Poly(ethylene glycol)-poly(D,L-lactide) (PEG-PLA, PEG and PLA M.W.=5 kD) was synthesized following the reported method . The molecular weight and polydispersity of PEG-PLA were also characterized by gel permeation chromatography and 1H-NMR. Doxorubicin-HCl for injection (2 mg/ml) was purchased from Bedford Laboratories (Adriamycin®, Bedford, OH), and treated with triethylamine to obtain the hydrophobic Doxo. Phenyl ether (99%), benzyl ether (99%), 1,2-hexadecanediol (97%), oleic acid (99%), oleylamine (>70%), tetrahydrofuran (THF), hexane, dimethyl sulfoxide (DMSO), and iron(III) acetylacetonate were purchased from Sigma-Aldrich Chemical Co. (Saint Louis, MO). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-5000] (ammonium salt) (DSPE-PEG) was purchased from Avanti Polar Lipids, Inc. (Alabaster, Al). All organic solvents are of analytical grade.
Preparation of Doxo-SPIO-micelles
Doxo-SPIO-micelles were fabricated according to the published procedure [18, 23]. In brief, highly crystalline and monodisperse SPIO were synthesized from Fe(acetyl acetonate)3 in an aryl ether . For the micelles, PEG-PLA was used as surfactant molecules for the encapsulation of SPIO and Doxo. Subsequently, SPIO with 7 nm diameter and Doxo were loaded into the polymeric micelles by a solvent-evaporation method . SPIO and Doxo were dissolved in 800 µl of THF and 200 µl of DMSO. Then, THF and DMSO were mixed together, and this mixture was added into water (3 ml) under sonication (LH700S, ULSSO HITECH Co.). After the sonication, the solution was shaken overnight to allow the organic solvent to evaporate. Finally, Doxo-SPIO-micelles were purified by Millipore centrifuge filtration (MW cut off: 30kD). The weight ratios (mg) of PEG-PLA:SPIO:Doxo were varied over three values: 10:2.5:2; 10:5:2; and 10:10:2.
Characterization of Doxo-SPIO-micelles
The hydrodynamic diameter (DH) of the Doxo-SPIO-micelles was estimated by dynamic light scattering method (DLS, Nano-ZS90, Malvern Panalytical Co. United Kingdom). Doxo-SPIO-micelles, made with different ratios of PEG-PLA:SPIO:Doxo (10:10:2, 10:5:2, 10:2.5:2), were introduced to DLS.
Transmission electron microscopy (TEM) imaging
To confirm the size and morphology of Doxo-SPIO-micelles, transmission electron microscopy (TEM) images for nanoparticles were obtained using an FE-TEM (JEM 2100F, JEOL Co. Japan). For Doxo-SPIO-micelles, formvar coated-copper grids were glow discharged using a vacuum coating unit. Each sample solution was dropped on to the glow discharged grid. After 2 minutes of standing, the excess solution was removed by blotting the grid against a filter paper. Negative staining (dark field) of sample was done by additional dropping of 2% phosphotungstic acid (PTA) solution to the grid. For SPIO, TEM samples were prepared by allowing a small drop of SPIO suspension in hexane to dry on carbon-coated copper grids without negative staining. All TEM images were obtained at an accelerating voltage of 120 kV.
Determination of drug loading content (DLC)
The DLC of the micelle was determined by UV-VIS analysis. At first, micelle solutions were frozen and lyophilized to yield the solid micelle samples. Then the dried samples were weighed and re-dissolved in a mixture of chloroform and DMSO (1:1, v/v) under bath-sonication for 30 minutes. The suspending SPIO were centrifuged down, and upper layer of solution was transferred to UV-VIS spectrometer (Spectra Max Plus 384 Microplate Reader, Molecular Devices, USA). The absorbance at 480 nm was measured to determine the Doxo content in the solution with a previously established calibration curve. The weight % of Doxo, entrapped in the core of micelle, was calculated from the dried weight of Doxo-SPIO-micelles and the amount of Doxo incorporated.
Estimation of thermodynamic properties of Doxo-SPIO-micelles
Critical micelle concentration (CMC) determination
The CMC of the polymer was estimated to investigate the effect of SPIO on the micelle stability. Following the reported method, a solution of pyrene in dichloromethane was dried by a stream of nitrogen . An aqueous solution of micelles was subsequently added to the dried film followed by gentle shaking for 72 h at 37°C. The final concentration of pyrene waFIG.0 × 10−8 mol·L−1 while that of PEG-PLA was varied from 0.1 to 100 µg·mL −1. The CMC was calculated by plotting the fluorescence intensity of pyrene (λem = 390 nm, λex = 333 nm) as a function of PEG-PLA concentration.
Evaluation of dissociation kinetics of Doxo-SPIO-micelles
A dissociation of micelles at a concentration below the CMC (100 µg·mL−1 and 8 µg·mL−1, respectively) was also evaluated. SPIO-loaded micelles and SPIO-free micelles encapsulating pyrene were prepared in HEPES buffer pH 7.4 at the polymer concentration of 1.5 mg·mL−1 according to the previously reported method. After micelle formation, the micelle solution was diluted, and the fluorescent intensity (λex = 333, λem = 390) was recorded at 37°C over time.
Estimation of thermodynamic fluidity of micelle core
In this study, we uploaded 1,3-di(1,1´-pyrenyl) propane (DPP) dye in the micelle core and estimated the molecular state of DPP (i.e., excimer formation) to examine the microenvironmental fluidity of the micelle core. DPP was dissolved in chloroform, dried, and reconstituted with ethyl acetate. Then DPP was mixed with the SPIO-loaded or SPIO-free micelles for 72 hrs under N2 gas. The final concentration of DPP was 1.8 × 10−7 M, while the polymer concentration was 100 µg·mL−1, above the CMC. The emission intensity of DPP excimer at 478 nm (Ie) and monomer at 377 nm (Im) was measured as a function of temperature (λex = 333 nm).
In vitro drug release study
Doxo-SPIO-micelles were transferred into dialysis tubes (MW cut-off: 50,000 Da, Spectrum Laboratories, USA). The tubes were immersed in 25 ml PBS (pH 7.4) or acetate buffered saline (pH 5.0) solutions. The release of Doxo from micelles was tested under mechanical shaking (100 rpm/min) at 37°C. At selected time intervals, buffered solution outside the dialysis bag was removed to estimate the released amount of Doxo and replaced with fresh buffer solution. Doxo concentration was calculated based on the fluorescence intensity (ex; 470 nm, em; 590 nm) with a previously established calibration curve. The error bars were obtained from triplicate samples.
Estimation of MR imaging properties of Doxo-SPIO-micelles
Estimation of magnetization properties of Doxo-SPIO-micelles
To help understand the MR properties, we measured the magnetization of Doxo-SPIO-micelles. Samples were prepared by pipetting 5 µL of solution containing Doxo-SPIO-loaded micelles into a glass cell (4.0 x 4.0 x 0.4 mm) and attached to the probe with a small amount of silicon grease. Magnetization measurements were conducted using an alternating gradient magnetometer (AGM, Princenton Measurements) at room temperature and up to 1.4 T.
Estimation of T2 relaxivity of Doxo-SPIO-micelles
MR sensitivity of Doxo-SPIO-micelles was measured at 0.55 T (23.4 MHz) on a Resonance Maran Ultra scanner at 37 oC (Oxford instruments, UK). T2 relaxation rates (1/T2, s−1) were measured using a CPMG (Carr Purcell Meiboom Gill) pulse sequence. T1 relaxation rates (1/T1, s−1) were measured using the INVREC pulse sequence with TE=5×T1. Linear regression analysis of 1/T1 vs total iron concentration yielded the T1,2 relaxivity (r1,2).
Phantom MR imaging of Doxo-SPIO-micelles
To evaluate the clustering effect of SPIO in the micelle on MR sensitivity, we measured T2 relaxation rates (1/T2, s−1) using a spin echo pulse sequence with TR = 6 s and TE varied from 9 ms to 150 ms (n = 8) on a 4.7 T Varian INOVA scanner. Linear regression analysis of 1/T2 vs. total metal concentration yielded the T2 relaxivity (r2).
Cellular uptake behaviors of Doxo-SPIO-micelles
Cellular uptake of different formula of Doxo-SPIO-loaded micelle was evaluated through flow cytometry analysis using H1299 non-small cell lung carcinoma cells. H1299 cells were seeded in 6-well plates (300,000 cells/well) in 2 ml DMEM with 10% FBS and incubated for 24 hrs, followed by co-incubation with Doxo-SPIO-loaded micelles at the Doxo concentration of 10 µg/ml for 2hrs. After the treatment, the cells were washed three times with PBS.
In vivo pharmacokinetic and biodistribution study
The enhanced thermodynamic stability of micelles through incorporation of SPIO was clearly distinguished from above experiments. In vivo pharmacokinetic stability of Doxo-SPIO-micelles was also identified in our experiment. Doxo-SPIO-micelles, Doxo-micelles and free Doxo were injected via a lateral tail vein into 20–22 g of BALB-C mice at a normalized dose (2.5 mg·kg−1 as Doxo). 50 uL of blood was collected from the retroorbital plexus and immediately centrifuged at 2000 g for 2 min. Doxo concentration in plasma was measured using a microplate fluorescence reader (Spectra Max M5, Molecular devices, CA) after organic solvent extraction .
For the body distribution study, each organ was collected and washed with the PBS after sacrificing of mice treated with Doxo-SPIO-micelles, Doxo-micelles, and Doxo (control). For quantification of Doxo content in the kidneys, liver, lungs, spleen, muscle and heart after the injection, mice were euthanized for imaging. Organs were collected, washed with PBS. Organs collected from 3 of Doxo treated mice served as control. Fluorescence image was performed and analyzed with in vivo imaging system (FOBI, NeoScience Co. Republic of Korea).
Quantitative data were presented as the mean±standard deviation and comparisons were carried out using t-text analysis. Statistical significances were described at (*P<0.1, **P<0.05, ***p<0.01).