Structure and properties of fe3o4 nanoparticles coated by pla-Peg copolymer with and without loading of curcumin - Phan Quoc Thong

Journal of Science and Technology 54 (1A) (2016) 268-276 STRUCTURE AND PROPERTIES OF Fe3O4 NANOPARTICLES COATED BY PLA-PEG COPOLYMER WITH AND WITHOUT LOADING OF CURCUMIN Phan Quoc Thong 1, 2, * , Ha Phuong Thu 1 , Le Thi Thu Huong 3 , Luu Huu Nguyen 2 Nguyen Xuan Phuc 1 1 Institute of Materials Science, 18 Hoang Quoc Viet, Cau Giay, Ha Noi, Viet Nam 2 Khanh Hoa University, Khanh Hoa, 01 Nguyen Chanh, Nha Trang, Khanh Hoa Viet Nam 3 Vietnam National University of Agriculture, Trau Quy, Gia Lam, Ha Noi, Viet Nam * Email: phankh4@yahoo.com Received: 31 August 2015; Accept for publication: 25 October 2015 ABSTRACT Nanoparticles (NPs) of poly(lactide)-polyethylene glycol (PLA-PEG) with PLA:PEG (3:1, w/w) component ratio was prepared by ring opening polymerization of Lactide for preparation of Fe3O4@PLA-PEG and Fe3O4@PLA-PEG/Cur nanosystem. The Cur (curcumin) loaded Fe3O4 could be used as multi-functional nano drugs beneficiating both the image contrast enhancement and locally controlled heating. The size, shape, surface bonding of Fe3O4@PLA-PEG, Fe3O4@PLA-PEG/Cur nanosystems were determined by Field Emission Scanning Electron Microscopy (FE-SEM), Transmission Electron Microscopy (TEM), Thermal Gravity Analysis (TGA), Fourier Transform Infrared Spectroscopy (FT-IR); while magnetic characteristics by Vibrating Sample Magnetometer (VSM). The research suggests that Fe3O4@PLA-PEG and Fe3O4@PLA-PEG/Cur nanosystems exhibit properties of great potential for biomedical applications, both for diagnosis and treatment purposes. Keywords: PLA-PEG copolymer, Fe3O4 nanoparticles, Fe3O4@PLA-PEG, Fe3O4@PLA- PEG/Cur. 1. INTRODUCTION Magnetic nanoparticles play an important role for biomedical applications such as: target drug delivery [1 - 3], contrast enhancement in Magnetic Resonance Imaging (MRI) [4 - 5] or cancer hyperthermia [5]. Surface functionalization of magnetic nanoparticles is necessary in order to improve dispersion and some biochemical characteristics [6]. While organic materials are commonly used for functionalization purposes, polymeric micelles are regarded, by themselves, as multifunctional materials for drug delivery and diagnostic imaging [7]. Nanostructures formed from the copolymer with hydrophilic and hydrophobic self-assembly Structure and properties of Fe3O4 nanoparticles 269 separate chains can create supramolecular core-shell structures (10 – 100 nm) dispersed in water. The hydrophobic core of the micelles have the ability to load the hydrophobic agent, and the hydrophilic shell helping the nanoparticles stabilized in water [8]. It has been reported recently that Fe3O4 nanoparticles incorporated in the polymer micelle has enhanced biocompatible and prolonged the present time of the systems in the blood circulation [9]. In this report, we present the research of a multifunctional system consisting of Fe3O4 nanoparticles synthesized by co-precipitation method coated with a copolymer of co(lactide)- polyethylene glycol (PLA-PEG) and loaded with curcumin to form Fe3O4@PLA-PEG/Cur drug delivery nanosystem dispersed in water. The structure and physicochemical properties of the system were characterized by multiple methods of analyzing. 2. MATERIALS AND METHOD 2.1. Materials Mono lactide acid (LA), polyethylene glycol - 2000 (PEG 2000), Tin (II) 2-ethylhexanoate were purchased from Sigma (St. Louis, MO, USA); FeCl3, FeCl2.4H2O, NH4OH, toluene, Dichlomethan (DCM, C2H2Cl2), Methanol (CH3OH), Ethanol (C2H5OH) were purchased from Merck (Germany); curcumin was purchased from Indian. Double distilled water was used in all the experiments. 2.2. The synthesis of PLA-PEG copolymer and Fe3O4 nanoparticles PLA-PEG copolymer with different PLA:PEG mass ratios were synthesized by ring opening polymerization reaction between lactic monomer and polyethylene glycol (PEG) in the presence of catalyst tin (II) 2-ethylhexanoate [10]. PLA-PEG copolymer was dissolved in DCM (1 mg/ml) and stirred for 24 hours, then H2O was added to form a liquid mixture that was stirred for another 24 hours to disperse PLA-PEG copolymer from DCM to H2O. DCM solvent was then evacuated to obtain dispersion of PLA-PEG copolymer in H2O (1 mg/ml). Fe3O4 nanoparticles were synthesized by co-precipitation method [11] according to the following equation: 2Fe 3+ + Fe 2+ + 8OH - → Fe3O4 + 4H2O Fe3O4 nanoparticles were dispersed into water and surface functionalized by PLA-PEG copolymer. 2.3. Preparation of Fe3O4@PLA-PEG and Fe3O4@PLA-PEG/Cur nanosystems Defined amount of Fe3O4 nanoparticles dispersed in water was slowly dropped into PLA- PEG copolymer solution, stirred for 24 hours to obtain Fe3O4@PLA-PEG nanoparticles with concentration of Fe3O4 nanoparticles various as 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml and fixed PLA-PEG copolymer concentration of 0.3 mg/ml. The obtained magnetic fluid solutions were denoted correspondingly as 1F.3P, 2F.3P, 3F.3P, 4F.3P and 5F.3P. Then, curcumin dissolved in C2H5OH 4 mg/ml was slowly dropped into 3F.3P solution, stirred for 48 hours, and then ethanol was allowed to vapour to obtain Fe3O4@PLA-PEG/Cur nanoparticles solutions. Phan Quốc Thông, Hà Phương Thư, Lê Thị Thu Hương, Lưu Hữu Nguyên, Nguyễn Xuân Phúc 270 2.4. Measurement and characteristic of sample Size and size distribution of nanoparticles were characterized by TEM (GEOL, Japan). Structure of the particles was determined by FT-IR spectroscopy (Shimadzu FT-IR Prestige-21, Japan). Saturation magnetization (Ms) was measured by a homemade vibrating sample magnetometer (VSM). The percent by mass of PLA-PEG and curcumin in nanoparticles system was determined by Thermal Gravity Analysis (TGA, Shimadzu DTG-60H, Japan). 3. RESULTS AND DISCUSSION 3.1. Size and structure of nanoparticles Figure 1. TEM image of nanoparticles Fe3O4 (a), Fe3O4@PLA-PEG (b) and Cur/Fe3O4@PLA-PEG (c). TEM image of Fe3O4, Fe3O4@PLA-PEG and Cur/Fe3O4@PLA-PEG samples are presented on Figure 1. The pictures show that the nanoparticles are spherical with a single core in each particle. The Fe3O4 functionalized by PLA-PEG and PLA-PEG/Cur nanoparticles are fairly uniform in size, of average diameter of about 20 nm, which is of a few nanometers larger than that of the Fe3O4 nanoparticles (15 nm by Fig. 1a). As indicated by the images in Figs 1b and 1c, the functionalization resulted in formation of core-shell structures, so that the coating by the copolymer in both the cases with and without curcumin occurred via single core encapsulation process. The formation of such a core-shell is assumed as the following: after being penetrated into the center of PLA-PEG nanoparticles (of about 50 nm in size [10]) the Fe3O4 nanoparticles attract PLA component to form strong binding on the core particle surface, so that the outmost size of the then copolymer particles become reduced to about 20 nm. For Fe3O4@PLA-PEG/Cur nanoparticles, curcumin molecules - due to their hydrophobic property are assumed also to penetrate into the copolymer particles center and absorbed on the Fe3O4 particles surface to even enhance the biding of Fe3O4 nanoparticles with coating layer, and making the diameter of the whole system remain almost the same as that of the case without curcumin. Figure 2. FT-IR spectra of nanoparticles Fe3O4, Fe3O4@PLA-PEG and Fe3O4@PLA-PEG/Cur. Structure and properties of Fe3O4 nanoparticles 271 3.2. FT-IR spectra As shown in FT-IR spectra of Fe3O4 and Fe3O4@PLA-PEG nanoparticle samples (Fig. 2), the peak at 632 cm -1 , originated from stretching of Fe-O bond of Fe3O4 nanoparticles, shifts to 628 cm -1 in the Fe3O4@PLA-PEG spectrum. Simultaneously, a peak at position 1384 cm -1 appearing on FT-IR spectrum of Fe3O4@PLA-PEG nanoparticles proves that ferromagnetic nanoparticles have been coated with the copolymer. In FT-IR spectrum of Fe3O4@PLA-PEG/Cur sample, there are peaks at 1276 cm -1 and 1589 cm -1 suggesting that Fe3O4@PLA-PEG/Cur nanoparticles system was successfully synthesized by the combination of ferromagnetic nanoparticles and curcumin to form drug loaded multifunctional system. 3.3. Effect of PLA-PEG to stability and saturation magnetization of Fe3O4@PLA-PEG nanoparticles Fe3O4 nanoparticles with a concentration of 1 mg/ml to 5 mg/ml were functionalized by PLA-PEG 0.3 mg/ml to investigate the influence of Fe3O4 and PLA-PEG concentration to stability of the resulted dispersion and Ms of nanoparticle systems. Results are presented in Table 1. Table 1. Influence of Fe3O4 over PLA-PEG concentration on dispersion stability of Fe3O4@PLA-PEG nanoparticles. Sample Component Dispersion stability (day) Fe3O4 (mg/ml) PLA-PEG (mg/ml) 1F.3P 1 0.3 < 1 2F.3P 2 0.3 < 7 3F.3P 3 0.3 > 90 4F.3P 4 0.3 < 20 5F.3P 5 0.3 < 1 Figure 3. Magnetic hysteresis curves of Fe3O4 nanoparticles functionalized by PLA-PEG of various concentration. Phan Quốc Thông, Hà Phương Thư, Lê Thị Thu Hương, Lưu Hữu Nguyên, Nguyễn Xuân Phúc 272 As shown in stability of 3F.3P, 4F.3P, 5F.3P, with increasing concentration of Fe3O4 nanoparticles, the proportion of PLA-PEG copolymer will decrease, leading to a decrease of hydrophilic component (PEG), and therefore the stability of dispersion of the Fe3O4/PLA-PEG nanoparticles will decrease [12, 13]. However, when the concentration of PLA-PEG/Fe3O4 rises too much (in 1F.3P and 2F.3P samples), it is possible to form clusters of Fe3O4@PLA-PEG nanoparticles [13] which results in a decrease in dispersion stability of Fe3O4/PLA-PEG nanoparticles. Thus, the most suitable ratio for stable purpose of PLA-PEG:Fe3O4 in Fe3O4@PLA-PEG nanoparticles is 0.3:3 (mg/ml) (3F.3P sample stable for more than 90 days). Magnetic hysteresis curve measured at room temperature for Fe3O4 nanoparticles coated by various mass ratios of PLA-PEG are shown in Figure 3. The as-measured saturation magnetization values Ms as-me , and those calculated based on the nominal percentage of nonmagnetic polymer mass, Ms nom , for the sample series are gathered in Table 2. As can be noticed from the last column of Table 2, not only no reduction in saturation magnetization caused by the functionalization has been observed in any of the coated samples; but even a significant increase (up to 10 %) of magnetization is evidenced. Table 2. Magnetization Ms as-me and Ms nom of Fe3O4 and Fe3O4@PLA-PEG nanoparticles. Sample Component % Fe3O4 Ms as-me Ms nom Fe3O4 (mg) PLA-PEG (mg) Fe3O4 Fe3O4 0 100 64.4 64.4 1F.3P 1 0.3 76.9 53.0 68.9 2F.3P 2 0.3 87 59.2 68.1 3F.3P 3 0.3 90.9 64.5 71 4F.3P 4 0.3 93 64.2 69 5F.3P 5 0.3 94.4 65.1 68.9 In other words, our experiment indicated that the coating with an appropriately biodegradable polymer (PLA-PEG) may restore somehow the magnetization reduction of the core Fe3O4 nanoparticles that was resulted during nanoparticle synthesis in general, and their co- precipitation synthesis [14]. On the other hand, the accompany of the highest saturation magnetization with the highest stability in 3F.3P sample suggests that dispersion stability could be a crucial cause for getting highly magnetization restoration. 3.4. Magnetic properties of curcumin loaded system To investigate the influence of curcumin loading, two compositions of Fe3O4: PLA-PEG of 3:0.3 (3F.3P) and 5:0.3 (5F.3P) were used as the base composition. The optimal amount of curcumin was found to be of 0.7 mg/ml, and the samples then were denoted, correspondingly as 3F.3P.7C and 5F.3P.7C. The nanoparticle samples of Fe3O4, Fe3O4@PLA-PEG and Fe3O4@PLA-PEG/Cur were analyzed by TGA (diagrams shown in Figure 4) in order to estimate experimentally the mass contribution of nonmagnetic coating materials of PLA-PEG and curcumin in the samples. Table 3 summarizes the TGA-determined mass percentage m ex along with the nominal mass m nom for the coated samples with and without curcumin loading. The data indicate that, except for one sample, the mass percentages determined experimentally by TGA are in good agreement with those used in nominal composition. Structure and properties of Fe3O4 nanoparticles 273 Figure 4.TGA diagram for nanoparticle samples of 3F.3P (a), 3F.3P.7C (b), 5F.3P (c), 5F.3P.7C (d). Figure 5. Magnetization hysteresis curves measured for Fe3O4, Fe3O4@PLA-PEG, Fe3O4@PLA-PEG/Cur sample. Influence of curcumin loading on the magnetic properties was studied for the stable samples 3F.3P and 3F.3P.7C. The saturation magnetization obtained from measured curves in Fig. 5, Ms as-me for the core Fe3O4 as well as coated Fe3O4@PLA-PEG and Fe3O4@PLA-PEG/Cur samples are gathered in Table 5, along with the corresponding values calculated by use of nominal (Ms nom ) and experimental (Ms exp ) percentage of nonmagnetic mass. As clearly shown, the Ms value of Fe3O4 nanoparticles after functionalization by PLA-PEG and loading curcumin tends to rise with the increase of at least 10 %. This observation consistent with that reported by Y. Piñeiro-Redondo et al [14]. Phan Quốc Thông, Hà Phương Thư, Lê Thị Thu Hương, Lưu Hữu Nguyên, Nguyễn Xuân Phúc 274 Table 3. Mass percentage of nonmagnetic material experimentally determined by TGA versus the nominal values. Sample Component mFe3O4 % m nom % m ex % Fe3O4 (mg) PLA-PEG (mg) Cur (mg) Fe3O4 Fe3O4 0 0 100 0 0 3F.3P 3 0.3 0 90.9 9.1 13.5 3F.3P.7C 3 0.3 0.7 75 25 24.95 5F.3P 5 0.3 0 94.34 5.66 5.677 5F.3P.7C 5 0.3 0.7 83.33 16.67 14.29 Table 4. Saturation magnetization as measured and after subtraction of nonmagnetic coating mass. Sample msample (μg) Ms as-me (emu/g) Ms nom (emu/g) Ms exp (emu/g) Fe3O4 10.5 64.4 64.4 64.4 3F.3P 13.8 64.5 71 74.5 3F.3P.7C 15.4 52.9 70.5 70.5 On the other hand, our observation suggested that the curcumin loaded Fe3O4@PLA- PEG/Cur nanoparticles exhibit higher dispersion stability than that of Fe3O4@PLA-PEG. This result is significant to open up many towards new application for Fe3O4 nanoparticles in biomedical fields such as hyperthermia treatment, magnetic resonance imaging (MRI), as well as study in effects of physical mechanism on their magnetization saturation. 4. CONCLUSION In this study, we have successfully synthesized Fe3O4 nanoparticles and functionalized by PLA-PEG to form single core Fe3O4@PLA-PEG core-shell nanoparticles, then loading curcumin to form multifunctional drug delivery nano system Fe3O4@PLA-PEG/Cur. Saturation magnetization of Fe3O4 nanoparticles after being functionalized is higher than that of uncoated Fe3O4 nanoparticles. These results suggest enormous potential of applications of the nano system Fe3O4@PLA-PEG as well as Fe3O4@PLA-PEG/Cur in biomedical fields, especially in the diagnosis and treatment of cancer. Acknowledgement. This work was done with the support of funding of basic research subject oriented application code ĐT.NCCB-ĐHƯD.2012-G/08, NAFOSTED subject code 106.99-2012.43 and Khanh Hoa University. REFERENCES 1. Durán J. D. G., Arias J. L., Gallardo V., Delgado A. V. - Magnetic colloids as drug vehicles, J. Pharm. Sci. 97 (2008) 2948-2983. Structure and properties of Fe3O4 nanoparticles 275 2. Purushotham S, Ramanujan R.V. - Thermoresponsive magnetic composite nanomaterials for multimodal cancer therapy, Acta Biomaterial 6 (2010) 502-510. 3. Zhang J., Misra R. D. K. - Magnetic drug targeting carrier encapsulated with thermosensitive smart polymer: core-shell nanoparticle carrier and drug release response, Acta Biomaterial 3 (2007) 838-850. 4. Zhang L., Xue H., Gao C., Carr L., Wang J., Chu B., et al. - Imaging and cell targeting characteristics of magnetic nanoparticles modified by a functionalizable zwitterionic polymer with adhesive 3,4-dihydroxyphenyl-l-alanine linkages, Biomaterials 31 (2010) 6582-6588. 5. Kluchova K, Zboril R, Tucek J, Pecova M, Zajoncova L, Safarik I, et al. - Superparamagnetic maghemite nanoparticles from solid-state synthesis e their functionalization towards paroral MRI contrast agent and magnetic carrier for trypsin immobilization, Biomaterials 30 (2009) 2855-2863. 6. Shultz M. D., Reveles J. U., Khanna S. N., Carpenter E. E. - Reactive nature of dopamine as a surface functionalization agent in iron oxide nanoparticles, J. Am. Chem Soc. 129 (2007) 2482-2487. 7. Zhang L., Eisenberg A. - Multiple morphologies and characteristics of “crew-cut” micelle-like aggregates of polystyrene-b-poly(acrylic acid) diblock copolymers in aqueous solutions, J Am. Chem. Soc. 118 (1996) 3168-3181. 8. Lodge T. P., Pudil B., Hanley K. J. - The full phase behavior for block copolymers in solvents of varying selectivity, Macromolecules 35 (2002) 4707-4717. 9. Torchilin V. P. - PEG-based micelles as carriers of contrast agents for different imaging modalities, Adv. Drug Delivery Rev. 54 (2002) 235. 10. Phan Q. T, Nguyen H. N., Nguyen X. P., Do H. M., Ha P. T. - Impact of PLA/PEG ratios on Curcumin solubility and encapsulation efficiency, size and release behavior of Curcumin loaded poly(lactide) poly(ethylenglycol) polymeric micelles, International Journal of Drug Delivery 6 (2014) 279-285. 11. Shen T., Weissleder R., Papisov M., Bogdanov A., Brady T. J. - Monocrystalline iron oxide Nano compounds (MION): Physicochemical properties. Magn. Reson. Med. 29 (1993) 599. 12. Kawamori T., Lubet R., Steele V. E., Kelloff G. J., Kaskey R. B., Rao C. V., Reddy B. S. - Chemopreventive effect of curcumin, a naturally occurring anti-inflammatory agent, during the promotion/ progression stages of colon cancer, Cancer Res. 59 (1999) 597- 601. 13. Karunagaran D., Rashmi R., Kumar T. R. - Induction of apoptosis by curcumin and its implications for cancer therapy. Curr Cancer Drug Targets 5 (2005) 117–129. 14. Piñeiro-Redondo et al. - The influence of colloidal parameters on the specific power absorption of PAA-coated magnetite nanoparticles, Nanoscale Research Letters 6 (2011) 383. Phan Quốc Thông, Hà Phương Thư, Lê Thị Thu Hương, Lưu Hữu Nguyên, Nguyễn Xuân Phúc 276 TÓM TẮT CẤU TRÚC VÀ TÍNH CHẤT CỦA HẠT NANO Fe3O4 BỌC COPOLYMER PLA-PEG CÓ VÀ KHÔNG MANG CURCUMIN Phan Quốc Thông1, 2, *, Hà Phương Thư1, Lê Thị Thu Hương3, Lưu Hữu Nguyên2, Nguyễn Xuân Phúc1 1 Viện Khoa học vật liệu, Viện HLKHCNVN, 18 Hoàng Quốc Việt, Cầu Giấy, Hà Nội 2Trường Đại học Khánh Hòa, 01 Nguyễn Chánh, Nha Trang, Khánh Hòa 3 Học viện Nông nghiệp Việt Nam, Trâu Quỳ, Gia Lâm, Hà Nội * Email: phankh4@yahoo.com Hạt Nano copolymer poly(lactide)-polyethylene glycol (PLA-PEG) với tỷ lệ trọng lượng PLA:PEG 3:1 đã được điều chế bằng phương pháp mở vòng lactic sử dụng cho mục đích chế tạo hệ Nano từ tính cấu trúc lõi-vỏ Fe3O4@PLA-PEG và hệ mang thuốc đa chức năng Fe3O4@PLA- PEG/Cur. Hạt Nano Fe3O4 đã chức năng hóa có thể sử dụng như hệ phân phối thuốc đa chức năng, vừa có khả năng trị bệnh vừa có khả năng chẩn đoán hình ảnh. Kích thước, hình dạng, bề mặt của hệ Nano Fe3O4@PLA-PEG, Fe3O4@PLA-PEG/Cur được đặc trưng bằng: kính hiển vi điện tử quét FE-SEM, kính hiển vi điện tử truyền qua TEM, phân tích nhiệt trọng lượng (TGA), phổ hồng ngoại (FT-IR); các đặc trưng từ đo trên hệ từ kế mẫu rung (VSM). Kết quả nghiên cứu này cho thấy hệ Nano Fe3O4@PLA-PEG và Fe3O4@PLA-PEG/Cur có những đặc tính và tiềm năng to lớn ứng dụng trong y sinh, đặc biệt trong chẩn đoán và điều trị ung thư. Từ khóa: copolymer PLA-PEG, hạt Nano Fe3O4, Fe3O4@PLA-PEG, Fe3O4@PLA-PEG/Cur.