All reagents used in the present investigation were of analytical grade. FeCl3 anhydrous (99.98%), propylene glycol and diethyl amine were provided by Fisher Scientific (Pittsburgh, PA, USA) and used as received. Rhodamine B and methotrexate were provided by Merck Chemicals (Beeston, Nottingham, UK) and used without further purification. Pure ethyl alcohol and tetraethyl orthosilicate (TEOS) were provided by Aldrich Chemical Co. MilliQ (St. Louis, MO, USA)water (18.2 MΩ.cm at 25°C) was used for all experiments.
Synthesis of hollow magnetic Fe3O4 microspheres
For a typical synthesis, 2 mmol of FeCl3 were dissolved in 10 mL of propylene glycol; the solution was magnetically stirred at room temperature for 10 min followed by soft ultrasonic treatment for 5 min. Next, 1 mmol of ferrocene was added, and the solution was stirred at room temperature for 2 h. Finally, 3 mL of diethyl amine were added to the mixture. The solution was placed into the Teflon-lined stainless steel autoclave of 30 mL capacity and maintained at 180°C for 24 h. Lower synthesis temperatures (100 to 150°C) were tested although the synthesized hollow magnetite microspheres were not stable, and the degradation was produced in a few days, generating compact magnetite nanoparticles of no more than 10 nm diameter. The use of FeCl3/ferrocene as precursor is required to induce a faster self-assembling of nanoparticles into hollow microspheres.
After cooling to room temperature, the black sediment is collected and washed in water by five centrifugation (6,000 rpm, 10 min)-redispersion cycles. Next, the black sediment was resuspended in ethanol and washed by two centrifugation (6,000 rpm, 5 min)-redispersion cycles. Finally, the microspheres were suspended in ethanol and dried overnight at 60°C. Hollow Fe3O4 microspheres were maintained in sealed containers before characterization.
Preparation of magnetic SiO2@Fe3O4 microspheres
The schematic procedure used to synthesize SiO2@Fe3O4 hybrid hollow microspheres is illustrated in Figure , and the detailed synthesis is described as follows.
Schematic representation of the synthesis of SiO2@hollow magnetite microspheres.
The silica shell has been prepared by using a modified Stobe-Fink-Bohn method [33
], which consists of two steps: (i) the hydrolysis of TEOS (Si(C2
) in ethanol, in presence of ammonium hydroxide as catalyst, and (ii) the polymerization phase, where the siloxane (Si-O-Si) bonds are formed and anchored on the hollow magnetite surface. In a typical synthesis, 50 mg of hollow Fe3
microspheres were added to 20 mL of ethanol. The mixture was homogenized in an ultrasound bath for 10 min. Next, 0.5 mL of deionized water and 0.5 mL of ammonium hydroxide (36%) were added into the flask under vigorous mechanical stirring to prevent particles from settling. Temperature was termostatized at 20°
C for at least 20 min, and after this period, 0.5 mL of TEOS was added dropwise to the reaction mixture over the course of 5 min under constant stirring in fumehood. After addition, reaction mixture was vigorously stirred for 30 min. Next, the solvent of the reaction mixture is evaporated at 60°
C overnight. The residue is washed twice in distilled water and finally in ethanol and allowed to dry in vacuum at room temperature.
Encapsulation efficacy within hollow microparticles
To evaluate the potential application of these hollow spheres as drug carriers, three different infiltrations of an organic compound were tested. Rh-B was chosen as test molecule for different reasons. First of all, the size of this molecule is not too small (as occurs with the most of drugs used for therapeutic treatments). On the other hand, this compound is soluble in polar solvents and shows a very high fluorescence quantum yield that can be useful to evidence the presence of very low amounts of this compound, even at trace levels. In this way, Rh-B at three different concentrations (0.05, 0.1, and 0.2 mg/mL) in ethanol were prepared. The infiltration consisted in adding the appropriate amount of Rh-B solution into an erlenmeyer flask containing 5 mg of hollow particles. This mix was mechanically stirred, and the result for each Rh-B concentration was evaluated at different time period, namely 1, 2, 4, 6, 8, 9, and 12 h. These infiltrations were developed at 20°C and 40°C. After infiltration, the particles were centrifuged (1,000 rpm, 5 min) and washed three times with MilliQ water. The particles loads with Rh-B were dried overnight at 60°C. To determine the amount of Rh-B storage in the hollow microspheres, thermogravimetry (TG) was employed to directly measure the weight loss of as-prepared product.
The release kinetics was studied in aqueous solution by controlling the pH and temperature of the solvent by the dialysis bag method. The dialysis bag was soaked in water for 3 h before use. The dialysis bag retained the magnetite microspheres allowing free Rh-B to diffuse into the solution of study. To monitor the Rh-B release by pH and temperature effect, solutions at different times were analyzed by fluorescence spectroscopy. The pH of solution was adjusted using acetate 0.01 M (pH = 3.7) and phosphate 0.01 M (pH = 7.4) buffers. All load and release tests developed on Rh-B were also tested on MTX, a drug used in some cancer treatments.
X-ray powder diffraction patterns (XRD) were collected using an X'Pert PRO X-ray diffractometer (PANalytical, The Netherlands) in Bragg-Brentano goniometer configuration. The X-ray radiation source was a ceramic X-ray diffraction Cu anode tube type Empyrean of 2.2 kW.
Raman spectra were collected using a micro-Raman Renishaw RM2000 single grating spectrograph, equipped with 532 and 785 nm excitation sources. Raman spectra were acquired in the spectral range of 3,200-100 cm-1. The acquisition time for each measurement was 20 s and a defocused laser power level in the range of 10 to 60 mW was used to prevent the possible thermal effects on the samples. Infrared spectra were obtained in the 4,000-400 cm-1 region by using a Bruker Optics IFS 66 series FT-IR spectrometer (Bruker Optik Gmbh, Ettlingen, Germany). The variation in magnetization and coercivity of the hollow magnetite samples was determined by using a Lake Shore-7400 vibrating sample magnetometer (VSM) (Lake Shore Cryotronics Inc, Westerville, OH, USA) at room-temperature.
Field emission scanning electron microscopy (FE-SEM) images were obtained using a JEOL JM-6400 microscope. High Resolution Transmission electron microscopy (HRTEM) images were recorded on a JEOL 3000 with an acceleration voltage of 300 kV.
X-ray photoelectron spectroscopy (XPS) measurements were performed on an ESCALAB 220i-XL spectrometer (VG-Scientific, East Grinstead, UK, by using the non-monochromated Mg Ka (1,253.6 eV) radiation of a twin-anode, operating at 20 mA and 12 kV in the constant analyzer energy mode, with a PE of 50 eV. In order to remove charging shifts and deal with Fermi edge coupling problems, binding energies were corrected using the peak of the C-(C, H) component coming from contamination carbon (set to 284.6 eV). The samples were pressed on to a molybdenum support in an argon-filled glove box and then were put into the preparation chamber to pump for approximately 24 h at 60°C under a pressure of about 10-7 Pa to minimize surface contamination. Small amounts of activated carbon fine powder were added to the samples to improve their conductivity. The vacuum during spectra acquisition was better than 5 × 10-9 mbar.
TG analysis data were obtained with a TGA Q-500 instrument (TA Instruments)under inert atmosphere of nitrogen at a heating rate of 20°
, from 100 to 600°
C. The specific surface area, the pore volume, and the pore size distribution of the hollow magnetite microspheres, were measured using a Micromeritics ASAP 2020. The micropore volume, WMP [cm2
/g], was measured using the Barrett-Joyner-Halenda (BJH) approach [35