Abstract
In this report, magnetic Fe3O4 nanoparticles were functionalized with chitosan-grafted-poly(ethylene glycol) methyl ether (CTS-mPEG) for paclitaxel (PTX) delivery. The Fe3O4 nanoparticles were prepared via the chemical coprecipitation method and then coated with CTS-mPEG (Fe3O4@CTS-mPEG) by a simple method. The formation of Fe3O4@CTS-mPEG was characterized by several methods including proton nuclear magnetic resonance spectroscopy, Fourier transform infrared, and X-ray diffraction. Furthermore, the superparamagnetic properties of Fe3O4@CTS-mPEG were demonstrated by a vibrating sample magnetometer; the saturation magnetization reached 23 emu g–1. The sizes and morphologies of Fe3O4 and Fe3O4@CTS-mPEG nanoparticles were determined by transmission electron microscopy. The result indicated that Fe3O4@CTS-mPEGs were nearly spherical in shape with an average diameter of 20 nm, compared with the 12-nm Fe3O4 particles. Especially, PTX was effectively loaded into the coated nanoparticles, 86.9±3.4% for drug loading efficiency, and slowly released up to 120 h. These results suggest the potential applications of Fe3O4@CTS-mPEG in the development of stable drug delivery systems for cancer treatment.
1 Introduction
Magnetic nanoparticles (MNPs) open up a new range of feasible therapeutic possibilities for developing safe and effective drug delivery systems (DDSs) due to their advantages of synthesis, characterization, and tailoring the functional properties of nanoparticles (NPs) for drug delivery uses [1], [2], [3], [4], [5], [6], [7], [8], [9]. Moreover, MNPs can be manipulated by the outside magnetic field for many applications for pharmaceutical and biomedical analysis [10]. Magnetic iron oxide nanoparticles (SPIO NPs), a kind of new functional materials, have numerous advantages such as biocompatibility, chemical stability, and superparamagnetic behavior, allowing them to be the primary choice for biological and biomedical applications [11]. However, there are still several drawbacks of SPIO NPs, including weak physiological stability, fast blood clearance from the circulation, and lack of target specificity, which may limit their potential clinical application [12].
Surface modification, one of the most outstanding approaches, has been studied for the purpose of overcoming the disadvantages of SPIO NPs, including amphiphilic molecules, bifunctional polymeric ligands, or biomolecules [13]. Among different types of synthetic and natural magnetic macromolecules for stabilizing magnetic NPs, protein and polysaccharides were found to be more promising based on their excellent characteristics of biocompatibility and biodegradability. Chitosan (CTS) has attracted attention for improving the dynamic stability of MNPs caused by its many health benefits, including high biocompatibility, relevant biodegradability, and low toxicity. According to the combined effect of two active groups (amino and hydroxyl groups) along the backbone of CTS, strategies for chemical modification of CTS are shown to be valuable tools for developing novel biocompatible materials with tailored chemo-physical properties. Despite the success of CTS in enhancing the stability of MNPs, CTS-grafted MNPs are mostly absorbed into the blood circulation, which is a major obstacle for pharmaceutical applications, especially in DDSs [14], [15], [16]. Therefore, polyethylene glycol (PEG), an extremely hydrophilic polymer, has been commonly employed to optimize the stability of DDSs in the bloodstream by increasing the water solubility of CTS. As a result, PEG grafted onto the CTS chain not only improves the biocompatibility of CTS but also avoids the absorption of protein and evades from the reticuloendothelial system. More importantly, this conjugation can be used to increase the solubility of water-insoluble drugs for controlled drug delivery carriers [17], [18], [19]. For instance, Najafabadi and co-workers reported a simple new method of methoxyl polyethylene glycol (mPEG) conjugated CTS (mPEG-CTS) for enhancing the solubility of CTS and controlling ibuprofen delivery. The result showed that the release of ibuprofen from mPEG-CTS NPs were much lower than CTS alone. This study demonstrated that mPEG-CTS NPs can be a promising candidate for water-insoluble DDSs [20]. Additionally, Qu et al. successfully developed magnetic nanocarriers for controlled 10-hydroxycamptothecin (HCPT) delivery. In detail, PEG chains were conjugated to the magnetic particles (CTS-Fe3O4) to enhance the biocompatibility of PEG-CTS-Fe3O4. The result of this study indicated that HCPT was released in a controlled manner up to 48 h. As a consequence, PEG-CTS-Fe3O4 could possibly be utilized as a stable MNP for controlling HCPT delivery in cancer treatment [21].
In this study, magnetic nanocarriers for paclitaxel (PTX) was prepared by using Fe3O4 NPs as cores and mPEG-CTS as polymer outer layers (PTX/Fe3O4@CTS-mPEG). In addition, the structural and morphological characterizations of the obtained samples were determined by proton nuclear magnetic resonance (1H NMR) spectroscopy, Fourier transform infrared (FT-IR), X-ray diffraction (XRD), and transmission electron microscopy (TEM). Particularly, either the drug loading or drug release behavior of PTX/Fe3O4@CTS-mPEG was 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
CTS (100–300 kDa), 4-nitrophenyl chloroformate (PNC, 96%), iron(III) chloride hexahydrate (FeCl3.6H2O, 97%), iron(II) chloride tetrahydrate (FeCl2.4H2O, 99%), mPEG (5 kDa), and tetrahydrofuran (THF) were obtained from Sigma-Aldrich (USA). 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 CTS-mPEG
CTS-grafted-mPEG (CTS-mPEG) was synthesized by utilizing PNC as a cross-linking agent, as previously reported, with modification [22], [23]. Briefly, 0.8 g mPEG was melted under vacuum at 65°C, followed by adding 0.05 g PNC under constant stirring for 6 h, and the temperature was then cooled down to 40°C. After that, 20 ml THF and 10 ml deionized water were added into the solution to remove unreacted mPEG and PNC. At that period of time, CTS solution was prepared at pH 5 by using HCl solution (1 m). The mPEG-PNC solution was slowly dropped into the CTS solution and the reaction was then kept at room temperature for 24 h. The solution was dialyzed by using a dialysis membrane (MWCO 12–14 kDa; Spectrum Laboratories Inc., USA) and lastly lyophilized to obtain CTS-mPEG.
2.3 Preparation of Fe3O4 and Fe3O4@CTS-mPEG MNPs
Fe3O4 NPs were prepared by the chemical coprecipitation method as described previously with some modifications (Scheme 1) [21]. Initially, an 80 ml mixture of 0.2 m FeCl3.6H2O and 0.1 m FeCl2.4H2O (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 vigorous stirring for 1 h until the pH reached 10. The color of the solution changed to dark black. Thereafter, the precipitate was isolated by using a super magnet bar and then rinsed with deionized water several times.
Fe3O4@CTS-mPEG MNPs were formed by adding Fe3O4 solution (154 mg Fe3O4 dissolved in 50 ml deionized water) into CTS-mPEG solution (300 mg CTS-mPEG dissolved in 50 ml deionized water) at room temperature under ultrasonication for 6 h. During this process, CTS-mPEG was adsorbed onto the surface of Fe3O4 NPs, and the obtained substance was centrifuged and lyophilized for further use.
2.4 Characterization
The chemical structures of the polymers corresponding to the synthetic procedure were analyzed by 1H NMR (Bruker Avance 500; Bruker Co., USA). The sizes and crystalline structures of Fe3O4 and Fe3O4@CTS-mPEG were assessed by Rigaku D/Max-2550 V diffractometer with Cu Ka radiation (λ=0.15405 nm, 40 kV, 40 mA) at a scanning speed of 4°/min in the 2θ range from 30° to 70°. Moreover, the magnetization curves of MPNs were recorded at –15 to 15 kOe at room temperature using EV11 vibrating sample magnetometer (EV11 VSM, USA). The sizes and morphologies of Fe3O4 and Fe3O4@CTS-mPEG MNPs were confirmed by TEM (JEM-1400 TEM; JEOL, Tokyo, Japan). For the purpose of investigating the presence of CTS-mPEG on the surface of Fe3O4 NPs, FT-IR analysis (Nicolet Nexus 5700 FT-IR; Thermo Electron Corporation, Waltham, MA, USA) of bare Fe3O4, CTS-mPEG, and Fe3O4@CTS-mPEG was carried out with KBr pellets in the 400–4000 cm–1 range.
2.5 PTX-loaded Fe3O4@CTS-mPEG MNPs, PTX loading contents, and in vitro PTX release
In order to prepare PTX-loaded Fe3O4@CTS-mPEG MNPs, 10 mg PTX was dissolved in methanol and 100 mg Fe3O4@CTS-mPEG MNPs was dissolved in deionized water. The PTX solution and Fe3O4@CTS-mPEG solution were mixed together, sonicated for 60 min for 24 h, and then dialyzed with distilled water to remove free drug and methanol. The resulting solution was freeze-dried to obtain the PTX-loaded Fe3O4@CTS-mPEG MNPs. The PTX loading contents in Fe3O4@CTS-mPEG 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@CTS-mPEG 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):
In vitro release of PTX from Fe3O4@CTS-mPEG 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; Spectrum Laboratories Inc., USA) 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 lyophilization of the collected medium, the amount of PTX released from Fe3O4@CTS-mPEG MNPs was determined using high performance liquid chromatography. Furthermore, the same procedure was repeated at 45°C, pH 7.4, to examine the hyperthermic effect of Fe3O4@CTS-mPEG MNPs.
3 Results and discussion
The synthesis of CTS-mPEG was performed by conjugating mPEG-PNC with the CTS backbone. The conjugation ratio of PNC to mPEG was approximately 98%, as reported in our previous studies. CTS-mPEG was analyzed by 1H NMR (Figure 1), and as consequence the 1H NMR spectrum of the CTS-mPEG conjugate presented peak at 4.74 ppm for D2O. The typical peak at 3.4–3.94 ppm (H-c, H-d, H-e, H-f, H-2, and H-3) was assigned to methylene protons of CTS saccharide units and repeat units in mPEG. Peaks that appeared at 2.95 ppm (H-b) and 3.37 ppm (H-1) were attributed to -CH-NH- from CTS and -OCH3 from mPEG, respectively. The presence of all these resonance signals demonstrated that the CTS-mPEG composite was successfully synthesized.
After the preparation of Fe3O4 NPs by chemical coprecipitation, the obtained magnetic NPs were coated with CTS-mPEG to form Fe3O4@CTS-mPEG, which was then characterized by XRD, VSM, FT-IR, and TEM. As shown in Figure 2, the X-ray pattern of both Fe3O4 [Figure 2(i)] and Fe3O4@CTS-mPEG [Figure 2(ii)] showed characteristic adsorption peaks of Fe3O4, which were marked by their respective indices [(220), (311), (400), (422), (511), and (440)]. These six diffraction peaks comprise the standard pattern for crystalline magnetite with a spinal structure [24]. Moreover, the insignificant influence of CTS-mPEG on the core of samples was also indicated by XRD data. Fe3O4 NPs still maintained their structure after polymeric coating. The crystallite sizes D (311) of Fe3O4 and Fe3O4@CTS-mPEG particles were 2.53 and 2.52 Å, while the particle sizes were 12 and 20 nm calculated by the Debye-Scherrer method, respectively.
The magnetization curves of (i) Fe3O4 and (ii) Fe3O4@CTS-mPEG are shown in Figure 3. The size of magnetite NPs is a crucial factor in their magnetic properties. If the size is small enough, such nanostructures have superparamagnetic properties. The saturation magnetization value (Ms) of Fe3O4 NPs (68.9 emu g–1) indicated the good crystal structure of iron oxide Fe3O4 NPs. Additionally, the Ms (23 emu g–1) of Fe3O4@CTS-mPEG MNPs was lower than that of Fe3O4 particles because of the outer non-magnetic layer coated on Fe3O4 NPs, CTS-mPEG. Consequently, Fe3O4@CTS-mPEG NPs may be more suitable for magnetic separation for DDSs [25].
The FT-IR spectra of (i) CTS-mPEG, (ii) Fe3O4 NPs, and (iii) Fe3O4@CTS-mPEG are shown in Figure 4. The spectrum of CTS-mPEG [Figure 4(i)] showed the characteristic infrared bands of CTS, including amide I and N-H bending vibration at 1630 and 3419 cm–1, respectively. In addition, the increased intensity of the peaks at around 2924 and 1100 cm–1 showed the CH2 groups and C-O-C stretch of mPEG, respectively. Importantly, a new strong peak that appeared at 1720 cm–1 was assigned to the carbonyl band. The characteristic peaks of Fe3O4 at 571 and 578 cm–1 could be obtained in Figure 4(ii) and (iii). The presence of Fe3O4 NPs was identified by the O-H stretch vibration at 3416 and 3420 cm–1. As shown in Figure 4(iii), there was a strong shift between Fe-O stretching (570–578 cm–1) and N-H bending vibration (1642–1630 cm–1) of Fe3O4 because of the existence of CTS-mPEG. This result confirmed that the NH2 groups of CTS were effectively bound together with iron ions. In the FT-IR of Fe3O4@CTS-mPEG, the band at 1400 cm–1 was assigned to the C-O alcoholic groups of CTS. Further, the peaks at around 2925 and 3430 cm–1 were attributed to the C-H stretching band and the amino groups of CTS-mPEG, respectively. These results showed that CTS-mPEG was prepared and attached onto the Fe3O4 NP surface.
Figure 5 exhibits the shape of (A) Fe3O4 and (C) Fe3O4@CTS-mPEG MNPs, and their particle size distributions. The obtained NPs still maintain the morphological property of Fe3O4 particles except that the structure and size of Fe3O4@CTS-mPEG were looser and larger than those of Fe3O4 NPs, respectively. Moreover, the average particle size of Fe3O4 and Fe3O4@CTS-mPEG MNPs was found to be 12 and 20 nm in diameter, respectively. These results implied that the surface of Fe3O4 NPs was covered with the CTS-mPEG layer.
MNPs have mostly been used to control drug release with an external magnetic field [26]. The in vitro release of PTX from Fe3O4@CTS-mPEG was carried out in PBS (pH 7.4) at 37°C and 45°C (Figure 6). The coated NPs showed a sustained release profile up to 96 h. The cumulative release amount of PTX from Fe3O4@CTS-mPEG at 37°C over 48 h was only 34.9%, compared with 37.5% and 41.2% of HCPT from PEG (5000)-CTS-Fe3O4 and PEG (2000)-CTS-Fe3O4, respectively [21]. The release behavior of PTX could be explained by the flexible and hydrophilic PEG chains on the surface of Fe3O4@CTS-mPEG, which enhance the diffusion resistance of PTX. However, after incubation with PBS at 45°C, the cumulative release amount of PTX was 54.6%, compared with 59.9% and 65.8% of HCPT from PEG (5000)-CTS- Fe3O4 and PEG (2000)-CTS-Fe3O4, respectively [21]. The release behavior of PTX in the obtained NPs at 37°C and 45°C was significantly different. In other words, the diffusion rate of PTX from Fe3O4@CTS-mPEG NPs was facilitated by high temperatures. According to these results, Fe3O4@CTS-mPEG might be conveniently available for both hyperthermia (42–45°C) and chemotherapy. Such combination therapy is very effective when using an external alternating magnetic field for magnetic targeted drug delivery. Overall, Fe3O4@CTS-mPEGs may serve as stable MNPs with dual therapeutic effects in cancer treatment.
4 Conclusion
In this study, CTS-mPEGs have been successfully conjugated and coated onto Fe3O4 NPs with 20 nm diameter. The water-insoluble PTX was effectively loaded into Fe3O4@CTS-mPEG NPs and slowly released up to 96 h. Furthermore, the coated NPs also showed their potential applications in cancer hyperthermia (42–45°C) therapy. These results suggest the promising potential of Fe3O4@CTS-mPEG NPs as a stable magnetic delivery system with dual therapeutic effects (hyperthermia and chemotherapy) for the treatment of cancer.
About the authors
Dong Quy Hoang received her PhD in polymer science and engineering from Sungkyunkwan University, Korea. She is mainly engaged in the study of the synthesis of polymers, biopolymers, composites-biocomposites, and flame-retardant polymers.
Tuong Vi Tran received her Bachelor of Biotechnology degree from Ho Chi Minh City International University, Vietnam, in 2015. She now works as a research assistant with Dr. Dai Hai Nguyen at the Institute of Applied Materials Science-Vietnam Academy of Science and Technology. Her major research is related to synthesis and characterization of nanomaterials such as chitosan nanogels and gelatin hydrogels as drug delivery systems for cancer therapy.
Ngoc Quyen Tran was born in 1979. He received a PhD degree in the Republic of Korea in 2011. He is currently a researcher at the Institute of Applied Materials Science-Vietnam Academy of Science and Technology. He is also an invited lecturer at TraVinh University and Lac Hong University. His teaching and studying areas are biochemistry and biomaterials for biomedical applications.
Cuu Khoa Nguyen was born in 1960. He received a PhD degree in Russia in 1997. He returned to Vietnam and worked at the Institute of Applied Materials Science-Vietnam Academy of Science and Technology. He was appointed associate professor in 2010. His teaching and study areas are materials and biomaterials for practical applications.
Thi Hiep Nguyen has a PhD in medical science and 10 years of experience in tissue engineering and regenerative medicine. The main focus of Hiep’s research is regeneration of skin and bone, and her work has covered a full range from fabricating biomaterials to in vitro and in vivo studies. Hiep attended Soonchunhyang University in 2012, and moved to work at the International University (IU), Vietnam National University-Ho Chi Minh City (VNU-HCM) until present. She currently has a senior lecturer position at IU, leads the tissue engineering and regenerative group, and manages her own laboratory. Hiep is running two projects funded by Vietnam National University and Office of Naval Research Global (ONRG). She has published 20 articles in international journals and 7 articles in domestic journals. Hiep has attended more than 40 conferences thus far.
Minh Dung Truong was born in 1985. He is a PhD candidate at the Department of Molecular Science and Technology in Ajou University, Korea. His major research area is stem cell application in tissue engineering.
Dai Lam Tran graduated from Belorussian State University (in the former USSR) with a master’s degree in solid state chemistry in 1994. Then, he received his PhD in physical chemistry (surface-interface) from the University of Paris VII, Paris, France, in 2003. He was a lecturer at Hanoi University of Technology from 1998 to 2008. He has been an Associate Professor at IMS since 2009. His research focus is on nanofabrications, characterizations, and applications of nanobiomaterials in drug delivery systems and biosensors.
Le Van Thu worked as a researcher at the Laboratory of Special Materials, Institute of Chemistry-Biology and Professional Documents, Ministry of Public Security. He received his bachelor’s degree in material chemistry from VNU University of Science in 2003, and his master of science in physiochemical-theoretical chemistry from VNU University of Science in 2007. In 2012, he received his PhD in physiochemical-theoretical chemistry from Vietnam Academy of Science and Technology. Nearly 60 of his articles and reports related to polymer composite and nanocomposites have been published in national journals, international journals, proceedings of national and international scientific conferences, and workshops. His present research concerns nanocomposite and polymer composite materials.
Dai Hai Nguyen was born in 1984. He received a PhD degree from Ajou University-Republic of Korea in 2013. Currently, he works as a researcher at the Institute of Applied Materials Science-Vietnam Academy of Science and Technology. He is also an invited lecturer in TraVinh University and Ho Chi Minh City University of Natural Sciences.
Acknowledgments
This research was funded by Vietnam National University, Ho Chi Minh City (VNU-HCM) under grant no. C2016-18-09.
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