In the past decade, magnetic micro and nanoparticles have attracted much attention because of their unique magnetic properties and widespread application in different fields. Magnetic nanoparticles are efficient, and readily available, and have high surface-area resulting in high catalyst loading capacity , chemical sensing [2-6], information storage [7, 8] and medical diagnostics [9–11] and outstanding stability to heterogeneous supports for catalysts preparation. Accordingly, the application of magnetic nano particles has become an interesting filed of research besides other advantages of nano particles [12, 13]. A series of functionalized magnetic nanoparticles (MNPs) involves iron oxide (Fe3O4/γ-Fe2O3) magnetic nanoparticles coated with silica and Gold shells. . Importantly, Core-shell nanoparticle provides both enhanced biocompatibility and stability of the surface and imparts the surfaces with the desired biological or chemical, catalytic interfacial reactivities [15, 16]. The successes of these synthetic approaches discover the potential applications of FSG [17-20].
In pharmaceutical and medical aspects, novel drug delivery systems such as polymers, liposomes, and Monoclonal antibodies open new views in cancer treatment . Traveling of Magnetic nanoparticles, such as Fe3O4-SiO2 core-shell through the bloodstream and tendency to accumulate into the surrounding tumor tissue is important. . One significant aspect is the development of compounds that can deliver a high boron payload to the tumors . Hence, considerable successes have been obtained on the liposomal delivery system as a new targeting fact.
Liposomes are profitable drug delivery carriers that are able to selectively transfer a large amount of a broad variety of 10B agents to the tumor tissue. For this purpose, for carrier system development, folate receptors was considered as an appropriate substrate that has a potential to attach to tumor compared with normal cells. During the investigation of its applicable behavior, we have found that the muscle cancer in BALB/c mice model possesses a unique advantage for treatment of tumors [24-27].
The main problem dealing with muscle cancer is that the tumor and normal cells are mixed without a map of the boron accumulation. As a result, in different tissue samples, the boron concentration in tissue composition is not accurate. The purpose of study was to perform biodistribution studies of FSG in the muscle cancer in BALB/c mice model. In this model, we have examined different sites surrounding healthy organs in the BALB/c mice model. The optimization of selective boron delivery to cancer cells is an interesting area of research and must be as a starting point to contribute and optimize its therapeutic efficacy. Using a novel drug containing 10B compound in smart release and administering to a patient is a novel method in the treatment of the cancerous tumors. The reduce production cost as well as minimum side effects in treatment are other advantages of the applied drug and method.
In the present article, 41.1 mg of the liposome-encapsulated FSG was delivered to a tumor site. Results demonstrated that the highest concentration of drug in the tumor tissue (27 mg/L) occurs at a period of 45 min to 3 h. Hence, Specific method of inductively coupled plasma (ICP) analysis was applied for samples of the tumor, normal (muscle) tissue, liver, heart, lung, spleen, skin, stomach, brain, bone, kidney and blood to check the boron distribution in different tissue portions.
MATERIALS AND METHODS
Preparation of Fe3O4-SiO2@Gold (FSG) nanoparticles
Preparation of the Fe3O4 nanoparticles (FMNs)
Fe3O4 nanoparticles were synthesized using a modified protocol. FeCl3 and FeCl2 solution in HCl with the optimum mole fraction ratio of Fe3+: Fe2+ in 2:1 (Fig. 1) was added to a flat bottom balloon separately and stirred using a magnetic stirring bar.
To prepare water-soluble and dispersed FMNs, the solutions of FeCl3.6H2O (1 M) and FeCl2.4H2O (2 M) were prepared by dissolving the iron salts in 2 and 1 M HCl solutions in a 250 ml flask. Then, the solutions were mixed at 25 °C. Then, ammonia solution (1 M, 100 ml) was added droplet. Then, particles were filtered and washed several times. The pH of the solution was adjusted up to 10. The stirring of the solution was continued for 1 h until the stable black FMNs were appeared. The ferrite powder was finally dried in vacuum oven at 80 °C for 24 h to complete removal of water. The reaction mechanism is explained as below.
Preparation of the Fe3O4/SiO2 magnetic nanoparticles (FSMNs)
The core/shell Fe3O4@SiO2 was prepared according to the Stober process  with some modifications. The obtained MNPs powder (1g) were dispersed in a mixture of 100 mL of ethanol, 20 mL of distilled water by the ultrasonic treatment for 30 min and 5.0 mL of 28 wt % concentrated ammonia aqueous solution (NH3.3H2O) with vigorous stirring, followed by the addition of 2 mL of Tetra Ethyl Ortho Silicate (TEOS) was dropwise added to this dispersion. The resulting dispersion was kept stirred mechanically under N2 protection for 12 h at room temperature. The obtained Fe3O4@SiO2 nanoparticles were separated with a magnet and washed repeatedly with ethanol and water to remove nonmagnetic byproduct.
Activation of FSMNs
Then, the obtained MNPs powder (1g) was dispersed in methanol (100 mL) and toluene (30 mL) by the ultrasonic treatment for 30 min before 3-Amino Propyl Tri Ethoxy Silane (APTES), (99%, 1 mL) was added to the mixture. After mechanically agitation for 12 h, the suspended substance was separated by a magnet. The settlement product was redispersed in ethanol by sonication and isolated with magnetic decantation for 5 times. The precipitated product (APTES-MNPs) was dried at room temperature under vacuum. The reactions mechanism is explained as below.
Preparation of Fe3O4-SiO2@Gold nanoparticles
The synthesis of Fe3O4 nanoparticles starts according to process in which Fe(acac)3 is reduced using 1,2-hexadecanediol in the presence of oleic acid. Following Fe3O4 make as nanoparticles with the favorite sizes, Gold is deposited on to the surface of Fe3O4 by reduction of Au(CH3COO)3 using 1,2-hexadecanediol in the presence of oleic acid at higher temperature (180–190 ºC). The resulting Fe3O4-SiO2@Gold nanoparticles can be separated by centrifugation from the uncoated Fe3O4 nanoparticles.
This approach can produce highly monodisperse Fe-oxide-SiO2-Gold nanoparticles.
Synthetic procedure of liposome-encapsulated FSG
The synthetic procedure of liposome-encapsulated FSG using polyethylene glycol (PEG) as a precursor mentioned in the literature  allows a solubility of the drug in the blood and binding of a ligand to the liposome. Furthermore, folate ligand bonded to the PEG. As demonstrated in Fig. 3, the liposome can encapsulate aqueous solutions of sodium salts anions and join lipophilic boron-containing nanodrug components embedd-ed within the bilayer membrane . For this purpose, 1 M solution of fructose in water or ethanol and 1 M solution of boric acid containing 10Boron were provided. Also, adjusting and controlling the solution pH around 3-4 was performed. Then, some carbonate salt of solid calcium (dissolved in a little amount of deionized water) was added to the solution. As a result of this process, the biphasic solution was formed and the lower phase was separated. Nanodrug has several boron atoms in each molecule that caused to easily penetrate to the membrane of the tumor cell.
Model of cancer: Biodistribution and boron analysis studies
The digestion of animals samples was carried out according procedure as discussed in the literature . Over time, to identify the optimal injection protocol for delivery of boron to the tumor tissue, performed experiments were analyzed. Following the appropriate experiments, the data were presented and boron concentration was individually analyzed.
In the present study, several samples of tumor and cancerous tissues around tumor tissues were taken from each animal to informant the distribution of boron in the tumor, blood, and normal tissues . Boron concentration values in the tumor, blood, and normal (muscle) tissues were obtained as post-administration to calculate the ratio of boron concentration in tumor/blood, normal tissue/blood and tumor/normal tissue for each of the compounds . Fig. 4 shows an overall schematic of the entrance process of the drug and the process of BNCT used in the prior investigation.
RESULTS AND DISCUSSION
In order to investigate the morphology and the structure of FSG nanoparticle, the prepared particles were analyzed.
Qualitative and Quantitative Analysis
XRD analysis of FSG nanoparticles
X-ray diffraction pattern (JCPDS, card number 01-072-1243) for the (a) Fe3O4 nanoparticles (b) Fe3O4-SiO2 nanoparticles (c) Fe3O4-SiO2@Gold nanoparticles showed in Fig. 5. The XRD pattern showed the characteristic peaks at 2θ of 9.2◦, 11◦, 31.8◦, and 45.4◦ and the size of Fe3O4 nanoparticles obtained was 9.80 nm. Furthermore, the data showed diffraction peaks at 2θ° = 38.2°, 44.2°, 64.5°, 77.8°, and 81.7°, which can be indexed to planes of Gold.
FTIR analysis of FSG nanoparticles
The reactions were confirmed using the FTIR analysis. The FTIR spectra of (a) Fe3O4 nanoparticles (b) Fe3O4-SiO2 nanoparticles and (c) Fe3O4-SiO2@Gold nanoparticles showed in Fig. 6. In contrast to the spectrum of Fe3O4-SiO2 nanoparticles, the spectrum of the Fe3O4-SiO2@Gold nanoparticles shows a bond at 3400 cm-1, that could be assigned to N-H stretching. The bonds at 1020 cm-1 also could be attributed to the stretching of C-H groups of alkyls in the Fe3O4-SiO2@Gold nanoparticles. The magnetite spectrum shows a bond at 573 cm-1, that could be assigned to magnetite vibrations. The bonds at 3440 cm-1 also could be attributed to the stretching of O-H groups of hydroxyls in the magnetic nanoparticles. Furthermore, the bonds at 1614 and 1391 cm-1 that appeared in the spectrum of the Fe3O4-SiO2 nanoparticles could be attributed to formation of carboxyl group bond to Fe3O4 in Fe3O4-SiO2 nanoparticles. In addition, the bonds at 1621, 1091 and 955 cm-1 could be attributed to H-O-H stretching, Si-O-Si stretching and Si-OH stretching in magnetite-silica nanoparticles.
TEM study of FSG nanoparticles
TEM data supports Fe3O4-SiO2@Gold core shell nanoparticles forms. There are two major findings from the morphological comparison. Firstly, the particles after coating with Silica and Gold seem much darker and secondly, for the particles after coating with SiO2 and Gold, the average particle size changed from 9.8 nm to 15 nm. In Fig. 6, the TEM data prepare an important piece of evidence for Fe3O4-SiO2@Gold core-shell forms.
Biodistribution and boron analysis studies: in-vivo and in-vitro treatment
In this section, the experiments conducted which were related to in-vivo and in-vitro conditions of the biological tests. Studying the effects of drug-containing 10B on the muscle cancer and the corresponding Dose-Responses curves were performed. The predetermined concentration was 20-35 mg 10B/g. The experiments were performed dealing with the biological distribution of the designed drug in different times of injection, biological distribution of various doses of the drug and sufficient accumulation of the designed drug-containing 10Boron aiming influential treatment of the tumor.
As mentioned above, the drug with concentrations of 15, 25, 35 and 45 mg was selected to inject to the animal. The results of ICP data indicate that the drug concentration of 35 mg into the cell culture medium maximized the accumulation of the drug within 24 h incubation of the drug introduced to the environment (Fig. 8). The reason can be attributed to the representation of the toxicity of the synthesized composition using MTT assay method.
To perform in-vivo treatment in testing animal, the toxicity of the liposome-encapsulated FSG was analyzed. Fig. 9 shows the diagram of toxicity of the liposome-encapsulated FSG determined by MTT assay method .
As can be seen, in the initial time, the number of live cells is not considerably changed; because of using the unit dietary compound to drug synthesis. While with respect to time, the number of tumor cells is gradually decreased. After 24 h, these reductions were 79, 64, 50, 80 and 36 % for various boron compounds at concentrations of 5, 10, 25, 50 and 100 mg/L, respectively. After 48 h, the results were similar to those obtained after 24 h with a slight differences .
Boron biodistribution studies were essential to preclinical design and subsequent clinical research protocols of BNCT. In particular, this analysis identifies the optimum time post-administration of the boron carrier to perform neutron therapy . To date, dose calculations were based on boron content values in blood, tumor, and normal tissues obtained beforehand from biodistribution studies. in the case of animal models, calculations were based on the mean values obtained from biodistribution studies in separate portions of tissues . The tabulated diagrams and data corresponding to the mean value ± standard deviation of the ratio for each sample are shown in Fig. 10.
The results showed that the highest concentration of drug in the tumor tissue occurred at a period of 45 min to 3 h. Also, after drug injection, its concentration initially in the blood reached to the highest value; while it may reach to the lowest value within 24 h .
Generally, the results of boron concentration in the tumor tissue showed that after 3 h of drug injection to the tumor, the drug concentration reached to its highest level (27 mg/L) due to the excessive amount of sugar transmitters such as GLUT5 in the tumor cells. It can be concluded that the transfer of FSG was carried out by the GLUT5 transmitter to the tumor . Since, the number of these transmitters in the cancerous tissue is greater than other tissues, selective accumulation of drug in the cancerous tissue occurred.
The drug concentration in the tumor tissue increased with respect to time due to the drug release to the tumor tissue through the blood . With the accumulation of drug in the tumor, its concentration increased after 6 h of injection due to the sugar consumption of the drug by tumor cells and then, the amount of boron in the tumor tissue reduced. These results were consistent with the results obtained by other researchers regarding the excessive amount of sugar transmitters in tumor tissue . Furthermore, Table 1 shows the absolute boron concentration values for the blood, tumor and normal tissues. The boron concentration (BC) in the blood, compared with normal tissue, is often used as the cited phrase to calculate the BC in the tumor for BNCT. By injecting the drug, its concentration in the blood reached to the highest value at initial time, and by transferring drug to other tissues and organs at long duration of time, its concentration ultimately decreased and reached to the lowest level within 24 h after injection . In this article, the ratio of BC in normal tissue to that in blood, i.e. N/B ratio, remains about ~1.6 at 45 min post-administration .
Fig. 11 shows a graphical representation of the biodistribution of the 10B-drug in different tissues of the model of the animal body at different time, after injection (41.1 mg of 10Boron complex per every 30 g of the animal weight). The concentration of boron in the kidneys tissue initially increased and reached to the highest value of 17 mg/L within 3 h after injection. The kidneys contain glomeruli that are blood filters. Hence, these data are consistent with the results of Edward’s study that highlights the expression of GLUT5 in the kidney glomerular tissue. In liver tissue, the accumulation of the drug reached to the highest value. The results of the ICP analysis for heart tissue were similar to the results obtained for normal tissue due to the muscular structure of the heart. The results of drug distribution studies on spleen and lung tissues were interesting. Furthermore, the results show that the drug concentration was high at initial time after injection to the lung and spleen tissues due to the high rate of blood donation in the tissues. With respect to time, it is observed that the concentration of drug in the tissues significantly reduced and reached to its minimum value (2 mg/L) within 24 h. The concentration of the drug in the skin tissue at the initial time has reached to the highest value and then, rapidly reduced to 1.5 mg/L for duration of 3 h. This result can be attributed to the high distribution of blood capillary network inside the skin tissue . At the initial time after injection, the concentration of boron in the skin tissue increased and then, decreased with respect to time with blood circulation. The study of drug concentration in stomach tissue shows its highest concentration after 3 and 6 h of injection and then, follows descending trend to the value of 4 mg/L within 24 h after injection. The reason for high concentrations of boron in the stomach tissue can be attributed to its muscle structure and high blood donation at a time interval of 3 to 6 h. The highest amount of boron in the brain tissue occurred within 3 h after injection. The concentration of boron in the bone tissue increased to 10 mg/L after 3 h with a high rate.
Considering high importance of the tissues in the case of breast cancer, it can be concluded that the mean concentration of boron considerably decreased and its value falls within the range of 6 to 10 mg/L, similarly for tumor, lung, spleen, and skin tissues.
There are many challenging issues for the application and characterization of the detailed core-shell FSG nanostructures. Results demonstrated that the coupling of the biological and catalytic interfacial reactivity and the magnetic activity of core-shell nanoparticles is an effort to further broaden the rapidly-emerging field of core@shell magnetic. In this study, the time-course biodistribution of boron delivered by liposome-encapsulated FSG in the BALB/c mice muscle cancer model was reported for the first time. In summary, it was demonstrated that the combined administration of FSG offers the most similarity in practicable targeting of different tumors; because of these boron carriers have different uptake mechanisms and properties. Furthermore, it was possible to selectively transfer the amounts of therapeutic boron to BALB/c mice tumor and achieve ratios between tumor and blood that was compatible with the treatment. The results of ICP data indicated that the drug concentration of 35 mg in the cell culture medium maximized the accumulation of the drug for 24 h incubation after the drug introduced to the environment. Also, the highest concentration of drug (27 mg/L) in the tumor tissue occurs at a period of 45 min to 3 h due to the excessive amount of sugar transmitters such as GLUT5 in the tumor cells. In summary, the results of the present study demonstrated that the therapeutic potential of boron-bearing liposomes led to large boron biodistribution values [45-50]. The results can be considered as a guideline for further understanding boron targeting processes associated with the agents studied.
The authors gratefully acknowledge the staffs of University of Isfahan, central laboratory in Isfahan University of Technology and Nano Technology Department, Agricultural Biotechnology Research Institute of Iran for their assistance on this research.
CONFLICT OF INTEREST
The authors declare that there are no conflicts of interest regarding the publication of this manuscript.