Bragg's angle in degree; β is the excess line broadening (radiant). The crystallite size of γ-Fe2O3 NPs was found to be 7.8 nm (Supporting information, Fig. S11). Crystalline nature of SPIONs and solid state of doxorubicin in NPs was observed by XRD. As shown in Fig. 4, diffraction patterns for DOX, SPIONs, mb NPs, mbSP NPs, mbSPD NPs and mb NPs, DOX, SPIONs physical mixture were recorded. XRD pattern of DOX showed major crystalline peaks at 2θ of 20.56°, 21.94°, 22.55°, 23.40°, 25.03°, 26.27° and 30.03° which indicates crystalline nature of drug. Drug crystallinity was observed in physical mixture suggesting its crystalline state in physical mixture. While in XRD patterns of mbSPD NPs, characteristics peaks of DOX were not observed, indicating encapsulation of DOX in amorphous form inside NPs. Low crystallinity indicates, low lattice energy is required for drug solubilization. This low lattice energy in amorphous form helps the drug for its faster release and dissolution from NPs [27]. 3.3.2. Fourier transform infrared spectroscopy (FTIR) studies of SPIONs FTIR study was carried out to confirm the formation of iron oleate complex precursor and SPIONs formed after thermal treatment of iron

oleate complex. Iron oleate complex showed its typical characteristic vibrational frequencies at 1600, 1520, and 1443 cm−1, which are related to carboxylate groups. While bands at 1709 and 619 cm−1 corresponds to C_O and Fe\\O stretching vibrations respectively. Sharp bands at 2926 and 2854 cm−1 are attributed to\\CH2 asymmetric and symmetric stretching in oleic acid (Supporting information Fig. S12). Thus FTIR spectra confirm the formation of iron oleate complex precursor and SPIONs. 3.3.3. Dynamic light scattering (DLS) studies The size of SPIONs was measured by DLS and found to be 11.39 ± 1.23 nm with PDI 0.025 ± 0.56, while average size for mbSPD NPs was 172.03 ± 2.12 nm with PDI 0.09 ± 0.07. Zeta potential of mbSPD NPs was found to be −33.7 ± 0.78 mV, as shown in Fig. 5(A). The increase in size (compared to TEM and AFM) was due to contribution of solvation layer at the surface of NPs (hydrodynamic size). Thus NPs with small size and low PDI monodisperse in water phase without any aggregation. Moreover PEGylation and negative zeta potential keeps NPs away from RES system [28]. 3.3.4. High resolution transmission electron microscopy (HRTEM) studies HRTEM studies were conducted to observe the shape, size and morphology of NPs. Through HRTEM observations, SPIONs were found to be monodisperse in nature and spherical shape with average size of 9.2 ± 0.69 nm, measured for 200 particles (Figs. 5(B) and 6(A)). Atomic lattice

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magnetic content found with TGA studies is in good agreement with results found with AAS studies.

Fig. 8. Thermogravimetric analysis of (A) SPIONs, (B) mbSPD NPs.

fringes and selected area diffraction (SAED) pattern reveals high crystallinity of SPIONs, as depicted in Fig. 6(B, E). In case of HRTEM observation of mbSPD NPs, (Fig. 6(D)), SPIONs were successfully encapsulated in polymer NPs with polymer sheath of ~ 22 nm and size of these nanoclusters was ~130 nm. As shown in Fig. 6(C), mbD NPs have similar size observation as in case of mbSPD NPs.

3.3.5. Scanning electron microscope and atomic force microscopy studies (SEM and AFM) SEM showed monodispersed and uniform spherical morphology of mbSPD NPs with size ~ 140 nm, (Fig. 7(A)). Size and morphology was also studied using AFM, (Fig. 7(B,C)), and the results are consistent with observations obtained from TEM and SEM. Particle size analysis was done using NanoScope Analysis software v1.40r1 and size of SPIONs and mbSPD NPs was found to be 11 nm and 123 nm respectively.

3.3.6. Thermogravimetric analysis studies On the basis of TGA we can determine the magnetic content of NPs. As shown in Fig. 8, magnetic content of oleic acid coated SPIONs was found to be ~47%. This is due to presence of multilayer oleic acid ligands on SPIONs, while magnetic content for mbSPD NPs was reduced to ~13%, which is attributed to dense coating of mbPLGA polymer on SPIONs. The

3.3.7. Alternating gradient magnetometer studies (AGM) Magnetization studies was evaluated using AGM (Micromag 2900, from Princeton Measurement Corp.), as shown in Fig. 9, SPIONs showed high saturation magnetization of 72.28 emu/gm. This is attributed to annealing of SPIONs at 320 °C. The increased magnetization due to annealing of SPIONs at different temperatures has been reported earlier [29, 30]. High magnetization and crystallinity of SPIONs are ideal properties for hyperthermia treatment [31]. Saturation magnetization for mbSPD NPs was reduced to 25 emu/gm, this loss of magnetization was attributed to the introduction of thick mass of mbPLGA polymer over the surface of SPIONs. Magnetization curves for both SPIONs and mbSPD NPs showed complete reversibility at 300 K with no hysteresis. Hysteresis loop of SPIONs depicts typical superparamagnetic nature with negligible coercivity and no magnetic remanence as expected from their size [32]. Coating of SPIONs with mbPLGA polymer did not affect the superparamagnetic nature of SPIONs. 3.3.8. Loading and encapsulation studies Loading of SPIONs in mbSPD NPs and mbSP NPs was determined as per their Fe content using AAS and was found to be 15% and 16.5% with encapsulation efficiencies of 65% and 71.67% respectively. The high loading of SPIONs was due to hydrophobic oleic acid coating on SPIONs which can be easily encapsulated in hydrophobic mbPLGA polymer. DOX loading and encapsulation efficiency was determined using HPLC. The loading was found to be 4% and 6.9%, for mbSPD NPs and mbD NPs respectively. While encapsulation efficiency was found to be 59% and 69% for mbSPD NPs and mbD NPs. Decreased loading of DOX in case of mbSPD NPs was due to concurrent high SPIONs loading in NPs. 3.4. Stability studies of NPs Stability study of NPs is an important parameter with respect to clinical applications of NPs. The nanoformulation should have uniform dispersion in aqueous media without any agglomeration which helps in their easy cell penetration. It is important to study the stability in different media as NPs aggregation cause changes in cell-nanoparticle interactions [33]. The stability of mbSPD NPs was studied by measuring hydrodynamic size and PDI using DLS in different media such as water, PBS 7.4, DMEM media, DMEM with 5% FBS, PBS 7.4 with 10 mmol GSH and 20 μM GSH. The redox potential in cancer cells cytosol and nuclei contains 100–1000 times higher concentration of GSH than extracellular matrix and body fluids including blood (0.5–10 mmol versus 2–20 μM GSH). This high redox potential inside the cells is kept reduced by NADPH and glutathione reductase, which helps in triggered release of drug from redox sensitive NPs in this environment [11]. As shown in Fig. 10, NPs were found to be stable in all media retaining particle size below 200 nm with low PDI. This is due to the presence of polyethylene glycol units in NPs which provides “stealth” nature to NPs giving steric stabilization in aqueous media [34]. Size and PDI of NPs in 20 μM GSH did not change significantly, which reveals the stability of these NPs in biophase, while significant difference of size in 10 mmol GSH solution suggesting aggregation of NPs. This can be attributed to breakdown of s-s linkages between the polymer backbone and destabilization of NPs [35]. 3.5. In Vitro drug release

Fig. 9. Magnetization curves of (A) SPIONs, (B) mbSPD NPs.

DOX release behaviour form mbSPD NPs was investigated under different conditions such as, PBS 7.4 and PBS 7.4 with and without 10 mmol GSH and 20 μM GSH. As shown in Fig. 11, DOX cumulative percentage release at physiological pH 7.4 buffer showed maximum ~16% within 4 h with no significant release over 24 h. Similar observations were observed with 20 μM GSH condition with maximum release of


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Fig. 10. Stability study of mbSPD NPs in different media (Mean ± SD, n = 3. Where *** indicates p b 0.001).

~14% in first 4 h and no significant release over the next 24 h. The low concentration of GSH is present in blood and extracellular milieu (2–20 μM) [36,37] was not able to break disulfide linkages in polymer backbone, as discussed in Section 3.4. However in the presence of PBS pH 7.4 with 10 mmol GSH, which simulates the cytosol GSH concentration of cancerous cells, burst release of DOX ~65% was observed within 4 h with maximum release of ~85% over 24 h. This significant release of DOX is attributed to breakage of disulfide linkages in polymer backbone by reversible disulfide-thiol exchange reaction between disulfide bonds of polymer and thiol groups of GSH. Thus due to efficient breakage of disulfide groups, higher DOX release can be anticipated in cancer cells.

Fig. 11. Drug release profile of mbSPD NPs in PBS 7.4 with and without 20 μM GSH and 10 mM GSH (Mean ± SD, n = 3. Where *** indicates p b 0.001).

3.6. Hemocompatibility of NPs Blood contact with foreign intruders such as NPs can cause various haemolytic alterations in body. Blood compatibility is an essential criterion for safety and toxicity of NPs. Hemocompatibility of NPs was determined using hemolysis and coagulation study. 3.6.1. Hemolysis study The interaction of NPs with blood was studies by hemolysis study. NPs can interact with RBCs and cause damage which releases haemoglobin from erythrocytes. The released haemoglobin can be measured by spectrophotometer at wavelength of 540 nm. According to earlier studies, nanoformulation is considered to be nontoxic, if hemolysis value is b 20% [38,39]. The charge on the NPs surface and hydrophobicity plays key role in interaction with RBCs. These interactions can be reduced by decreasing charge density and increasing hydrophilic surface coating over NPs surface [40]. NPs with concentration range of 10–80 μg of Fe/mL and 2.5–21 μg of DOX/mL were incubated in RBC stock for 1 h at 37 °C. As depicted in Fig. 12, at all concentration range percent hemolysis was below 10%, indicating hemocompatibility of NPs. This can be attributed to the presence of polyethylene glycol chains and negative charge density over the surface of NPs [41]. 3.6.2. Coagulation study After entering into blood circulation NPs may interact with different coagulation factors and alter their function. These adverse reactions are called NPs induced coagulopathies, causing life threatening toxicities

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Fig. 13. Extent of protein adsorption for NPs (mean ± SD, n = 3). Fig. 12. Hemocompatibility of NPs (mean ± SD, n = 3. Where *** indicates p b 0.001).

such as deep vein thrombosis, intravascular thrombosis etc. which may lead to multiple organ failure and death [42]. The PT and aPTT is related to extrinsic and intrinsic coagulation pathway respectively. The normal range values for PT is 11–14 s, whereas for aPTT, it is 27–40 s. Any changes in these values are direct indication of nanoparticle induced coagulation pathway. mbSPD NPs in the range of 10–80 μg of Fe/mL and 2.5–21 μg of DOX/mL were incubated in blood for 1 h and measurement was done for PT and aPTT values. As shown in Table 3, there was no apparent change in PT and aPTT observed and all the results are within normal range, which shows NPs did not cause any interference with coagulation pathway. Thus NPs are found to be biocompatible with respect to blood coagulation system. 3.7. Protein adsorption study Protein adsorption is an important parameter to determine the stability and circulation half-life of NPs. The NPs with hydrophobic surface tend to adsorb blood proteins on their surface which leads to macrophage recognition and opsonisation leading to NPs elimination form blood circulation. Albumin is the most abundant protein in body and to simulate physiological blood condition, NPs were incubated in 4% of BSA solution for 24 h. As shown in Fig. 13, extent of protein adsorption for NPs was found below 15%. This low protein adsorption was attributed to PEG chain in NPs. The presence of PEG chain in polymeric NPs provides stealth nature, which helps to avoid opsonisation and protein adsorption on nanoparticle surface [43]. 3.8. NPs cells interactions To study the NPs cells interactions, fluorescence microscopy and light microscopy Prussian blue staining method was used. The uptake study was carried out on L929, a normal mouse fibroblast cell line which do not overexpress folate receptors on their surface [44,45] and Table 3 Coagulation studies of mbSPD NPs with respect to PT and aPTTa. NPs concentration (μg of Fe/mL)

PT (s)

Control 10 15 30 50 80

12.53 12.43 12.23 12.30 12.53 12.37

a

Mean ± SD, n = 3.

aPTT (s) ± ± ± ± ± ±

0.40 0.45 0.31 0.36 0.38 0.42

30.80 32.93 32.90 32.93 33.20 33.20

± ± ± ± ± ±

0.61 0.67 0.35 0.45 0.35 0.36

cell lines which overexpresses folate receptors such as HeLa, cervical cancer cell line [46,47] and MDA-MB-231, breast adenocarcinoma cell line [48,49]. Prussian blue staining, for cells showed higher uptake of mbSPD NPs in HeLa and MDA-MB-231 than L929 cells (Fig. 14(A)). This higher uptake is attributed to the receptor mediated endocytosis in folate receptor overexpressing cells. The observations obtained from Prussian blue staining are well corroborated with fluorescence imaging, as shown in Fig. 14(B), after 12 h incubation of mbSPD NPs, DOX fluorescence intensity in HeLa and MDA-MB-231 cells was found to be higher than L929 cells due to higher uptake of NPs into folate receptor overexpressed cells. Low DOX fluorescence intensity in L929 cells is attributed to nonspecific uptake of these NPs. Higher uptake of folate conjugated NPs in folate overexpressing cell lines than folate nonexpressing cells, has also been explored previously by our group [45]. Thus folate targeted NPs can efficiently target folate receptor overexpressing cancer cells. In control experiment (Supporting information Fig. S14) for non-targeted mbPSD NPs decrease in DOX fluorescence was observed. Similar observation was found in folate receptor binding competition assay (Supporting information Fig. S15), where cells were treated with free folic acid before culturing with mbPSD NPs. Thus results indicate that NPs uptake was mediated by folate receptors present on the cell surface [20]. 3.9. Anticancer efficacy of NPs under optimum RF conditions Heating of SPIONs under RF, depends upon their SAR value. High SAR value indicates efficient heating under RF. The mbSPD NPs showed high SAR value 404.67 W/g of SPIONs. As shown in SAR graph (Supporting information Fig. S13), therapeutic temperature for hyperthermia (42–46 °C) was reached within 10 min of RF exposure with 50 W power. The temperature increase in RF conditions is result of Neel and Brownian relaxation mechanism. Heat generated due to Brownian relaxation process is anticipated due to friction of SPIONs with surrounding medium while in case of Neel relaxation heat is generated due to fluctuations in directions of magnetic dipoles. SPIONs with size b30 nm acts as single domain system where Neel relaxation is higher than Brownian relaxation process in RF exposure [50]. The SPIONs with single domain system are ideal candidates for hyperthermia treatment, moreover high RF favours smaller particles for optimum rotation of their magnetic dipoles than larger particles to produce heat [51]. Anticancer efficacy for single and combined therapy of hyperthermia and chemotherapy was performed on L929, HeLa and MDA-MB-231 cell lines at optimum RF conditions, 50 W for 10 min, where temperature was raised to ~ 43 °C for hyperthermia treatment. The temperature in 96 well plate after RF treatment was determined using sterile digital probe thermometer, which showed increase in temperate range


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Fig. 14. Cellular uptake of mbSPD NPs in L929, HeLa and MDA-MB-231 Cells, (A) Prussian blue imaging using light microscopy after 12 h incubation of NPs, where iron is stained by Prussian blue and cells were counterstained with nuclear red fast at 40×. (B) Fluorescence microscopy images after 12 h incubation of NPs at 40×. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

(41–43 °C). RF exposure to cell culture media and PBS can lead to extra heat generation due to their elemental contents, as reported in previous studies [52,53]. Thus to avoid this excess heat during cellular experiments we have replaced cell culture media with 40% FBS in MilliQ water. This special media did not produce extra heat for 15 min under RF exposure at 50 W (Supporting information Fig. S13). We have conducted different trials and found that, viability of cells did not get affected when 40% FBS in Milli Q water was added to cells for 10 min as depicted in Fig. 15(E). The anticancer efficacy under RF conditions was carried out on L929, HeLa and MDA-MB-231. As shown in Fig. 15(A, B, C and D) cytotoxicity of blank NPs (mb NPs) with optimum RF exposure and mbSP NPs without RF exposure was analysed, both types of NPs showed no significant cytotoxicity indicating their biocompatible nature. In L929 cells all NPs showed percent viability N 80% indicating their non-toxicity and biocompatibility towards noncancerous cells and folate receptor non-expressing cells. As shown in Fig. 15(A), irrespective of the targeting ligand attached, mbSPD NPs and mbD NPs showed some toxicity at higher concentration of 50–80 μg/mL. This was attributed to nonspecific cellular uptake of NPs. Toxicity for mbD NPs was found to be greater than mbSP NPs, due to higher efficacy of DOX to kill cells than mbSP NPs after their RF treatment. In HeLa cells and MDA-MB-231 cells, mbSP NPs, mbD NPs and mbSPD NPs showed concentration dependant toxicity under RF treatment. In both cell lines, combined therapy of hyperthermia and chemotherapy (mbSPD NPs) have very significant cytotoxicity compared to individual hyperthermia and chemotherapy treatments. As shown in Fig. 15(B,C), both cell lines, HeLa and MDA-MB-231 showed cell death of 94% and

95% at higher concentration of combined therapy for mbSPD NPs (80 μg of Fe/mL and 21 μg of DOX/mL). This higher cell death in these folate receptor overexpressed cells was attributed to receptor mediated endocytosis of folate targeted NPs, which imparts combined toxicity of SPIONs under RF exposure and DOX. As shown in Fig. 15(E), only RF treatment control experiment on cells did not affect cell viability. Thus heat generated by SPIONs loaded formulation is the main cause of cell death under RF treatment. Analysis of combined treatment of hyperthermia and chemotherapy using Valeriote's method showed synergistic nature. 4. Conclusion In conclusion, the developed nanoformulation exhibited higher cellular uptake and cytotoxic potential in folate receptor overexpressing cancer cells with significantly reduced toxicity to normal cells. Moreover, triggered drug release from the nanocarriers in high redox potential of cancer cells could enhance their therapeutic potential. In short, the developed nanosystem can be utilized as a combinatorial therapeutic carrier in cancer therapy, achieving better pharmacological effect at lower dose of drug and minimising side effects of chemotherapy. Acknowledgments We are thankful to NRF (Nanoscale research Facility) at IIT Delhi for AFM and AGM studies. Mr. Chetan Nehate is thankful to Indian Institute of Technology Delhi, (Teaching assistantship for entry number 2012BMZ8114) India and Department of Biotechnology (DBT), India,

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Fig. 15. MTT assay of NPs on (A) L929, (B) HeLa and (C) MDA-MB-231 after RF treatment, (D) Viability of cells for mbSP NPs without RF treatment, (E) Viability of cells with only RF treatment (Mean ± SD, n = 3. Where, *** indicates p b 0.001, ** indicates p b 0.01, * indicates p b 0.05).

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