Study on fabrication and effectiveness evaluation of multifunctional nanosystem (polymer - Drug - Fe3O4 - folate) on cancer cells

MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY ...*** LE THI THU HUONG Study on fabrication and effectiveness evaluation of multifunctional nanosystem (polymer-drug-Fe3O4-folate) on cancer cells Major: Electronic materials Code: 9440123 SUMMARY OF MATERIALS SCIENCE DOCTORAL THESIS Hanoi – 2018 This thesis was finished at Institute of Mataerials Science and Graduate university of Science and Technology, Vietnam Academy of Science and Technology Supervisor 1: Dr. Ha Phuong Thu Supervisor 2: Prof. Dr. Nguyen Xuan Phuc Reviewer 1: Reviewer 2: Reviewer 3: . This thesis will be defended against Board of thesis defense at Graduate university of Science and Technology – Vietnam Academy of Science and Technology at , Date .. It can be found at: - Library of Graduate university of Science and Technology - Vietnam National Library 1 INTRODUCTION 1. The urgency of the thesis Today, the development of science and technology has made great strides in biomedical science but human beings are still facing many diseases, most notably cancer. There are many cancer drugs available on the market. However, the biggest disadvantage of many cancer drugs is that they are less soluble in water or more readily excreted. Besides, the selectivity of these drugs is not high, and more or less affects healthy tissues and results in side effects including nausea, diarrhea, anemia or reduced immunity of the body. This is due to most treatments not only affect the tumor locally, but also affect a large part of the body's normal tissues and organs. To overcome the shortcomings of the method above, researchers have applied nanotechnology, using nanometer-sized materials as a vehicle to deliver cancer-specific drugs such as Curcumin, Paclitaxel, Doxorubicin. to the tumor safely. In addition, magnetic nanomaterials have been studied extensively for cancer screening, cancer diagnosis by magnetic resonance imaging (MRI), thermotherapy by increasing tumor temperature under magnetic field, and especially tumor targeting by magnets... Magnetic nanoparticles and anti-cancer drugs could be encapsulated in the shells of natural or synthetic polymers such as dextran, modified dextran, chitosan, modified chitosan, alginate, PLA-TPGS, PLA-PEG ... to become nano stable systems. The surface of these system can be added a number of target factors such as folate, aptamer, tranferin, lectin and antibody. Such a multifunctional nanosystem will increase the effect on certain cancer cells, partly addressing the need for chemotherapy to be highly selective for cancer cells. The benefits of the material utility are: reducing the dose of the drug, focusing on the tumor position, avoiding to affect the healthy cells and therefore minimizing adverse side effects on patients. From the above mentioned issues, it is possible to use a multifunctinal nanosystem consisting of Fe3O4 nanoparticles coated with modified chitosan, modified dextran, alginate or copolymers and attached folate as a vehicle for Curcumin (Cur) or Doxorubicin (Dox) to safely target the cancerous tumor. Based on that fact, the thesis "Research and make the effect of polyunsaturated (polymer-drug- Fe3O4-folate) on cancer cells" was done. 2 2. The objectives of the thesis - Manufacturing multifunctional nanoparticles including: Fe3O4 nanoparticles (magnetical properties) coated with biocompatible polymers, attaching drugs (Cur, Dox) and targeted folate factor (optical properties)) that are well dispersed in water, able to target the cancer. - Experiment and evaluate the effect of the nanoparticles on cancer cell lines such as HT29; HeLa; HepG2 ... and on experimental animals 3 . The main contents of the thesis - Synthesis of multifunctional nanocomposite materials containing curcumin and doxorubicin based on Fe3O4 nanoparticles coated with natural polymers (O-carboxylmethylchitosan and alginate). - Characterization of the materials by modern physicochemical methods: FTIR, UV-VIS, fluorescence spectrum, XRD, VSM, TGA, SEM, TEM ... - Determine the effect of multifunctional nanoparticles on cancer cell lines: Hep-G2, HeLa, LU-1, ... and in mice. Chapter 1. OVERVIEW In this chapter, we review the issues involved in the synthesis of multifunctional nanoparticles and the effect assessment of these systems on cancer cells. Multifunctional systems consist of Fe3O4 nanoparticles coated with polymer, drugs loading and folate attaching. In details, this part provides an overview of the properties, synthesis methods and applications of Fe3O4 nanoparticles. Especially, the issues that need to be addressed in order to use Fe3O4 nanoparticles in biomedical field were clearly shown. The nature and applicability of natural polymers commonly used (O-carboxyl methyl chitosan, alginate, dextran) were discussed while characteristics and some studies using the drug substances: curcumin and Doxorubicin were presented. In addition, the method of folate attachment to the nanoparticles and the targeted effect of this agent were overviewed. Chapter 2. CONDITION AND EXPERIMENTAL METHOD 2.1. Synthesis of multifunctional nanosystems Multifunctional nanomaterials were synthesized through the procedures shown in Figure 2.1. The magnetic nanoparticles (Fe3O4) were synthesized by co-precipitation of Fe 2+ and Fe 3+ at 1:2 molar ratio with normal apparatus 3 [41] or using microwave technique on Sineo-Uwave 1000 apparatus. Fe3O4 nanoparticles were then coated with OCMCS (1 mg/ml) or alginate at different concentrations. In the next step, Curcumin or Doxorubicin was introduced into the system by adsorption interaction with the magnetic core or reaction with the polymer shell. Ultimately, the optimized drug delivery system was chosen to incorporate folate-targeting factor or CdTe quantum dots. Figure 2.1: Synthesis procedures of multifunctional nanosystems 2.2. Characterization The characteristics of the systems were determined by modern methods: X-ray diffraction (XRD), infrared spectroscopy (FTIR), UV-Vis spectroscopy, fluorescence spectroscopy, thermal analysis, scanning electron microscopy (SEM), tranmittance electron microscopy (TEM). Drug encapsulating efficacy, drug loading content, and drug release profiles were determined by UV-Vis spectroscopy. The cytotoxicity of the samples was determined according to the method of Skehan and Likhiwitayawuid [171, 172]. Atomic Absorption Spectrum (AAS) method was used to quantify Fe present in mouse tissues. In vivo experiments: 4 7-10 mm tumor – bearing mice were divided into 4 groups, each group of 6 mice, including: control group (mice with untreated tumors) and groups treated with FA, FAD, FADF, respectively. In each treatment cycle, the drug was injected directly into the tumor at 50 l/mouse. At 40 minutes post injection, the mouse was fixed in a plastic tube and put into a RDO-HFI coil of a magnetic field with frequency of 178 kHz and strength of 90 Oe for 30- minute time. Two consecutive cycles separated by 3 days. Changes in tumor size were recorded before each treatment. These information was used to assess the therapeutic effects of Doxorubicin loading magnetic nanoparticles on model mouse with lung cancer. Data analysis: Excel 2010, OriginPro 8 hoặc SPSS 22.0. Chapter 3: CURCUMIN LOADING OCMCS COATED Fe3O4 NANOPARTICLES 3.1. Synthesis of nano Fe3O4 nanoparticles (NPs) Fe3O4 nanoparticles ware successfully synthesized by co-precipitation method (Fe-O bond characterized by absorption peaks at 575 cm -1 on infrared spectra), reverse spinel structure (with typical peaks in XRD diagram), saturation magnetization of 70.5 emu/g, superparamagnetic property with Mr and Hc  0 and average size of 15 nm. 3.1.2. Microwave synthesized Fe3O4 NPs 3.1.2.1. Magnetic properties Magnetic remanance Mr and coercivity Hc of fabricated samples were  0, indicating that the material were superparamagnetic (Table 3.1). Thus, microwave technique did not change this property of the materials. The saturation of the M5 sample was the highest compared to the other samples, reaching 69 emu/g. This value is not much different than the Fe3O4 sample prepared under normal conditions (70.5 emu/g). Table 3.1: Magnetic parameters of microwave synthesized Fe3O4 NPs Sample M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 Ms (emu/g) 53,9 56,2 56,7 63,0 69,0 64,6 59,6 60,6 60,7 62,7 64,5 Hc 2,5 14 4 2,5 0 20 0 18 2 2 21 5 Thus, M5 is the best magnetic sample. 3.1.2.2. X-ray diffraction The XRD diagriam of the M5 sample showed full series of Fe3O4 typical peaks and no strange peaks, indicated that M5 formed with a single-phase spinel structure. This result confirms that M5 is the best sample in term of crystal structure. 4.2.1.3. IR spectra In Fig. 3.4, it can be seen that the microwave-assisted synthesized Fe3O4 samples showed the characteristic peak for the Fe-O bond at about 570 cm -1 . In some samples, however, a lower intensity peak at 630 cm -1 was observed, corresponding to the presence of Fe2O3 in these samples [174]. The spectra reveal that M5 is the highest purity sample with only one characteristic peak with high intensity. Thus, through the magnetometry, crystal structure and infrared spectra, we selected the M5 sample for further studies. 3.2. Effect of curcumin amount on curcumin loading systems (FOC1- FOC5) The amount of curcumin varying from 20 to 100 mg was investigated to determine the effect of the curcumin amount on magnetic properties as well as the stability of the systems (evaluated by measuring the zeta potential of the systems). The results are presented in Table 3.2. Table 3.2: Properties of curcumin loading systems Sample FOC1 FOC2 FOC3 FOC4 FOC5 Mass of curcumin (mg) 20 40 60 80 100 Ms (emu/g) 54,9 52,9 49,0 35,3 25,8 Zeta potential(mV) 40,2 32,6 30,4 18,2 8,1 The saturation magnetization measurement of FOC1-5 showed that when the amount of curcumin increased from 20 to 100 mg, the saturation magnetization of the samples decreased, especially in the samples FOC4 and FOC5. The Zeta potiential of FOC1-5 samples were positive because the magnetic particles were coated with O-carboxylmethyl chitosan polymer (Oe) Mr (emu/g) 0,5 1,0 0,2 0,4 0 1,7 0 2 0,1 0,2 1,9 6 with many NH2 functional groups on the surface. The change in Zeta potential of these samples is similar to that in saturation magnetization. Zeta potential values of FOC1-3 are greater than 30 mV showing that these samples could maintain stable state [170]. Meanwhile, Zeta potential values of FOC4 and FOC5 are significantly lower than those of above samples (less than 20 mV). In order to ensure that the multifunctional system carries the largest range of curcumin loaded and retains its magnetic properties, we use a curcumin mass of 60 mg for other related synthesis procedure. This curcumin amount is also used to prepare FOCF system. The actual curcumin contents of the systems are quantified by thermal analysis (Section 3.3.4). 3.3. FOC and FOCF NPs 3.3.1. IR spectra Infrared spectra of FOC and FOCF were compared with the infrared spectra of each component: Fe3O4, OCMCS, Curcumin and folic acid. The transfer of characteristic peaks proves that the system has been successfully synthesized. 3.3.2. Flourescence spectra Curcumin is a natural fluorescence compound. After receiving stimulation by radiation at 442 nm, the FOC solution emits fluorescence spectrum at a maximum wavelength of 515 nm.In comparison with the fluorescence spectrum of curcumin in ethanol/water (1:1) with a maximum at 542 nm, fluorescence of FOC exhibits a blue shift (27 nm shift towards short wavelength region). This is due to the interaction of the curcumin molecule with Fe3O4/OCMCS. In terms of intensity, FOC solution fluoresces much less weakly than free curcumin does. This is due to the presence of Fe3O4 in the sample which reduces the fluorescent ability of curcumin [132]. 3.3.3. FeSEM The surface morphology of the FOC and FOCF systems was determined through SEM images. The results show that the size of these particles is about 30 nm, which is larger than the size of the original Fe3O4 particle (about 20 nm), suggesting that curcumin and folic acid adsorbed onto the surface of Fe3O4 nanoparticles. 3.3.4. Thermal analysis Figure 7 show the DrTGA, TGA and DTA curves of FOC and FOCF samples. The TGA curves showed wto steps of weight loss of FOC and three 7 steps of weight loss of FOCF sample and then there was no change in weight of samples when continuing increase temperature. The weight loss for the first step of each sample at around 100 o C is attributed to quantitative mass losses of water present in the samples. All the other steps are endothermal, that can be explained by the decomposition of OCMCS, curcumin or folic acid. As mentioned above, the weight of OCMCS in the sample was very small, so the weight loss was almost attributed to the weight of curcumin or folic acid in the samples. The second step for weight loss of FOC corresponds to the third steps of FOCF at temperature range of 360 and 430 o C and can be assigned as the decomposition of curcumin. Therefore, the second weight loss step at around 299 o C of Fe3O4/OCMCS/Cu/Fol must be the loss due to the decomposition of folate. The result also show that in the first sample the mass of curcumin and Fe3O4 account for 45% and 48% the total mass while the mass of folic acid, curcumin and Fe3O4 are 26%, 25% and 46%, repectively total mass of Fe3O4/OCMCS/Cu/Fol sample. Based on this data, curcumin-loading capicity was calculated and found to be about 0.95 mg and 0.54 mg per mg of Fe3O4 in FOC and FOCF NPs. Despite of the decrease, FOCF is a good loader of curcumin as compared to other studies [134, 176, 177] and can be used as selective orientation drug deliverer. Figure 3.14 shows the structure of FOC and FOCF, in which curcumin is adsorbed on the surface of Fe3O4 particles. Figure 3.1: Structural models of FOC and FOCF 3.3.5. XRD diagrams and magnetic properties 8 30 40 50 60 70 2theta ( o ) (a)Fe3O4 (b) Fe3O4/OCMCS/Cur (c) Fe3O4/OCMCS/Cur/Fol (a) (b) (c) (200) (311) (400) (422) (511) (440) -15000 -10000 -5000 0 5000 10000 15000 -80 -60 -40 -20 0 20 40 60 80 -25 -20 -15 -10 -5 0 0.0 0.5 1.0 1.5 (c) Fe 3 O 4 /OCMCS/Cur/folic (b) Fe 3 O 4 /OCMCS/Cur (a) Fe 3 O 4 M s ( e m u /g ) H (Oe) (a) (b) (c) Figure 3.15: XRD diagrams of (a) Fe3O4, (b) FOC and (c) FOCF Figure 3.16: Hysterisis loops of (a) Fe3O4, (b) FOC and (c) FOCF The XRD patterns of FOC and FOCF show no difference from that of the Fe3O4 nanoparticles (Figure 3.15). It was clear that there were six diffraction peaks corresponding to six faces of (200), (311), (400), (422), (511) and (440) which were characteristic for single phase spinel structure of Fe3O4. These facts indicate that the two systems have not changed their crystal line structure during the encapsulation process. Magnetization measurements also provided evidence that the Fe3O4 nanoparticle encapsulated in maintained its crystalline structure (Figure 3.16). The magnetic properties of FOC and FOCF NPs was measured by VSM. The saturated magnetization of the FOC and FOCF NPs was about 53 emu/g, which was about 20 emu/g lower than that of free Fe3O4 due to the adsorption of curcumin or folic acid in the surface of Fe3O4. Although the magnetism has decreased, nanoparticles can still be adsorbed quickly and firmly by the magnet. On the other hand, it is well known that magnetic particles less than 30 nm will demonstrate the characteristic of superparamagnetism, which can be verified by the magnetization curve. The remanence (Mr) and coercivity (Hc) for FOC and FOCF NPs in the figure were close to zero, exhibiting the characteristic of superparamagnetism [169]. 3.3.6. Magnetic inductive heating effect The results of induction heating are presented in Table 3.4. When the iron oxide concentration decreases, both the saturated Ts temperature and the initial heating rate dT/dt (determined at t = 0) decrease. Particles concentrations of 0.3 mg/ml or more resulted in saturated temperatures of up to 42 °C and higher after 10 minutes. 9 Retention time of 10 minutes and possibly longer can be established by maintaining the magnetic field conditions. Because cancer cells can undergo apoptosis within the range of 42-46 °C [73], FOCF can be used to treat cancer by thermotherapy. Table 3.4: Induction heating parameters of curcumin loading samples Concentration (mg/ml) FOC FOCF Ts (1500 s) dT/dt Ts (1500 s) dT/dt 0.1 45.5 0.02 38.6 0.01 0.3 50.0 0.03 44.2 0.02 0.5 54.6 0.04 54.7 0.03 0.7 58.6 0.06 58.9 0.04 1 64.3 0.09 67.5 0.06 3.3.9. Cytotoxicity Curcumin Combination: Red - actine; blue – cell nucleus; green – curcumin Figure 3.21: Fluorescence of HT29 cells under normal conditions (control) and in 15 hour incubation with FOC Fluorescent images showed cellular uptake of curcumin into HT29 cell (green color) when incubated with FOC (Fig. 3.21). The cause of the green signal here is that curcumin is capable of spontaneous fluorescence when 10 stimulated with Argon lasers. There is no signal in control sample. This finding also demonstrates that incorporating curcumin into the nano-carrier does not affect the ability of the curcumin to enter the cell, the nanoparticle that ensures the release of curcumin into the cell. 3.3.10. Biodistribution Biodistribution of FOC and FOCF on different mouse organs are shown in figure 3.23. In Sarcoma 180 tumors, FOCF was present significantly higher than FOC after injection of 2.5 h. After 5 h, the folate attached system was still present higher than that of FOC without folate. From the heating curves, it is possible to reveal that the higher the Fe3O4 content, the higher the heat and saturated temperature, so as the Fe3O4 concentration increases with the folate-targeting element, the FOCF system can be more efficiently used to cure cancer. In addition, when a magnet was applied to the back of the mouse treated with FOCF, after 5 hours, the amount of magnetic nanoparticles appearing in the mouse kidney and spleen remained higher than in organs of the mouse treated with FOCF. This suggests that folate and magnetic fields may contribute to prolong the retention time of magnetic particles in the body, and may therefore be more likely to be transported to the tumor. Chapter 4: DOXORUBICIN LOADING ALGINATE COATED Fe3O4 NANOPARTICLES 4.1. Effect of alginate concentration on Dox loading capacity and system properties Unlike curcumin, Dox is a good soluble drug in water. Therefore, Dox is difficult to interact with the hydrophobic surface of Fe3O4. To encapsulate Dox on the multi-functional system, it is necessary to attach Dox to the particle surface by chemical bonding. Research has shown that the polymer shell is the decisive factor in the ability of Dox to carry Fe3O4 nanoparticles [143]. 4.1.1. IR and flourescence spectra The Fe-O bond of Fe3O4 in FA4, FA10, and FA4D samples is characterized by the peak in the 570 cm -1 region. Compared with pure Fe3O4 (575 cm -1 ), the wave number of Fe-O oscillations in alginate-coated samples decreased (566, 574 and 563 cm -1 , respectively, on the spectrum of FA4, 11 FA10, FAD). The coating process of alginate on the surface