Magnetic Mixed Micelles Composed of a Non-Ionic Surfactant and Nitroxide Radicals Containing a d-Glucosamine Unit: Preparation, Stability, and Biomedical Application

Metal-free magnetic mixed micelles (mean diameter: < 20 nm) were prepared by mixing the biocompatible non-ionic surfactant Tween 80 and the non-toxic, hydrophobic pyrrolidine-N-oxyl radicals bearing a d-glucosamine unit in pH 7.4 phosphate-buffered saline (PBS). The time-course stability and in vitro magnetic resonance imaging (MRI) contrast ability of the mixed micelles was found to depend on the length of the alkyl chain in the nitroxide radicals. It was also confirmed that the mixed micelles exhibited no toxicity in vivo and in vitro and high stability in the presence of a large excess of ascorbic acid. The in vivo MRI experiment revealed that one of these mixed micelles showed much higher contrast enhancement in the proton longitudinal relaxation time (T1) weighted images than other magnetic mixed micelles that we have reported previously. Thus, the magnetic mixed micelles presented here are expected to serve as a promising contrast agent for theranostic nanomedicines, such as MRI-visible targeted drug delivery carriers.


General
The surfactant 2 (Tween 80 (DKS, Kyoto, Japan)) was used as received. Unless otherwise noted, solvents and reagents were reagent grade and used without further purification. Tetrahydrofuran (THF) (Kanto Chemical, Tokyo, Japan) used for EPR spectroscopy or Grignard reactions was distilled from sodium/benzophenone ketyl under argon. EPR spectra were recorded on JEOL JES-RE2X (JEOL, Tokyo, Japan) and Bruker EMX Plus (Bruker Biospin, Rheinstetten, Germany). FT-IR spectrometry was performed with a Shimadzu IRSpirit instrument (Shimadzu, Kyoto, Japan) using a dry KBr powder. Elementary analysis was performed with a Yanaco CHN Corder MT-6 instrument (Yanaco, Kyoto, Japan) at the Center of Organic Elemental Microanalysis of Kyoto University. Scheme S1. Synthesis of 7n (n = 14, 16, and 18).
Similarly, 714 and 716 were prepared in 52% and 74% yields, respectively, as yellow solids. Subsequently, 718 (175 mg, 0.193 mmol) was dissolved in a mixture of H2O, MeOH and trimethylamine (2 mL each) at 25 °C and the mixture was stirred overnight at 25 °C . The resultant solution was acidified with 5 w% H2SO4 to pH 6-7 and extracted three times with CH2Cl2 (15 ml × 3). The combined organic layer was dried over MgSO4 and filtered, and the solvent was evaporated under reduced pressure. The residual solid was purified by flash column chromatography on silica gel eluting with CHCl3/MeOH (90:10 v/v) to give 418 as a yellow solid (84 mg, 0.114 mmol, 59%). Similarly, 414 and 416 were prepared in 41% and 54% yields, respectively, as yellow solids. The existence of functional groups in 4n (n =14, 16, and 18) was confirmed by FT-IR spectroscopy ( Figure  S1). The HPLC analyses of products 4n (n = 14, 16, and 18) verified the high purity of each diasteromer mixture ( Figure S2

Preparation of Magnetic Mixed Micelles 2/4n (n = 14, 16, and 18) in PBS
As a typical example, 2/414 (10 mM for each component) was prepared in PBS (FUJIFILM Wako Pure Chemical, Osaka, Japan) as follows. To a glass vial containing 414 (13.67 mg, 20 μmol) was added 3 (10 mM) dissolved in PBS (2.0 mL). The mixture was subjected to sonication (Branson Model 5800, frequency 40 kHz) (Branson, Danbury, USA) for more than 10 min at 25 °C to give a white suspension. Then the suspension was heated for more than 10 min at 90 °C using a water bath to form a clear yellow dispersion of 2/414, which was passed through a 0.45 μm membrane filter. 2/416 and 2/418 (10 mM each), and 2/414 (40 mM each) in PBS were prepared by the same procedure.

DLS Measurement (Table 1 and Figure 2)
The mean diameter of mixed micelles was determined using a UPA-UT151 instrument (MicrotrackBEL, Osaka, Japan) at 25 °C . After the samples were passed through a 0.45 μm φ disposable membrane filter, the particle size was measured in PBS using an attached cuvette. The mean diameter was calculated on volume and number weighted averages from five measurements for each sample. Only the volume average size distributions are described in this paper. The polydispersity index was not available using the present instrument.

Determination of the rotation diffusion coefficient (Dz) (Figure 4)
The EPR spectra of 2/414 (a ratio of 1:0.01 of 2-414) were recorded at 100 K and in the temperature range 263-298 K on a Bruker EMX Plus spectrometer (X-band). The variable temperature unit (Bruker) was used to control temperature. Microwave powers were chosen carefully, especially in the low-temperature measurement, to prevent the signal saturation arising from the prolonged spinlattice relaxation time of a radical magnetization vector. Modulation amplitudes were kept below 1/2 to 1/3 times of peak-to-peak line widths. For the experiment at 298 K, the samples were placed into glass capillary tubes (~1 mm φ). For the experiment at 100 K, the samples in the plastic tubes (~ 4 mm φ) were dipped quickly in liq. N2 to be frozen. The rotation diffusion coefficients and angles (beta and gamma) that define the position of the rotation axis of the probe in the g-tensor frame were determined by nonlinear-least squares simulation of the experimental ESR spectra, i.e., by minimizing the discrepancy (sum of squared deviations) between the experimental and the simulated spectra. The errors of determined parameters were estimated based on the covariance matrix at the point of minimum. The magnetic parameters of the radicals (principal values of g-and hfc-tensors) were determined independently from the spectra recorded at 100 K. The representative result of the simulation of several high-temperature spectra is presented in Figure S3. The detailed EPR fitting procedure was described in our previous paper [5].

Measurement of the Reduction Resistance of the Mixed Micelles to a Large Excess of Ascorbic Acid (Figure5)
To 2/414 (10 mM each) in PBS (1.5 mL) was added 20 equiv of ascorbic acid. The mixture was transferred to a capillary tube (1 mm φ) immediately, followed by the measurement of EPR spectra during the process of the radical reduction. The spectra were collected with the following parameters; 1.00 mW of microwave power, 0.079 mT of modulation, 0.10 s of time constant, and 120 sec of sweep time. This experiment was performed twice and the high reproducibility was confirmed.

Evaluation Method of In Vitro Cytotoxicity (Figure 6a)
HeLa cells (8,200 cells in 100 μL per well) were seeded in a 96-well tissue culture plate with Dulbecco's modified Eagle's medium (DMEM) (FUJIFILM Wako Pure Chemical, Osaka, Japan) containing 10% fetal bovine serum (FUJIFILM Wako Pure Chemical, Osaka, Japan) and 1% penicillin/streptomycin (FUJIFILM Wako Pure Chemical, Osaka, Japan) and grown for 24 h at 37 °C with 5% CO2. Then, each well was treated with a PBS dispersion (10 μL) of P2 or 2/414. The initial dispersion of the mixed micelles containing 2.5 mM of 2 and 414 and diluted dispersion of 1.2, 0.62, 0.31, 0.16, 0.078, 0.039, and 0.020 mM were applied to the cells before incubation. After incubation for 24 h at 37 °C under 5% CO2, the compounds shown above were removed and then cell toxicity was assayed using CCK-8 kit according to the manual provided by the kit manufacturer (Dojindo Molecular Technologies, Kumamoto, Japan). Toxicology Test (Figure 6b) The protocols for the animal experiments were approved by the Shiga University of Medical Science (approval number: 2018-4-1) and were carried out in accordance with the National Institutes of Health Animal Care and Use Protocol (NIH, Bethesda, MD, USA). Female ICR mice (aged 7 weeks, body weight 32-34 g) were supplied from Japan SLC Inc. (Shizuoka, Japan) and nine mice were used for the toxicological experiments. Three mice were housed per cage in climate-controlled, circadianrhythm-adjusted rooms, and were allowed access to food and water ad libitum.

In Vivo
To evaluate the toxicity of the mixed micelles to an animal, 200 μL of 2/414 (40 mM each), or controls 2/3 (40 mM) in PBS or PBS were intravenously administered to nine mice which were separated into three groups under anesthesia with 1.5% isoflurane. The body weight of each mouse (30-32 g) before injection was normalized to 100 in order to make valid comparisons, and the body weight changes of three different ICR mice were monitored for 2/414, 2/3 (employed as in vivo MRI contrast agent in our previous report [5]) and PBS over one month. The mean body weight values of three measurements each day were plotted with the standard deviation represented by error bars.

Evaluation Method of the In Vitro MRI Experiment (Figure 3)
In vitro MRI measurement was conducted on a 7-Tesla preclinical scanner (BioSpec 70/20 USR; Bruker BioSpin MRI GmbH, Ettlingen, Germany). The initial PBS dispersion of 2/4n (10 mM each) and diluted dispersions of 5.0, 2.5, and 1.2 mM were prepared. These mixed micelles were transferred to plastic tubes and fixed in a sample holder. The MR relaxometry was conducted at 25 °C . MR phantom images were obtained by a rapid acquisition with relaxation enhancement (RARE) pulse sequence with variable repetition time (TR) (echo time (TE) = 11 ms, rare factor = 2, repetition time (TR) = 5,000, 3,000, 1,500, 800, 400, 200, 100, and 50 ms, field of view = 80 × 40 mm 2 , acquisition matrix size = 256 × 128, and slice thickness = 2.00 mm). In the acquired images, the region-of-interest (ROI) was set on each tube and the mean signal intensities within the ROI were measured for the TR-varied images. T1 values were calculated by fitting a saturation recovery equation to the plot of signal intensity versus TR using the image sequence analysis tool in ParaVision 5.1 (Bruker BioSpin).

In Vivo MRI Experiment (Figure 7)
All animal procedures for in vivo MRI measurement were conducted in accordance with the guidelines of animal experiments of Kyoto University (approval number: 30−A−9). Male ICR mice (n = 2, aged 7 weeks, body weight 32-34 g; JAPAN SLC. Inc., Shizuoka, Japan) were used. After the induction of anesthesia with isoflurane, mice were placed in a cradle in a prone position. The anesthesia was kept with an inhalation of 2% isoflurane in air at 1.4 L/min through a face mask. Respiratory rate and rectal temperature were continuously measured using a pressure-sensitive respiration sensor and thermistor temperature probe, respectively, and monitored using a monitoring system (Model 1025, MR-compatible Small Animal Monitoring and Gating System; SA Instruments, Inc., NY, USA) with a dedicated software (PC-SAM V.5.12; SA Instruments). The body temperature was maintained by a flow of warm air using a heater system (MR-compatible Small Animal Heating System, SA Instruments).