METHYLENE BLUE LOADED SILICA ENCAPSULATED MAGNETITE NANOPARTICLES: A POTENTIAL DRUG DELIVERY VECTOR FOR PHOTODYNAMIC THERAPY

International Journal of Nanoscience Vol. 1, No. 1 (2007) 1–4 World Scientific Publishing Company

METHYLENE BLUE LOADED SILICA ENCAPSULATED MAGNETITE NANOPARTICLES: A POTENTIAL DRUG DELIVERY VECTOR FOR PHOTODYNAMIC THERAPY NIDHI ANDHARIYA Department of Physics, Bhavnagar University Bhavnagar, Gujarat 364022, India [email protected] BHUPENDRA CHUDASAMA School of Physics & Materials Science, Thapar University Patiala, Punjab 147004, India [email protected] R V UPADHYAY P.D. Patel Institute of Applied Sciences, Charotar University of Science & Technology Changa, Gujarat 364022, India [email protected] R V MEHTA Department of Physics, Bhavnagar University Bhavnagar, Gujarat 364022, India [email protected] Received Day Month Year Revised Day Month Year In this article, we describe synthesis of a novel drug delivery vector (DDV) for photodynamic therapy (PDT). The DDV consists of a magnetite core surrounded by a thin layer of functionalized silica. These core-shell structures are loaded with a photosensitizer (PS) drug “Methylene Blue” (MB). Magnetite nanostructures are produce by the well-established chemical co-precipitation technique and encapsulated in silica shell by modified process of hydrolysis and condensation of tetraethyl orthosilicate (TEOS). MB is grafted into the pores of silica shell by demethylation reaction. Reaction kinetics has been established for tunable loading of PS in DDV. Physical and chemical properties of composite nanostructures are determined by X-ray diffraction (XRD), dynamic light scattering (DLS), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR) and vibrating sample magnetometry (VSM). Amount of PS loading in DDV is measured by UV-visible spectroscopy. Smaller size, biocompatibility, tunable loading of PS and capabilities of magnetic guidance, makes this DDV, a potential candidate for the treatment of malignant tumors by PDT.

Keywords: Photodynamic therapy; magnetite, photosensitizer, core-shell nanostructures, silica

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1. Introduction One of the concerns of public about the modern day medicine is „side effects‟. Therapies targeting only the diseased cells are the solution for this1,2. The targeted delivery of drugs is one of the main challenges that all cancer therapies are dealing with35 . Most of the cancer therapies extend the life of patients by few months instead of really targeting the cancer cells and cure them6. Photodynamic therapy is a noninvasive medicinal modality for the treatment of cancers7-10. The therapy can be applicable to both neoplastic and non-neoplastic disease7,8. PDT is based on the concept that certain therapeutic molecules called photosensitizers can be preferentially localized in malignant tissues, and when these PSs are activated with appropriate wavelength of light, they pass on their excess energy to surrounding molecular oxygen. This results into the generation of reactive oxygen species, such as free radicals and singlet oxygen (1O2), which are toxic to cells and tissues11-14. This leads to a number of biological effects including damages to proteins, nucleic acids, lipids, and other cellular components. This often results into cell death and possible activation of the immune system14-16. Its advantage lies in the inherent dual selectivity. First, selectivity is achieved by a preferential localization of PS in the target tissue, and second, the photo-irradiation and subsequent photodynamic action can be limited to a specific area of interest. Because the PS is nontoxic without light exposure, only the irradiated areas will be affected, even if the PS does infiltrate normal tissues. Considerable work has been done in the field to understand the process and to maximize the efficacy using animal models14. These pre-clinical and clinical studies recently resulted in the approval of first PS drug (Photofrin)for the treatment of selected tumors17. Equal biodistribution of PS in the body after intravenous injection significantly lowers the performance of PS in PDT as the PS dose, time window for treatment (after injection of PS) and energy density depend on the balance between concentration of drug in tumor tissues and in normal tissues at the time of light irradiation7. If PS is loaded in a DDV, which can be effectively targeted towards a tumor site, and have preferential binding ability with tumor tissues then it will cause absolute damage to tumor cells when exposed to light. Hence, the

required dose of PS and time of treatment would decrease significantly. The aim of the present research is to design and fabricate a magnetic drug delivery vector, loaded with a PS drug MB, which can effectively be guided to a desired sight by means of external magnetic field. 2. Materials and methods 2.1 Synthesis of MB loaded silica encapsulated magnetite nanoparticles Chemicals: FeCl3.6H2O and FeSO4.7H2O were obtained from s.d. fine-chem Ltd. Ammonium hydroxide solution (25% NH3), HPLC grade water, acetone and methylene blue were purchased from Merck. Absolute ethanol was procured from Hayman Ltd. TEOS (99%) and tetramethylammonium hydroxide (TMAH) were obtained from Aldrich. Aqueous solutions were prepared in HPLC grade water. Magnetite nanoparticles were synthesized by coprecipitation technique18,19. Stoichiometric aqueous mixture of ferric (5 mM) and ferrous (2.5 mM) ions were prepared using ferric and ferrous salts. To this 20 mM aqueous solution of NH4OH was added drop wise under continuous stirring. The pH of the slurry was maintained at 10. After continuous stirring for 20 min at room temperature (300 K), the black precipitates were magnetically decanted and washed several times with warm water. Finally, water wet slurry of nanoparticles was equally divided in two parts. One part was preserved in ethanol, while the other was dispersed in a mixture, containing 30 mL methanol and 15 L TMAH by ultrasonication. At the same time, an ethanol-water mixture was prepared by diluting 240 mL absolute ethanol with 60 mL HPLC grade water. TEOS (0.05 mL) was added under vigorous stirring and pH was adjusted to 11 with NH4OH. After 20 min of sonication, both suspensions were mixed in a round bottom flask. 10 mL aqueous suspension of MB (2.67 mM) was added and pH was again adjusted to 12 with the help of NH4OH. The demethylation reaction was carried out for 22 h at 300 K. MB loaded Fe3O4-SiO2 core-shell nanostructures have been isolated from the solution by magnetic decantation. This DDV were washed with absolute ethanol until traces of MB were observed in UV-visible spectrum of supernatant. Same experiment was repeated by varying the pH of the demethylation reaction from 11 – 13 to understand its effect on the loading of MB in DDV.

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2.2 Characterization of DDV

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Structural investigation of DDV was carried out on Bruker D8 Advanced X-ray diffractometer. TEM image was recorded on JEOL (model GEM 200) transmission electron microscope. Hydrodynamic size of samples were determined by DLS. These measurements were carried out on Microtrac particle size analyzer (model Nanotrac). Magnetic properties were determined at 300 K on a indigenously build VSM. UV-visible spectroscopy was used to confirm the loading of drug in nanostructures. Spectrums were recorded on Elico biospectrophotometer (model BL 198).

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3. Results and discussions

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X-ray diffractograms of magnetite nanoparticles and MB loaded DDV are shown in Figure 1. Six peaks are observed in the diffractogram of magnetite nanoparticles, which corresponds to the face centered

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Figure 1. X-ray diffraction pattern of (a) magnetite nanoparticles and (b) MB loaded DDV inverse spinel structure. The space group of the system is Fd3m and lattice parameter calculated from the highest intense reflection (311) is 8.37 Å. Results are consistent with those reported in the powder diffraction database (PDF card No #751610). Assuming the first order diffraction, the crystallite size obtained from the highest intense reflection (311) using classical Sheerer formula is 8.4 nm. X-ray diffraction pattern of DDV (figure 1b) is almost similar to that of magnetite nanoparticles with a marked difference at 23. A broad hump is observed at 23. This hump is the characteristic signature of the presence of amorphous silica in nanostructures20. It is not possible to determine the size of DDV from the line broadening as silica is in the amorphous state.

50 nm Figure 2. TEM image of (a) bare Fe3O4 nanoparticles and (b) MB loaded DDV. Inset shows the size distribution histogram obtained from DLS measurements. TEM image of bare Fe3O4 nanoparticles and MB loaded silica encapsulated magnetite nanospheres is shown in figure 2. Image shows that DDV has nearly spherical morphology. Size distribution histogram of DDV obtained from DLS measurements is also shown as an inset in figure 2b. The average size and polydispersity index obtained from DLS are 49.8 nm and 0.16, respectively. Magnetization of magnetite nanoparticles and MB loaded DDV as a function of applied magnetic field is shown in figure 3. The saturation magnetization (Ms) of magnetite nanoparticles is 64 emu/g, which is 70% of its bulk value (92 emu/g). Reduction in Ms might be due to the decrease in particle size accompanied by an increase in surface area, and is consistent with the results observed by Mohapatra et al.21. The Ms value of MB loaded DDV is 35 emu/g. The observed 45% decrease in Ms of DDV with respect to magnetite nanoparticles is mainly due to the presence of diamagnetic shell of silica on magnetite nanoparticles and partially due to the encapsulated MB. No remanence or coercivity has been observed, which indicates that both magnetite nanoparticles and MB loaded DDV are superparamagnetic. The observed magnetization for DDV is considerably higher than reported by other groups20-23. This might be due to the prevention of creation of clusters of magnetite and exclusion of formation of structures of free silica during hydrolysis and condensation.

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This article describes synthesis protocols of a novel DDV for PDT having capabilities of magnetic guidance and tunable loading of PS.

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Figure 3. Magnetization curve of (a) magnetite nanoparticles and (b) MB loaded DDV

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Encapsulation of MB in DDV is confirmed by UVvisible spectroscopy. Figure 4 shows the UV-visible spectrum of MB loaded DDV. A broad hump centered at 657 nm is observed, which corresponds to the characteristic absorption of MB molecule in water. The effect of pH on the drug loading is shown in figure 5. Drug loading capacity was determined by the method explained by Zhang et al24. As can be seen from the figure, loading of MB in DDV increases as the pH increases, attains a maxima at pH 12

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