Summary of materials science doctoral thesis: Study on fabrication and effectiveness evaluation of multifunctional nanosystem (polymer-drug-Fe3O4-folate) on cancer cells

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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.

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Nội dung Text: Summary of materials science doctoral thesis: Study on fabrication and effectiveness evaluation of multifunctional nanosystem (polymer-drug-Fe3O4-folate) on cancer cells

MINISTRY OF EDUCATION VIETNAM ACADEMY AND TRAINING 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
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. 1
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 Fe2+ and Fe3+ at 1:2 molar ratio with normal apparatus 2
[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: 3
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 53,9 56,2 56,7 63,0 69,0 64,6 59,6 60,6 60,7 62,7 64,5 (emu/g) Hc 2,5 14 4 2,5 0 20 0 18 2 2 21 4
(Oe) Mr 0,5 1,0 0,2 0,4 0 1,7 0 2 0,1 0,2 1,9 (emu/g) 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 5
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 Fe 3O4 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 Fe 3O4 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 6
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 100oC 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 430oC and can be assigned as the decomposition of curcumin. Therefore, the second weight loss step at around 299oC 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 7
(a)Fe3O4 80 (311) (b) Fe3O4/OCMCS/Cur (440) (a) 60 1.5 (c) Fe3O4/OCMCS/Cur/Fol (b) 1.0 (511) 40 (c) (200) (400) 0.5 (422) Ms (emu/g) (c) 20 0.0 -25 -20 -15 -10 -5 0 0 -20 (c) Fe3O4/OCMCS/Cur/folic (b) (b) Fe3O4/OCMCS/Cur -40 (a) Fe3O4 (a) -60 -80 -15000 -10000 -5000 0 5000 10000 15000 30 40 50 60 70 o 2theta ( ) H (Oe) Figure 3.15: XRD diagrams of (a) Figure 3.16: Hysterisis loops of (a) Fe3O4, Fe3O4, (b) FOC and (c) FOCF (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 Fe 3O4 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. 8
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 FOC FOCF (mg/ml) 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 9
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 Fe 3O4 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, 10
FA10, FAD). The coating process of alginate on the surface of Fe 3O4 has been achieved. In addition, the shift in the wave numbers of characteristic peak for organic bonds proves that doxorubicin has been encapsulated into the nanoparticles. Figure 4.2 is the FA4D fluorescence spectrum versus that of free Dox. Chemical bonding between Dox and the nanoparticles may be confirmed by fluorescence peak position of FA4D (17 nm shift compared to free Dox) [131]. In addition, the fluorescence intensity of FA4D decreased sharply because of the fluorescence suppression effect of Fe3O4 present in the sample [180]. 4.1.2. Loading content (LC) and Encapsulation efficiency (EE) The drug encapsulating efficiency (EE) of the samples is shown in Table 4.1. The data show that alginate concentration is an important factor influencing Dox loading performance. The higher the alginate concentration, the greater the Dox EE. This phenomenon can be explained by the formation of chemical bonds between Dox and alginate on the surface of Fe 3O4 nanoparticles. However, due to increased alginate content from FA2D to FA10D, the total mass increase so the drug loading content (LC) does not increase continuously. The maximum drug loading content was reach at FA4D, so we chose this sample for further bioassay. Table 4.1: EE and LC values Sample FA2D FA4D FA6D FA8D FA10D EE (%) 61,2±0,5 78,5±0,3 85,0±0,9 87,2±0,8 90,8±0,7 LC (%) 17,89±0,15 18,96±0,07 17,49±0,19 15,62±0,14 14,41±0,11 4.1.3. Size distribution and TEM images The size distribution of the nanosystems determined by the DLS spectrum depends a great deal on the alginate concentration. Prior to carrying Dox, Fe3O4 particles coated with FA4 and FA8 alginate had the hydrodynamic sizes of 18 nm and 91 nm. High concentration of alginate in FA8 forms a thicker coating layer and expands the hydrodynamic size of the FA8 particles due to the hydrophilicity of alginate. Loading Dox in FA4D and FA8D significantly increased the particle size (255 and 480 nm respectively), while the size distribution was also wider than FA4 and FA8. Corresponding to the amount of Dox, FA8D contains more Dox than FA4D 11
so the increase in its particel size is also greater than FA4D. FA4D is more appropriate than FA8D for biomedical applications. The TEM image shows that the particle size varies from 6 to 13 nm with the average size about 9.3 nm corresponding to the size calculated from the X-ray diffraction method (Figure 4.5) according to the Scherrer equation (D = K / (cos) 8 nm) [161]. 4.1.4. XRD diagrams and magnetic properties FA2 (311) 60 Ms (emu g ) Fe3O4 FA4 -1 FA6 FA4D 40 FA8 (220) (511) FA10 (400) FA4D 20 Lin (cps) (440) FA8D (422) H (Oe) 0 -10000 -5000 0 5000 10000 Ms (emu g ) -1 -20 4 3 -40 2 1 30 40 50 60 70 -60 o 0 2theta ( ) H (Oe) -40 -20 0 Figure 4.5: XRD diagram of Fe3O4 and Figure 4.6: Tính chất từ của các hệ hạt FA4D Characteristic peaks of Fe3O4 crystals in FA4D samples are fully present. These peaks have relatively low intensity compared to free Fe 3O4 due to the presence of organic components (alginate and dox) in the system. Table 4.2: Magnetic parameters of alginate-coated samples Sample FA2 FA4 FA6 FA8 FA10 FA4D FA8D Fe3O4 Fe3O4 [176] [177] Ms 61.2 69.5 65.3 65.8 63.9 51.6 33.9 43 25 (emu/g) Hc (Oe) 18 14 12 13 17 50 52 45 108 Mr 1.5 1.4 1.0 1.1 1.6 4.1 3.2 2 6 (emu/g) In figure 4.6, both magnetic remanance and coercivity of the samples are approximately zero, demonstrating that the nanoparticle systems are superparamagnetic. In addition, the Hc and Mr values of FA4D and FA8D were significantly higher than that of non-Dox samples. This may have been due to Dox presence that altered the magnetic anisotropy [186]. Saturation magnetization decrease as alginate concentration increase (from FA4 to 12
FA10). The saturation magnetization of FA4 are the highest in this sample range. The cause of this phenomenon is due to the alginate content, a non- magnetic substance, increasing in samples. For FA2, the lowest saturation in the range observed in this sample can be explained by the fact that at low alginate concentrations this polymer does not fully cover the magnetic particle surface, so that they can partially oxidize by the air during the sample drying and becomes Fe2O3 which is lower in magnetization [164]. The saturation magnetization of the 2 Dox loading samples, FA4D and FA8D, significantly decreased compared with the non-drug samples (51.6 and 33.9 emu/g respectively), indicating that Dox was present in the samples with significant amount. The deeper reduction of the FA8D vs. FA4D is a result of the greater Dox loading in this system. However, the values of the saturation magnetization of FA4D and FA8D are large enough to be easily and quickly separated from the reaction media by external magnetic fields. 4.1.5. Magnetic inductive heating effect The magnetic inductive hyperthermia results of samples with different concentrations of Fe3O4 particles (range from 0.5 to 3.0 mg ml-1 in term of Fe3O4) of FA4 and FA4D and the same concentration (3.0 mg ml -1 in term of Fe3O4) of FA8 and uncoated Fe3O4 in deionized water measured in the same field conditions, namely of a frequency of 178 kHz and amplitude of 80 Oe are shown on Table 4.3. The magnetic induction heating characteristics observed for the material (figure 4.7 (a) and (b)) are concentration dependences of saturation temperature Ts (estimated at heating time of t = 1500 s). It is found that upon decreasing of NPs concentration by adding more and more water, both Ts and dT/dt of the sample decrease. All the samples can reach the temperature up to 42°C and even higher for 20 min. The temperature retention could prolong when the heating conditions were held. Because cancer cells may be killed in the temperature range of 42– 46oC [73], we therefore note that the systems, both DOX loading nanoparticles FA4D and FA4 is able to act as a good thermoseed for cancer hyperthermia application. The heating characteristics of FA4 and FA4D shows little change while the saturation magnetizations of the 2 samples are so different (as shown in figure 4.6). This can be explained by the interaction between the particles in the aqueous medium (as magnetic induction heated) is altered to become solid form (for magnetization measurements). Thus, 13
FA4D can become a potential combination of chemotherapeutic and hyperthermia cancer treatment. Table 4.3: Magnetic induction heating of FA4, FA4D, FA8 and Fe3O4 samples Conc. T1500s SAR ILP Sample dT/dt (mg/ml) (oC) (W/g) (nHm2.kg-1) 0.5 49.7 0.03 225.7 1 52.1 0.03 129.6 FA4 2 62.1 0.04 85.7 3 68.4 0.06 85.0 11.6 0.5 48.1 0.02 150.5 1 50.5 0.03 112.9 FA4D 2 59.2 0.04 73.2 3 65.1 0.05 66.9 9.2 FA8 3 62.1 0.05 64.1 8.8 Fe3O4 3 60.1 0.07 103.1 14.1 4.1.6. Thermal analysis Figure 4.9 shows the thermal analysis results of FA4 and FA4D. It can be seen from the figure that both samples lose mass in the temperature range of 80 to 550oC. Around 100oC, there is the same small mass loss (about 2%) of water present in the 2 samples while the next steps of mass loss are extremely different. FA4 shows two exothermal peaks but loses only 12% its mass during the heating process. This can be explained by the decomposition of alginate in the sample. On the other hand, FA4D shows only one peak in Heat flow diagram at 380oC corresponding to a mass loss of 24%, that is double to those of FA4. This mass loss step of FA4D also can be matched with the disappearance of organic components in the sample and can support for the formation of a complex between DOX and Alginate in the sample. Figure 4.10 shows the general structure of Fe3O4 nanoparticles coated with alginate with doxorubicin loading (FA4D or FAD) and folate attached (FADF). In these systems, Dox is binded to the alginate shell by chemical interaction 14
(a) 20 o 380 C TG% FA4 20 0 0 Heatflow FA4 16 -5 -5 16 HeatFlow (/µV) o 479 C 581 C o HeatFlow (/µV) 12 -10 12 TG (/%) -10 TG (/%) 8 -15 -15 8 TG% FA4D -20 4 Heatflow FA4D -20 4 -25 0 100 200 300 400 500 600 700 800 -25 0 100 200 300 400 500 600 700 800 Furnace temperature (/°C) (b) Furnace temperature (/°C) Figure 4.9: Thermal Analysis Diagrams of FA4 (a) and FA4D (b) . Figure 4.1: Structural models of FAD and FADF 4.1.7. In vitro drug release The in vitro release process of DOX from the FA4D in neutral (pH 7.4) and acidic (pH 5) medium (shown in figure 4.11) are both gradual release. In the first 12 hours, the rate of drug release is maximal and reaches 21% and 29.5% at 12h, at pH 7.4 and pH 5 respectively. The drug release from nanoparticles was slower at pH 7.4 than at pH 5.0. After 120 hours, approximately 61% of the total drug was released in pH 5.0 conditions, in comparison with a 42% release rate in pH 7.4 conditions. The DOX release from the FA4D nanoparticles may be achieved by the degradation of alginate layer through hydrolysis process. The hydrolysis process increases in acidic solutions leading to a higher percentage of release at pH 5 compared to that at neutral conditions. Because the environment in cancer tumors is acidic, this indicates that the nano drug system is suitable for tumor treatment. 4.1.8. Cytotoxicity Because DOX is highly toxic [140], the released amount of DOX is enough to treat cancer cells, as indicated by low IC50 values of FA4D on the 15
cell lines (figure 4.12(b)). All the IC50 values are smaller than 5 g ml-1, and much less than those of DOX loaded PLA-TPGS nanoparticles that we reported before. Difference in cytotoxicity of FA4D and DOX loaded PLA- TPGS can be resulted from the synergic effect of Fe3O4 nanoparticles and anticancer activity of DOX on the cell accumulation. Recently, sodium alginate–polyvinyl alcohol–bovine serum albumin coated Fe3O4 nanoparticles were synthesized and used as DOX delivery system. However, this complicate system exhibits toxicity on Hep-G2 cell lines only at high range of DOX concentration (200-1000 g ml-1). This range is much larger than DOX concentrations used in this study indicating that our optimized drug delivery system show better anticancer activity in this cell line. In addition, cytotoxicity of FA4D and free DOX on different cell lines were compared. The IC50 values of FA4D are higher than those of free DOX can be a result of the slow release process of DOX from the nanoparticles [147]. It was also reported that the impact of nanoparticles loaded with doxorubicin on cell survival depended on just a certain extent of DOX concentration and the main factor that affects the toxicity is the time. In another report, the IC 50 on some cancer cells of DOX loaded chitosan coated Fe3O4 also decrease with time. The lower IC50 values of the Hep-G2, LU-1, RD and FL cell lines compared to that of normal cells (Vero cell line) indicate that the nanoparticles express higher toxic effect on cancer cells than healthy cells. Therefore, the drug delivery systems suggest a safer chemotherapy for cancer treatment in the way of decrease the toxicity for normal cells. (a) (b) 1.5 1.41 100 1.30 FA4D 1.2 1.20 Hep-G2 DOX 80 LU-1 0.96 Cell survival (%) IC50 (g/ml) RD 0.9 60 0.72 FL Vero 0.6 0.60 40 0.39 0.3 20 0.21 0.16 0.11 0 0.0 0.2 01 05 25 Hep-G2 LU-1 RD FL Vero Concentration (g/ml) Cell line Figure 4.12: Dose-response curve and comparisons of FA4D and free Dox IC50 4.2. Effect of microwave-assisted synthesized Fe3O4 core on system properties 16
Microwave technology is used to synthesize Fe3O4 nanoparticles with many advantages, most notably that this technique allows shortening of reaction time and in large scale. In this section, we investigated the microwave-assisted synthesis of Fe3O4 nanocore at different conditions with microwave technique and compared the cell killing efficiency of multifunctional systems with the microwave-assisted synthesized Fe3O4 core with normal coprecipitated Fe3O4 particles. To assess induction magnetic heating effect and interaction of the nanosystems with biological systems, we fabricated FA and FAD samples with components and methods similar to those of FA4 and FA4D from the Fe3O4 M5 particles fabricated by microwave technique. 4.2.1. Material characteristics and magnetic inductive heating efffect Both FA and FAD samples are highly stable, exhibiting a large zeta potential value. The heating curve exhibits a similar trend compared to the sample in the conventional co-precipitation conditions. Comparison of saturation temperature (determined at 1500 s) of FA and FAD (table 4.6) with corresponding results of FA4 and FA4D (Table 4.3) found no significant difference in heating effect of microwave assisted synthesized sample compared to conventional synthetic conditions. 4.2.2. Cytotoxicity Comparison results of the IC50 values of microwave assisted synthesized systems and conventional co-precipitate systems are presented in Table 4.7 . Table 4.7: IC50 of the microwave samples and conventional samples Cell line Hep- LU-1 RD FL Vero HeLa G2 Dox1 0,21 0,39 0,11 0,16 1,30 - 2 Dox 0,18 0,35 - - 1,34 0,25 FA4D 0,72 0,96 0,60 1,20 1,41 - FAD 0,67 1,02 - - 1,43 0,81 FADF 0,44 0,87 - - 1,39 0,68 1 A control sample was used to determine the IC50 of FA4D 2 A control sample was used to determine the IC50 of FAD and FADF The results in Table 4.7 show that the FAD affecting pattern on Hep-G2, LU-1 and Vero cell lines was not significantly different from that of FA4D. 17
This shows that the use of microwave technique to fabricate Fe3O4 meets the material requirement as well as interaction with the biological system. The preservation of this material or biological interaction of FAD versus FA4D may be due to the intrinsic nature of the microwave technique used is still coprecipitation. The advantage of this technique is simple operation, and short reaction time. Especially, this technique allows the synthesis of Fe 3O4 nanoparticles in large scale. Therefore, in subsequent studies to synthesize Dox loading folate or quantum dot attaching nanosystem and in vivo test specimens, we used Fe3O4 particles prepared by microwave technique. 4.3. Folate attached (FADF) or CdTe loaded (FADQ) NPs 4.3.1. IR spectra Infrared spectra demonstrate the existence of folic acid in the FADF system. 4.3.2. Flourescence spectra The fluorescence spectrum of FADF versus folic acid has a clear shift in the peak of emission (from 420 nm to 428 nm). The peak at 428 nm is far shifted from the peak of Dox, suggesting that in the two fluorescents, folic acid predominates in FADF samples. This result again confirms the presence of folic acid in the system. On the other hand, while the fluorescence spectrum of the FAD sample is much lower than that of Dox, the fluorescence intensity of FADF does not change much compared to either folic acid or pure dox. In the case of FADF, the fluorescence intensity of this sample was slightly lower than that of folic acid, allowing FADF to be used as a fluorescence probe to observe the interaction of the nanosystems with biological systems. 70000 50000 FAQ 0.05 mg Fe3O4/ml 580 nm 60000 folic 50000 FAQ 0.1 mg Fe3O4/ml FAQ 0.2 mg Fe3O4/ml 420 FADF S1 (Counts) 40000 FAQ 0.4 mg Fe3O4/ml 40000 428 FAQ 0.8 mg Fe3O4/ml FAD 30000 DOX 20000 30000 10000 cuong do 0 450 500 550 600 650 700 20000 20000 FAQD 0.05 mg Fe3O4/ml FAQD 0.1 mg Fe3O4/ml 612 nm FAQD 0.2 mg Fe3O4/ml FAQD 0.4 mg Fe3O4/ml 10000 10000 FAQD 0.8 mg Fe3O4/ml 0 400 450 500 550 600 650 700 0 450 500 550 600 650 700 a) buoc song (nm) b) Wavelength (nm) Figure 4.17: Fluorescence spectra of FAD, FADF compared to folic acid and dox (a) and samples containing CdTe quantum dots (b) 18