pH- and thermosensitive thin lipid layer coated mesoporous magnetic nanoassemblies as a dual drug delivery system towards thermochemotherapy of cancer.

Acta Biomaterialia xxx (2014) xxx–xxx

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pH- and thermosensitive thin lipid layer coated mesoporous magnetic nanoassemblies as a dual drug delivery system towards thermochemotherapy of cancer Lina Pradhan a,b, R. Srivastava c,⇑, D. Bahadur b,⇑ a b c

Centre for Research in Nanotechnology and Sciences, IIT Bombay, Mumbai 400076, India Department of Metallurgical Engineering and Materials Science, IIT Bombay, Mumbai 400076, India Department of Biosciences and Bioengineering, IIT Bombay, Mumbai 400076, India

a r t i c l e

i n f o

Article history: Received 25 July 2013 Received in revised form 27 March 2014 Accepted 10 April 2014 Available online xxxx Keywords: Mesoporous Nanoassemblies Dual drug Lipids Thermochemotherapy

a b s t r a c t A new pH-sensitive and thermosensitive dual drug delivery system consisting of thin lipid layer encapsulated mesoporous magnetite nanoassemblies (MMNA) has been developed which can deliver two anticancer drugs simultaneously. The formulation of lipid layer used is 5:2:2:2 w/w, DPPC: cholesterol:DSPE-PEG2000:MMNA. The structure, morphology and magnetic properties of MMNA and lipid coated MMNA (LMMNA) were thoroughly characterized. This hybrid system was investigated for its ability to carry two anticancer drugs as well as its ability to provide heat under an alternating current magnetic field (ACMF). A very high loading efficiency of up to 81% of doxorubicin hydrochloride (DOX) with an 0.02 mg mg1 loading capacity and 60% of paclitaxel (TXL) with an 0.03 mg mg1 loading capacity are obtained with LMMNA. A sustained release of drug is observed over a period of 172 h, with better release, of 88:53% (DOX:TXL), at pH 4.3 compared to the 28:26% (DOX:TXL) in physiological conditions (pH 7.4). An enhanced release of 72 and 68% is recorded for DOX and TXL, respectively, during the first hour with the application of an ACMF (43 °C). A greater in vitro cytotoxic effect is observed with the two drugs compared to them individually in HeLa, MCF-7 and HepG2 cancer cells. With the application of an ACMF for 10 min, the cell killing efficiency is improved substantially due to simultaneous thermo- and chemotherapy. Confocal microscopy confirms the internalization of drug loaded MMNA and LMMNA by cells and their morphological changes during thermochemotherapy. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction During the past decade, dual drug delivery systems (DDDS) have invoked remarkable interest for use in a broad range of therapeutic applications. This is because certain limitations, like poor solubility of hydrophobic drugs [1,2], limited loading capacity of drug and poor colloidal stability, can be overcome by using DDDS, while they also have other advantages, including biodegradability [3], the minimization of side effects [4–7] and increased circulation time in vivo [8,9]. Researchers have investigated a number of DDDS, including poly(lactic-co-glycolic acid) (PLGA)–mesoporous silica nanoparticles [10], chitosan-containing PLGA nanoparticles [11], polymersomes [12], lipid–polymer hybrids [13], polymeric magnetic nanoparticles [14], nanoparticle–aptamer bioconjugates [1], magnetic mesoporous silica nanoparticles [15] and mesopor⇑ Corresponding authors. Tel.: +91 22 2576 7746 (R. Srivastava). Tel.: +91 22 2576 7632 (D. Bahadur). E-mail addresses: [email protected] (R. Srivastava), [email protected] (D. Bahadur).

ous silica nanoparticles [16]. It has also been recently shown that the combination of two drugs shows synergistic effects, prevents more disease recurrence [1,17,18] and increases tumor regression capabilities compared to individual drugs in clinical studies [19,20]. Additionally, there are reports on synergistic effects towards cancer treatment using chemotherapy (using a single drug) along with thermal therapy [5,21]. It is known that drug release can be triggered by external stimuli, such as an AC magnetic field, an electric field, ultrasound, temperature and pH [21– 24], which can enhance the efficacy of simultaneously released drugs in a synergistic manner. Superparamagnetic particles under an applied alternating current magnetic field (ACMF) can produce local heat that may further enhance drug release from its carrier. In addition, the heat so produced can simultaneously help in more cancer cell death due to thermal therapy. Thus, treatment with drugs in conjunction with heat can act as a combinatorial thermochemotherapy. With thermochemotherapy in mind, we have designed a new drug delivery system comprising a thin lipid layer encapsulating mesoporous magnetite (Fe3O4) nanoassemblies

http://dx.doi.org/10.1016/j.actbio.2014.04.011 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Pradhan L et al. pH- and thermosensitive thin lipid layer coated mesoporous magnetic nanoassemblies as a dual drug delivery system towards thermochemotherapy of cancer. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.04.011

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L. Pradhan et al. / Acta Biomaterialia xxx (2014) xxx–xxx

(MMNA) and demonstrated its use as a dual drug carrier where the drug release can be triggered by a change either in pH or in temperature by the application of an ACMF (Fig. 1). The thin lipid layer is advantageous for drug delivery in that it: (i) prevents the agglomeration of particles; (ii) increases colloidal stability; (iii) is able to encapsulate both hydrophilic and hydrophobic drugs; and (iv) can control the drug release efficiency. The MMNA is encapsulated with a lipid formulation consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol (Chol) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[amino (polyethylene glycol)-2000] (DSPE-PEG2000). The formulation of LMMNA is made such that it is sensitive to both temperature and pH. Moreover, the functional PEG groups of DSPE-PEG2000 increase the circulation time and stability. The lipid layer protects the surface, improves the biocompatibility and prevents the agglomeration of MMNA in its aqueous phase [4,5,9]. These nanoassemblies, by virtue of their thermo- and pH sensitivity, may be used as stimuli responsive drug release systems in tumor tissue. In addition, these may be investigated not only as dual dug carriers but also as dual therapeutic agents [25–27]. They also have the potential to be used as contrast agents in magnetic resonance imaging [28]. 2. Materials and methods Iron(III) chloride hexahydrate (FeCl36H2O) and iron(II) chloride tetrahydrate (FeCl24H2O) were purchased from Sigma–Aldrich. Ethylene glycol (EG), ethylenediamine (EDA) and anhydrous ethanol were from Merck and sodium acetate was from Himedia. Doxorubicin hydrochloride (DOX) and paclitaxel (TXL) were obtained from Sigma Aldrich. DPPC, Chol, (DSPE-PEG2000) and N-(NBD-aminohexanoyl)-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine sodium salt (N-NBD) were purchased from Sigma Aldrich. MilliQ water was used in all the experiments. 2.1. Synthesis of aqueous stable MMNA Aqueous stable MMNA were prepared by a solvothermal method [2,3,29]. In brief, 1 g of FeCl36H2O and FeCl24H2O (2:1

wt ratio) was dissolved in 30 ml of EG. The mixture was stirred vigorously for 1 h at 80 °C to obtain a homogeneous solution, followed by the addition of 2 g of sodium acetate and 7 ml of ethylenediamine (EDA). The solution temperature was slowly increased and maintained at 160 °C for 1 h. The temperature was then raised up to 180 °C and maintained for 6 h, before being allowed to cool down to room temperature. The solids were separated from the black solutions with a magnet and washed several times with water and ethanol (50:50 v/v) simultaneously. 2.2. Characterization X-ray diffraction (XRD) analysis was carried out on a Philips 40 powder diffractometer PW3040/60 with Cu Ka (1.5406 Å) radiation. The surface charges of MMNA and LMMNA were measured using a zeta plus zeta potential analyzer (Brookhaven Instruments) at room temperature. The size and morphology of the MMNA were analyzed using high-resolution transmission electron microscopy (JEOL JAM- 2100F, 200 kV). Further, the surface morphology of LMMNA was characterized by atomic force microscopy (AFM; Digital Instruments, Nanoscope IV). The magnetic properties of MMNA and LMMNA were carried out using a vibrating sample magnetometer (VSM, Model 7410, Lake Shore) at room temperature. The Brunauer–Emmett–Teller (BET) surface area of the MMNA was measured on an ASAP 2020 analyzer (Micromeritics, USA). The thermal ability of MMNA and LMMNA was measured by applying an ACMF (EASY HEAT, EZLI5060, Ambrell, UK). The transmission electron microscopy (TEM) image was collected with an FEI Tecnai G2 BioTwin D312 microscope. Laser scanning confocal microscopy images were recorded using an Olympus Model IX 81 inverted confocal microscope. Flow cytometry analysis was carried out using BD FACSAria instrument. 2.3. Preparation of DOX loaded MMNA To prepare the MMNA loaded with the anticancer drug DOX, a total of 100 mg of MMNA particles was dispersed in 5 ml of MilliQ water and sonicated for 10 min. Different amounts (0.5, 1, 2, 4, 6, 8 and 10 mg ml1 concentrations) of MMNA particles were then prepared from the stock (100 mg in 5 ml) in Eppendorf tubes (all the

Fig. 1. The overall concept of the present study: the pH-sensitive and thermosensitive LMMNA is a dual drug delivery system containing the drugs doxorubicin (DOX, in mesopores) and paclitaxel (TXL, in lipid layer). Drug release is triggered by an ACMF applied to the tumor cells. In this paper, the formulation and in vitro characterization of dual drug loaded LMMNA with an ACMF are reported for the thermochemotherapy of cancer.



loading was done in triplicate) with serial dilutions and sonicated for 2 min. A fixed amount of DOX (50 lg ml1) was added to each suspension of MMNA. After shaking overnight in a water bath at room temperature, the drug loaded MMNA (termed DOX-MMNA) were separated from the free drug molecules by centrifugation at 6720g for 10 min. The drug loaded particles were stored at 4 °C until further use. The concentration of drug in the supernatant was measured from the fluorescence spectra of DOX using a calibration curve with a dilution series. The amount of drug incorporated into the MMNA was estimated by subtracting the amount of supernatant having free drug from the total amount of drug initially added. The percentage of entrapment efficiency was calculated using the following relation [30,31]:

% entreapment efficiency ¼

Ic Sc 100 Ic

ð1Þ

where Ic and Sc are respectively the initial concentration of DOX added and the DOX content of the supernatant. 2.4. Preparation of TXL encapsulated thermosensitive LMMNA LMMNA was prepared by the thin film hydration method [30–32]. Different compositions of phospholipids (DPPC, Chol and DSPE-PEG2000) were used for a thin layer supported on the surface of MMNA. The thin lipid layer consisting of DPPC/Chol/DSPEPEG2000 (5:2:2 w/w) were prepared by dissolving 5 mg of DPPC, 2 mg of cholesterol and 2 mg of DSPE-PEG2000 in 3 ml of chloroform/methanol (2:1 v/v) in a round bottom flask. In a typical synthesis of lipid covered DOX-MMNA (labeled DOX-LMMNA), 2 mg of preloaded DOX-MMNA (DOX 40 lg) was added to the preheated lipid solutions and fully mixed. This was then heated at 50 °C for 5 min to ensure that all the lipids were in liquid phase. The organic solvent was removed by a rotary evaporator and the sample further dried under a vacuum overnight to remove any trace solvent remaining. The dried lipid films were hydrated with phosphate-buffered saline (PBS, pH 7.4; Hi-Media) at 45 °C for 10 min. In order to prepare the dual drug encapsulated LMMNA, TXL (100 lg) was added during the solution phase of lipid formation over DOX-MMNA. A similar procedure was followed after the addition of TXL. The unencapsulated TXL with the extra lipids in suspension was separated by a magnet. The encapsulation efficiency of DOX and TXL in LMMNA (termed DOX:TXL-LMMNA) was determined using a UV–visible spectrophotometer (Super Aurius CE3021, Cecil Instruments, UK), following the addition of 1% Triton X-100, at wavelengths of 490 and 230 nm. A calibration curve was plotted (not shown) with a serial dilution of known concentrations of DOX and TXL in 1% Triton X-100 in PBS for quantification of unknown drug amounts. The encapsulation efficiency of drugs was calculated based on the ratio of the amount of drug encapsulated in the LMMNA (final formulation ratio 5:2:2:2 w/w, with 5 mg of DPPC, 2 mg of Chol, 2 mg of DSPE-PEG2000 and 2 mg of preloaded DOX-MMNA) to the initially amount of drug added during the loading process [30,33]. The loading capacity of drug in LMMNA was calculated using following relation:

Loading capacity ¼

amount of drug incorporated in LMMNAðmgÞ LMMNA massðmgÞ ð2Þ

2.5. pH-sensitive and thermosensitive dual drug (DOX-TXL) release Drug release studies were carried out at different pHs such as physiological (pH 7.4) and acidic (pH 4.3). Four milligrams of dual drug (DOX and TXL) loaded LMMNA was dispersed in each of 1 ml of PBS (pH 7.4) and 1 ml of acetate buffer (pH 4.3). The dispersed

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solution was loaded into two different dialysis membrane bags (Himedia, 150, LA401). Each of the bags was then kept separately in 20 ml of PBS solution and gently stirred at room temperature. At different time intervals 1 ml of the release (PBS) solution was taken out and replaced with 1 ml of fresh PBS to maintain a constant volume. The amount of released DOX and TXL was quantified with UV–visible absorption at wavelengths of 490 and 230 nm, respectively. The absorption spectra of DOX and TXL were scanned over a wide range, from 200 to 600 nm. For thermosensitive drug release, 2 mg of DOX:TXL loaded LMMNA was dispersed in 2 ml of PBS in Eppendorf tubes and kept at 37 and 43 °C in separate water baths. In another set of experiment, the samples were kept under an ACMF at temperatures of 37 (175 Oe, 250 kHz) and 43 °C (293 Oe, 250 kHz). Every 5 min, 200 ll of the release solution was collected and replaced with 200 ll of fresh preheated PBS to maintain the volume. The absorption of the samples was measured using a UV–visible spectrophotometer to calculate the amount of drugs released (DOX:TXL). 2.6. Measurement of heating ability of MMNA and LMMNA by the specific absorption rate (SAR) The heating ability of MMNA and LMMNA was measured by the time-dependent SAR method. A sample (0.5–1 mg), dispersed in 1 ml of MilliQ water in an Eppendorf tube, was placed at the center of a 6 cm diameter (four turns) coil connected to the measuring instrument. Samples were heated for 600 s with a desired current (450 A) at 250 kHz and the increase in temperature was recorded every minute. The SAR value was calculated using the following formula:

SAR ¼ C

DT 1 Dt mmag

ð3Þ

where C is the specific heat capacity of water (Cwater = 4.186 J g1 K1), DT/Dt is the initial slope of the time-dependent temperature curve and mmag is the mass fraction of magnetic material present in the sample [34]. 2.7. Cell culture Human cervical cancer (HeLa), breast cancer (MCF-7) and human hepatocellular carcinoma (HepG2) cell lines were obtained from the National Centre for Cell Sciences, Pune, India. All of the cell types were cultured in minimal essential medium (MEM; HiMedia, Mumbai, India) supplemented with 10% fetal bovine serum (Hi-Media) and 1% antibiotic antimycotic solution (Hi-Media) in a humidified incubator at 37 °C, 5% CO2. 2.7.1. Cytotoxicity study with DOX:TXL loaded LMMNA compared to DOX loaded MMNA Cytotoxicity was determined by Sulforhodamine B (SRB; Sigma Aldrich) assay [35]. HeLa, MCF-7 and HepG2 cancer cells were seeded at a density of 1 104 cells in 200 ll MEM per well in 96-well microtiter plates. After 24 h of incubation, DOX loaded MMNA and DOX:TXL loaded LMMNA samples (0.03–1 mg ml1) were added to cells, with a further set of cells kept blank as controls. The plates were incubated for a further 24 h in the humidified incubator at 37 °C, 5% CO2. The cells were washed thrice with PBS and fixed with 100 ll of 10% trichloroacetic acid for 1 h at 4 °C. The plate was washed slowly with water and dried with an air drier. Thereafter, 100 ll of 0.06% SRB (prepared in PBS) was added to the cells, which were then incubated for 30 min at room temperature. The cells were washed thoroughly with 1% acetic acid to dissolve the unbound dye. The live cell-bounded dye was extracted with 10 mM Tris buffer solution (pH 10.5) and kept for 20 min at


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room temperature. The cell viability was measured from the absorbance of the SRB bound to the cells (kabs = 560 nm) using a plate reader. Cell viability (%) was calculated using the following formula:

% cell viability ¼

absorbance of treated sample 100 absorbance of control

ð4Þ

2.7.2. Intracellular localization study of LMMNA by TEM The HeLa cells treated with LMMNA were thoroughly washed with PBS and fixed with 1 ml of 2.5% glutaraldehyde for 1 h at room temperature. The pellet was rinsed with PBS three times and fixed with 100 ll of 1% osmium tetroxide solution for 1 h, then rinsed with PBS a further five times followed by washing with water twice. The rinsed pellet was then stained with 1% aqueous uranyl acetate for 1 h to increase the fine contrast and any excess uranyl acetate was washed off with water. Next, the cells were dehydrated in a graded series of ethanol (50, 70 and 90%, for 10 min each) and finally the cells were kept in 100% ethanol overnight. The following day, the ethanol was removed and 1 ml of 100% propylene oxide was added, left for 1 h and removed. A 500 ll volume of a 50:50 mixture of propylene oxide and resin was added to the pellet and kept overnight. The resin was then polymerized at 60 °C for 48 h and any extra propylene oxide was evaporated. Ultrathin sections were prepared using an ultramicrotome and placed on a grid [36]. 2.7.3. Confocal microscopy The effect of drug loaded LMMNA (tagged with N-NBD) on HeLa, MCF-7 and HepG2 cells was determined by confocal images. For imaging, cells were seeded on cover slips on a 30 mm plate with 1 ml of MEM medium and incubated in 37 °C. After 24 h incubation, the cells were treated with or without samples at a concentration of 0.5 mg ml1. The treated and untreated cells were washed twice with PBS. The adhered cells on the cover slips were fixed with 4% paraformaldehyde for 10 min at room temperature and later washed with PBS. Finally, nucleus was stained by 40 , 6-diamidino-2-phenylindole (DAPI; Sigma Aldrich) for 10 min at room temperature. The extra DAPI was washed with PBS from cover slip. Further, cover slip containing cells was mounted over a clean glass slide. The samples were observed by confocal microscopy [37]. 2.7.4. In vitro hyperthermia study with dual drug loaded LMMNA under an ACMF The synergistic cytotoxic effects (thermochemotherapy) of dual drug loaded LMMNA were evaluated with HeLa, MCF-7, and HepG2 cells in the absence and presence of an ACMF. For this study, cells (1 106) were seeded with 1 ml of complete (10% v/v Fetal Bovine Serum and 1% v/v penicillin-Streptomycin mixture) MEM medium in a 30 mm Petri dish and incubated overnight. The medium was then discarded and a 0.5 mg ml-1 concentration of DOX:TXL loaded LMMNA sample was added to the cells with complete MEM medium. Control (untreated) and treated cells were incubated for 6 h in a humidified incubator at 37 °C. One set (in triplicate) of plates containing treated adherent cells was completely enclosed in parafilm and exposed to an ACMF (293 Oe, 250 kHz) for 5 and 10 min at 43 °C under sterile conditions. Another set (in triplicate) of treated plates was kept in the incubator but not subjected to an ACMF. After field exposure, the plates were transferred back to the incubator and left overnight. After incubation, the control and treated cells were collected by trypsinization. Cell viability was determined using trypan blue dye exclusion [5,32]. 2.7.5. Cell cycle distribution analysis by flow cytometry The cell cycle was studied by flow cytometry with propidium iodide (PI; Sigma Aldrich). HeLa, MCF-7 and HepG2 cells were cultured in MEM supplemented with 10% fetal bovine serum and

were maintained in a humidified incubator. For the cell cycle assays, all cells were seeded in 30 mm Petri plates and allowed to attach for 24 h. After 24 h of incubation, 0.5 mg ml1 DOX:TXL loaded LMMNA sample in fresh medium was added to the cells. The plate of adhered cells was then allowed to incubate for 6 h before being placed under an ACMF, transferred again to the incubator and left overnight. In the next step, the plate was washed with PBS four times and the cells were collected by the trypsinization method. The collected cells were fixed with 1 ml of 70% chilled ethanol and incubated for 1 h at 4 °C. Later, the cells were washed with PBS and resuspended in a staining buffer containing 100 lg ml1 of PI (Sigma) and 10 lg ml1 of RNAase A (Sigma). The cell suspensions were incubated for 30 min at 37 °C in the dark. The DNA content of the cells was measured using a flow cytometer and 10,000 events were analyzed for each group. The percentage of cells in each cell cycle phase was determined using Flowjo software. 2.7.6. Statistical analysis Each experimental value was expressed as the mean ± standard deviation of the mean (SD) of results from three independent experiments at three different times. An unpaired, two-tailed t-test was used to evaluate significant differences between a pair of groups. Levels of significance were p < 0.05 (⁄), p < 0.01 (⁄⁄) and p < 0.001 (⁄⁄⁄). The statistical analysis was completed using GraphPad Prism 6 software. 3. Results and discussion 3.1. XRD and zeta potential The XRD patterns of the MMNA and LMMNA samples are shown in the supporting information (ESI Fig. S1). The average crystallite size of the magnetite nanoparticles estimated by the Scherer formula is found to be 9 nm. The zeta potential values of MMNA and LMMNA were measured to be 35.6 and 23.65 mV, respectively, at physiological pH (7.4). A more negatively charged MMNA is suitable for efficient drug (DOX, which has NH+3 group) conjugation. MMNA functionalized with lipid protects the surface of the MMNA and decreases the number of negative charges on LMMNA. This suggests that there is good colloidal stability at pH 7.4. Moreover, a previous report states that the negative surface charge, unlike positively charged particles, effectively avoids opsonization, a process whereby plasma immunoproteins bind to the particle surface, leading to recognition by monocytes and tissue specific macrophages that clears the nanoparticles from the circulating blood [38]. Thus the anionic surface charge may increase the particle circulation time. 3.2. TEM, AFM, magnetization and porosity studies Fig. 2A–E shows the TEM images of MMNA and LMMNA and their size distribution. Fig. 2A shows spherically shaped particles, which are aggregates of small nanoparticles of Fe3O4. After mixing the lipids with MMNA, the particles increase in aggregate size, as shown in Fig. 2B. From Fig. 2C (a magnified image of a single LMMNA), there is evidence of a thin layer over the MMNA which confirms the lipid coating (indicated with yellow arrows). Fig. 2(D and E) show the average assembly size to be 73 and 92 nm for MMNA and LMMNA, respectively. The increase in assembly size up to 92 nm from 73 nm with the lipid coating suggests the approximate thickness of the lipid layer to be 20 nm. In this case, the average size of LMMNA increased with inceasing aggregate size, confirming the successful immobilization of the lipid coating. Further, the surface morphology and the size of LMMNA were observed by AFM images (see ESI Fig. S2). The



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Fig. 2. TEM images of (A) MMNA (inset magnified image) and (B) LMMNA; (C) magnified image of a single LMMNA shows the thin lipid layer (marked with yellow arrows) on the surface of MMNA; (D, E) size distribution of MMNA and LMMNA.

magnetization of MMNA and LMMNA was measured by VSM (see ESI) and the BET surface area of MMNA was observed by ASAP 2020 analyzer (see ESI). Fig. 3A–D shows the TEM images of MMNA in acidic conditions (pH 4.3) after 0, 24 and 48 h respectively. After 24 h, the MMNA starts to disassemble (Fig. 3B) and degrade. The degradation is clear after 48 h, as shown in Fig. 3(C and D) and ESI Fig. S3. With the increase of time, the MMNA may fully degrade in mild acidic condition. The degradation of particles is important for a drug delivery system, pH dependent drug release and their use in other biological applications [3,39].

liquid media and increases the heat generation properties. Subjecting the magnetic nanoparticles to an ACMF results in heat generation by virtue of the Neel and Brownian relaxation mechanisms. The Brownian relaxation requires free rotation of nanoparticles, which is enhanced due to greater dispersibility and stability provided by the lipid coating [41]. Thus, both the dispersion and stability properties of the magnetic nanoparticles are responsible for the higher heating rate. Therefore, it was possible to achieve a hyperthermic temperature with a much lower concentration (0.5 mg ml1) of LMMNA than reported previously (40 mg ml1 [5] and 48 and 80 mg ml1 [46]).

3.3. Evaluation on heating ability of MMNA and LMMNA

3.4. Loading behavior of DOX into MNNA and DOX:TXL within LMMNA

Fig. 4 shows the time-dependent heating efficiency of superparamagnetic bare MMNA and LMMNA under an ACMF (376 Oe, 250 kHz). The heating ability is estimated based on the SAR. The SAR depends on a number of parameters, including the magnetic field strength, frequency (both were kept fixed in our case), concentration and suspension stability in aqueous medium [40–43]. The concentration of 0.5 mg ml1 of bare MMNA could not attain 43 °C in 600 s, while the same concentration of LMMNA shows that a hyperthermic temperature of 43 °C was reached within the same time (Fig. 4). The SAR values of MMNA and LMMNA were estimated from the initial slopes (not shown) of the heating curves, and the observed values were 169.5 and 192 W g1 for 0.5 and 1 mg ml1 MMNA, respectively. These corresponding values for LMMNA were 303.4 and 1046 W g1 for 0.5 and 1 mg ml1, respectively. From this figure, it can be seen that the lower concentration of LMMNA exhibits better heating capacity compared to MMNA. The higher SAR values for LMMNA [43,44] are possibly because of the better suspension stability of the lipid modified MMNA in an aqueous environment [45]. The PEG functional group of the lipid layer enhances the dispersibility and stability of LMMNA in

Owing to its good aqueous dispersibility and its mesoporous nature (for details, see ESI), MMNA can be used to carry high payloads of hydrophilic drugs. The ionized form of DOX is more hydrophilic due to its NH+3 group, which also helps the efficient conjugation of DOX with MMNA. Fig. 5(A and B) shows the entrapment of DOX (hydrophilic as well as fluorescent) in MMNA. Fig. 5A clearly shows the sequential decrease in fluorescence intensity of DOX with increasing amount of MMNA. This is due to the increasing amount of DOX entrapment with increasing MMNA, which leads to decreasing fluorescence intensity in the supernatant. Fig. 5B shows the entrapment (loading) efficiency of DOX (50 lg in each case) in different concentrations of MMNA, with a maximum efficiency of 99% (49 lg) being obtained at 10 mg ml1. Such a high entrapment efficiency may be attributed to the porous nature of MMNA, which gives it a large surface area. In addition, the particle surface is negatively charged and hence the particles readily and efficiently attract positively charged DOX due to electrostatic interaction. Fig. 5C shows the encapsulation of DOX and TXL in LMMNA. During the experiment, DOX loaded MMNA were encapsulated with lipids and then instantaneously mixed


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Fig. 3. TEM images of time-dependent acid-degradable MMNA at pH 4.3 after (A) 0 h, (B) 24 h and (C) 48 h; (D) magnified image (red circle) of (C) showing the disintigration of MMNA after 48 h.

Fig. 4. Temperature vs. time graphs for the suspensions with different concentrations of MMNA and LMMNA under an applied ACMF (250 kHz, 376 Oe) for 10 min. The results are expressed as mean ± SD (n = 3).

with TXL for entrapment with the lipids on the surface of the MMNA. The encapsulation efficiencies of DOX and TXL in LMMNA are 81 ± 2 and 60 ± 3%, respectively. The loading capacities of DOX and TXL are 0.02 ± 0.0004 and 0.03 ± 0.0018 mg mg1, respectively. This indicates that the thin layer acts as a molecular hedge that helps retain the drugs inside the layer and maintain a high encapsulation efficiency of dual drug in LMMNA [47].

conducted in a water bath (WB) and under an ACMF at 37 °C (physiological temperature) and 43 °C (hyperthermic temperature) at pH 7.4. Fig. 6A shows the cumulative release curves of DOX:TXL at pH 7.4 and 4.3. During 24 h, 28% of DOX and 17% TXL was released from LMMNA at pH 4.3, and this significantly increases up to 65% (DOX, p < 0.05) and 37% (TXL, p < 0.05) during the next 30 h. The cumulative release of both drugs significantly increased, up to 88% (DOX, p < 0.01) and 53% (TXL, p < 0.01), at pH 4.3 over a period of 172 h. However, at pH 7.4, much lower cumulative release of these drugs, of 28 ± 2% (DOX) and 26 ± 3% (TXL), is observed during the same period. These results suggest that more drugs are released if the pH is decreased because of increased protonation of –NH2 groups on DOX from degradation of MMNA and release of TXL through the permeable membrane of the lipid layers at acidic pH [48,49]. Fig. 6B and C show the comparative cumulative release profiles of DOX and TXL, respectively, in a WB and under ACMF conditions at physiological (37 °C) and hyperthermic (43 °C) temperatures during 1 h at physiological pH. For both drugs, there is a clear indication of significantly higher release (p < 0.05) under an ACMF. Thus, these results suggest that the applied ACMF helps the diffusion of drugs through bond breaking by the strong mechanical force and disruption of the soft thin layer membrane of LMMNA due to local heat generated by the magnetic particles under the AC field, thereby enhancing the drug release from LMMNA [23,39,50,51]. Moreover, the heat thus generated enhanced the effective cellular death of tumor cells due to the synergistic effect of thermochemotherapy. 3.6. In vitro cytotoxicity studies

3.5. Drug release behavior of DOX and TXL from LMMNA We have examined the in vitro release profiles of drugs (DOX:TXL) from LMMNA at physiological pH (7.4) and at acidic pH (4.3; a tumoral environment). The release studies were also

The cytotoxicity of bare MMNA, DOX-LMMNA and DOX:TXLLMMNA was evaluated by SRB assays in HeLa, MCF-7 and HepG2 cancer cell lines. Fig. 7A–C shows the cytotoxicity effect with different concentrations of individual (DOX) and dual (DOX and



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Fig. 5. (A) Fluorescence spectra of DOX solution (50 lg in 1 ml solution), which decrease (i–vii) with increasing amounts of MMNA (0.5, 1, 2, 4, 6, 8 and 10 mg ml1); (B) the entrapment efficiency (%) of DOX into MMNA and (C) the encapsulation efficiency (%) with loading capacity (mg mg1) of DOXand TXL in LMMNA (5:2:2:2 ratio). The results are expressed as mean ± SD (n = 3).

Fig. 6. Cumulative release profiles of DOX and TXL from LMMNA (A) depending on pH (7.4 and 4.3), and (B, C) at different temperatures (37 and 43 °C) in a WB and with an applied ACMF for 1 h (pH 7.4). The differences in DOX and TXL release at pH 4.3 (at 50 and 172 h intervals) are significant at ⁄p < 0.05 and ⁄⁄p < 0.01 as determined by anunpaired, two-tailed t-test. The differences in DOX and TXL release between the WB and the AC field at 30 and 60 min (43 °C) intervals are significant at ⁄p < 0.05,⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001. The results are expressed as mean ± SD (n = 3).

TXL) drug loaded LMMNA compared to unloaded MMNA during 24 h. The results exhibit a significantly higher cytotoxicity effect with DOX:TXL-LMMNA compared to DOX-LMMNA in the three cancer cell lines at concentrations of 0.5 and 1 mg ml1 (p < 0.01). The unloaded MNNA does not show any cytotoxic effect (up to 1 mg ml1) with the different types of cells, suggesting good biocompatibility. In contrast, treatment of cells with DOX:TXLLMMNA at 0.5 mg ml1 (which contains DOX(10):TXL(15) lg ml1) leads to antiproliferative activity with living cells up to 50% (HeLa), 51% (MCF-7) and 52% (HepG2). However, the DOX-LMMNA exhibits greater viability of cells, with 68% (HeLa), 66% (MCF-7) and 68% (HepG2) cells being viable at a concentration of 0.5 mg ml1 (which contains 10 lg ml1 of DOX), respectively (Fig. 7D). Thus, it can be concluded that the antiproliferative activity against cancer cells can be enhanced by using the simultaneous delivery of two anticancer drugs compared to a single drug. This is because multiple drugs, as compared to an individual drug, in a single delivery system can attack the same cell at different cell growth phases, and can compete for the cellular compartments and increase the cytotoxic effect on the cell. In view of the above, we subjected DOX:TXL-LMMNA (0.5 mg ml1, which causes 50% antiproliferative activity) to further studies. 3.7. Intracellular localization of LMMNA The cellular uptake pathway of LMMNA in HeLa cells was observed by TEM using a Microtome (Fig. 8). Fig. 8A shows a single cell and Fig. 8(B and C) are a magnified portion of Fig. 8A which

shows the localization of LMMNA in the cell. Intracellular trafficking of LMMNA occurs by the endocytosis pathway after contact between LMMNA and the plasma membrane of cells, as shown in Fig. 8(D and E). The particles are found to form aggregates within the endosome in the cytoplasmic region (marked with yellow dotted circles in Fig. 8(D and E). This indicates the uptake of LMMNA by the cell. The locations of LMMNA can be observed in close proximity to the nuclear region (which is indicated by a blue dotted circle) in Fig. 8E. The above result implies that the PEG group of the lipid layer helps make the interaction between LMMNA and the cell surface more efficient. It is anticipated that enhanced LMMNA internalization in the cells will encourage further studies on drug delivery systems [52]. 3.8. LMMNA and single/dual drug uptake evaluation by a confocal study The cellular uptake of LMMNA was evaluated using N-NBD, which labels the hydrophobic lipid layers. Fig. 9 shows the intracellular uptake of N-NBD-LMMNA, DOX-LMMNA and N-NBDDOX:TXL-LMMNA in HeLa and MCF-7 cells (HepG2 cells are not shown here). This is compared with untreated cells as a control for both HeLa and MCF-7 cells. Fig. 9A shows the control for HeLa cells, with the blue color indicating the nuclear region of cells, which was stained with DAPI (in all cases). The presence of red (due to N-NBD) fluorescence indicates the presence of a lipid layer on the MMNA, which are distributed throughout the cytoplasm, and is shown in Fig. 9B for HeLa cells.


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Fig. 7. In vitro cytotoxicity effect of MMNA, DOX:LMMNA and DOX:TXL-LMMNA up to 1 mg ml1 with different drug contents after 24 h in (A) HeLa, (B) MCF-7 and (C) HepG2 cells. (D) The same dose (0.5 mg ml1) of MMNA, DOX-LMMNA and DOX:TXL-LMMNA with the same drug contents (DOX-10 lg ml1 and DOX:TXL-10:15 10 lg ml1) in three cell lines. For each cell line, differences between the no drug and DOX-LMMNA groups, and the DOX-LMMNA and DOX:TXL-LMMNA groups, are significant at ⁄⁄ p < 0.01 and ⁄⁄⁄p < 0.001, as determined by an unpaired, two-tailed t-test. The results are expressed as mean ± SD (n = 3).

The presence of DOX encapsulated LMMNA (without tag) in HeLa cells is observed by the green color emitted from DOX (Fig. 9C). Fig. 9D shows the presence of both green (DOX) and red (N-NBD tagged LMMNA), indicating that DOX and TXL (nonfluorescent) encapsulated LMMNA are efficiently internalized in the cytoplasm of the HeLa cells. The presence of TXL could not be directly observed from fluorescent emission. However, its presence can be confirmed from changes in cell morphology, as seen in Fig. 9D, wherein the cell membrane was observed to become more rounded with DOX:TXL-LMMNA but no significant change was observed in DOX-LMMNA treated HeLa cells (Fig. 9C). Fig. 9E–H shows similar effects on MCF-7 cells. In all cases, the inset images are magnified. This suggests that the release of dual drugs occurred inside the cytoplasm and helped to increase the cytotoxicity effect due to their combined action, and supports our observations in the previous sections. 3.9. Evaluation of in vitro hyperthermia with DOX:TXL-LMMNA The simultaneous cytotoxic effect of hyperthermia and chemotherapy due to DOX:TXL-LMMNA (0.5 mg ml1) was evaluated under an applied ACMF. Fig. 10A shows the cytotoxicity on HeLa,

MCF-7 and HepG2 cells under hyperthermic conditions (43 °C) under an applied ACMF. It takes 900 s to reach the hyperthermic temperature at 259 Oe (Fig. 10B). The cell death due to DOX:TXLLMMNA (0.5 mg ml1, which was found to prevent 50% of the proliferation of cells when tested with the above three types of cancer cells) was evaluated in the presence of an ACMF for 5 and 10 min after attaining 43 °C (259 Oe, 250 kHz). During the 5 min treatment, killing efficiencies of 67 ± 2% (HeLa), 65 ± 1% (MCF-7) and 69 ± 2% (HepG2) were observed. Interestingly, the respective killing efficiencies of cells increased to 88 ± 1, 89 ± 1 and 84 ± 2% with an exposure time of 10 min under similar conditions. However, such conditions (0.5 mg ml1 of DOX:TXL-LMMNA) without an ACMF induces less killing efficiency, of 48 ± 1, 49 ± 1 and 48 ± 1% for HeLa, MCF-7 and HepG2 cells, respectively, but both of the AC field treatment durations (i.e. 5 and 10 min) showed significant differences compared with the no AC field group (p < 0.001). Such an increase in cell death with the application of an oscillating magnetic field may be due to the enhanced drug release from LMMNA and the simultaneous thermotherapy. Moreover, heat generated along with strong magneto-mechanical stimuli through an applied ACMF can cause damage to the cell membrane and nuclear DNA [5,39,53]. Thus this



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Fig. 8. TEM images of cellular uptake and localizations in HeLa cell after incubation with LMMNA for 24 h at 37 °C. (A) Cell morphology with internalization of LMMNA; (B) magnified image of the area indicated by the green box in (A); (C) magnified image of (B), showing the nanoassemblies in cells; (D) labeled (dark blue arrow) LMMNA bound to the plasma membrane and taken up by the endocytosis pathway, surrounded by endosomal membrane after internalization into the cell (marked with a yellow dotted circle); and (E) an enlarge area of the nuclear envelope (marked with dotted dark blue line and red arrows).

Fig. 9. Confocal images demonstrating intracellular uptake of N-NBD-LMMNA, DOX-LMMNA without N-NBD labeling and DOX:TXL-LMMNA with N-NBD labeling in HeLa and MCF-7 cells after 24 h incubation at 37 °C. (A) Control for HeLa cells (the top left inset image is of the nuclear region, which is stained blue with DAPI). (B) Cells treated with NNBD-LMMNA (the top right inset image shows the red–blue color combination that indicates the presence of lipid membrane lebeled with N-NBD). (C) Cells treated with DOX-LMMNA without N-NBD (the green color in the top right inset image shows the presence of DOX). (D) Cells treated with N-NBD-DOX:TXL-LMMNA, showing the presence of DOX and N-NBD (top left inset image), which are bound to the cytoplasm and nucleus sites, and showing signicant changes of cell morphology after dual drug treatment compared to individual drug treatment. (E–H) Similar changes in MCF-7 cells (all inset images are magnified). All scale bars are 50 lm.

thermal stress enhanced the killing efficiency of cancer cells as these are more sensitive to heat than normal cells when exposed to temperature of 42–45 °C [46]. Therefore, the application of thermal therapy with dual drug systems at very low concentration (0.5 mg ml1 of DOX:TXL-LMMNA) enhances the inhibition of cell growth compared tothe previously reported values which are 1.25 mg ml1 concentration of TXL containing magneto liposomes [5], 1.2 mg ml1 DOX loaded peptide like shell cross-linked magnetic nanocarriers [54], 48 mg ml1 magnetic mesoporous silica without drug [46], 20 mg ml1 tamoxifen citrate loaded folic acid and b-cyclodextrin functionalized superparamagnetic iron oxide

nanoparticles [50] and 10 mg ml1 magnetic-core silica nanoparticles [51]. Fig. 11 shows the confocal microscopy images of cells after treatment with DOX:TXL-LMMNA in the presence (for 10 min) or absence of an ACMF followed by 24 h of incubation. In all cases, the presence of green (for DOX) and red (for N-NBD tagged lipid membrane) in the cytoplasm and blue (for DAPI, nuclear staining) in the nuclear region is clearly observed. Fig. 11A shows a confocal image of HeLa cells after treatment with DOX:TXL-LMMNA in the absence of an ACMF, whereas Fig. 11B shows a corresponding image under the application of an ACMF for 10 min with


10


Fig. 10. (A) Cell death efficiency of HeLa, MCF-7 and HepG2 cells with DOX:TXL-LMMNA (0.5 mg ml1) after 5 and 10 min incubation at 43 °C in the absence/presence of an ACMF. The differences between the no AC field control group and the groups treated with an AC field for 5 and 10 min are significant at ⁄⁄⁄p < 0.001, as determined by an unpaired, two-tailed t-test. (B) The heating ability (43 °C) of DOX:TXL-LMMNA (0.5 mg ml1) at 259 Oe. The results are expressed as mean ± SD (n = 3).

Fig. 11. Confocal fluorescence images of HeLa, MCF-7 and HepG2 cancer cells after treatment with N-NBD-DOX:TXL-LMMNA (0.5 mg ml1) in the presence/absence of an ACMF (for 10 min) and 24 h of incubation. In all cases (A, C and E), the effects of N-NBD-DOX:TXL-LMMNA are shown in the absence of an ACMF, with the red color coming from N-NBD, the green from DOX and the blue from DAPI. It is demonstrated that greater accumulation of drug occurs in the cytoplasm. (B, D and F) The effects after exposure to an ACMF, where the cell morphology has become rounded in shape, the greatest amount of cytoplasm has disrupted and more drug has accumulated in the nucleus. All inset images are magnified.

DOX:TXL-LMMNA. Similarly, Fig. 11(C and D) and Fig. 11(E and F) show the effect of DOX:TXL-LMMNA in the absence and presence of an ACMF on MCF-7 and HepG2 cells, respectively. After the ACMF treatment, significant changes in cell morphology are observed in all cases. The cells are seen to become rounded in structure with membrane shrinkage after exposure to an ACMF. The cytoplasm regions are mostly disrupted due to accumulation of appreciable amount of drugs in the nucleus after 24 h. Thus, heat and dual drug administration contribute simultaneously and synergistically to cause maximum cell death. The apoptosis in the cell cycle due to the synergistic effect of thermochemotherapy was also determined from flow cytometric analysis (details see ESI).

4. Conclusions In summary, we have successfully developed novel pH- and temperature-sensitive thin lipid layer coated mesoporous magnetic nanoassemblies for dual drug delivery and hyperthermia applications under an external ACMF. The spherical shape, narrow

size distribution, high magnetization, good stability, thin layer of lipid on the surface of single MMNA and its good heating ability (after lipid coating at low concentration) were confirmed by different characterization methods. In this system, hydrophilic DOX was loaded within mesopores of MMNA and hydrophobic TXL was encapsulated within the lipid layer of LMMNA. This hybrid material demonstrates the high loading efficiency of the two drugs with a sustained release profile based on an acidic pH of 4.3. The release could be further enhanced with the application of an ACMF. The LMMNA are readily internalized by cancer cells through endocytosis. The enhanced intracellular uptake of DOX:TXL-LMMNA resulted into higher cytotoxicity than a single drug loaded system (DOX-LMMNA) on HeLa, MCF-7 and HepG2 cells. The cytotoxicity effect due to drugs released from LMMNA was further enhanced with the application of an ACMF at 43 °C. This is confirmed by a flow cytometry study, with appreciable inhibition of cell growth occurring through the arrest of the G1 phase of the cell cycle at low drug concentrations. Overall, this newly developed magnetic nanoassembly based dual drug system shows promising in vitro results in enhancing therapeutic efficacy due to the combined effects of



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