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

Abstract

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.

1. Introduction

Non-invasive imaging of living tissue is of great importance in the medical field. The magnetic resonance imaging (MRI) method is one of the most frequently used and important imaging techniques in clinical medicine. In fact, the use of MRI contrast agents plays a crucial role in accurately evaluating physiological and pathological changes. The majority of MRI contrast agents approved by the US Food and Drug Administration (FDA) are gadolinium-based contrast agents (GBCAs) such as Magnevist (a Gd^III complex agent) [1,2,3]. Although they are used on a daily basis, this modality still faces many challenges [4,5,6,7,8]. For example, people with moderate to advanced kidney failure are in danger of developing nephrogenic systemic fibrosis through the use of GBCAs. Thus, it is urgently required to exploit novel agents that exhibit adequate contrast enhancement with a very low risk.

Metal-free magnetic nanoparticles containing nitroxide radicals as a spin source have attracted great interest since the 1980s [9] because of their lack of toxicity, despite the imaging ability being less compared to Gd^III complex agents [10] and their having less reduction resistance to antioxidants such as ascorbic acid and glutathione [11]. However, the reduction resistance should potentially improve through the molecular design and/or the micelle construction of nitroxide radicals [12,13,14,15,16,17]. In this context, we have recently prepared metal-free magnetic mixed micelles comprised of a surfactant, Brij 58 (1) or Tweens 80 (2), and pyrrolidine-N-oxyl radical 3, namely 1/3 or 2/3 (Figure 1), according to a simple experimental procedure [18,19]. These micelles showed high colloidal stability, reduction resistance to ascorbic acid, and contrast enhancement in the T₁-weighted MRI in phosphate-buffered saline (PBS) in vitro and in vivo. The mixed micelle 2/3 was found to be much less toxic than 1/3. Furthermore, additional hydrophobic fluorophores or drugs were stably encapsulated inside the mixed micelles. Although passive targeting can be expected due to the micelle size (10–20 nm), the micelles that we prepared did not possess any active targeting site for tumor.

Herein, we report on the novel metal-free mixed micelles including nitroxide radicals 4_n conjugated with a d-glucosamine unit as a tumor targeting site, because d-glucosamine derivatives are well-known to accumulate in tumor cells [20,21,22,23]. The obtained magnetic mixed micelles showed little toxicity, excellent in vitro MRI contrast ability, and high stability in the presence of an excess amount of ascorbic acid. When applied to in vivo imaging for healthy mice, bright MRI contrast enhancement was observed in the liver.

2. Results and Discussion

2.1. Preparation, Stability and In Vitro MRI Contrast Ability of 2/4_n

The nitroxide radicals 4_n (n = 14, 16, and 18 in Figure 1, Figures S1 and S2) were synthesized by condensation of the racemic benzoic acid derivatives of the nitroxide radicals (a 1:1 mixture of (R,R) and (S,S) enantiomers) [24,25] and d-tetraacetylglucosamine [26,27,28], followed by deacetylation (Schemes S1 and S2 in the Supplementary Information).

The mixed micelles 2/4_n (Figure 1) were prepared at a concentration of 10 mM for each component in the PBS according to the procedure described in the Supplementary Information. The stability of the micelles was found to depend on the length of the alkyl chain (n = 14, 16, and 18) in the radicals 4_n (

Table 1. Mean diameters and colloidal stability of the mixed micelles 2/4_n (n = 14, 16, and 18) in phosphate-buffered saline (PBS) at 30 °C.

Micelle	2/414	2/416	2/418
Diameter by DLS	16 nm a	13 nm a 92 nm c	14 nm a 45 nm c
Colloidal stability	Dispersion a	Dispersion a	Dispersion a
	Precipitates b
	Dispersion d	Dispersion c	Dispersion c

^a Immediately after preparation. ^b After 24 h of preparation. ^c After 6 days of preparation. ^d After heating the precipitates.

and Figure 2). The 2/4₁₆ and 2/4₁₈ were formed as a clear dispersion immediately after preparation and their mean diameter gradually increased up to 92 and 45 nm after one week, respectively (Table 1). The micelle 2/4₁₄ collapsed within one day to give white precipitates of 4₁₄ after 24 h. From these results, summarized in Table 1, the relative stability of the micelles 2/4_n in PBS was in the following order: 2/4₁₈ > 2/4₁₆ > 2/4₁₄. The similar dependence of the micellar stability on the alkyl chain length in the nitroxide radicals 4_n was also observed in the cases of the mixed micelles 1/3 and 2/3 [18,19]. The mean diameters of the resulting magnetic mixed micelles 2/4_n in PBS were determined to be 13 to 16 nm by DLS analysis (Table 1 and Figure 2). Their mean diameters fell in a range of 10–100 nm, which is required for the most prolonged blood circulation time.

Importantly, the once-precipitated sample of 2/4₁₄ was revived to the original clear dispersion with the same diameter (16 nm) by just heating it with full reproducibility (Table 1). The micelle 2/4₁₄ turned out to be easily available as a clear dispersion even after the long-term preservation of the precipitated sample.

The dependence of the alkyl chain length in 4_n on the longitudinal relaxivity (r₁) of 2/4_n was determined from the relaxation time (T₁) as a function of the concentration at 25 °C by using an MRI machine at 7.0 T. Sufficiently bright T₁-weighted MR phantom images were obtained at a concentration of 10 mM of the magnetic mixed micelles 2/4₁₄, 2/4₁₆, and 2/4₁₈ as compared with that of the control PBS (panel A, E, and I in Figure 3a). This result implies that 2/4_n may show a distinct MRI contrast enhancement in vivo in this concentration or higher. The linear regression analysis yielded r₁ = 0.14, 0.13, and 0.11 mM⁻¹s⁻¹ for 2/4₁₄, 2/4₁₆ and 2/4₁₈, respectively (Figure 3b). That is, the MRI contrast ability of the micelle 2/4_n in PBS was in the following order: 2/4₁₄ > 2/4₁₆ > 2/4₁₈. The mixed micelle 2/4₁₄ was used for further experiments for the following two reasons: (1) 2/4₁₄ exhibited superior in vitro MRI-enhanced ability to those of 2/4₁₆ and 2/4₁₈, and (2) the clear dispersion was fully revived in a reproducible manner by simply heating the precipitated sample. Although the stability of the 2/4₁₄ was less than that of 2/4₁₆ and 2/4₁₈ as mentioned above (Table 1), we gave priority to the MRI-enhanced ability over the stability.

These r₁ values were much larger than those of micelles 2/3 and 1/3 (r₁ = 0.07, and 0.09 mM⁻¹s⁻¹, respectively, at 7.0 T), which we reported previously, although they were much lower than those of Gd^III complex agents [6]. These experimental results could be interpreted in terms of the slower rotation diffusion of 4₁₄ than that of 3 inside the mixed micelles [29,30,31]. In order to compare the rotation diffusion mobility between radicals 4₁₄ and 3 inside the micelles, the electron paramagnetic resonance (EPR) spectra of radical 4₁₄ or 3 in the micelles consisting of a 1:0.01 molar ratio of surfactant 2 and 4₁₄ or 3 were measured in the temperature range 263–298 K and then were numerically simulated as described in the Supplementary Information (Figure 4 and Figure S3, and Table S1). The temperature dependence of the rotation diffusion mobility was successfully described by Arrhenius law with the values of activation energy (E^a_z) shown in

Table 2. The effective activation energy of the rotation diffusion of 4₁₄ in 2/4₁₄ and 3 in 2/3.

Mixed Micelle	Eaz/kJmol−1
2/414	21.1 ± 1.0
2/3a	18.4 ± 0.4

^a Previously reported value [19].

, indicating that 4₁₄ showed slower rotational diffusion inside the micelle to produce a highly enhanced MRI compared to 3. The better r₁ of 2/4₁₄ than 2/4₁₆ and 2/4₁₈ mentioned above might be interpreted by the slower rotation diffusion mobility of 4₁₄ than 4₁₆ and 4₁₈.

2.2. Reduction Resistivity of 2/4₁₄ in the Presence of Ascorbic Acid

The concentration of ascorbic acid in the healthy adult serum was reported to be kept in the range of 14.9–52.8 μM by a daily intake of ascorbic acid (60 mg) [32]. When nitroxide radicals were applied to the in vivo MRI measurement, radical reduction occurred and resulted in a significant decrease in the MRI contrast [33,34,35]. For example, 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) derivatives, such as 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPONE) and 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL), were reduced rapidly (half-life (τ_1/2) < 2 min) to the corresponding hydroxylamines in the presence of ascorbic acid [36]. In our molecular design, we expected that the interplay between four long hydrophilic tails in 2 and four neighboring substituents in 4₁₄ should enhance the reduction resistance to ascorbic acid sterically. The decay of 4₁₄ in 2/4₁₄ in response to a large excess of ascorbic acid (20 equiv based on 4₁₄) in PBS was monitored by EPR spectroscopy (Figure 5). As expected, the τ_1/2 of 2/4₁₄ (30 min) was almost comparable to that of 2/3 (33 min) and much longer than that of 1/3 (7 min) [19].

2.3. Biomedical Application of 2/4₁₄

Since biocompatibility is a prerequisite of using the magnetic mixed micelles as an MRI contrast agent, the cancer cell viability of 2/4₁₄ was assessed by the CCK-8 assay at the initial concentration of 2.5 mM for 2 and 4₁₄ and compared with those of pure micelle of 2, designated as P2, (Figure 6a). Both P2 and 2/4₁₄ exhibited little cytotoxicity to HeLa cells at concentrations up to 2.5 mM, demonstrating that 2/4₁₄ is an appropriate candidate for in vivo experiment. In addition, the body weights gradually increased in the healthy Institute of Cancer Research (ICR) mice over one month after injection of 2/4₁₄, 2/3 and PBS (Figure 6b). It was concluded that mixed micelles 2/4₁₄ can serve as a bio-compatible MRI contrast agent similar to 2/3.

Finally, the in vivo MRI experiment using 2/4₁₄ was performed for healthy ICR mice. Bright MRI contrast enhancement was observed in the liver in both coronal and sagittal planes over 1 h with high reproducibility (Figure 7). This result reveals that the magnetic mixed micelle 2/4₁₄ is effective as an in vivo T₁-weighted MRI contrast agent. The prolonged MRI enhancement observed for 2/4₁₄ is attributed to the high resistance to reducing agents as described above.

3. Conclusions

We prepared highly robust and biocompatible metal-free magnetic mixed micelles which are composed of non-ionic surfactant 2 and hydrophobic nitroxide radical 4_n in PBS. The time-course stability and in vitro MRI contrast ability of the mixed micelles was found to depend on the length (n) of the alkyl chain in the nitroxide radicals. In addition, the mixed micelle 2/4₁₄ showed a considerable reduction resistance to a large excess of ascorbic acid, little toxicity, and sufficient contrast enhancement in the T₁-weighted MRI in vivo. Such highly biocompatible magnetic mixed micelles composed of nitroxide radicals bearing a d-glucosamine unit are expected to be utilized as a low-molecular-weight cancer targeted MRI contrast agent in line with the theranostic applications of micelles, which have recently been attracting increasing interest [37,38,39].