Biodegradable gelatin decorated fe3o4 nanoparticles for paclitaxel delivery - Dai Hai Nguyen

Journal of Science and Technology 55 (1B) (2017) 7–12 BIODEGRADABLE GELATIN DECORATED Fe3O4 NANOPARTICLES FOR PACLITAXEL DELIVERY Dai Hai Nguyen Institute of Applied Materials Science, Vietnam Academy of Science and Technology 01 TL29, Thanh Loc Ward, District 12, Ho Chi Minh City, Vietnam *Email: nguyendaihai0511@gmail.com Received: 30 December 2016; Accepted for publication: 26 February 2017 ABSTRACT The objective of this study is to prepare biodegradable iron oxide nanoparticles with gelatin (GEL) for paclitaxel (PTX) delivery. In detail, Fe3O4 nanoparticles were prepared and then coated them with GEL (Fe3O4@GEL) conjugate by co–precipitation method. Furthermore, the formation of Fe3O4@GEL was demonstrated by Fourier transform infrared (FT–IR) and powder X–ray diffraction (XRD). The superparamagnetic property of Fe3O4@GEL was also showed by hysteresis loop analysis, the saturation magnetization reached 20.36 emu.g–1. In addition, size and morphology of Fe3O4@GEL nanoparticles were determined by transmission electron microscopy (TEM). The results indicated that Fe3O4@GEL nanoparticles were spherical shape with average diameter of 10 nm. Especially, PTX was effectively loaded into the coated magnetic nanoparticles, 86.7 ± 3.2 % for drug loading efficiency and slowly released up to 5 days. These results suggest that the potential applications of Fe3O4@GEL nanoparticles in the development of stable drug delivery systems for cancer therapy. Keywords: superparamagnetic iron oxide, gelatin, paclitaxel, drug delivery. 1. INTRODUCTION Iron oxide nanoparticles (Fe3O4 and γ–Fe2O3 NPs), one of the most prominent properties of magnetic nanoparticles (MNPs), has been commonly used in biomedical applications such as in vivo magnetic resonance imagining, magnetic–mediated hyperthermia for cancer treatment and tissue–specific delivery of therapeutic agents [1, 2]. Moreover, if the size of magnetic structure is small enough MNPs may have superparamagnetic properties, becoming magnetized in the presence of a magnetic field and showing no magnetization in the absence of magnetic field [3, 4]. However, MNPs tend to aggregate and form a larger cluster because of magnetic dipole– dipole attractions between NPs, which may limit their potential biomedical applications [5]. To overcome this drawback, MNPs were often surface modified with many materials such as silica, carbon, and biopolymers. These modifications are not only improving the chemical stability but also increase the biocompatibility of MNPs [6]. Gelatin (GEL), a protein derived from collagen, possesses numerous useful features such as high solubility, biodegradability, biocompatibility and pH–induced surface charge. It can be Biodegradable gelatin decorated Fe3O4 nanoparticles for paclitaxel delivery 8 widely used to bind with drug or poly(ethylene glycol) due to its multifunctional groups, like –NH2 and –COOH [7, 8]. Furthermore, fibronections, are large glycoprotein found at cell surface, in extracellular matrices and in blood plasma, have binding sites for a number of other macromolecules, including GEL [9]. Moreover, tumor cell phagocytosis can be significantly enhanced in the presence of GEL [10]. There is some research using GEL–coated MNPs for delivery of chemo– and bio–therapeutic agents. Babita Gaihre and co–workers developed GEL A– and B–coated MNPs as potential nanocarriers for magnetic doxorubicin (DOX) targeting. The results demonstrated that electrostatic interactions between DOX and the coated MNPs played a crucial role in the encapsulation efficiency of the DOX, and pH–responsive drug release of DOX–loaded particles [11]. In addition, Erxi Che reported a magnetic targeted drug delivery system based on magnetic mesoporous silica NPs (MMSN), which were surface coated by GEL layer, for sustained release of paclitaxel (PTX). The results showed that the biodistribution of the GEL–coated MMSN were altered by external magnet, and therefore the higher concentration of these nanocarriers detected in tumor tissues than normal tissues. According to the result of tumor reduction study, the tumor growth of S180 tumor–bearing mice treated with the PTX–loaded carriers were significantly delayed without obvious body weight loss [12]. These results suggested that the GEL–coated MMSN can be used as promising drug carriers for effective delivery of anticancer drugs in the treatment of cancer. In this study, we report the preparation and characterization of magnetite nanoparticles coated with GEL for PTX delivery system. GEL as polymeric outer layers were prepared and coated on Fe3O4 NPs (Fe3O4@GEL NPs) which was prepared by the co–precipitation method. The obtained samples were then characterized by transmission electron microscopy (TEM), Fourier transform infrared spectra (FT–IR), powder X–ray diffraction (XRD), and vibration sample magnetometer (VSM). Especially, either drug loading efficiency or drug release behavior of PTX–loaded Fe3O4@GEL NPs were also evaluated. This study is expected to improve the stability of magnetic NPs for controlled delivery systems in cancer therapy. 2. MATERIALS AND METHODS 2.1. Materials GEL type A was obtained from Sigma–Aldrich (St. Louis, MO, USA). Iron(III) chloride hexahydrate (FeCl3.6H2O, 97%), iron(II) chloride tetrahydrate (FeCl2.4H2O, 99%) and tetrahydrofuran (THF) were purchased from Merck (Germany). Ammonium Hydroxide (28– 30%) was obtained from Tianjin Bodi Chemical Co., Ltd. (China). PTX was supplied by Samyang Corporation (Seoul, Korea). All chemicals and solvents were of highest analytical grade and used without further purification. 2.2. Preparation of Fe3O4 and Fe3O4@GEL MNPs Fe3O4 NPs were prepared by the chemical co–precipitation method as described previously with some modifications. Initially, an 80 mL mixture of 0.2 M of FeCl3.6H2O and 0.1 M of FeCl2.4H2O (the molar ratio of Fe2+/ Fe3+ = 1:2) was added into the three–necked flask and constantly stirred under nitrogen. NH4OH solution (10 w/w%) was injected into the mixture and the reaction was maintained at room temperature under vigorously stirring for 1 h until pH reach to 10. The color of the solution changed to dark black. Thereafter, the precipitate was isolated by using a super magnet bar and rinsed with deionized water (deH2O) several times, sonicated and then freeze–dried to obtain Fe3O4 NPs. Dai Hai Nguyen 9 Fe3O4@GEL MNPs were formed by adding Fe3O4 solution (0.4 g of Fe3O4 dissolved in 25 mL of deH2O) drop–wise into GEL solution (0.1 g of GEL dissolved in 20 mL of deH2O) at room temperature under ultra–sonication for 1 h. During this process, GEL was adsorbed onto the surface of Fe3O4 NPs, and the obtained substance was dialyzed by dialysis membrane (MWCO 12–14 kDa, Spectrum Laboratories, Inc., USA) against deH2O for 36 hours at room temperature. The deH2O was changed 5–6 times a day and the resulting solution was then lyophilized to obtain Fe3O4@GEL. 2.3. Characterization The size and morphology of Fe3O4@GEL MNPs were confirmed by TEM (JEM–1400 TEM; JEOL, Tokyo, Japan) at National Key Lab for Polymer & Composite, HCMUT– VNUHCM. For the purpose of investigating the presence of GEL on the surface of Fe3O4 NPs, FTIR analysis (Nicolet Nexus 5700 FTIR, Thermo Electron Corporation, Waltham, MA, USA) of GEL, bare Fe3O4 and Fe3O4@GEL NPs was carried out with KBr pellets in 400–4000 cm–1 range at Department of Agricultural Chemistry, ICT–VAST. The sizes and crystalline structures of Fe3O4 and Fe3O4@GEL were assessed by Rigaku D/Max–2550 V diffractometer with CuKα radiation (λ = 0.15405 nm, 40 kV, 40 mA) at a scanning speed of 4°/min in the 2θ range from 30° to 70°, at Faculty of Basic Science, The College of Accounting and Finance. Moreover, the magnetization curves of these MNPs were recorded at –15 to 15 kOe at room temperature using EV11 vibrating sample magnetometer (EV11 VSM, USA) at Department of New Materials and Nano structured Materials, HCMIP–VAST. 2.4. PTX loaded Fe3O4@GEL MNPs, PTX loading contents and in vitro PTX release In order to prepare PTX–loaded Fe3O4@GEL MNPs, 10 mg PTX was dissolved in methanol and 100 mg Fe3O4@GEL MNPs was dissolved in deionized water. The PTX solution and Fe3O4@GEL solution were mixed together, sonicated for 60 min for 24 h, and then dialyzed with deH2O to remove free drug and methanol. The resulting solution was freeze–dried to obtain the PTX–loaded Fe3O4@GEL MNPs. The PTX loading contents in Fe3O4@GEL MNPs were analyzed using a Shimadzu LC–20A Prominence system (Shimadzu, Kyoto, Japan). The injected volume was 10 μL, and the mobile phase (acetonitrile/water = 60:40 v/v) was delivered at 1.00 mL/min. A reverse–phase Fortis C18 column (150×4.6 mm i.d., pore size 5 μm; Fortis Technologies Ltd., Cheshire, UK) was used, and column effluent was monitored with a UV detector at 227 nm. The calibration curve for quantification of PTX in Fe3O4@GEL MNPs was found to be linear over the standard PTX concentration range of 0–20,000 ng/mL with a high correlation coefficient of R2 = 0.998. The following equations were used to calculate the drug loading efficiency (DLE) and drug loading content (DLC): DLE (%) = weight of PTX in Fe3O4@GEL MNPs/ weight of PTX feed initially × 100 DLC (%) = weight of PTX in Fe3O4@GEL MNPs/ weight of Fe3O4@GEL and PTX × 100 In vitro release of PTX from Fe3O4@GEL MNPs was performed in phosphate buffer saline (PBS) containing 0.5 wt% Tween–80 (0.01 m, pH 7.4) at 37 °C using a dialysis method. One milliliter of this suspension (PTX content, 0.3 mg/mL) was transferred into a dialysis bag (MWCO = 12–14 kDa) and then immersed into 14 mL fresh medium at 37 °C. The samples were placed in an orbital shaker bath, which was maintained at 37 °C and horizontally shaken at 100 rpm. At predetermined time intervals, 14 mL of the released medium was withdrawn, filtered (pore size = 0.20 μm), and replaced with an equal amount of fresh medium. Following Bio 10 lyo wa nan VS Fig Be ag lay NP str asy ben O ob 34 spe Fe C– degradable philization s determined After pre oparticles w M. TEM im ure 1. The F sides, these gregation or er for impro F Fourier–tr s are shown etch coupled mmetrical d coupled w stretch). Ad tained and th 16 cm–1 and ctrum of F 3O4@GEL b O stretching gelatin deco of the collec using high paration o ere coated age of Fe3O e3O4@GEL MNPs still fusion. Thes ving the disp igure 1. (a) T Figure 2. F ansform inf in Figure with hydr stretching), ith CN stre ditionally, th e presence 3420 cm–1, e3O4, there ecause of th of GEL. Ad rated Fe3O4 ted medium performanc 3. RESU f Fe3O4 N with GEL, w 4@GEL N NPs were n maintained e results im ersibility of EM image a T–IR spectra rared (FT–I 2. The spec ogen bondin 1631 cm–1 ( tch), 1444 cm e character of Fe3O4 pa which were were a stron e existence o ditionally, t nanoparticl , the amoun e liquid chro LT AND D Ps by the hich was th Ps (a) and i early spheri the morpho plied that GE MNPs. nd (b) particle of (i) GEL, (i R) spectra o trum of GE g), 3085 cm C=O stretch –1 (CH2 ben istic peaks o rticles were detected in b g shift of C f GEL. The he peaks at es for paclita t of PTX re matography ISCUSSIO chemical en character ts particle s cal in shape logical pro L would be size distribu i) Fe3O4, and f (i) GEL, L show vibr –1 (alkenyl /HB couple d), 1240 cm f Fe3O4 at 5 identified by oth Figure 2 H2 asymm band appea around 1240 xel delivery leased from (HPLC). N co–precipit ized by TEM ize distributi with averag perty of Fe3 a promising tion of Fe3O4@ (iii) Fe3O4@G (ii) Fe3O4 a ation bands C–H stretc d with COO –1 (NH bend 71 cm–1 an the O–H s ii and iii. A etrical stretc ring at 1100 cm–1 and 14 Fe3O4@GE ation, the , FT–IR, X on (b) are s e diameter o O4 particles surface–hy GEL. EL. nd (iii) Fe3O at 3285 cm h), 2956 cm –), 1533 c ), and 1078 d 578 cm–1 tretching vib s compared hing (2917 cm–1 is relat 44 cm–1 cor L MNPs obtained RD, and hown in f 10 nm. without drophilic 4@GEL –1 (N–H –1 (CH2 m–1(N–H cm–1 (C– could be ration at with the cm–1) of ed to the responds Dai Hai Nguyen 11 to the NH and CH2 bend of GEL. In order words, all those characteristic bands of GEL are presented in the spectrum for Fe3O4@GEL either. These results confirmed that GEL was successfully attached onto the surface of Fe3O4 NPs. As shown in Figure 3a, the characteristic adsorption peaks for Fe3O4 NPs marked by their indices ((220), (311), (400), (422), (511) and (440)) could be observed in the X–ray diffraction patterns of either (i) Fe3O4 NPs or (ii) Fe3O4@GEL. These six diffraction peaks are the standard pattern for crystalline magnetite with spinal structure. The insignificant effect of the outer– modifiers on the core of samples were also indicated by XRD data, Fe3O4 NPs still maintained their structure after polymeric coating. Magnetization curves of (i) Fe3O4 and (ii) Fe3O4@GEL are shown in Figure 3b. The size plays a critical role in its magnetic properties. If the size is small enough, such nanostructures have superparamagnetic properties. The saturation magnetization values (Ms) of Fe3O4 NPs and Fe3O4@GEL were 69.01 emu.g–1 and 20.36 emu.g–1, respectively. These results demonstrated that both structures are superparamagnetic which allow for rapid and easy separation of a number of MNPs in a reaction mixture. More importantly, lower Ms of the coated Fe3O4 is the result of the non–layer coated on Fe3O4 NPs, GEL. As a result, after coating, Fe3O4@GEL NPs exhibited good magnetic separation ability. Figure 3. (a) XRD pattern and (b) hysteresis loops of (i) Fe3O4 and (ii) Fe3O4 @GEL NPs and (c) In vitro release profiles of free PTX (circle) and PTX from PTX–loaded Fe3O4@GEL (square). DLE is an important property in drug–loaded nanocarriers and directly affects the therapeutic effect of the system. The higher encapsulation capacity NPs have, the larger number of drug are released at the tumor site. In this study, the DLE of Fe3O4@GEL was found to be 86.7 ± 3.2 %. The result demonstrated that Fe3O4@GEL with the high DLE have the potential to be delivered more efficiently to tumor tissues. In vitro release profiles of free PTX and PTX from PTX–loaded Fe3O4@GEL were performed in order to evaluate the stability and release behavior of Fe3O4@GEL. As shown in Figure 3c, the prepared Fe3O4@GEL showed a long term stable drug release profile up to 5 days. The cumulative release amount of PTX in the initial 3 h was around 10 % as compared with 34% of free PTX. The initial release of PTX could be explained by the PTX molecules, which were absorbed into the outer GEL layer of Fe3O4@GEL. Within the first 24 h, 31 % PTX was released from Fe3O4@GEL, which was significantly smaller than this amount of free PTX, approximately 91 %. And for the last 5 days, total release amount of PTX from Fe3O4@GEL was around 44 %, compared with around 95 % of free PTX. The release behaviors of free PTX and PTX in the Fe3O4@GEL were significantly different. These results together confirmed that Fe3O4@GEL may serve as stable NPs for controlled drug delivery system. M ag ne tiz at io n (e m u/ g) Applied field (Oe) i) ii) -80 -60 -40 -20 0 20 40 60 80 -15 -5 5 15 0 5500 20 30 40 50 60 70 In te ns ity 2θ (degree) ii) i) PT X re le as e (% ) Time (h) 0 20 40 60 80 100 0 20 40 60 80 100 b) c)a) Biodegradable gelatin decorated Fe3O4 nanoparticles for paclitaxel delivery 12 4. CONCLUSION In this study, GEL have been successfully coated on Fe3O4 NPs with 10 nm in size and high saturation magnetization. The PTX–loaded Fe3O4@GEL showed a steady and sustained release profile in vitro up to 5 days. These results suggest that the PTX–loaded Fe3O4@GEL NPs may serve as stable delivery systems with dual therapeutic effects (hyperthermia combined with chemotherapy) for cancer therapy. Acknowledgement. This work was financially supported by a grant from the Viet Nam Academy of Science and Technology, Institute of Applied Materials Science, Ho Chi Minh. REFERENCES 1. Gregorio-Jauregui K. M., et al. – One–Step Method for Preparation of Magnetic Nanoparticles Coated with Chitosan, Journal of Nanomaterials 2012 (2012) 813958. 2. Nguyen, H. D., Nguyen, T. D., Nguyen, D. H., Nguyen, P. T. – Magnetic properties of Cr doped Fe3O4 porous nanoparticles prepared through a co–precipitation method using surfactant, Advances in Natural Sciences: Nanoscience and Nanotechnology 5 (3) (2014) 035017. 3. Qu J., Liu G., Wang Y., Hong R. – Preparation of Fe3O4–chitosan nanoparticles used for hyperthermia, Advanced Powder Technology 21 (4) (2010) 461–467. 4. Unsoy G., Yalcin S., Khodadust R., Gunduz G., Gunduz, U. – Synthesis optimization and characterization of chitosan–coated iron oxide nanoparticles produced for biomedical applications, Journal of Nanoparticle Research 14 (11) (2012) 1. 5. Qu, J. B., Shao, H. H., Jing, G. L., Huang, F. – PEG–chitosan–coated iron oxide nanoparticles with high saturated magnetization as carriers of 10–hydroxycamptothecin: preparation, characterization and cytotoxicity studies, Colloids and Surfaces B: Biointerfaces 102 (2013) 37–44. 6. Safari, J., Javadian, L. – Chitosan decorated Fe3O4 nanoparticles as a magnetic catalyst in the synthesis of phenytoin derivatives, RSC Advances 4 (90) (2014) 73–79. 7. Young, S., Tabata, Y., Mikos, A. G. – Gelatin as a delivery vehicle for the controlled release of bioactive molecules, Journal of Controlled Release 109 (1) (2005) 256–274. 8. Kushibiki, T., Matsuoka, H., Tabata, Y. – Synthesis and physical characterization of poly (ethylene glycol)–gelatin conjugates, Biomacromolecules 5 (1) (2004) 202–208. 9. Engvall, E., Ruoslahti, E. – Binding of soluble form of fibroblast surface protein, fibronectin, to collagen, International Journal of Cancer 20 (1) (1977) 1–5. 10. Coester C., Nayyar P., Samuel J. – In vitro uptake of gelatin nanoparticles by murine dendritic cells and their intracellular localisation, European Journal of Pharmaceutics and Biopharmaceutics 62 (3) (2006) 306–314. 11. Gaihre B., Khil M. S., Lee D. R., Kim H. Y. – Gelatin–coated magnetic iron oxide nanoparticles as carrier system: drug loading and in vitro drug release study, International Journal of Pharmaceutics 365 (1–2) (2009) 180–189. 12. Che E., Gao Y., Wan L., Zhang Y., Han N., Bai J., Li J., Sha Z., Wang S. – Paclitaxel/gelatin coated magnetic mesoporous silica nanoparticles: Preparation and antitumor efficacy in vivo, Microporous and Mesoporous Materials 204 (2015) 226–234.