- Email: [email protected]
dedoping method
Materials Chemistry and Physics 145 (2014) 27e35
Contents lists available at ScienceDirect
Materials Chemistry and Physics journal homepage: www.elsevier.com/locate/matchemphys
g-Fe2O3/polyaniline-lonidamine prepared by doping/dedoping method Han Huang, Li Zhang, Jinqing Kan* School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
g-Fe2O3/polyaniline-lonidamine synthesized by the doping-dedoping of polyaniline. The composite has superparamagnet, good conductivity and electrochemical activity. The composite can be concentrated by applying magnetic field. The g-Fe2O3/polyaniline-lonidamine can kill MadineDarby Canine Kidney (MDCK).
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 August 2013 Received in revised form 29 December 2013 Accepted 4 January 2014
A new conductive superparamagnetic nanocomposite, g-Fe2O3/polyaniline-lonidamine (g-Fe2O3/PANILND), was synthesized successfully by two-step doping method. The properties of g-Fe2O3/PANI-LND were tested by FT-IR, X-ray diffractometry (XRD), X-ray photoelectron spectroscopy (XPS), vibrating sample magnetometer (VSM), transmission electron microscopy (TEM), thermogravimetric analysis (TGA) and cyclic voltammetry (CV). The results show that the particle size of g-Fe2O3/PANI-LND with stable electric conductivity and high electrochemical activity was approximately 40 nm, and the optimal molar ratio of nLND : ngFe2 O3 =PANI was 1:1 for the synthesis of g-Fe2O3/PANI-LND. The g-Fe2O3/PANI-LND could be concentrated in a magnetic field and located to the region of interest, which meant to potentially provide effective treatment of tumor cells with less therapeutic doses and side-effects. Ó 2014 Elsevier B.V. All rights reserved.
Keywords: Chemical synthesis Electrical conductivity Polymers Composite materials Magnetic properties X-ray photo-emission spectroscopy (XPS)
1. Introduction In the past decades, polyaniline (PANI) has become one of the most important functional conductive polymers, because of ease of processability, environmental stability, the high and reversible nature of its conductivity, versatile redox behavior and protondoping effect [1e8]. Numerous studies have demonstrated that when combined with inorganic compounds through chemical
* Corresponding author. Tel.: þ86 514 87975590x9415; 87975590x8410. E-mail addresses: [email protected], [email protected] (J. Kan).
fax:
0254-0584/$ e see front matter Ó 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.matchemphys.2014.01.009
þ86
514
oxidative polymerization, PANI could improve the electrochemical activity and surface properties of the inorganic compound significantly with no effect on its own structure [9e12]. In addition to this, PANI can render conduction by protonation of the emeraldine base (EB) leading to emeraldine salt (ES) through acid-base chemistry. The positive charge in the PANI chains facilitates its incorporation of negatively charged ions via doping [13e16] thereby promising numerous potential applications in the synthesis of composite materials. Meanwhile, iron oxides of a small size are very interesting because of their special catalytic and suitable magnetic properties. Especially, magnetite (g-Fe2O3) is one of the most widely investigated magnetic nanomaterials because of its non-toxicity, thermal
28
H. Huang et al. / Materials Chemistry and Physics 145 (2014) 27e35
and chemical stability and easy-functionalization, and is being viewed as a valuable material for information storage, magnetic sensors, drug delivery, catalysts, bioseparation, etc. [17e21]. Lonidamine (LND), a dichlorinated derivative of indazole-3carboxylic acid, is a new non-conventional anticancer agent that selectively interferes with the energy metabolism of neoplastic cells by delaying their growth and inhibiting the repair processes [22,23]. The clinical study demonstrates that lonidamine has a deterrent effect on lung cancer, chronic lymphocytic leukemia, malignant melanoma, breast cancer, prostrate cancer and brain tumors [24e28]. In this work, we focus attention concerning the synthesis and properties of nanoscaled superparamagnetic conductive composite, g-Fe2O3/polyaniline-lonidamine (g-Fe2O3/PANI-LND). The gFe2O3 with a shell of PANI was doped with lonidamine to form gFe2O3/PANI-LND in the acid solution. The composite g-Fe2O3/PANILND can be driven by an applied magnetic field to the target site. The advantages of the method lie in that the functional groups of lonidamine for g-Fe2O3/PANI-LND are not changed, which meant that the drug like lonidamine also can keep its effectiveness after doped in g-Fe2O3/PANI, thereby indicating that g-Fe2O3/PANI-LND may have potential applications in targeted therapy. 2. Materials and methods 2.1. Materials Ferrous sulfate heptahydrate (FeSO4$7H2O), ferric chloride hexahydrate (FeCl3$6H2O), aniline, ammonium persulfate (APS), ethanol, ammonia and hydrochloric acid were all of analytical grade and obtained from Sinopharm Chemical Reagent Co., Ltd. Sodium dodecyl benzene sulfonate (SDBS) was purchased from BIOBASIC. Lonidamine (LND) was procured from Changzhou Rui Ming Pharmaceutical Co., Ltd. Aniline was distilled under reduced pressure prior to use. 1-ethyl-3-methylimidazolium ethylsulfate [EMIM][EtSO4] was synthesized as reported earlier [29]. Brittone Robinson (BR) buffer solutions of required pH were prepared by adding 0.04 M acetic acid and 0.04 M boric acid into 0.04 M orthophosphoric acid with appropriate amount of 0.2 M sodium hydroxide. Cell lines of canine kidney cells were provided by College of Animal Science of Yangzhou University. Double-distilled water was used throughout the experiments. 2.2. Preparation of magnetic nanoparticles, g-Fe2O3
g-Fe2O3 was prepared by coprecipitation [30e33]. Typically, FeCl3$6H2O (0.02 mol) and FeSO4$7H2O (0.01 mol) were dissolved in 20 and 10 mL volumes of double-distilled water, respectively. The two iron-salt solutions were then mixed together and dropped into
200 mL of 0.6 M ammonium hydroxide solution. The pH of the mixture was kept between 11 and 12 with the addition of concentrated ammonium hydroxide solution. After stirring for 30 min, the as-prepared g-Fe2O3 was separated from the solution by a strong magnet, washed with double-distilled water and dried at 85 C under vacuum. 2.3. Synthesis of magnetic conductive composite, g-Fe2O3/PANI The preparation of g-Fe2O3/PANI was based on a previously published method [34]. Briefly, a solution containing 3.65 mL of 0.04 mol aniline, 0.5 M HCl, 1.22 mL of [EMIM][EtSO4] and several drops of SDBS were added into the solution of g-Fe2O3 (0.02 mol). APS (0.04 mol) was also added as an initiator and the reaction was kept for 4e6 h at room temperature by stirring to obtain g-Fe2O3/ PANI. The product was filtered and washed with double-distilled water and ethanol until a colorless filtrate was obtained. The final product was dried in a vacuum oven at 65 C. 2.4. Preparation of g-Fe2O3/PANI-LND by two-step doping method Ammonia (0.5 M) was stirred after the addition of g-Fe2O3/PANI for 15e20 h. After filtration and washing with water and ethanol, the product was dried in a vacuum oven at 65 C. Then the product was mixed with different proportions of lonidamine in doubledistilled water with the pH adjusted to 2e4 by HCl. After stirred for 13e15 h, the solution was filtered and the final product was washed and dried under vacuum at 65 C. Fig. 1 was the schematic representation for the preparation of g-Fe2O3/PANI-LND. The overall synthesis process of g-Fe2O3/PANI-LND is shown in Fig. 2. As shown in Fig. 2, g-Fe2O3/PANI was prepared by mixing gFe2O3 and aniline with SDBS as dispersant and APS as initiator. The addition of [EMIM][EtSO4] facilitated the availability of doped PANI across wider pH range. After filtration and washing with doubledistilled water and ethanol, the product was mixed with ammonia which changed the doped g-Fe2O3/PANI into intrinsic gFe2O3/PANI. Then the intrinsic g-Fe2O3/PANI was doped with lonidamine to form the final product g-Fe2O3/PANI-LND by adding HCl. HCl was used to allow the interaction between Hþ with polyaniline chain to form g-Fe2O3/PANIþ, which was then doped with LND thereby leading to the formation of g-Fe2O3/PANI-LND following charge balance. 2.5. Characterization of g-Fe2O3, g-Fe2O3/PANI and g-Fe2O3/PANILND Infrared spectra were recorded on a pressed KBr pellet employing a TENSOR Model 27 FT-IR spectrometer with the
Fig. 1. Schematic representation for the preparation of g-Fe2O3/PANI-LND.
H. Huang et al. / Materials Chemistry and Physics 145 (2014) 27e35
29
Fig. 3. Schematic illustration of the applied magnetic field.
Piston 1, 2 and 3 were opened first and the initial solution was collected. Then the dripping solution flew through the applied magnetic field. During the process, the field intensity was measured by Teslameter (SHT-4A, China). The solutions in and flowing through the applied magnetic field were received in the centrifuge tubes and their concentrations were measured after drying and weighing.
3. Results and discussion Fig. 2. Synthesis process of g-Fe2O3/PANI-LND.
resolution of 4 cm1. X-ray diffraction (XRD, MAC) diagrams were collected using X-ray powder diffractometer. Chemical states of elements were measured by X-ray Photoelectron Spectroscopy (XPS, PHI 5000, VersaProbe, Japan) from 0 to 1100 eV under vacuum. Thermo-gravimetric analysis (TGA) was performed using a synchronous thermal analyzer (Netzsch STA 409 PC) at a heating rate of 10 C min1 from 30 to 800 C under nitrogen. The magnetization was measured by vibrating sample magnetometer (VSM, EV7, ADE, USA) in fields ranging from 8000 to 8000 Oe at room temperature. The morphology was examined by transmission electron microscopy (TEM, Tecnai-12, Philip Apparatus). Cyclic voltammetry (CV) was performed on a CHI 660 electrochemical workstation (Shanghai, China). The conductivity of the samples was measured by conventional four-probe technique on pressed pellets of the powder samples with an YJ8312 model current source (Shanghai Huguang Instrument Works, China) and digital multimeter (DDS-IIA, China).
3.1. FT-IR spectra of samples Fig. 4 shows the FT-IR spectra of g-Fe2O3, PANI, g-Fe2O3/PANI, gFe2O3/PANI-LND and lonidamine. In curve a, the peaks at 1623 cm1 and 598 cm1 are stretching vibrations (n) of FeeO and characteristic absorption peak of g-Fe2O3 respectively. In curve c, the peaks at 1556 cm1 and 1487 cm1 are the stretching vibrations of quinone and benzene respectively and the stretching vibration of CeN (nCeN) was observed at 1299 cm1. The absorption peak near 1141 cm1 results from the NeQeN (Q denotes quinoid ring) stretching mode and is an indication of electron delocalization in PANI, which means PANI, in curve b, c and d, are in the doped state. In addition, compared with curve c, curve d has a new absorption peak of the C]O stretching vibration (n) (nC]O) at 1716 cm1, which also appears at 1701 cm1 in case of lonidamine (curve e) thereby proving that lonidamine is doped into the chain of g-Fe2O3/ PANI-LND [38e41].
2.6. Cell experiments of g-Fe2O3/PANI-LND MadineDarby Canine Kidney (MDCK) cells [35e37] were cultured in Petri dishes at 37 C for 20 h while shaking gently in order to avoid accumulation. 1 mL of culture fluid was mixed with 20 mg of g-Fe2O3/PANI, g-Fe2O3/PANI-LND and lonidamine respectively. The mixture was sterilized under high-pressure steam and dried on the laminar flow. 20 mL of each former suspension was put on cell culture plates for 20 h at 37 C under 5% (V/V) CO2. The results were observed by microscope. 2.7. Simulation of applied magnetic field A simple applied magnetic field simulation model is illustrated in Fig. 3. 50 mg g-Fe2O3/PANI-LND was dispersed in 200 mL of ultrapure water with different concentrations of PVP in an infusion bottle by ultrasonication. The solution was dripped into test tubes via an infusion tube with a magnet placed in the middle of the infusion tube.
Fig. 4. FT-IR spectra of samples at room temperature. (a) g-Fe2O3, (b) PANI, (c) g-Fe2O3/ PANI, (d) g-Fe2O3/PANI-LND and (e) lonidamine.
30
H. Huang et al. / Materials Chemistry and Physics 145 (2014) 27e35
The FT-IR spectra shown in Fig. 5A, shows the successful doping of g-Fe2O3/PANI with lonidamine in various solvents. However, compared with water, acidic solution improves the conductivity of final product (g-Fe2O3/PANI-LND). In addition, HCl has small size and easily removed because of volatility. Considering the difficulty in purification, aqueous HCl (0.1 M) was chosen as the experimental solvent. The effect of lonidamine concentration on the FT-IR spectra of gFe2O3/PANI-LND is illustrated in Fig. 5B. With the molar ratio between LND and g-Fe2O3/PANI decreasing from 3:1 to 1:3, the transmittance of its characteristic oscillation (nC]O) at 1716 cm1 gradually decreased, the intensity of the characteristic peaks of quinone and benzene also gradually decreased, which verified that the doping effect of lonidamine was proportional to its concentration in the solution. 3.2. XRD and XPS patterns of samples The crystallinity of g-Fe2O3 prepared by previous method with some modifications was characterized by XRD analysis. As shown
Fig. 6. (A) XRD pattern and (B) XPS spectra of g-Fe2O3.
in Fig. 6A, the characteristic diffractions at 2q ¼ 30.2, 35.5, 43.2, 53.6, 57.0 and 62.7, correspond to the spinel structure of magnetite (g-Fe2O3, JCDPS, No. 39-1346). Fig. 6B shows representative XPS spectrum of the prepared product. Sharp photoelectron peaks
Fig. 5. (A) FT-IR spectra of samples at room temperature. (a) Lonidamine, (b) g-Fe2O3/ PANI, (c) g-Fe2O3/PANI-LND (double-distilled water), (d) g-Fe2O3/PANI-LND (0.1 M HCl) and (e) g-Fe2O3/PANI-LND (0.05 M H2SO4); (B) FT-IR spectra of g-Fe2O3/PANI-LND with different molar ratios between lonidamine and g-Fe2O3/PANI [nLND : ngFe2 O3 =PANI ¼ (a) 3:1, (b) 2:1, (c) 1:1, (d) 1:2 and (e) 1:3].
Fig. 7. XRD patterns of samples. (a) PANI, (b) g-Fe2O3, (c) g-Fe2O3/PANI, (d) g-Fe2O3/ PANI-LND and (e) lonidamine.
H. Huang et al. / Materials Chemistry and Physics 145 (2014) 27e35
Fig. 8. XRD of g-Fe2O3/PANI-LND with different molar ratios between lonidamine and g-Fe2O3/PANI [nLND : ngFe2 O3 =PANI ¼ (a) 1:3, (b) 1:2, (c) 1:1, (d) 2:1 and (e) 3:1].
appear at binding energies of 711 eV (Fe 2p3/2), 725 eV (Fe 2p1/2), and 531 eV (O 1s), along with a C 1s peak at 285 eV due to contamination from the XPS instrument itself. The photoelectron peaks at 711.5 and 725.4 eV are the characteristic doublets of Fe 2p3/ 2 and 2p1/2 core-level spectra of iron oxide, respectively, which is consistent with the oxidation state of Fe in Fe2O3. The inset shows that a new peak has appeared at 718.2 eV which could be assigned to a pure g-Fe2O3 [42]. The XRD patterns of PANI, g-Fe2O3, g-Fe2O3/PANI, g-Fe2O3/PANILND and lonidamine are shown in Fig. 7. In curve a, the two main peaks at 2q ¼ 15 and 2q ¼ 25 , assigned to the periodicity of the repeat unit of the PANI chain and the periodicity parallel to the polymer backbone chain [43]. The diffraction angles at 30.2 , 35.6 , 43.2 , 57.0 and 62.9 were characteristic diffraction peaks of gFe2O3 (curve b, c and d). A wide and low peak at 2q ¼ 21 occurred in curve a, which was due to g-Fe2O3 coated with PANI successfully. Compared curve c with curve d, the new diffraction peaks of gFe2O3/PANI-LND (17.4 , 25.0 , 26.0 and 27.2 ) correspond to characteristic diffraction peaks of lonidamine due to the dopant of lonidamine.
Fig. 9. TGA of samples: (a) g-Fe2O3/PANI, (b) g-Fe2O3/PANI-LND and (c) lonidamine.
31
Fig. 10. VSM of samples (a) g-Fe2O3, (b) g-Fe2O3/PANI and (c) g-Fe2O3/PANI-LND (inset: VSM of samples from 500 to 500 Oe).
As shown in Fig. 8, with increase in the concentration of lonidamine, the relative intensity of g-Fe2O3/PANI-LND diffraction peak at 35.6 has decreased, but that of the g-Fe2O3/PANI-LND diffraction peak at 11.4 , 17.4 , 25.0 , 26.0 and 27.2 increased, which is in consistence with the results of FT-IR spectra.
3.3. TGA of samples Fig. 9 illustrates the TGA curves of the g-Fe2O3/PANI, g-Fe2O3/ PANI-LND and lonidamine. Three-stage weight loss was observed at g-Fe2O3/PANI (curve a). The first step (about 100 C) indicates the loss of water in the polymer; the second step (about 230 C) might be attributed to the loss of acid dopant or volatile elements bound to the polyaniline chain, and the last step (460 C) was caused by skeleton bond decomposition of polyaniline chain [44,45]. Curve b shows that g-Fe2O3/PANI-LND quickly lost weight at 250 C, which was clearly related with lonidamine (curve c), which was further confirmed in the following VSM and TEM images.
Fig. 11. VSM of g-Fe2O3/PANI-LND with different molar ratios between lonidamine and g-Fe2O3/PANI [nLND : ngFe2 O3 =PANI ¼ (a) 1:3, (b) 1:2, (c) 1:1, (d) 2:1 and (e) 3:1].
32
H. Huang et al. / Materials Chemistry and Physics 145 (2014) 27e35
Fig. 12. TEM images of samples: (a)g-Fe2O3, (b)g-Fe2O3/PANI and (c) g-Fe2O3/PANI-LND.
3.4. VSM of samples Magnetizations of nanoparticles obtained from VSM are shown in Fig. 10. The results illustrate that the saturated magnetizations of g-Fe2O3, g-Fe2O3/PANI and g-Fe2O3/PANI-LND decreased from 66.3 emu g1 (curve a) to 32.2 emu g1 (curve b), and further to 19.6 emu g1 (curve c), which owed to the decrease in content of gFe2O3 because the magnetization is mainly dependent on the mass
fraction of g-Fe2O3 [46]. The inset picture clearly shows that the saturated magnetizations of g-Fe2O3, g-Fe2O3/PANI and g-Fe2O3/ PANI-LND are all almost zero without applied magnetic field, indicating that all the samples are superparamagnetic. As shown in Fig. 11, the coercive forces of curve a, b, c, d and e are almost zero, which indicated that the concentration of lonidamine had no effect on superparamagnetism of g-Fe2O3/PANI-LND. As the concentration of lonidamine is increased, the saturated magnetization of g-Fe2O3/PANI-LND is decreased, owing to the decrease in mass fraction of g-Fe2O3. The high intensity of magnetism indicates the decrease in the lonidamine content. For ensuring the magnetic strength of nanocomposite and to avoid the decrease of medical efficacy coming from lonidamine, the molar ratio of nLND : ngFe2 O3 =PANI ¼ 1:1 was selected. 3.5. TEM images of samples From the TEM images in Fig. 12, it is obvious that the diameter of
g-Fe2O3 particles ranges between 10 and 20 nm. With the encapsulation of polyaniline and doping of lonidamine, the sizes of gFe2O3/PANI and g-Fe2O3/PANI-LND increased and the average size of g-Fe2O3/PANI-LND was about 40 nm. After doping with lonidamine, the morphology did not change significantly. The results demonstrate that lonidamine was successfully doped into PANI, and the size of nanoparticles was within the superparamagnetic range (14e90 nm), which is consistent with the results obtained from the MeH curve.
Fig. 13. (A) CV curves for g-Fe2O3/PANI-LND from 0.7 V to 0.7 V at 30 mV s1. pH ¼ (a) 4, (b) 5, (c) 6 and (d) 7 (inset picture was electrode reaction for Nernst equation); (B) CV curves for g-Fe2O3/PANI-LND from 0.6 V to 1.0 V at pH ¼ 4 at scan rates (mV s1) of (a) 300, (b) 200, (c) 150, (d) 100, (e) 70, (f) 50 and (g) 30.
Fig. 14. Conductivity of g-Fe2O3/PANI-LND with different molar ratios between lonidamine and g-Fe2O3/PANI [nLND : ngFe2 O3 =PANI ¼ 1:3, 1:2, 1:1, 2:1 and 3:1].
H. Huang et al. / Materials Chemistry and Physics 145 (2014) 27e35
33
Fig. 17. Cell culture experiment of g-Fe2O3/PANI-LND (a) 5 mL, (b) 20 mL and (c) 100 mL.
3.7. Conductivity of g-Fe2O3/PANI-LND
Fig. 15. The relationship between the doping amount of lonidamine with different compositions of lonidamine and g-Fe2O3/PANI [nLND : ngFe2 O3 =PANI ¼ 1:3, 1:2, 1:1, 2:1 and 3:1].
3.6. CV curves for g-Fe2O3/PANI-LND Fig. 13A shows the CV plots of g-Fe2O3/PANI-LND in BR buffer at different pH. With increase in pH, both the anodic peak current of g-Fe2O3/PANI-LND and its corresponding potential decreased. The reason was that the doping level of PANI increased at low pH. Ac1=2 cording to the equation ip ¼ ð2:69 105 Þn3=2 AD0 C0* v1=2 , with the increasing concentration of compound A (inset picture), the value of ip increased. However, According to Nernst equation 2n þ 2n f ¼ f+B=A þ RT the nF lnf½CðBÞ$CðH Þ =½CðAÞ$CðLNDÞ g[47,48], lower concentration of Hþ should lead to the negative shift of the oxidation potential of PANI. Hence, the reaction equation is proposed and shown in the inset. The CV curves of g-Fe2O3/PANI-LND in BR buffer solution at different scan rates were illustrated in Fig. 13B. According to the 1=2 equation ip ¼ ð2:69 105 Þn3=2 AD0 C0* v1=2 , with the decrease in scan rate, both the oxidation and the reduction peak currents of gFe2O3/PANI-LND decreased.
As shown in Fig. 14, the conductivity of g-Fe2O3/PANI-LND measured by four-probe method decreased with increasing concentrations of lonidamine, which was due to the poor conductivity of lonidamine. Considering the conductivity of g-Fe2O3/PANI-LND and the drug loading capacity of g-Fe2O3/PANI, the optimum molar ratio of lonidamine and g-Fe2O3/PANI was 1:1, which corresponds to the result of VSM. 3.8. Determination of doping amount of lonidamine The doping effect of lonidamine on the amount of g-Fe2O3/PANI in g-Fe2O3/PANI-LND was measured using combustion method. gFe2O3/PANI (W1) and g-Fe2O3/PANI-LND (W2) were added into the empty crucible (W3), respectively. The samples were burned in the electric stove till a constant mass with the color of each sample changed from dark blue to dark red. The total mass of the crucible and the residue were W4 and W5, respectively. The doping ratio of lonidamine (Y) was calculated by
Y ¼ W2 þ ½W1 ðW5 W3 Þ=ðW3 WÞ4 : As shown in Fig. 15, the doping amount of lonidamine increased with increasing concentration of lonidamine, which further confirmed that g-Fe2O3/PANI was doped well with lonidamine and g-Fe2O3/PANI acted as an excellent drug carrier. The results were consistent with those of MeH and conductivity of g-Fe2O3/PANILND. 3.9. Cell culture experiments
Fig. 16. Cell culture experiments of (A) g-Fe2O3/PANI and (B) lonidamine (a) 0 mL, (b) 20 mL and (c) 100 mL.
In principle, if the added material has no effect on MDCK cells, the cell-culture medium remains yellowish brown, otherwise it will become red. Fig. 16A shows the cell-culture media containing 0 mL (0 mg$mL1), 20 mL (0.4 mg$mL1) and 100 mL (2 mg$mL1) of gFe2O3/PANI in a, b and c, respectively. After culturing for 20 h, none of the solutions turned into red. This indicates that g-Fe2O3/PANI was not toxic for MDCK cells. Compared with Fig. 16A and B showed that lonidamine could kill MDCK cells, and the more it was added, the more significant the effect on MDCK cells would be. The results obtained from concentration gradient experiment of g-Fe2O3/PANI-LND are shown in Fig. 17. The solution (a) kept its original color, the solution (b) turned into a very pale red color. This is due to the low content of g-Fe2O3/PANI-LND in the solutions, which is very similar to earlier reported results [49]. However, when 100 mL (2 mg mL1) of g-Fe2O3/PANI-LND was added into solution (c), it becomes red color, which show that a massive number of MDCK cells have been killed in the solution. It is seen that only when the concentration of g-Fe2O3/PANI-LND is high enough, it has an efficient effect on killing MDCK cells at 37 C. The Micrographs of the above three solutions are shown in Fig. 18. The effect of different concentrations of g-Fe2O3/PANI-LND was investigated using optical microscopy and the results are shown in
34
H. Huang et al. / Materials Chemistry and Physics 145 (2014) 27e35
Fig. 18. Micrographs for effect of g-Fe2O3/PANI-LND concentration on cell-culture medium (a) 5 mL, (b) 20 mL and (c) 100 mL.
Fig. 18a, b and c for 5 mL (0.1 mg mL1), 20 mL (0.4 mg mL1) and 100 mL (2 mg mL1) concentrations respectively. It could be observed that the color of the cell-culture medium in a and b almost remain unchanged, while the color of the cell-culture medium turned to red in c, which confirmed that only cells in c could be killed by the g-Fe2O3/PANI-LND and the results are consistent with those shown in Fig. 16. 3.10. Effect of applied magnetic field The results of the magnetic drug targeting of g-Fe2O3/PANILND are shown in Fig. 19. It could be observed that the concentration of solution remaining in the magnetic field was higher than that of effluent liquid from the applied magnetic field when the strength of magnetic field was more than 0.15 T. Furthermore, when the strength of magnetic field was 0.312 T, the concentration of effluent liquid from the applied magnetic field was reduced to 0.058 g kg1 which was 53.2% of the original concentration (0.109 g kg1) while the concentration of solution remaining in the magnetic field was 0.171 g kg1 which was 1.57 times of the original concentration or 2.95 times of effluent liquid from the applied magnetic field. So it was confirmed that g-Fe2O3/PANILND could be focused on a defined area under the control of the applied magnetic field and its concentration could be reduced in other parts of solution, which might be used in the magnetic targeting therapy of tumor.
Fig. 19. Concentrations of receiver in different strengths of the applied magnetic field. (a) Solution remaining in the magnetic field and (b) effluent liquid from the applied magnetic field.
4. Conclusions A conductive superparamagnetic nanocomposite (g-Fe2O3/ PANI-LND) has been synthesized following a two-step method. Various characterizations of g-Fe2O3/PANI-LND have validated its good electrochemical activity and stable magnetic conductivity with the particle size of about 40 nm. The molar ratio of nLND : ngFe2 O3 =PANI ¼ 1:1 was found to be optimum for the synthesis of g-Fe2O3/PANI-LND. g-Fe2O3/PANI-LND can be partially concentrated in a defined area by applying magnetic field. g-Fe2O3/ PANI-LND further exhibited the capability of killing cells in their adsorbing position, which promised the potential application in magnetic targeting tumor therapy. Acknowledgments This project was supported by National Science Foundation of China (No. 20873119), and by the Priority Academic Program Development of Jiangsu Higher Education Institutions. Part of data was from Testing Center of Yangzhou University. References [1] P.P. Jeeju, S.J. Varma, P.A. Francis Xavier, A.M. Sajimol, S. Jayalekshmi, Mater. Chem. Phys. 134 (2012) 803e808. [2] T.B. Cao, L.H. Wei, S.M. Yang, M.F. Zhang, C.H. Huang, W.X. Cao, Langmuir 18 (2002) 750e753. [3] W. Wang, Q. Li, B. Wang, X.T. Xu, J.P. Zhai, Mater. Chem. Phys. 135 (2012) 1077e1083. [4] H. Zengin, G. Kalayci, Mater. Chem. Phys. 120 (2010) 46e53. [5] S. Ben-Valid, H. Dumortier, M. Décossas, R. Sfez, M. Meneghetti, A. Bianco, S. Yitzchaik, J. Mater. Chem. 20 (2010) 2408e2417. [6] A. Fattoum, Z. Ben Othman, M. Arous, Mater. Chem. Phys. 135 (2012) 117e 122. [7] J. Kan, X. Pan, C. Chen, Biosens. Bioelectron. 19 (2004) 1635e1640. [8] E.H. Kim, Y.J. Lee, J.G. Bang, K.J. Kim, S.J. Choe, Mater. Chem. Phys. 134 (2012) 814e820. [9] X.T. Zhang, J. Zhang, R.M. Wang, Z.F. Liu, Carbon 42 (2004) 1455e1456. [10] K. Singha, A. Ohlana, R.K. Kotnalab, A.K. Bakhshic, S.K. Dhawana, Mater. Chem. Phys. 112 (2008) 651e658. [11] P.S. Smertenkoa, O.P. Dimitrieva, S. Schraderb, L. Brehmerb, Synth. Met. 146 (2004) 187e196. [12] Z.Z. Wang, H. Bia, J. Liu, T. Sun, X.L. Wu, J. Magn. Magn. Mater. 320 (2008) 2132e2139. [13] S.J. Tian, J.Y. Liu, T. Zhu, W. Knoll, Chem. Commun. (2003) 2738e2739. [14] M.M. Ayad, E.A. Zaki, Eur. Polym. J. 44 (2008) 3741e3747. [15] J.H. Wu, Q.W. Tang, Q.H. Li, J.M. Lin, Polymer 49 (2008) 5262e5267. [16] A. Varela-Álvarez, J.A. Sordo, G.E. Scuseria, J. Am. Chem. Soc. 127 (2005) 11318e11327. [17] M.E. Khosroshahi, L. Ghazanfari, Mater. Chem. Phys. 133 (2012) 55e62. [18] J. Wang, Q.W. Chen, C. Zeng, B.Y. Hou, Adv. Mater. 16 (2004) 137e140. [19] R. Suresh, R. Prabu, A. Vijayaraj, K. Giribabu, A. Stephen, V. Narayanan, Mater. Chem. Phys. 134 (2012) 590e596. [20] T. Hyeon, S.S. Lee, J. Park, Y. Chung, H.B. Na, J. Am. Chem. Soc. 123 (2001) 12798e127801. [21] C. Caparrós, M. Benelmekki, P.M. Martins, E. Xuriguera, C.J.R. Silva, L.I.M. Martinez, S. Lanceros-Méndez, Mater. Chem. Phys. 135 (2012) 510e517.
H. Huang et al. / Materials Chemistry and Physics 145 (2014) 27e35 [22] M. De Lena, V. Lorusso, A. Latorre, G. Fanizza, G. Gargano, L. Caporusso, M. Guida, A. Catino, E. Crucitta, D. Sambiasi, A. Mazzei, Eur. J. Cancer 37 (2001) 364e368. [23] G. Giorgioni, S. Ruggieri, A. Di Stefano, P. Sozio, B. Cinque, L. Di Marzio, G. Santoni, F. Claudi, Bioorg. Med. Chem. Lett. 18 (2008) 2445e2450. [24] L. Milane, Z.F. Duan, M. Amiji, J. Nanomed. Nanotechnol. 7 (2011) 435e444. [25] Y. Mardor, O. Kaplan, M. Sterin, J. Ruiz-Cabello, E. Ash, Y. Roth, I. Ringel, J.S. Cohen, Cancer. Res. 60 (2000) 5179e5186. [26] P. Papaldo, M. Lopez, E. Cortesi, E. Cammilluzzi, M. Antimi, E. Terzoli, G. Lepidini, P. Vici, C. Barone, G. Ferretti, S. Di Cosimo, C. Nisticò, P. Carlini, F. Conti, L. Di Lauro, C. Botti, C. Vitucci, A. Fabi, D. Giannarelli, P. Marolla, J. Clin. Oncol. 21 (2003) 3462e3468. [27] S. Di Cosimo, G. Ferretti, P. Papaldo, P. Carlini, A. Fabi, F. Cognetti, Drug Today 39 (2003) 157e174. [28] P. Pacini, M. Rinaldini, R. Algeri, A. Guarneri, E. Tucci, G. Barsanti, B. Neri, P. Bastiani, S. Marzano, C. Fallai, Eur. J. Cancer 36 (2000) 966e975. [29] J.D. Holbrey, R.D. Rogers, in: R.D. Rogers, K.R. Seddon (Eds.), Ionic Liquids: Industrial Applications for Green Chemistry, E-Publishing, Inc., Washington, DC, 2002, pp. 12e14. [30] B.Z. Tang, Y.H. Geng, Q.H. Sun, X.X. Zhang, X.B. Jing, Pure Appl. Chem. 72 (2000) 157e162. [31] K. Mori, S. Kanai, T. Hara, T. Mizugaki, K. Ebitani, K. Jitsukawa, K. Kaneda, Chem. Mater. 19 (2007) 1249e1256. [32] K.J. Lee, J.H. An, J.S. Shin, D.H. Kim, H.S. Yoo, C.K. Cho, Curr. Appl. Phys. 11 (2011) 467e471. [33] Z.G. Wu, J.F. Gao, Micro Nano Lett. 7 (2012) 533e535.
35
[34] B.Z. Tang, Y.H. Geng, J.W.Y. Lam, B.S. Li, Chem. Mater. 11 (1999) 1581e1589. [35] T. Frensing, C. Seitz, B. Heynisch, C. Patzina, G. Kochs, U. Reichl, Vaccine 29 (2011) 7125e7129. [36] M. Mochizuki, Vaccine 24 (2006) 1744e1748. [37] J.A. Tree, C. Richardson, A.R. Fooks, J.C. Clegg, D. Looby, Vaccine 19 (2001) 3444e3450. [38] B.H. Jeon, S. Kim, M.H. Choi, I.J. Chung, Synth. Met. 104 (1999) 95e100. [39] P.S. Rao, S. Subrahmanya, D.N. Sathyanarayana, Synth. Met. 143 (2004) 323e 330. [40] Y. Lu, Y. Ren, L. Wang, X. Wang, C. Li, Polymer 50 (2009) 2035e2039. [41] N.A. Ogurtsov, Y.V. Noskov, K.Y. Fatyeyeva, V.G. Ilyin, G.V. Dudarenko, A.A. Pud, J. Phys. Chem. B 117 (2013) 5306e5314. [42] G.B. Sun, B.X. Dong, M.H. Cao, B.Q. Wei, C.W. Hu, Chem. Mater. 23 (2011) 1587e1593. [43] Q. Wang, Q. Yao, J. Chang, L.D. Chen, J. Mater. Chem. 22 (2012) 17612e17618. [44] W.F. Alves, E.C. Venancio, F.L. Leite, D.H.F. Kanda, L.F. Malmonge, J.A. Malmonge, L.H.C. Mattoso, Thermochim. Acta 502 (2010) 43e46. [45] H.J. Zhu, X. Yao, S.D. Hua, Polym. Compos. 34 (2013) 834e841. [46] B.F. Shi, J.W. Ren, X.H. Liu, G.Z. Lu, Y.Q. Wang, Synth. Met. 162 (2012) 1443e 1450. [47] H. Bagheri, E. Babanezhad, A. Es-haghi, J. Chromatogr. A 1152 (2007) 168e 174. [48] A.M. Massari, K.J. Stevenson, J.T. Hupp, J. Electroanal. Chem. 500 (2001) 185e 191. [49] F. Maranghi, A. Mantovani, C. Macri, A. Romeo, P. Eleuteri, G. Leter, M. Rescia, M. Spano, L. Saso, Contraception 72 (2005) 268e272.
Contents lists available at ScienceDirect
Materials Chemistry and Physics journal homepage: www.elsevier.com/locate/matchemphys
g-Fe2O3/polyaniline-lonidamine prepared by doping/dedoping method Han Huang, Li Zhang, Jinqing Kan* School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225002, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
g-Fe2O3/polyaniline-lonidamine synthesized by the doping-dedoping of polyaniline. The composite has superparamagnet, good conductivity and electrochemical activity. The composite can be concentrated by applying magnetic field. The g-Fe2O3/polyaniline-lonidamine can kill MadineDarby Canine Kidney (MDCK).
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 August 2013 Received in revised form 29 December 2013 Accepted 4 January 2014
A new conductive superparamagnetic nanocomposite, g-Fe2O3/polyaniline-lonidamine (g-Fe2O3/PANILND), was synthesized successfully by two-step doping method. The properties of g-Fe2O3/PANI-LND were tested by FT-IR, X-ray diffractometry (XRD), X-ray photoelectron spectroscopy (XPS), vibrating sample magnetometer (VSM), transmission electron microscopy (TEM), thermogravimetric analysis (TGA) and cyclic voltammetry (CV). The results show that the particle size of g-Fe2O3/PANI-LND with stable electric conductivity and high electrochemical activity was approximately 40 nm, and the optimal molar ratio of nLND : ngFe2 O3 =PANI was 1:1 for the synthesis of g-Fe2O3/PANI-LND. The g-Fe2O3/PANI-LND could be concentrated in a magnetic field and located to the region of interest, which meant to potentially provide effective treatment of tumor cells with less therapeutic doses and side-effects. Ó 2014 Elsevier B.V. All rights reserved.
Keywords: Chemical synthesis Electrical conductivity Polymers Composite materials Magnetic properties X-ray photo-emission spectroscopy (XPS)
1. Introduction In the past decades, polyaniline (PANI) has become one of the most important functional conductive polymers, because of ease of processability, environmental stability, the high and reversible nature of its conductivity, versatile redox behavior and protondoping effect [1e8]. Numerous studies have demonstrated that when combined with inorganic compounds through chemical
* Corresponding author. Tel.: þ86 514 87975590x9415; 87975590x8410. E-mail addresses: [email protected], [email protected] (J. Kan).
fax:
0254-0584/$ e see front matter Ó 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.matchemphys.2014.01.009
þ86
514
oxidative polymerization, PANI could improve the electrochemical activity and surface properties of the inorganic compound significantly with no effect on its own structure [9e12]. In addition to this, PANI can render conduction by protonation of the emeraldine base (EB) leading to emeraldine salt (ES) through acid-base chemistry. The positive charge in the PANI chains facilitates its incorporation of negatively charged ions via doping [13e16] thereby promising numerous potential applications in the synthesis of composite materials. Meanwhile, iron oxides of a small size are very interesting because of their special catalytic and suitable magnetic properties. Especially, magnetite (g-Fe2O3) is one of the most widely investigated magnetic nanomaterials because of its non-toxicity, thermal
28
H. Huang et al. / Materials Chemistry and Physics 145 (2014) 27e35
and chemical stability and easy-functionalization, and is being viewed as a valuable material for information storage, magnetic sensors, drug delivery, catalysts, bioseparation, etc. [17e21]. Lonidamine (LND), a dichlorinated derivative of indazole-3carboxylic acid, is a new non-conventional anticancer agent that selectively interferes with the energy metabolism of neoplastic cells by delaying their growth and inhibiting the repair processes [22,23]. The clinical study demonstrates that lonidamine has a deterrent effect on lung cancer, chronic lymphocytic leukemia, malignant melanoma, breast cancer, prostrate cancer and brain tumors [24e28]. In this work, we focus attention concerning the synthesis and properties of nanoscaled superparamagnetic conductive composite, g-Fe2O3/polyaniline-lonidamine (g-Fe2O3/PANI-LND). The gFe2O3 with a shell of PANI was doped with lonidamine to form gFe2O3/PANI-LND in the acid solution. The composite g-Fe2O3/PANILND can be driven by an applied magnetic field to the target site. The advantages of the method lie in that the functional groups of lonidamine for g-Fe2O3/PANI-LND are not changed, which meant that the drug like lonidamine also can keep its effectiveness after doped in g-Fe2O3/PANI, thereby indicating that g-Fe2O3/PANI-LND may have potential applications in targeted therapy. 2. Materials and methods 2.1. Materials Ferrous sulfate heptahydrate (FeSO4$7H2O), ferric chloride hexahydrate (FeCl3$6H2O), aniline, ammonium persulfate (APS), ethanol, ammonia and hydrochloric acid were all of analytical grade and obtained from Sinopharm Chemical Reagent Co., Ltd. Sodium dodecyl benzene sulfonate (SDBS) was purchased from BIOBASIC. Lonidamine (LND) was procured from Changzhou Rui Ming Pharmaceutical Co., Ltd. Aniline was distilled under reduced pressure prior to use. 1-ethyl-3-methylimidazolium ethylsulfate [EMIM][EtSO4] was synthesized as reported earlier [29]. Brittone Robinson (BR) buffer solutions of required pH were prepared by adding 0.04 M acetic acid and 0.04 M boric acid into 0.04 M orthophosphoric acid with appropriate amount of 0.2 M sodium hydroxide. Cell lines of canine kidney cells were provided by College of Animal Science of Yangzhou University. Double-distilled water was used throughout the experiments. 2.2. Preparation of magnetic nanoparticles, g-Fe2O3
g-Fe2O3 was prepared by coprecipitation [30e33]. Typically, FeCl3$6H2O (0.02 mol) and FeSO4$7H2O (0.01 mol) were dissolved in 20 and 10 mL volumes of double-distilled water, respectively. The two iron-salt solutions were then mixed together and dropped into
200 mL of 0.6 M ammonium hydroxide solution. The pH of the mixture was kept between 11 and 12 with the addition of concentrated ammonium hydroxide solution. After stirring for 30 min, the as-prepared g-Fe2O3 was separated from the solution by a strong magnet, washed with double-distilled water and dried at 85 C under vacuum. 2.3. Synthesis of magnetic conductive composite, g-Fe2O3/PANI The preparation of g-Fe2O3/PANI was based on a previously published method [34]. Briefly, a solution containing 3.65 mL of 0.04 mol aniline, 0.5 M HCl, 1.22 mL of [EMIM][EtSO4] and several drops of SDBS were added into the solution of g-Fe2O3 (0.02 mol). APS (0.04 mol) was also added as an initiator and the reaction was kept for 4e6 h at room temperature by stirring to obtain g-Fe2O3/ PANI. The product was filtered and washed with double-distilled water and ethanol until a colorless filtrate was obtained. The final product was dried in a vacuum oven at 65 C. 2.4. Preparation of g-Fe2O3/PANI-LND by two-step doping method Ammonia (0.5 M) was stirred after the addition of g-Fe2O3/PANI for 15e20 h. After filtration and washing with water and ethanol, the product was dried in a vacuum oven at 65 C. Then the product was mixed with different proportions of lonidamine in doubledistilled water with the pH adjusted to 2e4 by HCl. After stirred for 13e15 h, the solution was filtered and the final product was washed and dried under vacuum at 65 C. Fig. 1 was the schematic representation for the preparation of g-Fe2O3/PANI-LND. The overall synthesis process of g-Fe2O3/PANI-LND is shown in Fig. 2. As shown in Fig. 2, g-Fe2O3/PANI was prepared by mixing gFe2O3 and aniline with SDBS as dispersant and APS as initiator. The addition of [EMIM][EtSO4] facilitated the availability of doped PANI across wider pH range. After filtration and washing with doubledistilled water and ethanol, the product was mixed with ammonia which changed the doped g-Fe2O3/PANI into intrinsic gFe2O3/PANI. Then the intrinsic g-Fe2O3/PANI was doped with lonidamine to form the final product g-Fe2O3/PANI-LND by adding HCl. HCl was used to allow the interaction between Hþ with polyaniline chain to form g-Fe2O3/PANIþ, which was then doped with LND thereby leading to the formation of g-Fe2O3/PANI-LND following charge balance. 2.5. Characterization of g-Fe2O3, g-Fe2O3/PANI and g-Fe2O3/PANILND Infrared spectra were recorded on a pressed KBr pellet employing a TENSOR Model 27 FT-IR spectrometer with the
Fig. 1. Schematic representation for the preparation of g-Fe2O3/PANI-LND.
H. Huang et al. / Materials Chemistry and Physics 145 (2014) 27e35
29
Fig. 3. Schematic illustration of the applied magnetic field.
Piston 1, 2 and 3 were opened first and the initial solution was collected. Then the dripping solution flew through the applied magnetic field. During the process, the field intensity was measured by Teslameter (SHT-4A, China). The solutions in and flowing through the applied magnetic field were received in the centrifuge tubes and their concentrations were measured after drying and weighing.
3. Results and discussion Fig. 2. Synthesis process of g-Fe2O3/PANI-LND.
resolution of 4 cm1. X-ray diffraction (XRD, MAC) diagrams were collected using X-ray powder diffractometer. Chemical states of elements were measured by X-ray Photoelectron Spectroscopy (XPS, PHI 5000, VersaProbe, Japan) from 0 to 1100 eV under vacuum. Thermo-gravimetric analysis (TGA) was performed using a synchronous thermal analyzer (Netzsch STA 409 PC) at a heating rate of 10 C min1 from 30 to 800 C under nitrogen. The magnetization was measured by vibrating sample magnetometer (VSM, EV7, ADE, USA) in fields ranging from 8000 to 8000 Oe at room temperature. The morphology was examined by transmission electron microscopy (TEM, Tecnai-12, Philip Apparatus). Cyclic voltammetry (CV) was performed on a CHI 660 electrochemical workstation (Shanghai, China). The conductivity of the samples was measured by conventional four-probe technique on pressed pellets of the powder samples with an YJ8312 model current source (Shanghai Huguang Instrument Works, China) and digital multimeter (DDS-IIA, China).
3.1. FT-IR spectra of samples Fig. 4 shows the FT-IR spectra of g-Fe2O3, PANI, g-Fe2O3/PANI, gFe2O3/PANI-LND and lonidamine. In curve a, the peaks at 1623 cm1 and 598 cm1 are stretching vibrations (n) of FeeO and characteristic absorption peak of g-Fe2O3 respectively. In curve c, the peaks at 1556 cm1 and 1487 cm1 are the stretching vibrations of quinone and benzene respectively and the stretching vibration of CeN (nCeN) was observed at 1299 cm1. The absorption peak near 1141 cm1 results from the NeQeN (Q denotes quinoid ring) stretching mode and is an indication of electron delocalization in PANI, which means PANI, in curve b, c and d, are in the doped state. In addition, compared with curve c, curve d has a new absorption peak of the C]O stretching vibration (n) (nC]O) at 1716 cm1, which also appears at 1701 cm1 in case of lonidamine (curve e) thereby proving that lonidamine is doped into the chain of g-Fe2O3/ PANI-LND [38e41].
2.6. Cell experiments of g-Fe2O3/PANI-LND MadineDarby Canine Kidney (MDCK) cells [35e37] were cultured in Petri dishes at 37 C for 20 h while shaking gently in order to avoid accumulation. 1 mL of culture fluid was mixed with 20 mg of g-Fe2O3/PANI, g-Fe2O3/PANI-LND and lonidamine respectively. The mixture was sterilized under high-pressure steam and dried on the laminar flow. 20 mL of each former suspension was put on cell culture plates for 20 h at 37 C under 5% (V/V) CO2. The results were observed by microscope. 2.7. Simulation of applied magnetic field A simple applied magnetic field simulation model is illustrated in Fig. 3. 50 mg g-Fe2O3/PANI-LND was dispersed in 200 mL of ultrapure water with different concentrations of PVP in an infusion bottle by ultrasonication. The solution was dripped into test tubes via an infusion tube with a magnet placed in the middle of the infusion tube.
Fig. 4. FT-IR spectra of samples at room temperature. (a) g-Fe2O3, (b) PANI, (c) g-Fe2O3/ PANI, (d) g-Fe2O3/PANI-LND and (e) lonidamine.
30
H. Huang et al. / Materials Chemistry and Physics 145 (2014) 27e35
The FT-IR spectra shown in Fig. 5A, shows the successful doping of g-Fe2O3/PANI with lonidamine in various solvents. However, compared with water, acidic solution improves the conductivity of final product (g-Fe2O3/PANI-LND). In addition, HCl has small size and easily removed because of volatility. Considering the difficulty in purification, aqueous HCl (0.1 M) was chosen as the experimental solvent. The effect of lonidamine concentration on the FT-IR spectra of gFe2O3/PANI-LND is illustrated in Fig. 5B. With the molar ratio between LND and g-Fe2O3/PANI decreasing from 3:1 to 1:3, the transmittance of its characteristic oscillation (nC]O) at 1716 cm1 gradually decreased, the intensity of the characteristic peaks of quinone and benzene also gradually decreased, which verified that the doping effect of lonidamine was proportional to its concentration in the solution. 3.2. XRD and XPS patterns of samples The crystallinity of g-Fe2O3 prepared by previous method with some modifications was characterized by XRD analysis. As shown
Fig. 6. (A) XRD pattern and (B) XPS spectra of g-Fe2O3.
in Fig. 6A, the characteristic diffractions at 2q ¼ 30.2, 35.5, 43.2, 53.6, 57.0 and 62.7, correspond to the spinel structure of magnetite (g-Fe2O3, JCDPS, No. 39-1346). Fig. 6B shows representative XPS spectrum of the prepared product. Sharp photoelectron peaks
Fig. 5. (A) FT-IR spectra of samples at room temperature. (a) Lonidamine, (b) g-Fe2O3/ PANI, (c) g-Fe2O3/PANI-LND (double-distilled water), (d) g-Fe2O3/PANI-LND (0.1 M HCl) and (e) g-Fe2O3/PANI-LND (0.05 M H2SO4); (B) FT-IR spectra of g-Fe2O3/PANI-LND with different molar ratios between lonidamine and g-Fe2O3/PANI [nLND : ngFe2 O3 =PANI ¼ (a) 3:1, (b) 2:1, (c) 1:1, (d) 1:2 and (e) 1:3].
Fig. 7. XRD patterns of samples. (a) PANI, (b) g-Fe2O3, (c) g-Fe2O3/PANI, (d) g-Fe2O3/ PANI-LND and (e) lonidamine.
H. Huang et al. / Materials Chemistry and Physics 145 (2014) 27e35
Fig. 8. XRD of g-Fe2O3/PANI-LND with different molar ratios between lonidamine and g-Fe2O3/PANI [nLND : ngFe2 O3 =PANI ¼ (a) 1:3, (b) 1:2, (c) 1:1, (d) 2:1 and (e) 3:1].
appear at binding energies of 711 eV (Fe 2p3/2), 725 eV (Fe 2p1/2), and 531 eV (O 1s), along with a C 1s peak at 285 eV due to contamination from the XPS instrument itself. The photoelectron peaks at 711.5 and 725.4 eV are the characteristic doublets of Fe 2p3/ 2 and 2p1/2 core-level spectra of iron oxide, respectively, which is consistent with the oxidation state of Fe in Fe2O3. The inset shows that a new peak has appeared at 718.2 eV which could be assigned to a pure g-Fe2O3 [42]. The XRD patterns of PANI, g-Fe2O3, g-Fe2O3/PANI, g-Fe2O3/PANILND and lonidamine are shown in Fig. 7. In curve a, the two main peaks at 2q ¼ 15 and 2q ¼ 25 , assigned to the periodicity of the repeat unit of the PANI chain and the periodicity parallel to the polymer backbone chain [43]. The diffraction angles at 30.2 , 35.6 , 43.2 , 57.0 and 62.9 were characteristic diffraction peaks of gFe2O3 (curve b, c and d). A wide and low peak at 2q ¼ 21 occurred in curve a, which was due to g-Fe2O3 coated with PANI successfully. Compared curve c with curve d, the new diffraction peaks of gFe2O3/PANI-LND (17.4 , 25.0 , 26.0 and 27.2 ) correspond to characteristic diffraction peaks of lonidamine due to the dopant of lonidamine.
Fig. 9. TGA of samples: (a) g-Fe2O3/PANI, (b) g-Fe2O3/PANI-LND and (c) lonidamine.
31
Fig. 10. VSM of samples (a) g-Fe2O3, (b) g-Fe2O3/PANI and (c) g-Fe2O3/PANI-LND (inset: VSM of samples from 500 to 500 Oe).
As shown in Fig. 8, with increase in the concentration of lonidamine, the relative intensity of g-Fe2O3/PANI-LND diffraction peak at 35.6 has decreased, but that of the g-Fe2O3/PANI-LND diffraction peak at 11.4 , 17.4 , 25.0 , 26.0 and 27.2 increased, which is in consistence with the results of FT-IR spectra.
3.3. TGA of samples Fig. 9 illustrates the TGA curves of the g-Fe2O3/PANI, g-Fe2O3/ PANI-LND and lonidamine. Three-stage weight loss was observed at g-Fe2O3/PANI (curve a). The first step (about 100 C) indicates the loss of water in the polymer; the second step (about 230 C) might be attributed to the loss of acid dopant or volatile elements bound to the polyaniline chain, and the last step (460 C) was caused by skeleton bond decomposition of polyaniline chain [44,45]. Curve b shows that g-Fe2O3/PANI-LND quickly lost weight at 250 C, which was clearly related with lonidamine (curve c), which was further confirmed in the following VSM and TEM images.
Fig. 11. VSM of g-Fe2O3/PANI-LND with different molar ratios between lonidamine and g-Fe2O3/PANI [nLND : ngFe2 O3 =PANI ¼ (a) 1:3, (b) 1:2, (c) 1:1, (d) 2:1 and (e) 3:1].
32
H. Huang et al. / Materials Chemistry and Physics 145 (2014) 27e35
Fig. 12. TEM images of samples: (a)g-Fe2O3, (b)g-Fe2O3/PANI and (c) g-Fe2O3/PANI-LND.
3.4. VSM of samples Magnetizations of nanoparticles obtained from VSM are shown in Fig. 10. The results illustrate that the saturated magnetizations of g-Fe2O3, g-Fe2O3/PANI and g-Fe2O3/PANI-LND decreased from 66.3 emu g1 (curve a) to 32.2 emu g1 (curve b), and further to 19.6 emu g1 (curve c), which owed to the decrease in content of gFe2O3 because the magnetization is mainly dependent on the mass
fraction of g-Fe2O3 [46]. The inset picture clearly shows that the saturated magnetizations of g-Fe2O3, g-Fe2O3/PANI and g-Fe2O3/ PANI-LND are all almost zero without applied magnetic field, indicating that all the samples are superparamagnetic. As shown in Fig. 11, the coercive forces of curve a, b, c, d and e are almost zero, which indicated that the concentration of lonidamine had no effect on superparamagnetism of g-Fe2O3/PANI-LND. As the concentration of lonidamine is increased, the saturated magnetization of g-Fe2O3/PANI-LND is decreased, owing to the decrease in mass fraction of g-Fe2O3. The high intensity of magnetism indicates the decrease in the lonidamine content. For ensuring the magnetic strength of nanocomposite and to avoid the decrease of medical efficacy coming from lonidamine, the molar ratio of nLND : ngFe2 O3 =PANI ¼ 1:1 was selected. 3.5. TEM images of samples From the TEM images in Fig. 12, it is obvious that the diameter of
g-Fe2O3 particles ranges between 10 and 20 nm. With the encapsulation of polyaniline and doping of lonidamine, the sizes of gFe2O3/PANI and g-Fe2O3/PANI-LND increased and the average size of g-Fe2O3/PANI-LND was about 40 nm. After doping with lonidamine, the morphology did not change significantly. The results demonstrate that lonidamine was successfully doped into PANI, and the size of nanoparticles was within the superparamagnetic range (14e90 nm), which is consistent with the results obtained from the MeH curve.
Fig. 13. (A) CV curves for g-Fe2O3/PANI-LND from 0.7 V to 0.7 V at 30 mV s1. pH ¼ (a) 4, (b) 5, (c) 6 and (d) 7 (inset picture was electrode reaction for Nernst equation); (B) CV curves for g-Fe2O3/PANI-LND from 0.6 V to 1.0 V at pH ¼ 4 at scan rates (mV s1) of (a) 300, (b) 200, (c) 150, (d) 100, (e) 70, (f) 50 and (g) 30.
Fig. 14. Conductivity of g-Fe2O3/PANI-LND with different molar ratios between lonidamine and g-Fe2O3/PANI [nLND : ngFe2 O3 =PANI ¼ 1:3, 1:2, 1:1, 2:1 and 3:1].
H. Huang et al. / Materials Chemistry and Physics 145 (2014) 27e35
33
Fig. 17. Cell culture experiment of g-Fe2O3/PANI-LND (a) 5 mL, (b) 20 mL and (c) 100 mL.
3.7. Conductivity of g-Fe2O3/PANI-LND
Fig. 15. The relationship between the doping amount of lonidamine with different compositions of lonidamine and g-Fe2O3/PANI [nLND : ngFe2 O3 =PANI ¼ 1:3, 1:2, 1:1, 2:1 and 3:1].
3.6. CV curves for g-Fe2O3/PANI-LND Fig. 13A shows the CV plots of g-Fe2O3/PANI-LND in BR buffer at different pH. With increase in pH, both the anodic peak current of g-Fe2O3/PANI-LND and its corresponding potential decreased. The reason was that the doping level of PANI increased at low pH. Ac1=2 cording to the equation ip ¼ ð2:69 105 Þn3=2 AD0 C0* v1=2 , with the increasing concentration of compound A (inset picture), the value of ip increased. However, According to Nernst equation 2n þ 2n f ¼ f+B=A þ RT the nF lnf½CðBÞ$CðH Þ =½CðAÞ$CðLNDÞ g[47,48], lower concentration of Hþ should lead to the negative shift of the oxidation potential of PANI. Hence, the reaction equation is proposed and shown in the inset. The CV curves of g-Fe2O3/PANI-LND in BR buffer solution at different scan rates were illustrated in Fig. 13B. According to the 1=2 equation ip ¼ ð2:69 105 Þn3=2 AD0 C0* v1=2 , with the decrease in scan rate, both the oxidation and the reduction peak currents of gFe2O3/PANI-LND decreased.
As shown in Fig. 14, the conductivity of g-Fe2O3/PANI-LND measured by four-probe method decreased with increasing concentrations of lonidamine, which was due to the poor conductivity of lonidamine. Considering the conductivity of g-Fe2O3/PANI-LND and the drug loading capacity of g-Fe2O3/PANI, the optimum molar ratio of lonidamine and g-Fe2O3/PANI was 1:1, which corresponds to the result of VSM. 3.8. Determination of doping amount of lonidamine The doping effect of lonidamine on the amount of g-Fe2O3/PANI in g-Fe2O3/PANI-LND was measured using combustion method. gFe2O3/PANI (W1) and g-Fe2O3/PANI-LND (W2) were added into the empty crucible (W3), respectively. The samples were burned in the electric stove till a constant mass with the color of each sample changed from dark blue to dark red. The total mass of the crucible and the residue were W4 and W5, respectively. The doping ratio of lonidamine (Y) was calculated by
Y ¼ W2 þ ½W1 ðW5 W3 Þ=ðW3 WÞ4 : As shown in Fig. 15, the doping amount of lonidamine increased with increasing concentration of lonidamine, which further confirmed that g-Fe2O3/PANI was doped well with lonidamine and g-Fe2O3/PANI acted as an excellent drug carrier. The results were consistent with those of MeH and conductivity of g-Fe2O3/PANILND. 3.9. Cell culture experiments
Fig. 16. Cell culture experiments of (A) g-Fe2O3/PANI and (B) lonidamine (a) 0 mL, (b) 20 mL and (c) 100 mL.
In principle, if the added material has no effect on MDCK cells, the cell-culture medium remains yellowish brown, otherwise it will become red. Fig. 16A shows the cell-culture media containing 0 mL (0 mg$mL1), 20 mL (0.4 mg$mL1) and 100 mL (2 mg$mL1) of gFe2O3/PANI in a, b and c, respectively. After culturing for 20 h, none of the solutions turned into red. This indicates that g-Fe2O3/PANI was not toxic for MDCK cells. Compared with Fig. 16A and B showed that lonidamine could kill MDCK cells, and the more it was added, the more significant the effect on MDCK cells would be. The results obtained from concentration gradient experiment of g-Fe2O3/PANI-LND are shown in Fig. 17. The solution (a) kept its original color, the solution (b) turned into a very pale red color. This is due to the low content of g-Fe2O3/PANI-LND in the solutions, which is very similar to earlier reported results [49]. However, when 100 mL (2 mg mL1) of g-Fe2O3/PANI-LND was added into solution (c), it becomes red color, which show that a massive number of MDCK cells have been killed in the solution. It is seen that only when the concentration of g-Fe2O3/PANI-LND is high enough, it has an efficient effect on killing MDCK cells at 37 C. The Micrographs of the above three solutions are shown in Fig. 18. The effect of different concentrations of g-Fe2O3/PANI-LND was investigated using optical microscopy and the results are shown in
34
H. Huang et al. / Materials Chemistry and Physics 145 (2014) 27e35
Fig. 18. Micrographs for effect of g-Fe2O3/PANI-LND concentration on cell-culture medium (a) 5 mL, (b) 20 mL and (c) 100 mL.
Fig. 18a, b and c for 5 mL (0.1 mg mL1), 20 mL (0.4 mg mL1) and 100 mL (2 mg mL1) concentrations respectively. It could be observed that the color of the cell-culture medium in a and b almost remain unchanged, while the color of the cell-culture medium turned to red in c, which confirmed that only cells in c could be killed by the g-Fe2O3/PANI-LND and the results are consistent with those shown in Fig. 16. 3.10. Effect of applied magnetic field The results of the magnetic drug targeting of g-Fe2O3/PANILND are shown in Fig. 19. It could be observed that the concentration of solution remaining in the magnetic field was higher than that of effluent liquid from the applied magnetic field when the strength of magnetic field was more than 0.15 T. Furthermore, when the strength of magnetic field was 0.312 T, the concentration of effluent liquid from the applied magnetic field was reduced to 0.058 g kg1 which was 53.2% of the original concentration (0.109 g kg1) while the concentration of solution remaining in the magnetic field was 0.171 g kg1 which was 1.57 times of the original concentration or 2.95 times of effluent liquid from the applied magnetic field. So it was confirmed that g-Fe2O3/PANILND could be focused on a defined area under the control of the applied magnetic field and its concentration could be reduced in other parts of solution, which might be used in the magnetic targeting therapy of tumor.
Fig. 19. Concentrations of receiver in different strengths of the applied magnetic field. (a) Solution remaining in the magnetic field and (b) effluent liquid from the applied magnetic field.
4. Conclusions A conductive superparamagnetic nanocomposite (g-Fe2O3/ PANI-LND) has been synthesized following a two-step method. Various characterizations of g-Fe2O3/PANI-LND have validated its good electrochemical activity and stable magnetic conductivity with the particle size of about 40 nm. The molar ratio of nLND : ngFe2 O3 =PANI ¼ 1:1 was found to be optimum for the synthesis of g-Fe2O3/PANI-LND. g-Fe2O3/PANI-LND can be partially concentrated in a defined area by applying magnetic field. g-Fe2O3/ PANI-LND further exhibited the capability of killing cells in their adsorbing position, which promised the potential application in magnetic targeting tumor therapy. Acknowledgments This project was supported by National Science Foundation of China (No. 20873119), and by the Priority Academic Program Development of Jiangsu Higher Education Institutions. Part of data was from Testing Center of Yangzhou University. References [1] P.P. Jeeju, S.J. Varma, P.A. Francis Xavier, A.M. Sajimol, S. Jayalekshmi, Mater. Chem. Phys. 134 (2012) 803e808. [2] T.B. Cao, L.H. Wei, S.M. Yang, M.F. Zhang, C.H. Huang, W.X. Cao, Langmuir 18 (2002) 750e753. [3] W. Wang, Q. Li, B. Wang, X.T. Xu, J.P. Zhai, Mater. Chem. Phys. 135 (2012) 1077e1083. [4] H. Zengin, G. Kalayci, Mater. Chem. Phys. 120 (2010) 46e53. [5] S. Ben-Valid, H. Dumortier, M. Décossas, R. Sfez, M. Meneghetti, A. Bianco, S. Yitzchaik, J. Mater. Chem. 20 (2010) 2408e2417. [6] A. Fattoum, Z. Ben Othman, M. Arous, Mater. Chem. Phys. 135 (2012) 117e 122. [7] J. Kan, X. Pan, C. Chen, Biosens. Bioelectron. 19 (2004) 1635e1640. [8] E.H. Kim, Y.J. Lee, J.G. Bang, K.J. Kim, S.J. Choe, Mater. Chem. Phys. 134 (2012) 814e820. [9] X.T. Zhang, J. Zhang, R.M. Wang, Z.F. Liu, Carbon 42 (2004) 1455e1456. [10] K. Singha, A. Ohlana, R.K. Kotnalab, A.K. Bakhshic, S.K. Dhawana, Mater. Chem. Phys. 112 (2008) 651e658. [11] P.S. Smertenkoa, O.P. Dimitrieva, S. Schraderb, L. Brehmerb, Synth. Met. 146 (2004) 187e196. [12] Z.Z. Wang, H. Bia, J. Liu, T. Sun, X.L. Wu, J. Magn. Magn. Mater. 320 (2008) 2132e2139. [13] S.J. Tian, J.Y. Liu, T. Zhu, W. Knoll, Chem. Commun. (2003) 2738e2739. [14] M.M. Ayad, E.A. Zaki, Eur. Polym. J. 44 (2008) 3741e3747. [15] J.H. Wu, Q.W. Tang, Q.H. Li, J.M. Lin, Polymer 49 (2008) 5262e5267. [16] A. Varela-Álvarez, J.A. Sordo, G.E. Scuseria, J. Am. Chem. Soc. 127 (2005) 11318e11327. [17] M.E. Khosroshahi, L. Ghazanfari, Mater. Chem. Phys. 133 (2012) 55e62. [18] J. Wang, Q.W. Chen, C. Zeng, B.Y. Hou, Adv. Mater. 16 (2004) 137e140. [19] R. Suresh, R. Prabu, A. Vijayaraj, K. Giribabu, A. Stephen, V. Narayanan, Mater. Chem. Phys. 134 (2012) 590e596. [20] T. Hyeon, S.S. Lee, J. Park, Y. Chung, H.B. Na, J. Am. Chem. Soc. 123 (2001) 12798e127801. [21] C. Caparrós, M. Benelmekki, P.M. Martins, E. Xuriguera, C.J.R. Silva, L.I.M. Martinez, S. Lanceros-Méndez, Mater. Chem. Phys. 135 (2012) 510e517.
H. Huang et al. / Materials Chemistry and Physics 145 (2014) 27e35 [22] M. De Lena, V. Lorusso, A. Latorre, G. Fanizza, G. Gargano, L. Caporusso, M. Guida, A. Catino, E. Crucitta, D. Sambiasi, A. Mazzei, Eur. J. Cancer 37 (2001) 364e368. [23] G. Giorgioni, S. Ruggieri, A. Di Stefano, P. Sozio, B. Cinque, L. Di Marzio, G. Santoni, F. Claudi, Bioorg. Med. Chem. Lett. 18 (2008) 2445e2450. [24] L. Milane, Z.F. Duan, M. Amiji, J. Nanomed. Nanotechnol. 7 (2011) 435e444. [25] Y. Mardor, O. Kaplan, M. Sterin, J. Ruiz-Cabello, E. Ash, Y. Roth, I. Ringel, J.S. Cohen, Cancer. Res. 60 (2000) 5179e5186. [26] P. Papaldo, M. Lopez, E. Cortesi, E. Cammilluzzi, M. Antimi, E. Terzoli, G. Lepidini, P. Vici, C. Barone, G. Ferretti, S. Di Cosimo, C. Nisticò, P. Carlini, F. Conti, L. Di Lauro, C. Botti, C. Vitucci, A. Fabi, D. Giannarelli, P. Marolla, J. Clin. Oncol. 21 (2003) 3462e3468. [27] S. Di Cosimo, G. Ferretti, P. Papaldo, P. Carlini, A. Fabi, F. Cognetti, Drug Today 39 (2003) 157e174. [28] P. Pacini, M. Rinaldini, R. Algeri, A. Guarneri, E. Tucci, G. Barsanti, B. Neri, P. Bastiani, S. Marzano, C. Fallai, Eur. J. Cancer 36 (2000) 966e975. [29] J.D. Holbrey, R.D. Rogers, in: R.D. Rogers, K.R. Seddon (Eds.), Ionic Liquids: Industrial Applications for Green Chemistry, E-Publishing, Inc., Washington, DC, 2002, pp. 12e14. [30] B.Z. Tang, Y.H. Geng, Q.H. Sun, X.X. Zhang, X.B. Jing, Pure Appl. Chem. 72 (2000) 157e162. [31] K. Mori, S. Kanai, T. Hara, T. Mizugaki, K. Ebitani, K. Jitsukawa, K. Kaneda, Chem. Mater. 19 (2007) 1249e1256. [32] K.J. Lee, J.H. An, J.S. Shin, D.H. Kim, H.S. Yoo, C.K. Cho, Curr. Appl. Phys. 11 (2011) 467e471. [33] Z.G. Wu, J.F. Gao, Micro Nano Lett. 7 (2012) 533e535.
35
[34] B.Z. Tang, Y.H. Geng, J.W.Y. Lam, B.S. Li, Chem. Mater. 11 (1999) 1581e1589. [35] T. Frensing, C. Seitz, B. Heynisch, C. Patzina, G. Kochs, U. Reichl, Vaccine 29 (2011) 7125e7129. [36] M. Mochizuki, Vaccine 24 (2006) 1744e1748. [37] J.A. Tree, C. Richardson, A.R. Fooks, J.C. Clegg, D. Looby, Vaccine 19 (2001) 3444e3450. [38] B.H. Jeon, S. Kim, M.H. Choi, I.J. Chung, Synth. Met. 104 (1999) 95e100. [39] P.S. Rao, S. Subrahmanya, D.N. Sathyanarayana, Synth. Met. 143 (2004) 323e 330. [40] Y. Lu, Y. Ren, L. Wang, X. Wang, C. Li, Polymer 50 (2009) 2035e2039. [41] N.A. Ogurtsov, Y.V. Noskov, K.Y. Fatyeyeva, V.G. Ilyin, G.V. Dudarenko, A.A. Pud, J. Phys. Chem. B 117 (2013) 5306e5314. [42] G.B. Sun, B.X. Dong, M.H. Cao, B.Q. Wei, C.W. Hu, Chem. Mater. 23 (2011) 1587e1593. [43] Q. Wang, Q. Yao, J. Chang, L.D. Chen, J. Mater. Chem. 22 (2012) 17612e17618. [44] W.F. Alves, E.C. Venancio, F.L. Leite, D.H.F. Kanda, L.F. Malmonge, J.A. Malmonge, L.H.C. Mattoso, Thermochim. Acta 502 (2010) 43e46. [45] H.J. Zhu, X. Yao, S.D. Hua, Polym. Compos. 34 (2013) 834e841. [46] B.F. Shi, J.W. Ren, X.H. Liu, G.Z. Lu, Y.Q. Wang, Synth. Met. 162 (2012) 1443e 1450. [47] H. Bagheri, E. Babanezhad, A. Es-haghi, J. Chromatogr. A 1152 (2007) 168e 174. [48] A.M. Massari, K.J. Stevenson, J.T. Hupp, J. Electroanal. Chem. 500 (2001) 185e 191. [49] F. Maranghi, A. Mantovani, C. Macri, A. Romeo, P. Eleuteri, G. Leter, M. Rescia, M. Spano, L. Saso, Contraception 72 (2005) 268e272.