Colloids and Surfaces A: Physicochem Eng Aspects 384 (2011) 23–30 Contents lists available at ScienceDirect Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa Design of carboxylated Fe3 O4 /poly(styrene-co-acrylic acid) ferrofluids with highly efficient magnetic heating effect Tai Thien Luong a , Thu Phuong Ha b , Lam Dai Tran b,∗ , Manh Hung Do b , Trang Thu Mai b , Nam Hong Pham b , Hoa Bich Thi Phan b , Giang Ha Thi Pham c , Nhung My Thi Hoang c , Quy Thi Nguyen c , Phuc Xuan Nguyen b,∗ a Faculty of Chemistry, Hanoi National University of Education, Hanoi, Viet Nam Institute of Materials Science, Vietnam Academy of Science and Technology, Hanoi, Viet Nam c Faculty of Biology, Hanoi University of Science, Vietnam National University, Viet Nam b a r t i c l e i n f o Article history: Received 14 December 2010 Received in revised form 14 February 2011 Accepted 22 February 2011 Available online 26 March 2011 Keywords: Carboxylated Fe3 O4 ferrofluids Poly(styrene-co-acrylic acid) Magnetic heating effect a b s t r a c t Magnetic heating effect of Fe3 O4 ferrofluids, surface-carboxylated and encapsulated by poly(styreneacrylic acid) was studied Magnetic Fe3 O4 NPs were synthesized by coprecipitation method and characterized by X-ray diffraction (XRD), Field Emission Electron Microscope (FESEM) and Vibrating Sample Magnetometer (VSM) Spectroscopic data (Infra red (IR) and Proton Nuclear Magnetic Resonance (1 H NMR) spectra) confirmed that Fe3 O4 NP surface was carboxylated and capable for protein binding (e.g., Hepatitis B surface antibody (HBsAb)) Magnetic measurement by Physical Properties Measurement System (PPMS) and magnetic heating experiments revealed that the saturation temperature of the ferrofluids could be advantageously applied in hyperthermic treatment, namely it could achieve as high as 48.6◦ –57 ◦ C, with as low as 0.3–0.5 mg/ml of Fe3 O4 in the fluid, under magnetic field of 60 Oe and 236 kHz Under those AC field parameters, the specific loss power (SLP) was measured as 0.84 kW/g and Sarcoma 180 cells were killed after 70–80 min-treatment in ex vivo heating experiment, meaning that the synthesized ferrofluid may be considered as promising in hyperthermic cancer treatments © 2011 Elsevier B.V All rights reserved Introduction In the recent years, magnetic nanoparticles (NPs) have attracted a considerable attention of scientists working in the fields of medicine and biotechnology Thanks to their unique magnetic properties magnetic particles can be utilized exclusively in some special medical techniques, most notably, separation for purification and immunoassay, drug delivery and targeting, magnetic resonance imaging (MRI), and hyperthermia [1–4] Among the magnetic materials Fe3 O4 NPs are most frequently chosen because of the following reasons: (i) Fe3 O4 is biocompatible, (ii) Fe3 O4 NPs can be synthesized at large scale; (ii) the magnetization of Fe3 O4 NPs is significantly high, thus allowing these particles to be easily controlled by an external magnetic field Hyperthermia is a promising approach to cancer therapy However, the technical challenge with hyperthermia is to heat locally tumor region to the desired temperature without damaging the surrounding healthy tissues It involves the introduction of ferromagnetic or superparamagnetic particles into the tumor tissue and then irradiation with an AC magnetic field The heating of the cancer area containing magnetic NPs to the elevated temperatures (41–46 ◦ C) in an external AC magnetic field induces apoptosis of tumor cells The particles transform the energy of the AC magnetic field into heat and the transformation efficiency strongly depends on the frequency of the external field as well as the nature of the particles such as magnetism and surface modification [5–8] Theoretically, the production of heat by magnetic substance in an external alternating magnetic field may be caused by several loss processes [9,10] First, hysteresis mechanism of heat generation occurs during reversal of the magnetization and is represented by the area of hysteresis loop, related to anisotropy energy density K, particle size and microstructure The second mechanism of magnetic heating is related to thermal fluctuation when the particle size decreases to below the superparamagnetic critical value The energy is dissipated when the particle moment relaxes to its equilibrium orientation This so-called Néel relaxation is determined by the ratio of KV to the thermal energy kT N = exp KV kT , −9 ∼10 s (1) Neel relaxation is related to the loss power P as follows: ∗ Corresponding authors Tel.: +84 37564129; fax: +84 438360705 E-mail addresses: lamtd@ims.vast.ac.vn (L.D Tran), phucnx@ims.vast.ac.vn (P.X Nguyen) 0927-7757/$ – see front matter © 2011 Elsevier B.V All rights reserved doi:10.1016/j.colsurfa.2011.02.050 P(H, f ) = 2 f N H fMS V kT + f N2 (2) 24 T.T Luong et al / Colloids and Surfaces A: Physicochem Eng Aspects 384 (2011) 23–30 Thus, the Néel loss is proportional to the square of saturation magnetization, square of field amplitude Another loss type, related to the rotational Brownian motion, may arise in the case of particle-in-liquid systems (ferrofluids) In that case, the energy barrier for reorientation of a particle is determined by rotational friction within the suspension fluid of viscosity Á Brown relaxation is described as: P= B (mHf Árh3 kT Experimentals B kTV (1 + f [2 = B) 2 )] B (3) 2.1 Materials (4) In general case, when both Néel and Brown relaxation mechanisms are present, the effective relaxation time may be expressed as: N× B (5) eff = N+ B Experimentally, magnetic heating is described by temperature increase versus heating time curve Heating capacity is characterized by so called specific loss power (SLP), which is proportional to the initial slope of the heating curve, according to the following equation: SLP = C ms dT mi dt account serious public health problems, caused by hepatitis B (HBV) and hepatitis C viruses (HCV) (chronic liver diseases, cirrhosis and hepatocellular carcinoma) The main impact parameters on magnetic heating effect will be discussed and optimized Then, under those AC field parameters, preliminary ex vivo heating experiment with Sarcoma 180 cells was also carried out (6) where C and ms , are, respectively, the heat capacity and mass of the ferrofluid; mi is the mass of magnetic NPs Accordingly, SLP of the magnetic material should be as high as possible in order to reduce the dose being applied to the patient to a minimum level From another point of view, except for having such properties as high saturation magnetization, uniform particle size and superparamagnetic behavior of magnetic NPs, biomedical applications require magnetic NPs to be capped and/or surface functionalized by low-toxic, biocompatible layers which could provide a steric barrier to prevent nanoparticle agglomeration and avoid opsonization (the uptake by the reticuloendothelial system (RES), shortening circulation time in the blood and NP’s ability to target the drug to specific sites and reduce side effects) Secondly, these coatings offer a means to tailor the surface properties of NPs such as surface charge and chemical functionality so that bioactive substances (enzyme, antibody, protein and nucleic acid) could be bound to their surface with the aid of coupling reagents (glutaraldehyde, carbodiimide, N-hydroxysuccinimide) Therefore, functional groups (such as –COOH, –NH2 , –OH and –CHO) play an important role in conjugating polymer-coated magnetic NPs with biomolecules In this context, acrylic acid (AA), an inexpensive substance having quite low toxicity and possessing carboxylated groups was preferentially chosen as a functional/protective shell in the copolymeric encapsulation with styrene (St) [5–7] In this study, we not take upon ourselves to introduce novel magnetic core/protective polymeric shells but emphasize our efforts on designing stable fluids with low toxicity and tunable magnetic heating effect for hyperthermic treatment Namely, Fe3 O4 /poly(St-co-AA) ferrofluid was prepared by dispersion polymerization, a special precipitation polymerization occurring in a homogeneous system (ethanol/water medium) involving monomers (St, AA), stabilizer and initiator before reaction, with Fe3 O4 magnetic particle as a core and poly(St-co-AA) as a shell Fe3 O4 core was first obtained by coprecipitation and then encapsulated either by in situ or ex situ copolymerization with St and AA units.–COOH functionalized shell played dual role of stabilizing and biomolecule-linking roles To investigate whether the fluids could be used advantageously for biomolecular binding we used Hepatitis B surface antibody (HBsAb) as bioconjugating protein taking into Sodium hydroxide (NaOH), ferric chloride hexahydrate (FeCl3 ·6H2 O), ferrous chloride, tetrahydrate (FeCl2 ·4H2 O), acrylic acid (AA), styrene (St), ammonium persulfate ((NH4 )2 S2 O8 ), ammonia (NH3 ), hydrochloric acid (HCl), acetone ((CH3 )2 CO) were of analytical grade and used as received Phosphate buffer saline (PBS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), hepatitis B surface antibody (HBsAb) were purchased from Sigma and used as received 2.1.1 Preparation of magnetic Fe3 O4 nanoparticles Fe3 O4 nanoparticles were synthesized by co-precipitation method of Fe3+ and Fe2+ under alkaline condition [11] Briefly, coprecipitation reaction is as follows: FeCl2 + 2FeCl3 + 8NaOH → Fe3 O4 + 8NaCl + 4H2 O ml of M FeCl3 , ml of M FeCl2 and 44 ml of M HCl were transferred into a three neck flask The solution was vigorously stirred under nitrogen atmosphere, followed by dropwise addition of an aqueous solution of M NH3 into the flask until the black precipitate appeared The temperature was kept constant at 80 ◦ C After h reaction, Fe3 O4 nanoparticles were collected by magnetic field separation, washed with deionized water and acetone several times and dried overnight in a vacuum oven at 40 ◦ C 2.1.2 Synthesis of Fe3 O4 /poly(St-co-AA) ferrofluids Ferrofluids were synthesized by dispersion polymerization without or with Fe3 O4 magnetic particle as core and poly(St-co-AA) as shell corresponding to ex situ and in situ capping method, respectively In the ex situ capping protocol, 20 ml of St and AA (St/AA varied from 1/1 to 1/9) was added in three neck flask containing 200 ml of water, under nitrogen atmosphere, with vigorous stirring (700 rpm) and temperature (70 ◦ C), followed by adding 0.1 g of (NH4 )2 S2 O8 , serving as the initiator After h reaction, water and unreacted monomers were removed by vacuum evaporation to get pure poly(St-co-AA) In difference from ex situ, in in situ process, Fe3 O4 capping was carried out simultaneously during the dispersion copolymerization Conjugating ability of carboxylated Fe3 O4 /poly(St-co-AA) was verified by EDC activated reaction with HBsAb The resulting complex was then separated magnetically, washed carefully with 1× PBS and characterized by gel electrophoresis on miniPROTEAN cell system (Bio-Rad) at 40 mA until the Bromophenol blue line reached the lower limit of the gels 2.1.3 Characterization methods The phase structure of Fe3 O4 was studied by X-ray diffraction (SIEMENS D-5000) Field Emission Scanning Electron Microscope (FE-SEM) and Transmission Electron Microscope (TEM) images was analyzed by Hitachi S-4800 and JEM-1200EX (voltage: 80 kV, magnification: 100,000×), respectively Dynamic light scattering (DLS) was analyzed with Zetasizer 2000 instrument (Malvern, UK) The magnetic properties of powder and ferrofluid were measured with homemade vibrating sample magnetometer (VSM), Quantum Design Physical Properties Measurement System (PPMS), respectively and were evaluated in term of saturation magnetization and coercivity The molecular structure of Fe3 O4 /poly(St-co-AA) T.T Luong et al / Colloids and Surfaces A: Physicochem Eng Aspects 384 (2011) 23–30 25 Fig Heating experiment set-up was characterized by Infra red (IR) Nicolet 6700 FT-IR spectrometer (KBr pellets, 400–4000 cm−1 , with resolution of cm−1 ) and Proton Nuclear Magnetic Resonance (1 H NMR) 500 MHz Brucker spectrometer 2.1.4 Magnetic heating experiment In this experiment a generator (RDO HFI kW) was used to create an alternating magnetic field of amplitude from 40 Oe to 100 Oe at frequency of 236 kHz (Fig 1) Samples were dispersed in 0.09–0.9 ml of water and kept in a round-bottom-shaped glass holder Temperature variation range of -20–250 ◦ C or 0–200 ◦ C was measured online either by optical thermometer(Opsens) or Copper-Constantan thermocouple, respectively The measured period lasts from 20 to 30 Sarcoma 180 cells were collected from Swiss mice bearing 10 days of Sarcoma 180 ascite Cells then were washed three times with 1× PBS and centrifuged to a pellet Magnetic fluid was added to the cancer cells Cell viability was determined by trypan blue, according to the standard procedure, described elsewhere [12] Results and discussions 3.1 Characterization of non-encapsulated Fe3 O4 and Fe3 O4 /poly(St-co-AA) ferrofluids The influence of AA on the copolymeric formation and morphology was investigated It is worth noting that the function of carboxylic acid in AA monomer is multiple: first, it induces the formation of polymer particles, thereby increasing the polymerization rate; second, it stabilizes the growing particles and finally, it provides reactive sites for biomolecular binding (e.g., with the aim for sandwich-type immunoassay detection of hepatitis B Antigene (HBsAg), hepatitis B surface antibody (HBsAb) should be used as a probe) Thus, in this study, different ratios of St/AA (from 1/1 to 1/9) were preliminarily tested To quantitatively estimate COOH density, available for protein binding on the surfaces, XPS are usually required and will not be reported in this paper However, FE-SEM images showed that there was almost no significant difference in morphology in such broad ratio (St/AA) range For further investigation, the ration of St/AA as 1/9 was rationalized for maximizing number of binding sites for biomolecular conjugation Next, quite strong diffraction lines in XRD pattern indicated that Fe3 O4 particles have been well crystallized (Fig S1, supporting information) Six characteristic peaks for Fe3 O4 corresponding to (2 0), (3 1), (4 0), (4 2), (5 1) and (4 0) were observed in uncapped nano Fe3 O4 particles (JCPDS file PDF no 65-3107) On the basic of Sherrer’s formula crystalline size D was calculated and further confirmed by the TEM analysis Briefly, XRD, TEM and FE- SEM analyses indicated that Fe3 O4 particles had a spherical shape, a smooth surface morphology and estimated particle size of 20 nm (Fig 2A) Although not being fully investigated by TEM techniques, careful imaging at efficiently high magnification (FE-SEM) can provide a good evaluation about particle size and morphology The capping process which associated with the formation of cumbersome poly(St-co-AA) on the surface of Fe3 O4 leads to a significant increase in size of core–shell nanosphere (50–70 nm) compared to that of naked Fe3 O4 core and it can be easily visualized by FE-SEM images in Fig 2B DLS data correlated satisfactorily with FE-SEM results and also indicated that the particle size distribution was relatively narrow (Fig 2C) The discrepancy between FE-SEM and DLS data can be understood if taking into account the fact that FESEM images are taken in a dried state while DLS experiment was carried out in solution By comparison of IR spectra of AA, St, bulk Fe3 O4 (figures not shown) and Fe3 O4 /poly(St-co-AA) (Fig 3A) it can be seen that poly(St-co-AA) has summarized all characteristic bands of both St and AA units On the spectrum of Fe3 O4 /poly(St-co-AA), the presence of free carboxyl group on the surface was confirmed from the C O stretching band (1702 cm−1 ) as well as a plateau of OH stretching band at ca 3015 cm−1 The origin of this plateau could be also assigned to C–H stretching of benzene ring, which initially appeared at 3030 cm−1 in St unit It can be also inferred that COOH was at the surface and the core is rich in St while the shell is rich in AA This feature is very important for effective conjugation of biomolecules on magnetic NP surface Next, the upshifting of characteristic absorption of Fe–O bonding from 575 cm−1 in bulk Fe3 O4 [13] to 652 cm−1 in Fe3 O4 /poly(Stco-AA) could bring a strong evidence to nanosize of Fe3 O4 particles in the fluids, because it could be a result of splitting into a larger number of bonds and rearranging inlocalized electrons of surface atoms of Fe3 O4 NPs [14] Also, considering position shifts of some characteristic protons, provided by H NMR spectra of St, AA, and poly(St-co-AA), polymeric encapsulation mechanism was better understood More details were given in Table S1 and Fig S2 of supporting information Further, the conjugation of HBsAb to Fe3 O4 /poly(St-co-AA) surface via covalent binding between –NH2 and –COOH moieties of HBsAb and Fe3 O4 /poly(St-co-AA), respectively, was clearly confirmed by disappearance of the C O stretching band (1702 cm−1 ) of carboxyl functional group on Fe3 O4 /poly(St-co-AA) and appearance of new bands at 1681 and 1100 cm−1 , due to C O and C–N stretching patterns, respectively, of the secondary amide linkage These fingerprint patterns unambiguously signified that the conjugation of HBsAb to Fe3 O4 /poly(St-co-AA) successfully took place by the covalent binding with amide linkage formation (Fig 3B and Table 1) [15,16] Successful covalent binding of HBsAb onto EDC 26 T.T Luong et al / Colloids and Surfaces A: Physicochem Eng Aspects 384 (2011) 23–30 Fig (A) FE-SEM (top) and TEM (bottom) images of naked Fe3 O4 powder (B) FE-SEM image of Fe3 O4 /poly(St-co-AA) ferrofluid with different ratios of St/AA (C) DLS size distribution of Fe3 O4 /poly(St-co-AA) ferrofluid (St/AA = 1/9) T.T Luong et al / Colloids and Surfaces A: Physicochem Eng Aspects 384 (2011) 23–30 27 A Transmittance (%) AA Fe 3O 4/poly(St-co-AA) 1700, ν C=O 3015, ν O-H 650 1633, ν C=C ν Fe-O 1702, νC=O 4000 350 300 250 200 1500 100 500 -1 W avenu mbers (cm ) B 0.20 Absorbance (a.u) Fig PPMS- magnetization curve of different Fe3 O4 /poly(St-co-AA) ferrofluids (St/AA = 1/9) 0.25 Fe 3O4/poly(St-co-AA) Fe 3O4/poly(St-co-AA) + HBsAb 1702 0.15 1681 0.10 1103 0.05 0.00 -0.05 2000 1800 1600 1400 1200 1000 800 -1 Wavenumbers (cm ) Fig (A) IR spectrum of Fe3 O4 /poly(St-co-AA) ferrofluid (B) IR spectra of Fe3 O4 /poly(St-co-AA) + HBsAb conjugate in the fingerprint region activated Fe3 O4 /poly(St-co-AA) ferrofluids was also confirmed by gel electrophoresis (Fig S3, supporting information) 3.2 Magnetic study of Fe3 O4 /poly(St-co-AA) ferrofluids The magnetic hysteresis loop, characterizing the response ability (magnetization, M) of magnetic materials to an external magnetic field (denoted by the magnetic field strength, H), provides the main magnetic parameters of the materials, which are saturation magnetization (Ms , it reflects the magnetizability of magnetic materials), coercive force (Hc , it characterizes the ability of magnetic materials to retain magnetization when the external magnetic field is removed) and magnetic remanence (Mr , it reflects the remaining magnetization of magnetic materials when an external magnetic field is removed) The superparamagnetic property (response to external applied magnetic field without retaining any magnetism after removal of the magnetic field) of Fe3 O4 NPs was documented by the hysteresis loop with almost immeasurable coercivity (a few of Oe) From magnetization curve, taken at room temperature (300 K) for naked Fe3 O4 NPs (Fig S4, supporting information) the saturation magnetization for Fe3 O4 NPs is estimated as ca 70 emu/g, slightly lower than that of bulk Fe3 O4 (ca 90 emu/g) This discrepancy in Ms can be resulted from the difference of particle size and/or surface modification processes as it was widely reported in the literature [17–22] The saturation magnetization of Fe3 O4 /poly(St-co-AA) fluid was shown in Fig As observed, under the same experimental conditions the fluid magnetization raised remarkably, from 0.09 to 0.20 emu/g when capping method was changed accordingly from ex situ to in situ Further, by using high power ultrasonic dispersion of Fe3 O4 NPs during 30 before capping, Fe3 O4 /poly(St-co-AA) ferrofluids with relatively high magnetization of 0.65 emu/g could be obtained (denoted as Fe3 O4 /poly(St-co-AA) is-opt, Fig 4) It was established that the linear interpolation between saturation magnetization of Fe3 O4 nanoparticles and ferrofluid could be used to calculate the content of Fe3 O4 in the later (Table 2) Effectively, since Fe3 O4 NPs and Fe3 O4 /poly(St-co-AA) ferrofluids have saturation magnetization of 70 emu/g and 0.65 emu/g, respectively, it can be inferred that the ferrofluids contain 0.9286 wt% of Fe3 O4 NPs, corresponding to 9.286 mg Fe3 O4 per gram of ferrofluid solution It is well known that the stability of magnetic NPs is a critical issue to be controlled, since Van der Waals’ forces and magnetic dipole–dipole interactions generated from residual magnetic moments tend to make the particles agglomerated and flocculated In our system, the presence of poly(St-co-AA) shell on the surface of Fe3 O4 NPs was expected to increase stabil- Table Main IR characteristic bands of St, AA, Fe3 O4 , Fe3 O4 /poly(St-co-AA) and Fe3 O4 /poly(St-co-AA) + HBsAb conjugate Possible assignments/ (cm−1 ) AA [14] St [14] Bulk Fe3 O4 [12] Nano Fe3 O4 Fe3 O4 /poly(St-co-AA) Fe3 O4 /poly(St-co-AA) + HBsAb C O (–COOH) O–H (–COOH) C C C–H (benzene) C C (benzene) Fe–O (Fe3 O4 ) C–O, amide II C–N, amide II 1701 2900 1632 – – – – – – – 1626 3030 1494 – – – – – – – – 573 – – – – – – – 652 – – 1702 3015 1633 3065 1439 650 – – – – 1633 3070 1450 650 1681 1103 28 T.T Luong et al / Colloids and Surfaces A: Physicochem Eng Aspects 384 (2011) 23–30 Table The magnetization and calculated content of Fe3 O4 in ferrofluid Sample code Description/synthetic mode Magnetic measurement apparatus Observed M(H) emu/g Calculated content of Fe3 O4 in g of magnetic solution (mg/1 g) Fe3 O4 Fe3 O4 /poly(St-co-AA)-es Fe3 O4 /poly(St-co-AA)-is Fe3 O4 /poly(St-co-AA)-is-opt Naked Fe3 O4 powder/co-precipitation Ferrofluid/ex situ copolymerization Ferrofluid/in situ copolymerization Ferrofluid/in situ copolymerization VSM PPMS PPMS PPMS 70 0.09 0.20 0.65 – 1.286 2.857 9.286 es, ex situ; is, in situ; opt, optimized sample 3.3 Magnetic heating of Fe3 O4 /poly(St-co-AA) ferrofluids As discussed above, NPs must be placed under an alternating magnetic field for a certain period of times in order to increase temperature to 42–46 ◦ C As the time period gets longer, the normal tissues surrounding tumor tissues will get more damages, so reduced heating time, equivalent to increased initial slope of heating curve (or SLP) is required For Fe3 O4 /poly(St-co-AA) ferrofluids, heating curves measured at fixed field strength (60 Oe and 236 kHz) with different concentrations of Fe3 O4 NPs (0.1–1 mg/ml) were represented in Fig 6A These heating curves clearly demonstrated that after a period of time of about 25 (1400 s) the ferrofluid temperature tends to saturated at some characteristic temperature Ts , as indicated in Table From this table, the linear dependence of dT/dt on c (equal to mi /ms ) was inferred (Fig S5, supporting information) As earlier reported, the linearity can be observed in quite broad range of concentration, namely for c = 0–50 mg/ml or 0–100 mg/ml [23] On the one side, the more the amount of the magnetic NPs or the stronger magnetic field (Fig 6B), the more the heat could be generated and the higher temperature could be reached However, it should be aware that magnetic NP dose, applied to the tumor region should be as low as possible in order to reduce the toxicity For above discussed reasons, the concentration and magnetic field strength should be optimized As indicated in Table 4, at frequency of 236 kHz and Fe3 O4 concentration of 0.5 mg/ml the temperature rise of Fe3 O4 /poly(St-co-AA) ferrofluid exhibits the highest value (43.5 ◦ C) for the field strength range of 40–80 Oe (1 mg/ml) (0.7 mg/ml) (0.5 mg/ml) (0.3 mg/ml) (0.1 mg/ml) 80 70 60 T ( oC) ity of Fe3 O4 /poly(St-co-AA) ferrofluid The magnetization curves, recorded for as-synthesized, day stored and 21 day stored ferrofluid showed a negligible decrease in magnetization (Fig 5) Indeed, taking into account the fact that the saturation magnetization (being a function of only H and T) is constant even when the particles are agglomerated or flocculated, other direct techniques such as small angle X-ray and/or neutron scattering should be applied to make sure that the stability of the synthesized ferrofluid was high 50 40 30 Fe3O4/poly(St-co-AA)-is-opt 200 400 600 800 1000 1200 1400 1600 t (s) 100 90 40 Oe 50 Oe 60 Oe 70 Oe 80 Oe 80 Fig Magnetic stability of the optimized Fe3 O4 /poly(St-co-AA) ferrofluid (St/AA = 1/9) Table Saturation temperature Ts , T and dT/dt of Fe3 O4 /poly(St-co-AA) ferrofluid at different values of Fe3 O4 concentration Fe concentration (mg/ml) dT/dt (◦ C/s) Ts (◦ C) 0.7 0.5 0.3 0.1 0.12 0.073 0.046 0.042 0.022 79.5 67.1 57 48.6 44.2 T = Ts − Tr (◦ C) T ( oC) 70 60 50 40 30 SLP (kW/g) 49.5 37.1 27 28.6 24.2 0.5 0.43 0.38 0.58 0.92 200 400 600 800 1000 1200 1400 1600 t (s) Fig (A) Heating curves of Fe3 O4 /poly(St-co-AA) ferrofluid at different values of Fe3 O4 concentration (B) Heating curves of Fe3 O4 /poly(St-co-AA) ferrofluid at different values of magnetic field strength T.T Luong et al / Colloids and Surfaces A: Physicochem Eng Aspects 384 (2011) 23–30 Table Comparison of main magnetic heating parameters with Ref [25] 100 Parameters This study Ref [25] Magnetic ferrofluid AC field parameters Fe3 O4 /poly(St-co-AA) H = 4.8 kA/m (60 Oe) f = 236 kHz Q = (H,f) = 1.13 109 (A/ms) Ferrofluid concentration ≤0.5 mg/ml Saturation temperature 44–57 ◦ C 0.84 kW/g SLP 80 Killed cell (%) 29 60 40 20 0 10 20 30 40 50 60 time (minutes) Fig Ex vivo heating experiment with Sacoma 180 cells performed for ferrofluid concentration of 0.4 mg/ml Under these optimized conditions, ex vivo magnetic heating experiment was performed on Sarcoma 180 cancer cells It can be observed that in the absence of AC field the cell could survive for over 72 h [12], while as illustrated in Fig 7, the presence of ferrofluid significantly inhibited more than 50% of cancer cells after ca.20 and totally killed them after 70–80 Although being preliminary, the results are promising Further experiments will be completed for searching optimal conditions for real, clinic hyperthermic treatment Next, it can be easily seen from Fig that SLP strictly followed square dependence on the field amplitude H, exhibiting superparamagnetic and single domain behavior of magnetic NPs in the Table Saturation temperature Ts , T and dT/dt of Fe3 O4 /poly(St-co-AA) ferrofluid at different values of magnetic field strength Fe concentration (mg/ml) H (Oe) dT/dt (◦ C/s) Ts (◦ C) 0.1 0.065 0.046 0.034 0.017 80 70 60 50 40 0.5 73.5 67.8 57 47 37.6 T = Ts − Tr (◦ C) SLP (kW/g) 43.5 37.8 27 17 7.6 0.84 0.54 0.38 0.28 0.14 Fe3 O4 /chitosan H = 30 kA/m (377 Oe) f = 80 kHz Q = (H,f) = 2.4 109 (A/ms) 20 mg/ml 53.7 ◦ C – ferrofluid Performing heating experiment at fixed frequency of f = 236 kHz and varied magnetic field strength from 60 to 100 Oe and measuring the hysteresis areas, S, a linear dependence of calculated SLP on the hysteresis area can be inferred (figure not shown) This finding suggests that the loss power comes both from the Néel relaxation process with significant contribution of the hysteresis mechanism [23] It should be emphasized that SLP data of our investigated samples vary quite broadly: from 0.14 up to 0.84 kW/g corresponding to 40 Oe and 80 Oe, respectively (Table 4) It should keep in mind that SLP values also depend on particle size and that parameter normally varies from one to another in different studies However, to the best of our knowledge, under the “quasi-similar” conditions, the found values of SLP are superior or at least comparable to the best results, recently reported by Hergt and Timko, respectively in Refs [10,24] Furthermore, under those optimized AC filed conditions, the ferrofluid exhibited highly efficient and easy controllable magnetic heating effect for hypertherimia at much less Fe3 O4 dose, with respect to earlier reported studies of Zhao et al [25], although not the same polymeric shell was used (Table 5) These results are very promising for further in vivo hyperthermic applications Besides the above main discussed parameters (concentration and magnetic field strength), there are other parameters that may affect magnetic heating such as particle size, coercivity, remanence but they were intensively studied in many other reports [26–29] and will not be repeated in this study Nevertheless, it might worth noting that under the same experimental conditions except for St/AA ratio, the dependence of T on this ratio was observed and plotted in Fig Effectively, St/AA ratio variation probably related to the viscosity change of Fe3 O4 /poly(St-co-AA) ferrofluids It is well known that the heat dissipated through Néel 110 100 90 0.10 80 o T (oC) dT/dt ( C/s) 0.08 0.06 70 60 1600 50 0.04 40 30 0.02 10 20 30 40 50 60 70 H (10 Oe) Fig Linear dependence of dT/dt on the square amplitude of the magnetic field H2 200 400 600 St/AA=1/1 St/AA=1/3 St/AA=1/5 St/AA=1/7 St/AA=1/9 800 1000 1200 1400 1600 t (s) Fig Heating curves of Fe3 O4 /poly(St-co-AA) ferrofluid at different values of St/AA ratio 30 T.T Luong et al / Colloids and Surfaces A: Physicochem Eng Aspects 384 (2011) 23–30 relaxation is not influenced by viscosity of the medium, whereas, Brownian relaxation is influenced greatly In that case, with increasing St/AA ratio, the viscosity of the medium becomes lower (the freedom of particle rotation is bigger), the heat dissipated by Brownian relaxation will increase To determine the relative contribution of Néel and Brown relaxation as well as to realize “effective” SLP values in in vivo experiment and thus find out the strategy that should be applied in in vivo magnetic heating treatments are beyond the aim of the present study and will be subjected to investigation in the next papers Conclusion In summary, a simple route for surface-carboxylated functionalization of carboxylated Fe3 O4 magnetic NPs to obtain Fe3 O4 /poly(St-co-AA) ferrofluids was developed Owing to carboxylated groups, Fe3 O4 /poly(St-co-AA) can be used in bioconjugation reaction A correlating discussion has been given on dependence of magnetic heating effect on AC field parameters and Fe3 O4 concentration The obtained fluid exhibits more efficient and tunable magnetic heating effect for hyperthermia at much less Fe3 O4 concentration, with respect to earlier reported results Nevertheless, further studies on fluid stability, heating rate efficiency (time exposure, Fe3 O4 dose, AC field parameters, toxicity) and in vivo experiments should be continued and will be reported in the upcoming paper Acknowledgements The authors are grateful for the financial support for this work by MOST application oriented basic research project (2009–2012, code: 04/02/742/2009/HÐ-ÐTÐL) and VAST key project on application of ferrofluid (2009–2010) Appendix A Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.colsurfa.2011.02.050 References [1] R Massart, E Dubois, V Cabuil, E Hasmonay, Preparation and properties of monodisperse magnetic fluids, Journal of Magnetism and Magnetic Materials 149 (1995) 1–5 [2] X Liu, Y Guan, H Liu, Z Ma, Y Yang, X Wu, Preparation and characterization of magnetic polymer nanospheres with high protein binding capacity, Journal of Magnetism and Magnetic 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(s) Fig (A) Heating curves of Fe3 O4 /poly(St-co-AA) ferrofluid at different values of Fe3 O4 concentration (B) Heating curves of Fe3 O4 /poly(St-co-AA) ferrofluid at different values of magnetic. .. correlating discussion has been given on dependence of magnetic heating effect on AC field parameters and Fe3 O4 concentration The obtained fluid exhibits more efficient and tunable magnetic heating effect. .. established that the linear interpolation between saturation magnetization of Fe3 O4 nanoparticles and ferrofluid could be used to calculate the content of Fe3 O4 in the later (Table 2) Effectively,