Dual roles of Curcumin Encapsulated Alginate/­Fe3O4 Nanoparticles in MRI Enhancement and Hyperthermia for Cancer Treatment

doi:10.21203/rs.3.rs-1843318/v1

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Dual roles of Curcumin Encapsulated Alginate/Fe3O4 Nanoparticles in MRI Enhancement and Hyperthermia for Cancer Treatment

https://doi.org/10.21203/rs.3.rs-1843318/v1

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Background

Magnetite (Fe₃O₄) nanoparticles (MNPs) had a high potential in biomedical application, including hyperthermia and magnetic resonance imaging (MRI). The combination of MNPs and curcumin has been reported to have synergetic effect on cancer cell death. In this study, we aimed to identify the bioactivities of the nanoconjugates composed of superparamagnetic Fe₃O₄ nanoparticles coated by alginate and curcumin (Fe₃O₄/Cur@ALG) on cancer cells in both in vitro and in vivo models.

Results

The results showed that Fe₃O₄/Cur@ALG NPs enhanced the contrast of MRI in cell culture and Swiss mice bearing tumor. The autofluorescence signal of curcumin revealed the penetration of the nanoparticles into Sarcoma cells. Besides, this nanoconjugate synergistically inhibited the growth of Sarcoma 180 tumor through the magnetic heating and the anti-cancer effect of curcumin. These MNPs had had high biocompatibility with a low toxicity on mice at the concentration of Fe₃O₄ up to 120 mg/kg. Histological analysis indicated that the MNPs presented in the liver tissue, but not in the kidney, with the increase of AST value. Interestingly, Fe₃O₄/Cur@ALG NPs were cleared from blood after more than 10 minutes.

Conclusion

Our study revealed that Fe₃O₄/Cur@ALG had a high potential in medicinal application and should be further developed to use in cancer diagnosis and treatment.

magnetic nanoparticles

MRI

hyperthermia

cytotoxicity

curcumin

Possessing unique chemical and physical properties, nanoparticles (NPs) play an important role in various research fields, including electronics, energy, and biomedicine. Among the numerous kinds of NPs, magnetic NPs show great promise due to their unique properties in a magnetic field and no depth-penetration limit into the human body (Zhang et al. 2018; Sangaiya and Jayaprakash 2018). Magnetic NPs have many potential biomedical applications, which include magnetic drug targeting, hyperthermia, cell separation, and magnetic resonance imaging (MRI) (Mukherjee, Liang, and Veiseh 2020; Hilger and Kaiser 2012; Faustino-Rocha et al. 2013). These NPs have been showed effective in many cancer diagnoses and treatment research (Faustino-Rocha et al. 2013; Avasthi et al. 2021; Fernandes et al. 2021; Beola et al. 2020), or antibacterial activity with low mammalian toxicity (Taimoory et al. 2018).

Among the magnetic iron oxide nanoparticle family, magnetite (Fe₃O₄) (MNPs) is one of the most popular NPs because of its high chemical stability and better magnetic strength compared to other popular iron oxide nanoparticles, such as maghemite (γ-Fe₂O₃), and hematite (α-Fe₂O₃) (Devkota et al. 2014). Besides, MNPs has superparamagnetic properties at the nanoscale level. This make MNPs are by far the most iron oxide nanoparticles employed in biomedical applications (Devkota et al. 2014). Several methods of synthesizing MNPs have been developed, such as co-precipitation (Ghazanfari et al. 2016), solvothermal method (Ganapathe et al. 2020), wet-chemical reduction (Massart 1981), micro-/nano-emulsion (Faraji, Yamini, and Rezaei 2010), sonochemical or sonolysis method (Sukumaran et al. 2018), and latterly green method (Sánchez-Domínguez, Aubery, and Solans 2012). Other methods have been developed recently to control the production of gadolinium doped iron oxides nanocrystalline powders by the extraction-pyrolytic method (Blanco-Andujar et al. 2012) or increase the biocompatibility (Yew et al. 2020). Each method has its own advantages and disadvantages, however, all of them targets to synthesize nanoparticles with suitable characteristics involving in magnetic properties, dispersity, size, size distribution and shape controlling for applying in biomedicine. Particularly, the size and size distribution of MNPs have major roles in heating efficacy and MRI contrast (Avasthi et al. 2021; Narayanaswamy et al. 2021).

In addition, in terms of using MNPs for clinical applications, biocompatibility and toxicity are two major areas of concern. When entering inside the cell, MNPs can affect the nuclear activities or cause leakage or blockage of cell membrane which can lead to decrease cell proliferation, viability, and metabolic activity results (Song et al. 2018). Surface functionalization of magnetic NPs can be used to improve dispersion and biochemical characteristics of MNPs, including biocompatibility themselves (Shultz et al. 2007; Feng et al. 2018; Aksimentyeva et al. 2014). The most two common natural polymers with advantage properties having been used to coat Fe₃O₄ NPs for functionalization are alginate and chitosan (Markides, Rotherham, and Haj 2012). Besides, combinations of MNPs with anti-cancer drugs such as paclitaxel, doxorubicin, docetaxel, and curcumin have been considered as a promising strategy in development of MNPs as a multifunctional nanosystem in biomedical application (Aksimentyeva et al. 2014; Tian et al. 2017; Thambiraj, Vijayalakshmi, and Ravi Shankaran 2021; Liang et al. 2016; Beyene et al. 2021). Among these compounds, curcumin has attracted the most attention of scientists based on its anti-cancer activities and drug labelling capacity. Several publications have reported about the combination of MNPs and curcumin in inducing cancer cell death, cellular uptake of the compound, and bio-distribution monitoring of the NPs (Hilger and Kaiser 2012; Beyene et al. 2021; Justin et al. 2018). However, most of studies were performed in in vitro and very little in vivo experiments. It is known that little correlation has been made between in vitro and in vivo studies due to the inability of in vitro studies to mimic the complex environment in the body. Hence, many in vitro results have not been able to translate into in vivo models (Patil et al. 2015; C et al. 2015).

In this study, we aimed to identify the bioactivities of the nanoconjugates composed of superparamagnetic Fe₃O₄ NPs coated by alginate and curcumin (Fe₃O₄/Cur@ALG) on cancer cells in both in vitro and in vivo models. The application of Fe₃O₄/Cur@ALG in the MRI contrast enhancement and hyperthermia treatment were demonstrated. The acute and sub-chronic toxicity and bio-distribution of the NPs in animals were also determined.

Structural and morphological analysis

In FTIR spectra of the Fe₃O₄@ALG and Fe₃O₄/Cur@ALG systems, all typical oscillations of functional groups in Fe₃O₄ nanoparticles, curcumin and polymer alginate appear with typical oscillations (Fig. 1A). In details, the carbonyl band (-C = O ester stretch) at 1630 cm-1, -OH stretching vibration at 3450 cm − 1, and the antisymmetric C-O-C stretch at 1042 cm-1 of polymer alginate were shifted to 1644, 3440 and 1092 cm − 1 in the FTIR spectrum of Fe3O4/Cur@ALG system, respectively. While the Fe-O vibration of iron oxide nanoparticles was shifted to 573 cm-1 confirmed the good bond between polymer alginate and the Fe₃O₄ core layer. Besides, these characteristic peaks were also shifted in the spectrum of Fe₃O₄/Cur@ALG where the corresponding wavenumber was 1637 cm-1 (C = O), 3442 cm-1 (-OH), 1021 cm-1 (C-O-C), and 594 cm-1 (Fe-O). Moreover, the peak characterizing for C = O bond, the O-H band, and C-O-C stretch of curcumin also can be seen in Fe₃O₄/Cur@ALG spectrum. These results revealed that the incorporation of curcumin compound on Fe₃O₄@ALG nanosystem was successfully implemented.

From the XRD diagram, the peaks are very clear, the main spectral clusters have high intensity, the background lines of the samples are quite flat, not mixed with strange phases, showing that the nanosystems produced are of good quality. The peaks at angular positions 2θ = 31^o, 36^o, 43^o, 53^o, 57^o, 63^o correspond to the crystal faces (220), (311), (400), (422), (440), (533) are the specific peaks for iron oxide nanoparticle nanocrystals (Fig. 1B).

The results also suggest that after coated with alginate and loaded curcumin, the crystal structure of iron oxide nanoparticles does not change, shown through the match of characteristic peaks at the characteristic crystal surfaces for Fe₃O₄ nanocrystals. Concurrently, from the X-ray diffraction scheme, we can calculate the average size of iron oxide nanoparticles is about 15 nm according to Scherrer's formula D = Kλ/(β cos θ), where K is a constant (K = 0.9 for Cu-K\({\alpha }\)), λ is the wavelength (0.15406 nm for Cu-K\({\alpha }\), and β is the full width at a half maxima of the (311) peak (Alexander and Klug 1950).

The morphology of the material was observed on a transmission electron microscope (TEM). Figure 1C and 1D showed the TEM image of the Fe₃O₄@ALG and Fe₃O₄/Cur@ALG systems. It was clear that the nanoparticles were round shaped and monodispersed with a diameter of about 12–15 nm, and the distribution of the particles is relatively uniform.

Furthermore, we evaluated the particle size in the liquid medium as well as the strength (zeta potential) of Fe₃O₄@ALG and Fe₃O₄/Cur@ALG. These liquid samples were measured using dynamic laser scattering spectroscopy (DLS) on a Zetasizer potentiometer. The distribution diagram of particle size and zeta potential is shown in Fig. 1E and 1F. The hydrodynamic dimensions of the Fe₃O₄@ALG and Fe₃O₄/Cur@ALG nanosystems are 92 and 98 nm, respectively. These sizes were obviously much larger than their particle sizes measured from the TEM image, but it could be explained by the polydispersity of the polymer alginate. Since the intensity of the scattered light depends on the square of the volume of the scattering substance according to the classical Rayleigh theory, resulting in larger particle sizes in the multiple dispersion state (Barhoum et al. 2018). In addition, the Zeta potentials of both Fe₃O₄@ALG and Fe₃O₄/Cur@ALG nanosystems were − 41.3 mV and − 38.5 mV, respectively. These Zeta potential values are due to the binding between the carboxyl groups of the alginate to the Fe₃O₄ core as well as the large value of the Zeta potential that creates an electrostatic repulsion between nanoparticles with the same charge. Such Zeta potentials proved that they have good stability and stability for possible applications in biomedical applications (Ma et al. 2007).

In vitro cytotoxicity, MR imaging, and magnetic heating of Fe₃O₄/Cur@ALG NPs on cancer cells

We could observe the penetration of Fe₃O₄/Cur@ALG NPs into Sarcoma cells based on the autofluorescence characteristic of curcumin after 48h of incubation. The higher concentration of NPs the more particles presented in the cell cytoplasm (Fig. 2A). It is worth noting that cells at high concentrations of Fe showed many blebs surrounding indicating the cell disruption (Fig. 2A).

Data analysis of cell viability assay showed that Fe₃O₄/Cur@ALG NPs had cytotoxicity on Sarcoma 180 cells at high doses. Nearly 49% of cell proliferation was inhibited at the dose of 1000 µg/ml of Fe, corresponded to 500 µg/ml of curcumin (Fig. 2B). The IC50 value of the nanoparticle was 897 ± 38 µg/ml for Fe, corresponded to 444.8 ± 42 µg/ml of curcumin. In the meantime, Fe₃O₄@ALG had less toxic to Sarcoma cells, with the cell growth at 1000 ug/ml of Fe was 65 ± 3.5% (Fig. 2A). The cytotoxic of Fe₃O₄/Cur@ALG could be caused by curcumin, an anti-cancer agent.

For the MRI enhancement assay, we performed in two conditions: Fe₃O₄/Cur@ALG NPs mixed with (1) 1% agarose and (2) cell-containing 1% agarose solutions. The condition 2 was set up to mimic the environment surrounded the nanoparticle after being injected into the body. From Fig. 2B, it could be seen the clear differentiations in the dark signal when comparing the treated wells to the control even from the lowest tested Fe concentration (10 µg/ml). The dark scale color progressively darkened with the increasing concentration of Fe₃O₄. In addition, dark signal in Sarcoma cell-containing agarose solution was stronger than in agarose only. Quantitative analysis of the MR images revealed that the MR signal intensity was statistically higher in the control compared to the nanoparticle-treated wells and the higher of Fe concentration the less signal intensity of MR images (p < 0.05) (Fig. 2C). These results indicated that Fe₃O₄/Cur@ALG NPs had ability to enhance the contrast of MR images.

Previous study of Devkota et al. showed that Fe₃O₄/Cur@ALG NPs could be an inductive heating agent when applying in an alternating magnetic field (Devkota et al. 2014). In this study we would like to check if this ability still presents in the mixture with the cells, and more important, if the hyperthermia caused by these NPs can kill cancer cells. Besides, we chose Fe₃O₄/Cur@ALG NPs but not Fe₃O₄@ALG NPs since the former showed more cytotoxicity to Sarcoma cell in vitro compared to later one. Temperature rise of the MNPs was examined with concentration range of MNPs from 100–2000 µg/ml. The magnetic field strength was fixed at 80 Oe and the frequency was 178 kHz. At the highest concentration, the temperature was 41.5^oC after 30 minutes of AMF (Fig. 4A). Along with this, the percentage of cell death reached 20%. Obviously, magnetic heating treatment significantly reduced the viability of Sarcoma 180 cells (Fig. 4B, C).

Figure 4. In vitro magneto-inductive heating of Fe₃O₄/Cur@ALG NPs induced the death of Sarcoma 180 cells. NPs were used at different concentrations ranged from 100 to 2000 µg/ml and applied with AMF irradiation for 30 minutes. (A) The temperature (^oC) values received when stimulating Fe₃O₄/Cur@ALG NPs with AMF. (B) Images of Sarcoma 180 cells stained with Blue Trypan after incubating with 2000 µg/ml of Fe₃O₄/Cur@ALG NPs and applying AMF. (C) Percentages of cell death (%) after 30 minutes of AMF irradiation. (D) Cell death (%) at different time-points after finishing 30- and 45-minutes AMF protocols at concentration of 2000 µg/ml Fe₃O₄. Data was presented as mean ± SD in triplicate repeated experiments, (*) p ˂ 0.05; (**) p < 0.01; (***) p < 0.001.

Interestingly, we observed that the cell death continued to significantly increase for following time after finishing the irradiation (p < 0.05). For the 30 minutes AMF protocol, the percentage of dead cells increased from 20.2% at time-point of 0 to 31.1% at the time-point of 60 minutes. For the 45 minutes AMF protocol, the cell death increased from 18.7–38.8%, respectively. This indicated that the increased time of field irradiation also induced further killing cell effect (Fig. 3D).

These results suggested that Fe₃O₄/Cur@ALG NPs have a potential to be used as inductive heating agent for the hyperthermia treatment of cancer cells. The results from in vitro assays encouraged us to evaluate the bioactivity of Fe₃O₄/Cur@ALG NPs in vivo.

In vivo MR imaging of tumor on mice model

We performed the MRI on mice bearing Sarcoma tumor at day 5 after the implantation of cancer cells, with the average size of tumors was 100 mm3. Fe₃O₄/Cur@ALG NPs were directly applied into the tumor with different concentrations of 50 µg/0.5 cm3 and 250 µg/0.5 cm3, then MR scanning was taken after 0, 10, and 30 minutes of the injection. It could be seen that the tumor regions were gradually darken and the dark areas were progressively enlarged to the entire of tumor post injection. Meanwhile it was hard to recognize the tumor site on un-treated mice. In addition, the higher concentration of Fe the darker signal received. Interestingly, even with the low concentration of Fe (50 µg/0.5 cm3), the contrast of MR images was still enhanced (Fig. 4A).

However, when NPs were applied by intravenous injection, we observed no difference in the image contrasts between the control and the treated mice (Fig. 4B). Nevertheless, our results indicated that Fe₃O₄/Cur@ALG NPs could induced the MRI contrast by directly applying into the tumor site.

In vivo magnetic heating of tumor on mice model

Hyperthermia treatment was performed on Swiss mice carrying Sarcoma 180 tumor. Fe₃O₄/Cur@ALG NPs were directly injected at the concentration of 250 µg/0.5 cm3 into the tumor at day 5 post-transplantation and applied in an alternative magnetic field of 178 kHz and 80 Oe, repeated 4 times with 48 h of interval. At this day, the average tumor size was 100 ± 22.3 mm3. We observed the significant differences in the size of tumor with the smallest in the mice treated with NPs and AMF, followed by NPs only, and then the control (Fig. 6A). There was a strong inhibition of tumor growth in mice treated with Fe₃O₄/Cur@ALG and AMF, especially from day 0 – day 8 post injection (POI) (Fig. 6B). At the end of experiment (day 20 POI), the size of tumor in NPs and AMF-treated mice increased about 400% compared to day 0 POI, meanwhile this value in the control was 1600% (Fig. 6B). Interestingly, in NPs-treated mice, the volume of tumor was also smaller than the untreated control, with the average size increased 1100% at day 26 POI (Fig. 6B).

Acute and sub-chronic toxicity of Fe₃O₄/Cur@ALG NPs

The acute toxicity assay of Fe₃O₄/Cur@ALG NPs was performed with 5 high doses ranged from 80–120 mg/kg. After 72 hours of intravenous injection, none of mice in 5 treated groups was died. There were no differences between the control and the injected one in term of their performance, behavior, and activity (Fig. 7A). This result means that Fe₃O₄/Cur@ALG NPs had no acute toxicity on mice. Since the LD50 value could not be defined, we chose the two doses 24 mg/kg and 17 mg/kg equal to 1/5 and 1/7 of 120 mg/kg, respectively, the highest dose used in the acute toxicity test, for the use in the sub-chronic toxicity test. In two treated groups, each mouse was injected 24 mg/kg or 17 mg/kg per day, in continuously 30 days. The body weight was measured every 5 days, the functional activities of liver and kidney was evaluated at the end of experiments. The results showed that, there was a light decline of 2–10% of the body weight in the treated mice compared to that of the control, however the difference was not significant (p > 0.05) (Fig. 7A). Notably, there were two mice died, one at day 15 and one at day 20 in both treat groups, accounted for 20%. Histological analysis indicated that Fe₃O₄/Cur@ALG NPs presented in the liver tissue, but not in the kidney, with a small amount. Through, the histological structures in these organs did not change compared to that in the control (Fig. 7B). That mean, the NPs tended to stay in the liver after a long time-treatment. This result was consistent with the value of AST and ALT tests. Data indicated that both AST and ALT values increased in the treated mice compared to the control, with the significant difference in AST value (p < 0.05), meanwhile the concentration of BUN and creatinine did not change (Fig. 7C). Thus, Fe₃O₄/Cur@ALG NPs accumulated in the liver and caused the increase of transaminase in this organ.

The lifetime of Fe₃O₄/Cur@ALG NPs in blood vessels

We determined the presence of Fe₃O₄/Cur@ALG NPs in the blood vessels of mice by two methods: MIH measurement and AAS analysis. After intravenous injection of 1000 µg magnetic NPs, mice were put in an AMF of 178 kHz and 80 Oe. By measuring the change of temperature in the blood samples draw from mice blood vessel follow time, we found that there was a rise of temperature in the blood sample in the treated mice at 10 minutes right after the injection, then the temperature declined to the comparable value measuring before NPs application to the blood, at the time-point of 2 h after injection and so on. Similar results were obtained by AAS, the concentration of Fe in the blood samples of treated mice increased at 10 minutes after injection, then decreased to the value of the control (Fig. 8). Taken together, it means that Fe₃O₄/Cur@ALG NPs were cleared from blood in a very short time of more than 10 minutes.

In 2014, Devkota et al. presented the engineering of multifunctional magnetic nanoconjugates composed of superparamagnetic Fe₃O₄ NPs coated by alginate and curcumin (Devkota et al. 2014). They showed that this nanosystem is very promising for applications in hyperthermia, and biomolecular detection. However, in this study, they only performed the application in vitro without any biological objectives. Hence, in this current study, we would like to check of this nanosytem still keep their bioactivities both in vitro and in vivo models.

In our hyperthermia experiment, the temperature of the MNPs after 30 minutes applying of an alternative magnetic field with 80 Oe and 178 kHz, at the highest Fe concentration of 2000 µg/ml, rise to 41.5^oC. This temperature was lower in comparison with the reported value of the same Fe concentration in the study of Devkota. The difference may come from the existence of the cancer cells in the heated solution. Previous studies also indicated that the temperature of the cell-containing solution is always lower compared to that in the solution consists of magnetic NPs only (Bhardwaj, Parekh, and Jain 2020; Luong et al. 2011; Linh et al. 2009). Even so, the temperature 41.5◦C still had potential to kill cancer cells, with 20% of Sarcoma cells death. Variation in results of cancer cell viability were obtained in reported the studies of in vitro hyperthermia(Linh et al. 2009; Bhardwaj, Parekh, and Jain 2020; Luong et al. 2011; Tomitaka, Yamada, and Takemura 2012). In these studies, the temperature was higher than 45^oC, but the cell viability after 30 minutes of AMF applying was less than that in our experiment (Tomitaka, Yamada, and Takemura 2012; Bhardwaj, Parekh, and Jain 2020; Luong et al. 2011; Linh et al. 2009). It means that beside the effect of hyperthermia, the cell death could be induced by the effect of curcumin in the NPs. Previous study proved that MIH could release curcumin from the nanoconjugates (Mukherjee et al. 2020; Luong et al. 2011), so that they could contribute to kill cancer cells. We also found that the cell death was increased by a domino effect after stopping the heating process. At the time of 60 minutes after heating, the cell death was raised up to 31.1% and 38.8% in the AMF of 30 minutes and 45 minutes, respectively. That means the extermination of Sarcoma 180 cells still occurred even the alternative field was stopped. Hyperthermia can lead cancer cell to apoptosis by the effect of heating (Tomitaka, Yamada, and Takemura 2012), and curcumin is proved to initiating programmed cell death (Song et al. 2018; Beyene et al. 2021). In this condition of our study, many apoptotic cells did not incontinently dead during heating treatments, but after the process finished. Therefore, for accurate assessment of the effectiveness of hyperthermia, the number of dead cells over time needs to be counted.

The results of hyperthermia treatment in mice bearing tumor were consistent with the in vitro test. The volume of Sarcoma 180 tumors was clearly inhibited in heated mice, especially during the time of heating process (D0-D8) with the no change in the size of tumor. This effect was kept after finishing the treatment until D14. This phenomenon clearly indicated the domino effect of hyperthermia. Although the tumor regrew after D14, but the progression rate was 4 times smaller than that of untreated mice. The regrowth of tumor suggested that it is necessary to re-treat after time to get more efficient in anti-tumor effect. Interestingly, for the mice injected with Fe₃O₄/Cur@ALG NPs but not heating, the tumor volume was also smaller than the control. This effect could belong to the function of curcumin since this compound was proved the role as anti-cancer substance (Beyene et al. 2021; Song et al. 2018). Taken together, our results revealed that Fe₃O₄/Cur@ALG NPs can efficiently killed cancer cells in both in vitro and in vivo models. The death of cancer cells may come from the synergetic effect of the combination of the thermal therapy and the chemotherapy of curcumin.

The time which nuclei take to return from an excited state to an equilibrium state is called relaxation time, which depends on the proton density in the tissues (Reimer and Balzer 2003). Magnetic NPs were considered as superparamagnetic iron oxide NPs which is potential to reduce T2 relaxation time and increase the negative image contrast (Reimer and Balzer 2003; Jordan et al. 1999). In our current study, data of MRI by Fe₃O₄/Cur@ALG expressed the compatible results for their nature as superparamagnetic iron oxide NPs. In the presence of curcumin, the nanosystem still had ability to enhance the image contrast, with the MR signal intensity significantly reduced even at the smallest Fe concentration of 10 µg/ml used in the experiment. Besides, whenever applying the NPs in the body for MRI diagnostic, it needs to consider them in the interaction with the cells of body because they could change the MRI signal. The results in our study mean that the presence of the cells did not affect the T2 MRI signal of Fe₃O₄/Cur@ALG. Our results were compatible with the other superparamagnetic iron oxide NPs which were reported about their ability in increasing MRI contrast (Khalkhali et al. 2015; Song et al. 2019). In addition, Fe3O4/Cur@ALG NPs showed the MRI effect when being directly injected in mice as well. At both concentrations of Fe, 50 µg/0.5 cm3 and 250 µg/0.5 cm3, the NPs increased the negative contrast of MR images at the tumor site, with the darken signal enlarged follow time and density of NPs. It worth to note that, the dark region in MRI could cause by the necrotic core in the tumor. Thus, we chose the 5th days old tumors, which were in the early of the logarithmic growth phase. Moreover, MRI image in un-treat mice revealed that there were no such necrotic cores in tested tumors. At the time of 30 minutes after the nanoparticle injection, the dark signal was maintained indicating that the nanoparticle still presented in the tumor site. Our results were consistent with the study of Sun et al. reported the remaining iron oxide nanoparticle-immobile alginate nanogels in tumor no shorter than 2 hours after injection (Khalkhali et al. 2015). However, when applying Fe₃O₄/Cur@ALG NPs via the tail vein, we could not observe the difference between the injected mice and the control after 1h, 6h, and 24h of injection. This may be due to the observation window in our experiment was not appropriate that the nanoparticle was already removed or not focused yet to the tumor. Nevertheless, our results suggested that these Fe₃O₄/Cur@ALG NPs could be applied for imaging guided therapy. For MRI diagnosis, the MNPs need to be modified to enhance the Enhanced Permeability and Retention (EPR) effect so that they could be intravenously used. Our study still had limitations. To know the remaining time of MNPs in the tumor, we should take the images after longer time, such as 1–2 hours post MNP injection. Taking MRI prior MNP injection should also be performed in every tested tumor to precisely determine the effect of MNPs in image contrast enhancement.

To apply on human, it is necessary to define the toxicity of the nanoparticle. The toxicity tests may help to determine whether the nanoparticle concentrations are high enough to cause adverse effects in organisms. The promising results of Fe₃O₄/Cur@ALG NPs in MR imaging and hyperthermia treatment prompted us to perform the acute and sub-chronic toxicity test. In the acute lethality test, the data showed that even at the highest dose could be used of 120 mg/kg, there were no mice died after 72h of treatment. That mean at this dose, the nanoparticle had no acute lethality on mice. We decided to choose two doses corresponded to 1/7 and 1/5 of the highest dose to test for the chronic toxicity. The purpose of sub-chronic toxicity test is to long-term measurement the effects of exposure to relatively lower, repeated and less toxic concentrations of compounds. After the intravenous injection every day in 30 days, corresponded to the total dose of 720 and 530 mg/kg/mice, 2/10 of mice were died at both used doses. For the body weight and daily activity of mice, there was no statistically significant difference between the control and the treated one (p < 0.05). Interestingly, even the mice get a large total amount of NPs, the histological analysis showed that there was only a small amount of Fe remaining in liver organ. Moreover, the tissue structures of liver and kidney were not changed in the injected mice compared to the control. To evaluate the function of liver, we measured the level of ALT and AST enzymes. These molecules are two popular indicators for the function test of liver (Jain et al. 2008). The obtained results agreed with the histological analysis with the significant increase of these enzymes in treated mice compared to the control. However, the ALT levels in treated mice were still in the normal range (25–60 U/L) (Alexiou et al. 2003). Besides, generally, AST levels three times the upper limit of the normal range (range 39–262 U/L) represent abnormal liver function. The changes seen in our studies were significantly below this limit. Particularly, after 10 minutes of injection, the levels of iron in blood of treated mice were turn back to the normal, indicating that the quick clearance of NPs form blood vessel. Previous studies mentioned about the toxicity of magnetic iron oxide NPs (IONPs) in vivo (Feng et al. 2018). Our results were similar with the report of Jain et al that the magnetic NPs made the increase of liver enzymes but still stay in the normal range. In the study of Feng et al., Polyethylenimine-coated IONPs exhibited dose-dependent lethal toxicity, with animal mortality of 100% at the tested concentration of 5 mg/kg, meanwhile there was no death mice in PEGylated-coated IONPs group at the same Fe concentration (Feng et al. 2018). It needs to be noted that in these studies, the NPs were injected only one time per treatment, which is much less frequent compared to our study with 30 times in 30 days. Taken together, the data clearly indicated that our Fe₃O₄/Cur@ALG NPs did not cause long-term effect to the animal when tested at high dosed in 30 days.

In conclusion, we have demonstrated the application of Fe₃O₄/Cur@ALG NPs in the MR imaging and hyperthermia treatment in both in vitro and in vivo models. These NPs showed the capacity of killing cancer cells and inhibit tumor growth by the synergetic combination of thermal and chemotherapy. The NPs also played the second function in enhancing the MRI contrast. More important, Fe₃O₄/Cur@ALG NPs were safe for the biomedical application. Further works on the elimination and bio-distribution of these NPs is necessary to the successful development of them in the clinical testing.

Materials

Curcumin, alginate, ferric chloride, and ferrous chloride tetra-hydrate were purchased from Sigma Aldrich. Solvents (NH₄OH (26% of ammonia), ethanol) were purchased from Merck (Germany). Distilled water was used for nanoparticle preparation experiments.

Synthesis of Fe3O4@alginate iron oxide nanoparticles

According to Ha et al. (Ha et al. 2012), Fe₃O₄ magnetic nanoparticles were made by co-precipitating ferric and ferrous hydroxide in alkaline conditions. All experiments employed distilled water, which will be referred to as water exclusively from here on. The reaction equation is as follows:

2Fe3⁺ + Fe2⁺ + 8OH− → Fe₃O₄ + 4H2O

In a typical process, 4 mmol (0.65 g) of FeCl₃ and 2 mmol (0.3977 g) of FeCl₂.4H₂O were dissolved in 50 ml of HCl 2M and poured to a 250 ml three-necked flask with a Teflon-coated magnetic stir bar. The mixture was aggressively agitated while under N₂ purge. Throughout the procedure, the temperature was fixed at 70°C. When 80 ml of 2M NH₄OH was added to the reaction mixture, a precipitate developed, turning the entire solution black and raising the pH to 9. After 30 minutes of churning, the product was rinsed numerous times with water until it reached a pH of 7. The finished product was washed with ethanol and dried at 60°C for 24 hours for characterization, or it was dispersed in water for further surface functionalization of Fe₃O₄, henceforth referred to as Fe₃O₄.

The Fe₃O₄ nanoparticles were distributed in water at a concentration of 3 mg/ml to coat with alginate. Next, 0.1 g of alginate was added to 10 ml of water and agitated for 2 hours until thoroughly dissolved. After that, 30 ml of the Fe₃O₄ suspension was dropped gently into the alginate solution at room temperature under ultrasonic conditions. After that, the Fe₃O₄ nanoparticles and alginate combination was magnetically swirled for 48 hours. Finally, uncoated particles were centrifuged off, yielding a magnetic fluid containing 5 mg/ml Fe₃O₄ nanoparticles coated with alginate (Fe₃O₄@ALG).

According to Ha et al. (Thu Ha et al. 2010), curcumin (Cur) was integrated into the magnetic system of Fe₃O₄@ALG nanoparticles. To begin, a 200 ml flask with a magnetic stir bar was filled with 20 ml of 0.1 g/ml Fe₃O₄@ALG magnetic fluid. 0.1 g of Cur was dissolved in 15 ml of ethanol and put into the Fe₃O₄@ALG magnetic fluid in a separate container. The mixture was agitated for 48 hours in a closed jar before the ethanol was removed. To eliminate un-encapsulated Cur, the resultant product was centrifuged at 5000 rpm for 5 minutes. The completed product (Fe₃O₄/Cur@ALG) was kept at room temperature.

Material characterization

The structural characterization of these materials was determined by the method of Fourier transform infrared spectroscopy (FTIR, SHIMADZU spectrophotometer) using KBr pellets in the wave number region of 400–4000 cm-1 and X ray diffraction (XRD) patterns by SIEMENS D5005 diffractometer using Cu-K\({\alpha }\) radiation at 0.15406 nm. The diffraction patterns were collected with 2θ in the range of 20o – 70o. Morphological characteristics of the material were observed in Transmission electron microscopy TEM (JEM 1010). Particle size distribution and zeta potential distribution of the sample was determined on the Zetasizer-Nano ZS device of Malvern - UK by dynamic laser scattering method.

Cell culture and cell viability assay

Sarcoma 180 cancer cells (American Type Culture Collection) were cultured in RPMI 1640 media (Gibco) containing 10% FBS (Gibco) and 1% penicillin/streptomycin (Invitrogen), 5% CO2, 37^oC. Cell proliferation assay MTT kit (Promega) was used to evaluate the cytotoxicity of Fe₃O₄@ALG and Fe₃O₄/Cur@ALG. 5.10⁶ cells were exposed with concentration range of Fe₃O₄ from 0.01 to 1000 µg/ml for 48 hours. Samples were measured by Laminator spectrometers at 570 nm wavelength. IC50 value was calculated following the guideline of MTT assay kit.

Immunofluoresence assay

Cells were grown on Labtek (Nunc) in culture medium and incubated with Fe3O4/Cur@ALG with concentration range of Fe3O4 from 0.01 to 1000 µg/ml for 48 hours. Removed the medium by centrifuge and wash the cells with PBS. Fixed cells with 4% paraformaldehyde (Gibco) and 2% sucrose (Sigma) for 15 min at 37^oC, permeabilized with 0.2% Triton X-100 (Sigma) for 10 min, blocked with 5 mg/ml BSA (Sigma), and incubated with mouse monoclonal anti-human alpha tubulin antibodies (Invitrogen) for 1h, then incubated with Alexa 546 conjugated anti mouse antibodies for 30 minutes. DNA was visualized with Hoechst 33342 (Invitrogen). Images were collected with a ZEISS 510 Laser Scanning Confocal (LSM) microscope.

In vitro magnetic resonance imaging (MRI)

Fe₃O₄/Cur@ALG were dispersed homogeneously in 1% agarose (Invitrogen) prepared in two solutions: (1) in phosphate buffer saline (PBS) (Gibco), and (2) in PBS containing 1×10⁶ Sarcoma 180 cells. MRI of samples was taken using a 3.0 T Phillips nuclear magnetic resonance spectrometer (USA). T2 weighted image was performed with the parameters setting as follows: TR 3000 ms, TE 80 ms, FA 90°. Brightness signal of MRI was converted into pixel by Image J software.

In vitro hyperthermia assay

10⁶ of Sarcoma 180 cells was dispersed in PBS solution contained Fe₃O₄/Cur@ALG with 5 concentrations of Fe₃O₄ ranged from 100–2000 µg/ml. Immediately, a generator (RDO HFI 5 kW) was used to create an alternating magnetic field of amplitude and frequency respectively of 80 Oe and 178 kHz. After the solution reached maximum temperature at each concentration of Fe₃O₄, the magnetic field was maintained for 30 and 45 minutes. Percentage (%) of cell viability was defined by Blue Trypan (Sigma) staining technique at 0, 10, 20, 30, 60 minutes after finished applying alternating magnetic field.

Animal experiments

Swiss mice (5–7 weeks old) were obtained from Standard Animal Research and Production Center, National Institute of Hygiene and Epidemiology, Vietnam. The animals were housed in clean polypropylene cages and maintained in an air-conditioned conventional animal house at 25^oC, 55% relative humidity and 12h light/dark cycle. All experiment protocols involving animals were approved by the Ethics Committee of Dinh Tien Hoang Institute of Medicine (IRB00010830 and IORG0009080) in accordance with the principles and procedures outlined in national regulations.

In vivo MRI

Male Swiss mice were subcutaneously injected with 2×10⁶ Sarcoma 180 cells per mouse in the left front. At day 5 of post-implantation, when the tumor size had reached the average volume of 100 mm³, the mice were anesthetized by an intraperitoneal injection of Thiopental (50µg/kg) (Rotexmedica, Germany). The Fe₃O₄/Cur@ALG nanoparticle solution were applied into each mouse by subcutaneously injection (at two Fe concentrations of 50 µg and 250 µg per tumor) or via the tail vein (at Fe concentration of 100 µg/mouse). The control mice were not transplanted with cancer cells and the tumor control of were not injected with NPs. The MR imaging was conducted on a commercial 3.0 T Phillips nuclear magnetic resonance spectrometer (USA) with the parameters set as follows: TR 3000 ms, TE 80 ms, FA 90° and 180^o, scan time 10 minutes, slice thickness 0.7 mm. The T2-weighted MR images were obtained after administration of NPs at time points of 0, 10 minutes, 30 minutes for the subcutaneously injection; and 1h, 6h, and 24h for the intravenously injection.

In vivo Hyperthermia

Tumor-bearing mice were prepared as mentioned in the in vivo MRI experiments. At day 5 post-implantation, after the anesthetization, mice were subcutaneously injected with Fe₃O₄/Cur@ALG NPs at the concentration of 300 µg per tumor. The injected mice were then randomly divided into 2 groups: with alternative magnetic field (AFM) irradiation (Fe₃O₄/Cur@ALG + AMF) and without AFM (Fe₃O₄/Cur@ALG). The control tumor-bearing mice were also set up without the injection of NPs. A field of 70 Oe amplitude, and 178 kHz frequency was applied to AFM mice to irradiate for the period of 40 minutes, starting at 15 min after the injection MNPs into the mice. Each therapy consisted of 3 sessions of AMF irradiation, with a 48h break between consecutive irradiations.

The treatment efficiency was described by observation of tumor volume V, which was calculated as [6]:

\(V=0.5 \times a \times {b}^{2} \left({\text{m}\text{m}}^{3}\right)\)

(2)

where a is tumor length, and b is tumor width. The measurement of tumors was performed by using a caliper every two days in the total of 26 days from the day of NP injection. Tumors were photographed with a ruler to illustrate the same magnification.

Toxicity test

For acute toxicity test, male Swiss mice (5 to 7 weeks) were intravenously injected with Fe₃O₄/Cur@ALG NPs at different doses (80, 90, 100, 110, 120 mg/kg, n = 5 for each dose), respectively. The mice were observed continuously for 72 hours. LD50 was calculated by method of Litchfield – Wilcoxon (Paumgartten et al. 1989).

For sub-chronic toxicity, mice were intravenously injected with Fe₃O₄/Cur@ALG NPs at two doses (24 and 17 mg/kg, n = 10 for each dose), respectively, every 48 hours in a total of 30 days. The control mice were injected with PBS. Mice were observed for any adverse reaction, such as death, change in body weight, food consumption, stool, motor activity. The body weight of mice was measured every 72 hours. On day 31, all the mice were sacrificed, and blood samples were obtained for serum chemistry analysis including alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine. Liver and kidney tissues were collected for histological analysis. The samples were fixed in 4% formalin for 48 hours then mass-casted with paraffin, cut into 7-µm thick slices, and stained with Hematoxylin-Eosin dye (Sigma, USA). The tissue characteristics and morphology were examined by pathologists under an optical microscope.

In vivo bio-distribution in blood

The in vivo bio-distribution of Fe₃O₄/Cur@ALG NPs in blood was determined by magnetic inductive heating experiments. A generator (RDO HF1 5kW) was used to create an AMF of amplitude of 80 Oe, and frequency of 178 kHz. Male Swiss mice (n = 5) were received 1000 µg of Fe₃O₄/Cur@ALG via the tail vein. After 10 minutes, 2 h, 12 h, 24 h, and 48h post-injection, blood was collected and put into a round-bottom shaped glass holder and applied to AMF. The sample temperature was measured by an optical thermometer (Opsens).

The concentration of Fe in blood of mice was also determined by atomic absorption spectrometry (AAS) method using Shimadzu 6300 AAS AA/AE Spectrophotometer. Blood samples were put into a Kjedahl bottle together with 10 ml of H₂SO₄ acid and mixed well. Heated the bottle to 450^oC for 15 minutes, and then added several drops of 70% perchloric acid while heating. Until the solution turned to transparent bright color, it was ready for AAS analysis. The iron content in the blood sample was calculated as: a = cV/m, where c is the concentration of Fe in the sample (kg/l), v is the volume of sample (l), and m is the weight of sample (kg).

Statistical analysis

All statistical analyses were performed using Graph Prism 9.1.0. Student ‘s t-test statistical analysis was performed to test the significance of the experimental data. In all cases, p < 0.05 was selected to indicate the statistical significance.

MNPs, Magnetite (Fe₃O₄) nanoparticles; MRI, magnetic resonance imaging; MR, Magnetic resonance; NPs, nanoparticles; FTIR, Fourier transform infrared spectroscopy; XRD, X ray diffraction; PBS, phosphate buffer saline; AFM, alternative magnetic field; ALT, aminotransferase, AST, aspartate aminotransferase, BUN, blood urea nitrogen; AAS, atomic absorption spectrometry; TEM, transmission electron microscope; POI, post injection; MIH, magnetic inductive heating.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Competing interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper

Funding: This research received no external funding

Author Contributions: XH Do, TMN Hoang, DT Nguyen, and TP Ha. conceived the study. TT To, DT Nguyen, KTV Bui, NH Pham, K Lam performed the experiments and the measurements. XH Do, TMN Hoang, PT Ha, and TT To analyzed the results and prepared the manuscript. XH Do revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Acknowledgments: The authors highly thank Prof. Xuan Phuc Nguyen for his kindly support and great advice in the experiment design and manuscript writing

Aksimentyeva, O. I., V. P. Savchyn, V. P. Dyakonov, S. Piechota, Yu Yu Horbenko, I. Ye Opainych, P. Yu Demchenko, A. Popov, and H. Szymczak. 2014. 'Modification of Polymer-Magnetic Nanoparticles by Luminescent and Conducting Substances', Molecular Crystals and Liquid Crystals, 590: 35–42.
Alexander, Leroy, and Harold P. Klug. 1950. 'Determination of Crystallite Size with the X-Ray Spectrometer', Journal of Applied Physics, 21: 137–42.
Alexiou, C., R. Jurgons, R. J. Schmid, C. Bergemann, J. Henke, W. Erhardt, E. Huenges, and F. Parak. 2003. 'Magnetic drug targeting–biodistribution of the magnetic carrier and the chemotherapeutic agent mitoxantrone after locoregional cancer treatment', J Drug Target, 11: 139–49.
Avasthi, A., C. Caro, E. Pozo-Torres, M. P. Leal, and M. L. García-Martín. 2021. "Correction to: Magnetic Nanoparticles as MRI Contrast Agents." In Top Curr Chem (Cham), 30.
Barhoum, Ahmed, M. Luisa García-Betancourt, Hubert Rahier, and Guy Van Assche. 2018. 'Chapter 9 - Physicochemical characterization of nanomaterials: polymorph, composition, wettability, and thermal stability.' in Ahmed Barhoum and Abdel Salam Hamdy Makhlouf (eds.), Emerging Applications of Nanoparticles and Architecture Nanostructures (Elsevier).
Beola, L., L. Asín, C. Roma-Rodrigues, Y. Fernández-Afonso, R. M. Fratila, D. Serantes, S. Ruta, R. W. Chantrell, A. R. Fernandes, P. V. Baptista, J. M. de la Fuente, V. Grazú, and L. Gutiérrez. 2020. 'The Intracellular Number of Magnetic Nanoparticles Modulates the Apoptotic Death Pathway after Magnetic Hyperthermia Treatment', ACS Appl Mater Interfaces, 12: 43474–87.
Beyene, Anteneh Marelign, Mohammad Moniruzzaman, Adhimoolam Karthikeyan, and Taesun Min. 2021. 'Curcumin Nanoformulations with Metal Oxide Nanomaterials for Biomedical Applications', Nanomaterials, 11: 460.
Bhardwaj, Anand, Kinnari Parekh, and Neeraj Jain. 2020. 'In vitro hyperthermic effect of magnetic fluid on cervical and breast cancer cells', Scientific Reports, 10: 15249.
Blanco-Andujar, Cristina, Daniel Ortega, Quentin A. Pankhurst, and Nguyen Thi Kim Thanh. 2012. 'Elucidating the morphological and structural evolution of iron oxide nanoparticles formed by sodium carbonate in aqueous medium', Journal of Materials Chemistry, 22: 12498–506.
C, N. Dong, J. A. Tate, W. C. Kett, J. Batra, E. Demidenko, L. D. Lewis, P. J. Hoopes, T. U. Gerngross, and K. E. Griswold. 2015. 'Tumor cell targeting by iron oxide nanoparticles is dominated by different factors in vitro versus in vivo', PLoS One, 10: e0115636.
Devkota, J., T. T. T. Mai, K. Stojak, P. T. Ha, H. N. Pham, X. P. Nguyen, P. Mukherjee, H. Srikanth, and M. H. Phan. 2014. 'Synthesis, inductive heating, and magnetoimpedance-based detection of multifunctional Fe3O4 nanoconjugates', Sensors and Actuators B: Chemical, 190: 715–22.
Faraji, M., Y. Yamini, and M. Rezaei. 2010. 'MAGNETIC NANOPARTICLES: SYNTHESIS, STABILIZATION, FUNCTIONALIZATION, CHARACTERIZATION, AND APPLICATIONS', JOURNAL OF THE IRANIAN CHEMICAL SOCIETY(JICS), 7: 1–37.
Faustino-Rocha, A., P. A. Oliveira, J. Pinho-Oliveira, C. Teixeira-Guedes, R. Soares-Maia, R. G. da Costa, B. Colaço, M. J. Pires, J. Colaço, R. Ferreira, and M. Ginja. 2013. 'Estimation of rat mammary tumor volume using caliper and ultrasonography measurements', Lab Anim (NY), 42: 217–24.
Feng, Qiyi, Yanping Liu, Jian Huang, Ke Chen, Jinxing Huang, and Kai Xiao. 2018. 'Uptake, distribution, clearance, and toxicity of iron oxide nanoparticles with different sizes and coatings', Scientific Reports, 8: 2082.
Fernandes, S., T. Fernandez, S. Metze, P. B. Balakrishnan, B. T. Mai, J. Conteh, C. De Mei, A. Turdo, S. Di Franco, G. Stassi, M. Todaro, and T. Pellegrino. 2021. 'Magnetic Nanoparticle-Based Hyperthermia Mediates Drug Delivery and Impairs the Tumorigenic Capacity of Quiescent Colorectal Cancer Stem Cells', ACS Appl Mater Interfaces, 13: 15959–72.
Ganapathe, Lokesh Srinath, Mohd Ambri Mohamed, Rozan Mohamad Yunus, and Dilla Duryha Berhanuddin. 2020. 'Magnetite (Fe3O4) Nanoparticles in Biomedical Application: From Synthesis to Surface Functionalisation', Magnetochemistry, 6: 68.
Ghazanfari, M. R., M. Kashefi, S. F. Shams, and M. R. Jaafari. 2016. 'Perspective of Fe3O4 Nanoparticles Role in Biomedical Applications', Biochem Res Int, 2016: 7840161.
Ha, Phuong Thu, Mai Huong Le, Thi My Nhung Hoang, Thi Thu Huong Le, Tuan Quang Duong, Thi Hong Ha Tran, Dai Lam Tran, and Xuan Phuc Nguyen. 2012. 'Preparation and anti-cancer activity of polymer-encapsulated curcumin nanoparticles', Advances in Natural Sciences: Nanoscience and Nanotechnology, 3: 035002.
Hilger, I., and W. A. Kaiser. 2012. 'Iron oxide-based nanostructures for MRI and magnetic hyperthermia', Nanomedicine (Lond), 7: 1443–59.
Jain, Tapan K., Maram K. Reddy, Marco A. Morales, Diandra L. Leslie-Pelecky, and Vinod Labhasetwar. 2008. 'Biodistribution, Clearance, and Biocompatibility of Iron Oxide Magnetic Nanoparticles in Rats', Molecular Pharmaceutics, 5: 316 – 27.
Jordan, Andreas, Regina Scholz, Peter Wust, Horst Fähling, and Felix Roland. 1999. 'Magnetic fluid hyperthermia (MFH): Cancer treatment with AC magnetic field induced excitation of biocompatible superparamagnetic nanoparticles', Journal of Magnetism and Magnetic Materials, 201: 413–19.
Justin, C., A. V. Samrot, D. S. P, C. S. Sahithya, K. S. Bhavya, and C. Saipriya. 2018. 'Preparation, characterization and utilization of coreshell super paramagnetic iron oxide nanoparticles for curcumin delivery', PLoS One, 13: e0200440.
Khalkhali, Maryam, Kobra Rostamizadeh, Somayeh Sadighian, Farhad Khoeini, Mehran Naghibi, and Mehrdad Hamidi. 2015. 'The impact of polymer coatings on magnetite nanoparticles performance as MRI contrast agents: a comparative study', DARU Journal of Pharmaceutical Sciences, 23: 45.
Liang, P. C., Y. C. Chen, C. F. Chiang, L. R. Mo, S. Y. Wei, W. Y. Hsieh, and W. L. Lin. 2016. 'Doxorubicin-modified magnetic nanoparticles as a drug delivery system for magnetic resonance imaging-monitoring magnet-enhancing tumor chemotherapy', Int J Nanomedicine, 11: 2021–37.
Linh, Pham Hoai, Nguyen Chi Thuan, Nguyen Anh Tuan, Pham Van Thach, Tran Cong Yen, Nguyen Thi Quy, Hoang Thi My Nhung, Phi Thi Xuyen, Nguyen Xuan Phuc, and Le Van Hong. 2009. 'Invitro toxicity test and searching the possibility of cancer cell line extermination by magnetic heating with using Fe < sub > 3</sub > O < sub > 4</sub > magnetic fluid', Journal of Physics: Conference Series, 187: 012008.
Luong, Tai Thien, Thu Phuong Ha, Lam Dai Tran, Manh Hung Do, Trang Thu Mai, Nam Hong Pham, Hoa Bich Thi Phan, Giang Ha Thi Pham, Nhung My Thi Hoang, Quy Thi Nguyen, and Phuc Xuan Nguyen. 2011. 'Design of carboxylated Fe3O4/poly(styrene-co-acrylic acid) ferrofluids with highly efficient magnetic heating effect', Colloids and Surfaces A: Physicochemical and Engineering Aspects, 384: 23–30.
Ma, H. L., X. R. Qi, Y. Maitani, and T. Nagai. 2007. 'Preparation and characterization of superparamagnetic iron oxide nanoparticles stabilized by alginate', Int J Pharm, 333: 177–86.
Markides, H., M. Rotherham, and A. J. El Haj. 2012. 'Biocompatibility and toxicity of magnetic nanoparticles in regenerative medicine', J. Nanomaterials, 2012: Article 13.
Massart, R. 1981. 'Preparation of aqueous magnetic liquids in alkaline and acidic media', IEEE Transactions on Magnetics, 17: 1247–48.
Mukherjee, S., L. Liang, and O. Veiseh. 2020. 'Recent Advancements of Magnetic Nanomaterials in Cancer Therapy', Pharmaceutics, 12.
Narayanaswamy, Venkatesha, Sangaraju Sambasivam, Alam Saj, Sulaiman Alaabed, Bashar Issa, Imaddin A. Al-Omari, and Ihab M. Obaidat. 2021. 'Role of Magnetite Nanoparticles Size and Concentration on Hyperthermia under Various Field Frequencies and Strengths', Molecules, 26: 796.
Patil, Ujwal S., Shiva Adireddy, Ashvin Jaiswal, Sree Mandava, Benjamin R. Lee, and Douglas B. Chrisey. 2015. 'In Vitro/In Vivo Toxicity Evaluation and Quantification of Iron Oxide Nanoparticles', International Journal of Molecular Sciences, 16: 24417–50.
Paumgartten, F. J., O. A. Presgrave, M. A. Menezes, F. F. Fingola, J. C. Freitas, R. R. Carvalho, and F. Q. Cunha. 1989. 'Comparison of five methods for the determination of lethal dose in acute toxicity studies', Braz J Med Biol Res, 22: 987–91.
Reimer, Peter, and Thomas Balzer. 2003. 'Ferucarbotran (Resovist): a new clinically approved RES-specific contrast agent for contrast-enhanced MRI of the liver: properties, clinical development, and applications', European Radiology, 13: 1266–76.
Sánchez-Domínguez, Margarita, Carolina Aubery, and Conxita Solans. 2012. "New Trends on the Synthesis of Inorganic Nanoparticles Using Microemulsions as Confined Reaction Media." In.
Sangaiya, P., and R. Jayaprakash. 2018. 'A Review on Iron Oxide Nanoparticles and Their Biomedical Applications', Journal of Superconductivity and Novel Magnetism, 31: 3397–413.
Shultz, Michael D., J. Ulises Reveles, Shiv N. Khanna, and Everett E. Carpenter. 2007. 'Reactive Nature of Dopamine as a Surface Functionalization Agent in Iron Oxide Nanoparticles', Journal of the American Chemical Society, 129: 2482–87.
Song, Wenxing, Xing Su, David Alexander Gregory, Wei Li, Zhiqiang Cai, and Xiubo Zhao. 2018. 'Magnetic Alginate/Chitosan Nanoparticles for Targeted Delivery of Curcumin into Human Breast Cancer Cells', Nanomaterials, 8: 907.
Song, X., M. Zhang, E. Dai, and Y. Luo. 2019. 'Molecular targets of curcumin in breast cancer (Review)', Mol Med Rep, 19: 23–29.
Sukumaran, S, MS Neelakandan, N Shaji, P Prasad, and VK Yadunath. 2018. "Magnetic nanoparticles: Synthesis and potential biological applications." In JSM Nanotechnology & Nanomedicine, 1068.
Taimoory, Seyedeh Maryamdokht, Abbas Rahdar, Mousa Aliahmad, Fardin Sadeghfar, Mohammad Reza Hajinezhad, Mohammad Jahantigh, Parisa Shahbazi, and John F. Trant. 2018. 'The synthesis and characterization of a magnetite nanoparticle with potent antibacterial activity and low mammalian toxicity', Journal of Molecular Liquids, 265: 96–104.
Thambiraj, S., R. Vijayalakshmi, and D. Ravi Shankaran. 2021. 'An effective strategy for development of docetaxel encapsulated gold nanoformulations for treatment of prostate cancer', Scientific Reports, 11: 2808.
Thu Ha, Phuong, Thi Minh Nguyet Tran, Hong Duong Pham, Quang Huan Nguyen, and Xuan Phuc Nguyen. 2010. 'The synthesis of poly(lactide)-vitamin E TPGS (PLA-TPGS) copolymer and its utilization to formulate a curcumin nanocarrier', Advances in Natural Sciences: Nanoscience and Nanotechnology, 1: 015012.
Tian, Jilai, Caiyun Yan, Kunliang Liu, Juan Tao, Zhenchao Guo, Jianping Liu, Yu Zhang, Fei Xiong, and Ning Gu. 2017. 'Paclitaxel-Loaded Magnetic Nanoparticles: Synthesis, Characterization, and Application in Targeting', Journal of Pharmaceutical Sciences, 106: 2115–22.
Tomitaka, Asahi, Tsutomu Yamada, and Yasushi Takemura. 2012. 'Magnetic Nanoparticle Hyperthermia Using Pluronic-Coated Nanoparticles: 'An In Vitro Study', Journal of Nanomaterials, 2012: 480626.
Yew, Yen Pin, Kamyar Shameli, Mikio Miyake, Nurul Bahiyah Bt Ahmad Khairudin, Shaza Eva Bt Mohamad, Takeru Naiki, and Kar Xin Lee. 2020. 'Green biosynthesis of superparamagnetic magnetite Fe3O4 nanoparticles and biomedical applications in targeted anticancer drug delivery system: A review', Arabian Journal of Chemistry, 13: 2287–308.
Zhang, H., X. L. Liu, Y. F. Zhang, F. Gao, G. L. Li, Y. He, M. L. Peng, and H. M. Fan. 2018. 'Magnetic nanoparticles based cancer therapy: current status and applications', Sci China Life Sci, 61: 400–14.

No competing interests reported.

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Dual roles of Curcumin Encapsulated Alginate/Fe3O4 Nanoparticles in MRI Enhancement and Hyperthermia for Cancer Treatment

Status:

Version 1

Abstract

Background

Results

Conclusion

Figures

Background

Results

Structural and morphological analysis

In vitro cytotoxicity, MR imaging, and magnetic heating of Fe₃O₄/Cur@ALG NPs on cancer cells

In vivo MR imaging of tumor on mice model

In vivo magnetic heating of tumor on mice model

Acute and sub-chronic toxicity of Fe₃O₄/Cur@ALG NPs

The lifetime of Fe₃O₄/Cur@ALG NPs in blood vessels

Discussion

Conclusions

Materials And Methods

Materials

Synthesis of Fe3O4@alginate iron oxide nanoparticles

Material characterization

Cell culture and cell viability assay

Immunofluoresence assay

In vitro magnetic resonance imaging (MRI)

In vitro hyperthermia assay

Animal experiments

In vivo MRI

In vivo Hyperthermia

Toxicity test

In vivo bio-distribution in blood

Statistical analysis

List Of Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1

Dual roles of Curcumin Encapsulated Alginate/­Fe3O4 Nanoparticles in MRI Enhancement and Hyperthermia for Cancer Treatment

Status:

Version 1

Abstract

Background

Results

Conclusion

Figures

Background

Results

Structural and morphological analysis

In vitro cytotoxicity, MR imaging, and magnetic heating of Fe3O4/Cur@ALG NPs on cancer cells

In vivo MR imaging of tumor on mice model

In vivo magnetic heating of tumor on mice model

Acute and sub-chronic toxicity of Fe3O4/Cur@ALG NPs

The lifetime of Fe3O4/Cur@ALG NPs in blood vessels

Discussion

Conclusions

Materials And Methods

Materials

Synthesis of Fe3O4@alginate iron oxide nanoparticles

Material characterization

Cell culture and cell viability assay

Immunofluoresence assay

In vitro magnetic resonance imaging (MRI)

In vitro hyperthermia assay

Animal experiments

In vivo MRI

In vivo Hyperthermia

Toxicity test

In vivo bio-distribution in blood

Statistical analysis

List Of Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1

Dual roles of Curcumin Encapsulated Alginate/Fe3O4 Nanoparticles in MRI Enhancement and Hyperthermia for Cancer Treatment

In vitro cytotoxicity, MR imaging, and magnetic heating of Fe₃O₄/Cur@ALG NPs on cancer cells

Acute and sub-chronic toxicity of Fe₃O₄/Cur@ALG NPs

The lifetime of Fe₃O₄/Cur@ALG NPs in blood vessels