Materials Science and Engineering C

Materials Science and Engineering C 30 (2010) 484 490 Contents lists available at ScienceDirect Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec Doxorubicin loaded PVA coated iron oxide nanoparticles for targeted drug delivery S. Kayal, R.V. Ramanujan School of Materials Science and Engineering, Nanyang Technological University, Singapore 639798, Singapore article info abstract Article history: Received 7 September 2009 Received in revised form 4 December 2009 Accepted 11 January 2010 Available online 20 January 2010 Keywords: Superparamagnetic magnetic nanoparticles Functionalization Conjugation Magnetic drug delivery Magnetic drug targeting is a drug delivery system that can be used in locoregional cancer treatment. Coated magnetic particles, called carriers, are very useful for delivering chemotherapeutic drugs. Magnetic carriers were synthesized by coprecipitation of iron oxide followed by coating with polyvinyl alcohol (PVA). Characterization was carried out using X-ray diffraction, TEM, TGA, FTIR and VSM techniques. The magnetic core of the carriers was magnetite (Fe 3 O 4 ), with average size of 10 nm. The room temperature VSM measurements showed that magnetic particles were superparamagnetic. The amount of PVA bound to the iron oxide nanoparticles were estimated by thermogravimetric analysis (TGA) and the attachment of PVA to the iron oxide nanoparticles was confirmed by FTIR analysis. Doxorubicin (DOX) drug loading and release profiles of PVA coated iron oxide nanoparticles showed that up to 45% of adsorbed drug was released in 80 h, the drug release followed the Fickian diffusion-controlled process. The binding of DOX to the PVA was confirmed by FTIR analysis. The present findings show that DOX loaded PVA coated iron oxide nanoparticles are promising for magnetically targeted drug delivery. 2010 Elsevier B.V. All rights reserved. 1. Introduction Corresponding author. Tel.: +65 67904342; fax: +65 67909081. E-mail address: ramanujan@ntu.edu.sg (R.V. Ramanujan). In the last decade, nanotechnology has developed to such an extent that it is possible to synthesize, characterize and tailor the functional properties of magnetic nanoparticles for biomedical applications [1 7]. This has led to enormous interest in magnetic nanoparticle based systems for cancer treatment [8 16]. The limitations of conventional chemotherapy include general systemic distribution of drug, lack of drug specificity to the tumor site, insufficient local drug concentration in the tumor and poor control over drug release. The general systemic distribution of chemotherapeutic agent results in deleterious side effects since the drug attacks the normal, healthy cells together with the tumor cells. Therefore, it is very important to selectively target chemotherapeutic agents to the tumor. This need has prompted a search for methods of drug delivery which can address this limitation and provide more effective cancer therapy. Magnetic drug delivery system using magnetic nanoparticle carriers targeted by an external magnetic field is a promising alternative to avoid the problems associated with conventional chemotherapy [8,10,11,16 18]. In magnetically targeted drug delivery, carriers comprising of coated magnetic nanoparticles loaded with anti-cancer drug are injected into the patient body via the human circulatory system. An external magnetic field is used to localize the drug loaded carriers at the tumor site and the drug can then be released from the carriers either via enzymatic activity or changes in physiological conditions such as ph, osmolality, or temperature [16] and be taken up by tumor cells. Alexiou et al. showed that this magnetic targeted drug delivery caused complete tumor remission in tumor bearing rabbits without any negative side effects and the applied dose of drug reduced to 20% of the regular systemic dose [8,19,20]. A Phase-I human clinical trial with 4-epidoxorubicin showed encouraging results of the physiological tolerance of magnetic drug targeting by patients [17]. Magnetic nanoparticles can also be used for hyperthermia treatment of cancer [21 23]. Hyperthermia is the heating of cells in the range of 41 47 C, which causes preferential death of tumor cells [24,25]. When magnetic nanoparticles are subjected to an alternating magnetic field, heat can be generated by Neel relaxation, Brownian relaxation and hysteresis losses [11]. Magnetic drug targeting using superparamagnetic iron oxide nanoparticles as carriers is of considerable amount research interest [17,18,26 32]. Iron oxide nanoparticles are biocompatible in the doses required for therapeutic use (FDA approved) and are sold commercially, currently in routine use as MRI contrast enhancement agents [33 35]. In vivo experiments on animal models have shown that iron oxides are suitable for drug delivery [16,36] and human clinical trials for drug delivery have been conducted with iron oxide based ferrofluids, the injected dosage being well tolerated by patients [17]. The most common synthesis processes of superparamagnetic iron oxide nanoparticles are based on the wet chemical coprecipitation of ferrous and ferric ions with sodium hydroxide or ammonia in aqueous solution or in microemulsion [37,38] or the partial oxidation of ferrous hydroxide gels [39].Magnetic nanoparticles have a tendency to agglomerate, hence magnetic nanoparticles are generally coated with surfactants [40] or polymers [41] to minimize aggregation. When the particles are injected into the bloodstream, they are surrounded by plasma proteins (hydrophobic surface), 0928-4931/$ see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2010.01.006

S. Kayal, R.V. Ramanujan / Materials Science and Engineering C 30 (2010) 484 490 485 this process is called opsonization [42]. Generally, particles with hydrophobic surfaces are more prone to opsonization due to hydrophobic hydrophobic interactions which causes rapid removal of opsonized particles by the reticulo-endothelial system (RES) [5,43]. However, particles which are more hydrophilic inhibit the plasma protein coating process (opsonization) [44]. Therefore, the role of hydrophilic coating is to provide stabilization of nanoparticles in biological suspension, functionalization at the surface with drugs and to increase circulation time by reducing immediate clearance of the carriers by RES. In this study, hydrophilic polymer polyvinyl alcohol (PVA) was chosen as the coating of the magnetic particles because of its biocompatibility, biodegradability and it can also be readily functionalized [10,45 47]. Doxorubicin drug (DOX) was used as an anti-cancer agent since it has high therapeutic index and gives better activity on solid tumor [48]. Several researchers have studied the synthesis and characterization of PVA coated iron oxide nanoparticles. Lee et al. [49] synthesized ultrafine Fe 3 O 4 particles (4 7 nm) by precipitation in aqueous PVA solution and showed that relative crystallinity and saturation magnetization (M s ) decrease with increasing PVA concentration. Chastellain et al. reported a multistep synthesis of PVA coated Fe 3 O 4 nanoparticles (5 20 nm) by coprecipitation method followed by a thermochemical treatment and demonstrated the colloidal stability at neutral ph [50]. Mohapatra et al. prepared polyvinyl alcohol phosphate (PVAP) coated magnetite nanoparticles having a broad size distribution (13 55 nm) which were superparamagnetic at room temperature [45]. Petri-Fink et al. developed functionalized super paramagnetic iron oxide nanoparticles (SPION) and showed the interaction of amino-pva-spion with human melanoma cells [46]. Hanessian et al. designed and synthesized drug-functionalized linkers which were coupled to amino- PVA-SPION and showed that this drug-functionalized-amino-pva- SPION was taken up by human melanoma cells [51]. In this work, our goal is to conjugate the DOX with various PVA coated iron oxide nanoparticle carriers and study the drug loading and release profile from these carriers. Iron oxide nanoparticles were synthesized by coprecipitation technique and coated with different weight % of PVA solution. XRD, TEM, TGA, FTIR, and VSM were used to characterize the synthesized nanoparticles. These magnetic carriers were loaded with the anti-cancer drug DOX and its in vitro loading and release profiles were studied. The novelty of this work is the binding of DOX to the PVA coated iron oxide nanoparticles, the study of drug loading and subsequent in vitro drug release in order to evaluate the suitability of DOX loaded PVA coated iron oxide nanoparticles as magnetic carriers for drug delivery. 2. Materials and methods 2.1. Materials All chemicals were used without further purification. Chemicals used for the synthesis of PVA coated iron oxide nanoparticles were ferrous chloride tetrahydrate (FeCl 2 4H 2 O, Aldrich), ferric chloride hexahydrate (FeCl 3 6H 2 O, Aldrich), sodium hydroxide (NaOH, Merck Inc.), polyvinyl alcohol (PVA, Fluka) with average molecular weight of 31,000 50,000 (99% degree of hydrolysis). Deionized water was used throughout the experiment. Doxorubicin hydrochloride (DOX, Aldrich) was used for the drug loading and release study. 2.2. Synthesis of PVA coated iron oxide nanoparticles PVA coated iron oxide nanoparticles were synthesized by precipitation of iron oxide at high ph and subsequently coated with PVA solution. A mixed solution of ferrous and ferric ions (molar ratio 1:2) was prepared by dissolving 0.15 mol FeCl 2 4H 2 O and 0.3 mol FeCl 3 6H 2 O in 30 ml aqueous medium. Iron oxide was precipitated by adding this mixed solution drop by drop to 100 ml of 0.4 M NaOH solution (ph=13) while stirring at 600 rpm with an impeller and purging with N 2, when the black precipitates of iron oxide were formed. The mixture was then continuously purged with N 2 for another 20min. Precipitates and supernatant liquid were separated by centrifugation. The precipitates were washed with deionized water (purged with N 2 ) under ultrasonication for 10 min and then separated by centrifugation at 5000 rpm for 10 min. Precipitates were dried after washing three times to carry out characterization. For coating of iron oxide nanoparticles, PVA solutions were prepared by dissolving dry PVA powder in deionized water at 55 C and iron oxide nanoparticles were added and mixed with the PVA solution using a magnetic stirrer for 12h. Coated particles were separated by the use of a permanent magnet and dried in the vacuum oven for characterization. Following the above synthesis method, 5 sets of samples were prepared by varying the concentration of PVA (Table 1), the uncoated sample is referred as S-1 and those prepared with 0.5%, 1%, 2% and 5% (by weight) PVA solution are referred as S-2, S-3, S-4 and S-5 respectively, in subsequent results and discussion. 2.3. Instrumentation A Shimadzu XRD-6000 X-ray diffractometer with Cu-K α radiation (wavelength=0.154056 nm) was used for X-ray analysis. Phase identification was performed by matching peak positions and relative intensities to reference JCPDS files. The Rietveld refinement of the XRD pattern was performed using TOPAS (version 4.1) software. The size and morphological characterization of the particles were carried out using a JEOL 2010 transmission electron microscope (TEM) operating at 200 kv. TEM samples were prepared by dispersing nanoparticles in acetone for 30 min by ultrasonic vibration. The aqueous dispersion was dropped on a carbon coated copper TEM grid with filter paper underneath to absorb the acetone and dried in vacuum. The amount of PVA attached to the magnetic particles was analyzed by thermogravimetric analyzer (TGA Q500) up to 600 C in air at a ramp rate of 10 Cmin 1. Fourier transform infrared (FTIR) spectra of uncoated, PVA coated, and DOX-conjugated PVA coated iron oxide nanoparticles were recorded by Nicolet Magna-IR 550 spectrometer at 4 cm 1 resolution. The FTIR spectra were measured in the 400 4000 cm 1 region with samples dispersed in KBr pellets. Magnetic properties were evaluated by a Lakeshore 7404 vibrating sample magnetometer (VSM), the applied field was in the range of 0 to 10kOe. The DOX concentration was measured by a UV vis (Shimadzu UV 1700) spectrophotometer equipped with quartz 1 cm optical length cuvettes (Hellma). 2.4. Doxorubicin (DOX) drug loading and release studies The water-soluble anti-cancer drug DOX was chosen as a model drug. The DOX loading was carried out by dispersing 5 mg of PVA coated iron oxide nanoparticles in 5 ml aqueous DOX solution (drug concentration=0.1 mg/ml) following the experimental procedure described by Kuznetsov et al. [52]. The mixture of PVA coated iron oxide nanoparticles in DOX was shaken in a rotary shaker (200 rpm) at 37 C for 26 h to facilitate DOX uptake. At fixed time intervals, the magnetic particles were removed from the liquid by means of a permanent magnet and the optical density of residual DOX in the supernatant was measured at 498 nm by UV vis spectrophotometer [53]. After the measurement, magnetic nanoparticles were redispersed for further DOX adsorption. Beyond a certain adsorption time, there were no further changes in the concentration of DOX since the loading capacity of the particles had reached saturation. The drug loading was determined as the difference between the initial DOX Table 1 Summary of sample sets synthesized by coprecipitation technique. Sample set S-1 S-2 S-3 S-4 S-5 PVA concentration (wt.%) 0 0.5 1 2 5

486 S. Kayal, R.V. Ramanujan / Materials Science and Engineering C 30 (2010) 484 490 concentration and the DOX concentration in the supernatant. The drug loaded magnetic nanoparticles were then magnetically separated and dried. The release profile was obtained by dispersing the dried drug loaded nanoparticles in 5 ml PBS buffer at 37 C. As in the uptake experiments, the concentration of DOX in the particle free liquid was determined at fixed time intervals by UV vis spectrophotometry. 3. Results and discussion Here we present structural, morphological and magnetic properties of uncoated and PVA coated iron oxide nanoparticles. The particles were characterized by XRD, TEM, TGA, FTIR and VSM. DOX loading and release studies of PVA coated iron oxide nanoparticles are also presented. 3.1. Structural and morphological properties Representative powder X-ray diffraction patterns of uncoated (S-1) and PVA coated iron oxide nanoparticles (S-3 and S-5) are presented in Fig. 1. From the XRD analysis, it has been found that the peaks correspond to the spinel structure of magnetite phase (Fe 3 O 4, reference JCPDS No. 82-1533). The peaks corresponding to reflection planes are indexed (Fig. 1). From the absence of (210) and (300) peaks in the XRD pattern, it can be concluded that separate maghemite (γ-fe 2 O 3 )isnot present in the samples. Interestingly, there is broadening of peaks from Fig. 1b to a with an increase in polymer concentration, the broadening of XRD peaks is predominantly attributed to the decrease in crystallite size. The mean crystallite size has been calculated using Scherrer's formula. The mean crystallite size of uncoated (S-1) sample is 19.3 nm and those of PVA coated iron oxide nanoparticles S-2, S-3, S-4, and S-5 are 14.6 nm, 7.9 nm, 5.6 nm and 4.5 nm respectively. Rietveld refinement of the XRD patterns of S-1 and S-5 using TOPAZ (version 4.1) software is shown in Fig. 2 and lattice parameter calculated (a=8.39 Å) is very close to that of magnetite (Fe 3 O 4 ) (lattice parameter (a) from the literature; Fe 3 O 4 : 8.396 Å and γ-fe 2 O 3 :8.35Å)[54]. Our XRD results agree well with the previous reports [29,45,49,50]. TEM micrographs of uncoated (S-1) and PVA coated iron oxide nanoparticles (S-4) are shown in Fig. 3. The particles are equiaxed with an average particle diameter in the range of 10 15 nm. PVA is responsible for image blurring due to film formation. In this work, we have synthesized iron oxide nanoparticles of narrow particle size distribution (10 15 nm) by the coprecipitation technique followed by coating with different weight percentage of PVA. In the context of drug delivery, a narrow particle size range such as that obtained in the present work is useful since uniform size particles offer Fig. 2. Rietveld refinement of X-ray powder diffraction pattern of (a) S-1 (uncoated iron oxide) and (b) S-5 (5 wt.% PVA coated iron oxide). Peak broadening occurs in PVA coated iron oxide nanoparticles due to decrease in crystallinity. equal probability of magnetic capture of drug loaded nanoparticles and are characterized by similar drug content. 3.2. PVA adsorption on iron oxide nanoparticles The amount of PVA adsorbed to the iron oxide nanoparticles was studied by thermogravimetric analysis (TGA). Fig. 4 shows the weight loss vs. temperature curves of pure PVA (inset), uncoated and PVA coated iron oxide nanoparticles heated up to 600 C in air. The inset shows that pure PVA degrades completely when heated up to 600 C. There is no significant weight loss in the TGA curve of uncoated iron oxide nanoparticles (Fig. 4a), whereas, there are distinct weight losses in the TGA curves of PVA coated iron oxide nanoparticles. The initial weight loss up to 100 C is due to the desorption of physically adsorbed water. The weight loss from 200 to 500 C is due to the dehydration reaction of OH groups in PVA chains and subsequent degradation of PVA releasing CO 2 gas. The weight loss due to PVA is presented in Table 2. PVA is known to adsorb nonspecifically on oxide surfaces through hydrogen bonding arising from the polar functional groups of PVA and the hydroxylated and protonated surface of the oxide [55]. Fig. 1. X-ray powder diffraction pattern of (a) S-5 (5 wt.% PVA coated iron oxide), (b) S-3 (1 wt.% PVA coated iron oxide) and (c) S-1 (uncoated iron oxide). The phase is magnetite (Fe 3 O 4 ), peaks corresponding to reflection planes are indexed. 3.3. FTIR study FTIR is an appropriate technique to establish the attachment of the polymer to the magnetic nanoparticles and conjugation of drug with the PVA coated magnetic nanoparticles. Fig. 5 shows the FTIR spectra of uncoated and PVA coated iron oxide nanoparticles. The series of characteristic IR bands are summarized in detail in Table 3 [56] and only the salient features are discussed below. In case of uncoated iron oxide, the band at 3394 cm 1 is assigned to stretching (ν) vibrations and the band at 1620 cm 1 is assigned to bending (δ) vibrations due to adsorbed water on the surface of the iron oxide nanoparticles. The band observed at 611 cm 1 corresponds to the stretching vibrations of M Th O M Oh, where M Th and M Oh correspond to the iron occupying tetrahedral and octahedral positions, respectively. In PVA coated iron oxide nanoparticles, the M Th O M Oh stretching band at 606 cm 1, the alcoholic O H stretching band at 3410 cm 1 are observed. The additional bands at 2912 cm 1 corresponding to C H stretching vibrations, at 1416 cm 1 corresponding to C C stretching vibrations, at 1092 cm 1 attributable to M O C (M=Fe) bond and at 850 cm 1 corresponding to CH 2 rocking are observed in PVA coated iron oxide

S. Kayal, R.V. Ramanujan / Materials Science and Engineering C 30 (2010) 484 490 487 Fig. 3. TEM micrographs of (a) S-1, corresponding to the uncoated iron oxide nanoparticles and (b) S-4, coated with 2 wt.% PVA. Particles are equiaxed with average size of 10 15 nm. Fig. 4. Weight loss vs. temperature TGA curves of (a) S-1 (uncoated iron oxide), (b) S-2 (iron oxide coated with 0.5 wt.% PVA), (c) S-3 (iron oxide coated with 1 wt.% PVA), (d) S-4 (iron oxide coated with 2 wt.% PVA) and (e) S-5 (iron oxide coated with 5 wt.% PVA) heated up to 600 C in air. The inset shows the TGA curve of pure PVA indicating complete degradation of PVA when heated up to 600 C. nanoparticles, confirming the attachment of PVA onto iron oxide nanoparticles which is also supported by thermal analysis (Section 3.2). The interaction between polymer coating and Fe 3 O 4 particles has been studied earlier [57 61]. Polymer interactions were studied in Fe 3 O 4 /polypyrrole and Fe 3 O 4 /polyaniline nanocomposites, where interactions exist between the lone pair electrons of the N atom in the polypyrrole chain or in the polyaniline chain with the 3d orbital of the Fe atom to form a coordinate bond [57,58]. Li et al. reported that oleic acid adsorption on the surface of Fe 3 O 4 nanoparticles could be due to hydrogen bonding or a coordination linkage [59]. Zhang et al. reported the attachment of polymethacrylic acid to Fe 3 O 4 nanoparticles via coordination linkages between the carboxyl groups and iron [60]. In the present work, we confirm by FTIR analysis (Fig. 5) that the attachment of PVA to iron oxide nanoparticles occurs via hydrogen Table 2 Weight loss due to PVA in thermogravimetric analysis (TGA) in air. Sample set Wt loss (%) S-2 (0.5 wt.% PVA coated) 11 S-3 (1 wt.% PVA coated) 14 S-4 (2 wt.% PVA coated) 18 S-5 (5 wt.% PVA coated) 23 Fig. 5. FTIR spectra of (a) uncoated and (b) PVA coated iron oxide nanoparticles. Bands at 2912, 1416, 1092 and 850 cm 1 in PVA coated iron oxide nanoparticles confirm the attachment of PVA to iron oxide nanoparticles. Table 3 Assignment of FTIR spectra of uncoated iron oxide, PVA coated iron oxide, pure DOX and DOX-conjugated PVA coated iron oxide shown in Figs. 5 and 6. Samples IR region or bands (cm 1 ) Description Uncoated iron oxide 3394 ν (H O) 1620 δ (H O H) ofadsorbed water 611 ν (M Th O M Oh ) PVA coated iron oxide 3410 ν (H O) 2912 ν (C H) 1624 δ (H O H) 1416 ν (C C) 1092 ν (M O C (M=Fe)) 850 CH 2 rocking 606 ν (M Th O M Oh ) Pure DOX 3450 ν (N H) 3330 ν (H O) 2932 ν (C H) 1730 ν (C O) 1618, 1521 δ (N H) 1410 ν (C C) 1280, 997 ν (C O C) 1070 ν (C O) 870, 805 ω (N H) DOX-conjugated PVA 3265 ν (N H), ν (H O) coated iron oxide 2910 ν (C H) 1712 ν (C O) 1630 δ (N H) 1409 ν (C C) 1250 ν (C O C) 1093 ν (C O) 600 ν (M Th O M Oh )

488 S. Kayal, R.V. Ramanujan / Materials Science and Engineering C 30 (2010) 484 490 Table 4 Saturation magnetization (M s ) of uncoated and PVA coated iron oxide nanoparticles. Sample set Saturation magnetization (emu/g) S-1 (uncoated) 42 S-2 (0.5 wt.% PVA coated) 33 S-3 (1 wt.% PVA coated) 28 S-4 (2 wt.% PVA coated) 23 S-5 (5 wt.% PVA coated) 19 coated iron oxide nanoparticles occurs via the interaction of NH 2 and OH groups of DOX with OH groups of PVA through hydrogen bonding which is consistent with previous report [62]. Fig. 6. FTIR spectra of (a) pure DOX and (b) DOX-conjugated PVA coated iron oxide nanoparticles. Conjugation of DOX to the PVA coated iron oxide nanoparticles occurs via the interaction of NH 2 and OH groups of DOX with OH groups of PVA. bonding between hydroxyl group of PVA and protonated surface of the oxide. FTIR was further extended to study the conjugation of DOX with the PVA coated iron oxide nanoparticles. FTIR spectra of pure DOX and DOX-conjugated PVA coated iron oxide nanoparticles are presented in Fig. 6 and characteristic peaks are tabulated (Table 3). FTIR spectrum of PVA coated iron oxide nanoparticles (Fig. 5) shows the alcoholic O H stretching band at 3410 cm 1. FTIR spectrum of pure DOX shows peaks at 3450 cm 1 due to N H stretching vibrations for the primary amine structure and at 3330 cm 1 due to O H stretching vibrations (Fig. 6). However, in case of DOX-conjugated PVA coated iron oxide nanoparticles, peaks due to N H stretching vibrations and O H stretching vibrations overlap, are broadened and shifted to the lower frequency range (~3265 cm 1 ). The bands observed at 870 cm 1 and 805 cm 1 due to N H wag in pure DOX diminish in the FTIR spectrum of DOX-conjugated PVA coated iron oxide nanoparticles. From this FTIR result, it can be interpreted that attachment of DOX to the PVA 3.4. Magnetic measurements Fig. 7 shows the room temperature magnetization curves of uncoated and PVA coated iron oxide nanoparticles. The absence of remanence in the hysteresis curves indicates that magnetic particles are superparamagnetic. The saturation magnetization (M s ) of uncoated sample (S-1) is 42 emu/g, less than that of bulk magnetite (88 emu/g) reported earlier [63 65]. As expected, the M s of PVA coated iron oxide nanoparticles decreases with increasing PVA concentration (Table 4). In this study, the observed saturation magnetization (M s ) of PVA coated iron oxide nanoparticles is comparable to the previous reports [29,45]. Our particles are superparamagnetic at room temperature which are useful in drug delivery as they do not retain magnetization before and after exposure to an external magnetic field, reducing the probability of particle aggregation due to magnetic dipole attraction [62,66]. The observed M s of uncoated Fe 3 O 4 nanoparticles is lower than that of bulk magnetite since M s generally decreases with a decrease in magnetic particle size [67]. For PVA coated iron oxide nanoparticles, the M s decreases with increasing PVA concentration (Fig. 7). This may be due to the dilution effect from adsorbed water and the hydroxyl content of PVA and the possibility of a small volume fraction of antiferromagnetic amorphous iron oxides. The reduced magnetization could also result from the small particle surface effect [68] which refers to the disordered alignment of surface atomic spins induced by reduced coordination and broken exchange between surface spins [45]. This surface effect is more Fig. 7. Magnetization vs. field curves measured at room temperature of (a) S-1, corresponding to uncoated iron oxide nanoparticles, (b) S-2, iron oxide coated with 0.5 wt.% PVA, (c) S-3, iron oxide coated with 1 wt.% PVA, (d) S-4, iron oxide coated with 2 wt.% PVA and (e) S-5, iron oxide coated with 5 wt.% PVA. Particles are superparamagnetic, M s decreases with increase in PVA concentration.

S. Kayal, R.V. Ramanujan / Materials Science and Engineering C 30 (2010) 484 490 489 Fig. 8. DOX loading on PVA coated iron oxide nanoparticles, DOX loading increases with increase in PVA concentration in the PVA coated iron oxide nanoparticles. prominent in small particles as the ratio of surface atoms to the interior atoms increases with a decrease in particle size. 3.5. Doxorubicin (DOX) drug loading and release study The DOX loading and release profiles of PVA coated iron oxide nanoparticle carriers are shown in Figs. 8 and 9, respectively. Initially there is a rapid adsorption of DOX, then the adsorption rate slows down and finally reaches the saturation value (Fig. 8). It has been found that higher PVA content results in higher drug adsorption, 35 µg, 41 µg, 47 µg, and 58 µg of DOX per mg of carrier was loaded in 26 h with 0.5%, 1%, 2%, and 5% PVA respectively. The drug release behavior of PVA coated iron oxide nanoparticle carriers was investigated in PBS buffer at ph of 7 and temperature of 37 C to maintain the experimental conditions similar to body fluid. The DOX release profiles from magnetic carriers coated with 0.5%, 1%, 2% and 5% PVA are presented in Fig. 9. The release profiles show that initially there is a rapid release until 6 h after which release slows down. A maximum of 45%, 33%, 25% and 17% of adsorbed drugs were released in 80 h from carriers coated with 0.5%, 1%, 2%, and 5% PVA respectively. The drug loading is attributed to the conjugation of NH 2 and OH groups in DOX to the surface active OH groups in PVA as shown in FTIR results (Fig. 6). When the PVA concentration increases (from 0.5 to 5 wt.%), the number of surface active OH groups increases, which results in higher drug adsorption (Fig. 8). Considering the conjugation of DOX with the surface active hydroxyl group ( OH) of PVA, there is an increased binding of DOX with increasing PVA concentration, therefore, the drug is likely to be released at a slower rate from the carriers with higher PVA content (Fig. 9). Our drug release profiles follow the Fick's law of diffusion for monolithic system [69]: M t M =1 6 1 π 2 n =1 n exp 2! Dn2 π 2 t R 2 where M t and M are cumulative amounts of drug released at time t and infinity, respectively; n is a dummy variable, D is the diffusion coefficient of the drug and R is the radius of particle. We have fitted the DOX release profile with above equation indicating that the DOX release from PVA coated iron oxide nanoparticles is Fickian diffusion-controlled process. We have calculated the diffusion coefficient (D) ofdox(1.5 10 18 cm 2 /s) which is close to previous report [70] (D of DOX for PEG-b-PCL and PEGb-PLA is 1.13 10 18 cm 2 /s and 1.82 10 18 cm 2 /s, respectively). The DOX release profile of our system is comparable to that of Kuznetsov et al. [52], where iron carbon adsorbent was loaded with DOX and approximately 25% of DOX was released in 24 h. Fig. 9. DOX release from PVA coated iron oxide nanoparticles, DOX release follows the Fickian diffusion-controlled process. In summary, magnetic carriers comprising of PVA coated iron oxide nanoparticles were synthesized, characterized and studied for anticancer drug loading and drug release. The important aspects of functionalization of magnetic iron oxide nanoparticles by PVA, conjugation of DOX with the PVA coated iron oxide nanoparticles and drug release showed that DOX loaded PVA coated iron oxide nanoparticles have potential to be used in magnetically targeted drug delivery. 4. Conclusions Magnetic carriers consisting of PVA coated superparamagnetic iron oxide (magnetite) nanoparticles were synthesized by precipitation of iron oxide and subsequently coated with polyvinyl alcohol (PVA). 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