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 INTRODUCTION 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-drugFe3O4-folate) on cancer cells" was done 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 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 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 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 [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: 7-10 mm tumor – bearing mice were divided into groups, each group of 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 30minute time Two consecutive cycles separated by 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 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  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 (Oe) Mr 0,5 1,0 0,2 0,4 (emu/g) 0 1,7 0 2 0,1 0,2 1,9 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 (FOC1FOC5) 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 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 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 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 80 (a)Fe3O4 (440) (b) Fe3O4/OCMCS/Cur (c) Fe3O4/OCMCS/Cur/Fol (511) (400) (422) (200) (a) 60 40 Ms (emu/g) (311) (c) (b) 20 1.5 (b) 1.0 (c) 0.5 0.0 -25 -20 -15 -10 -5 0 -20 (c) Fe3O4/OCMCS/Cur/folic (b) Fe3O4/OCMCS/Cur -40 (a) (a) Fe3O4 -60 30 40 50 o 2theta ( ) 60 70 Figure 3.15: XRD diagrams of (a) Fe3O4, (b) FOC and (c) FOCF -80 -15000 -10000 -5000 5000 10000 15000 H (Oe) 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 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 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 EE (%) FA2D 61,2±0,5 FA4D 78,5±0,3 FA6D 85,0±0,9 FA8D 87,2±0,8 FA10D 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 Lin (cps) (400) (440) (422) -10000 -5000 40 20 H (Oe) 5000 10000 -1 (511) 60 Ms (emu g ) (220) FA2 FA4 FA6 FA8 FA10 FA4D FA8D Fe3O4 FA4D -1 (311) Ms (emu g ) 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 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 -20 -40 30 40 50 o 2theta ( ) 60 70 -60 H (Oe) Figure 4.5: XRD diagram of Fe3O4 and FA4D -40 -20 Figure 4.6: Tính chất từ hệ hạt 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 Ms 61.2 (emu/g) Hc (Oe) 18 Mr 1.5 (emu/g) FA4 FA6 FA8 FA10 FA4D FA8D 69.5 65.3 65.8 63.9 51.6 33.9 Fe3O4 Fe3O4 [176] [177] 43 25 14 1.4 50 4.1 52 3.2 45 2 12 1.0 13 1.1 17 1.6 108 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 nonmagnetic 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 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 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 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 Sample FA4 FA4D FA8 Fe3O4 Conc (mg/ml) 0.5 0.5 3 T1500s (oC) 49.7 52.1 62.1 68.4 48.1 50.5 59.2 65.1 62.1 60.1 dT/dt 0.03 0.03 0.04 0.06 0.02 0.03 0.04 0.05 0.05 0.07 SAR (W/g) 225.7 129.6 85.7 85.0 150.5 112.9 73.2 66.9 64.1 103.1 ILP (nHm2.kg-1) 11.6 9.2 8.8 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 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) TG (/%) 479 C 581 C -10 o 16 12 -15 -20 -25 100 200 300 400 500 600 700 800 Furnace temperature (/°C) 380 C -5 16 -10 12 -15 TG% FA4D Heatflow FA4D -20 -25 (b) 20 HeatFlow (/µV) o o HeatFlow (/µV) -5 20 TG (/%) TG% FA4 Heatflow FA4 0 100 200 300 400 500 600 700 800 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 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 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 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 PLATPGS 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 Hep-G2 LU-1 RD FL Vero 80 60 40 0.72 01 05 Concentration (g/ml) 25 0.60 0.6 0.39 0.0 0.2 1.20 1.41 1.30 0.96 0.9 0.3 20 FA4D DOX 1.2 IC50 (g/ml) Cell survival (%) 100 0.21 Hep-G2 0.11 LU-1 RD Cell line 0.16 FL Vero 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 HepG2 Dox1 0,21 Dox 0,18 FA4D 0,72 FAD 0,67 FADF 0,44 LU-1 RD FL Vero HeLa 0,39 0,35 0,96 1,02 0,87 0,11 0,60 - 0,16 1,20 - 1,30 1,34 1,41 1,43 1,39 0,25 0,81 0,68 A control sample was used to determine the IC50 of FA4D 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 50000 cuong 420 428 30000 70000 60000 50000 40000 30000 20000 10000 450 20000 10000 10000 450 500 550 600 buoc song (nm) 650 700 b) 580 nm FAQ 0.05 mg Fe3O4/ml FAQ 0.1 mg Fe3O4/ml FAQ 0.2 mg Fe3O4/ml FAQ 0.4 mg Fe3O4/ml FAQ 0.8 mg Fe3O4/ml 20000 400 a) S1 (Counts) 40000 folic FADF FAD DOX 450 500 550 FAQD 0.05 mg Fe3O4/ml FAQD 0.1 mg Fe3O4/ml FAQD 0.2 mg Fe3O4/ml FAQD 0.4 mg Fe3O4/ml FAQD 0.8 mg Fe3O4/ml 500 550 600 650 700 612 nm 600 Wavelength (nm) 650 700 Figure 4.17: Fluorescence spectra of FAD, FADF compared to folic acid and dox (a) and samples containing CdTe quantum dots (b) 18 The FAQ and FADQ samples that contain CdTe have a fluorescent emission at 580 nm of CdTe In addition, FADQ samples consisting Dox exhibit fluorescence at 612 nm, similar to FAD 4.3.6 Passive and active Dox release by magnetic inductive heating effect The passive Dox release profile at 37 ° C from FADF was performed and gave the similar results as the FAD Changing the pH of the solution almost does not affect the FADF heating ability in the magnetic field At just the same temperature as the body temperature (about 37 oCm), Dox is released from 4.7 to 11.2% from FAD or FADF In magnetic field of 80 Oe, nanoparticles generate more heat (or reach higher temperature) than in magnetic field of 70 Oe The release of Dox from the nanoparicles also occurs faster, and the amount of Dox released larger Unlike conventional drug release, magnetic inductive heating effect makes the particles heated from inside of the particle and hence accelerates the release of the drug Thus, the magnetic field can be adjusted to control the speed and amount of drug release Some other studies have shown that it is possible to release the drug in an active way Table 4.9: Dox release profile when heated with different magnetic fields Time (s) 750 1500 2250 3000 750 1500 2250 3000 FAD pH FAD pH7.4 t % Dox t % Dox o o ( C) release ± ( C) release ± SD SD 30 30 70 Oe 39,27 11.2±1.3 40.23 6.0±0.7 43.08 28.8±0.8 44.52 25.3±1.3 45.32 49.5±0.9 46.05 37.9±1.8 45.87 56.3±1.5 46.81 41.5±1.2 80 Oe 46.13 30.1±1.5 45.56 21.5±0.5 51.25 53.5±0.6 50.78 38.8±2.1 51.98 67.6±0.9 51.43 49.4±1.7 52.16 78.1±1.6 51.83 56.4±1.2 FADF pH FADF pH 7.4 t % Dox t % Dox o o ( C) release ± ( C) release ± SD SD 30 30 37.63 42.35 43.68 44.30 10.9±0.4 29.1±0.6 46.7±1.7 51.6±1.0 38.11 43.48 44.99 45.71 4.7±1.0 24.4±0.7 37.2±1.3 39.4±2.0 45.87 50.19 51.16 51.24 28.2±2.0 48.0±1.4 62.7±0.9 74.2±1.8 46.22 51.74 52.10 52.31 20.6±0.9 35.4±0.9 45.1±1.7 51.0±1.5 As a result, both FADF and FADQ have suitable material properties for use in biological objects 4.3.7 Cytotoxicity 19 4.3.7.1 Cytotoxicity of FADF Compared to pure Dox, FADF is toxic on four Hep-G2, LU-1, Vero, and HeLa-lower cell lines (larger IC50) The reason can be that Dox was chemically binded to the surface of the nanoparticles, which slows down the action of the Dox on the cell Compared to FAD, FADF exhibited higher toxicity due to the fact that in this sample, the folate factor helped the sample to better target the cell However, both FAD and FADF with low IC 50 (less than μg/ml) showed good chemotherapy for the studied cell lines 1.4 CdTe FAQ FAQD 4.0 3.5 1.0 0.8 0.6 0.4 0.2 0.0 IC50>5 ug/ml 4.5 IC50 (g/ml) IC50 (gml) 1.2 DOX FAD FADF 3.0 2.5 2.0 1.5 1.0 Hep-G2 LU-1 Vero Dong te bao HeLa Figure 4.24: Cytotoxicity of Dox loading systems 0.5 0.0 Hep-G2 LU-1 RD Vero Figure 4.26: Cytotoxicity of CdTe loading systems 4.3.7.2 Cytotoxicity of CdTe loaded NPs For each cell line, the IC50 value of the FAQ is slightly higher than CdTe, meaning that when CdTe is attached to the Fe3O4 nanoparticles by the alginate polymer matrix, the CdTe toxicity decreases (Figure 4.26) The sample consisting of CdTe and Dox concurrently (FADQ) was the most effective in cancer treatment (IC50 values for Hep-G2, LU-1, RD and Vero were 1.34, 3.83, 1.35 and 2.13 μg/ml, respectively) thanks to the combination of cancer drug Dox and CdTe quantum dots As such, FAD, FADF, and FADQ can perform a variety of functions such as hyperthermia, fluorescence or chemotherapy FADQ demonstrates good cell killing ability and at the same time shows high toxicity to experimental animals (white mice) bearing tumors (dead or very weak rats after injection of 50 l/mouse) Therefore, only FA, FAD and FADF were further in vivo investigated on mice 4.3.8 Stability of FAD, FADF and FADQ in biophysical environment 20 FAD, FADF and FADQ were determined the stability in a solution containing 0.2 M salts and different pH values by Zeta potential measurement The results are shown in Table 4.12 Table 4.12: The Zeta potential (mV) of FAD, FADF and FADQ in solution of NaCl 0.2 M and different pH values pH FAD FADF FADQ -19.2 ± 1.9 -18.5 ± 2.1 -9.4 ± 2.1 -32.0 ± 2.6 -30.4 ± 1.7 - 13.8 ± 2.3 -33.2 ± 1.5 -32.2 ± 2.9 -15.6 ± 1.8 -43.1 ±1.2 -39.0 ± 2.5 -19.4± 1.4 The results in Table 4.12 show that Zeta potential of Dox loading alginate coated Fe3O4 samples were negative, even at pH This can be explained by the fact that the alginate shell of the magnetic particles is rich in COO- groups Similar results concerning alginate coated nanoparticles were also previously published [194] The Zeta potential of FADF is not significantly different from that of FAD In the pH range of to 9, FAD and FADF have good stability (with Zeta potential over 30 mV) The FADQ is less stable than FAD and FADF 4.3.9 In vivo evaluation 4.3.9.2 Distribution of Fe on mice’ organs Quantification of Fe in mice’ tissues and organs revealed that the levels of Fe in the tissues and in particular in the tumors (except in the blood) of the FADF mice were higher than those of the control group However, statistically significant differences were not observed Comparison of Fe content from the FAD group compared with the control group tended to be similar (p = 0.08) 4.3.9.3 In vivo treatment results by nanosystems associated with magnetic induction heating During treatment, the weight of the mice groups was not significantly different at each measurement point The mean tumor size at the time of group deviding was not significantly different between groups After treatment cycles, the tumor size of FADF group has been shown to be undeveloped The size of the tumor tends to go flatly This group had significantly smaller tumors than the other groups after cycles, corresponding to the fifth measurement point 21 (p