Design and Preparation of a Nanoprobe for Imaging Inflammation Sites
© The Author(s) 2012
Received: 6 September 2011
Accepted: 22 November 2011
Published: 9 February 2012
To image inflammation sites, we developed a novel nanoparticle, hydroxylamine-containing nanoparticle (HANP), which emits an intense electron spin resonance (ESR)-signal triggered by enzymatic oxidation reaction and pH-sensitive self-disintegration. The nanoparticle was prepared from an amphiphilic block copolymer, poly(ethylene glycol)-b-poly[4-(2,2,6,6-tetramethylpiperidine-1-hydroxyl)aminomethylstyrene] (PEG-b-PMNT-H), which spontaneously forms a core–shell type polymeric micelle (particle diameter = ca. 50 nm) in aqueous media. Because the PMNT-H segment in the block copolymer possesses amino groups in each repeating unit, the particle can be disintegrated by protonation of the amino groups in an acidic pH environment such as inflammation sites, which is confined to the hydrophobic core of HANP. Mixing HANP with horseradish peroxidase (HRP)/H2O2 mixture resulted in enzymatic oxidization of the hydroxylamines in the PEG-b-PMNT-H and converted the hydroxylamine to the stable nitroxide radical form in PEG-b-poly[4-(2,2,6,6-tetramethylpiperidine-1-oxyl)aminomethylstyrene] (PEG-b-PMNT), which shows an intense ESR signal. It is interesting to note that the ESR signal increased at a greater rate under acidic conditions (pH 5.6) than that under neutral conditions (pH 7.4), although the enzymatic activity of HRP under neutral conditions is known to be much higher than that under acidic conditions. This indicates that enzymatic oxidation reaction was accelerated by synchronizing the disintegration of HANP under acidic conditions. On the basis of these results, HANP can be used as a high-performance ESR probe for imaging of inflammation sites.
Inflammation is strongly related to various disorders and diseases such as rheumatoid arthritis, multiple sclerosis, and inflammatory bowel disease . Furthermore, tumors are known to develop at chronic inflammation sites, and inflammatory cells have been shown to be present in biopsied samples from tumors [2, 3]. A non-invasive imaging probe for detecting inflammation at an early and subclinical stage is important not only for decisions related to the necessity of therapy and subsequent prediction of outcomes but also for diagnostics of several diseases including cancer. To image an inflamed area, a probe with a high signal-to-background ratio is desirable. Promising strategies to improve the signal-to-background ratio include “specific accumulation of probe at the inflamed area” and “on–off regulation of signal”, in which the imaging probe ideally has no signal in the non-target tissue and is activated at the inflamed area. Nanoprobes are the candidates for specific accumulation at an inflamed area because they accumulate in inflammation sites due to an increased vascular permeability . Inflammation is a complex cellular event, during which various cytokines are released and excess reactive oxygen species (ROS) are generated by immune cells . Interstitial acidification is commonly associated with the course of inflammatory reactions against pathogenic microorganisms in peripheral tissues, where extracellular pH values as low as 5.5–7.0 have been observed [6–8]. For achieving “on–off regulation” of imaging probes at inflammation sites, nanoparticle-type probes capable of on–off signal regulation in response to an acidic pH and oxidation by ROS are desirable.
Among non-invasive imaging techniques, magnetic resonance imaging (MRI) and electron spin resonance imaging (ESRI) are two of the most powerful tools for visualizing specific and deep tissues. In particular, electron spin resonance (ESR) is highly sensitive [9, 10], and in vivo imaging has been achieved using L-band ESR instruments [11, 12]. Hydroxylamines such as 2,2,6,6-tetramethylpiperidine-1-hydroxyl are the candidates as ROS-sensitive probes and function as ESRI and MRI probes after hydroxylamines are oxidized by superoxide and peroxylnitrite or its decomposition products to corresponding nitroxide radicals, such as 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), with an intense ESR signal [13–15]. If hydroxylamine can accumulate in an inflamed area, it may be useful for imaging of inflammation sites. However, low-molecular-weight hydroxylamines cause several problems such as autoxidation before use, preferential renal clearance, and diffusion throughout the whole body.
We developed a nitroxide radical-containing nanoparticle (RNP) for use as a nanomedicine for oxidative stress injury [16–20] and as a bioimaging nanoprobe for MRI and ESRI , which is composed of a poly(ethylene glycol)-b-poly(methylstyrene) block copolymer possessing TEMPO moieties via an amine linkage (PEG-b-PMNT) . This PEG-b-PMNT forms core–shell-type micelles in the physiological environment; the cumulant average diameter of the RNP is about 40 nm, and it emits an intense ESR signal. The toxicity of RNP is extremely low due to the confinement of the 4-amino TEMPO moieties in the hydrophobic core of RNP . The RNP was confirmed to have long-term spin circulation in the blood due to the formation of polymeric micelles in the blood stream. Disintegration of RNP is caused by protonation of the amino groups in response to acidic pH, which typically occurs at inflammation sites, since amino groups are introduced into the hydrophobic segments of amphiphilic block copolymers . Along with the decreasing in pH, ESR signals of RNP gradually change from broad to sharp triplets. On the basis of these changes in ESR signals, we confirmed that phantom images showed remarkable on–off regulation in response to acidic conditions. However, RNP, as an imaging agent, exhibits undesirable background signals due to broad signal of RNP under physiological conditions.
2 Experimental Methods
2.1 Preparation of PEG-b-PMNT
PEG-b-PMNT block copolymer was prepared as previously reported [21, 22]. Briefly, MeO-PEG-b-poly(chloromethylstyrene) (PCMS) was synthesized by radical telomerization of chloromethylstyrene (CMS) using PEG possessing a methoxy group at the α-chain end and a sulfonyl group at the ω-chain end (MeO-PEG-SH) as a telogen. The polymer backbone of PEG-b-PCMS consisting of PEG with a molecular weight of 5,000 g/mol for the hydrophilic segment and 16 repeating units of PCMS for the hydrophobic segment (MW = 2,500), as determined using the 1H NMR data based on the Mn of PEG. To obtain PEG-b-PMNT, chloromethyl groups on the PCMS segment of the block copolymer were converted to nitroxide radicals via amination of MeO-PEG-b-PCMS with 4-amino-TEMPO in dimethyl sulfoxide (DMSO). After purification of the obtained PEG-b-PMNT, the substitution ratio of the modified TEMPO moieties per repeating unit of PCMS was 80%, as determined by ESR using a standard curve generated from 4-amino-TEMPO in chloroform (MW of PEG-b-PMNT = 9,000).
2.2 Preparation of the HANP
The HANP was prepared from MeO-PEG-b-PMNT by the dialysis method in the presence of hydrazine. MeO-PEG-b-PMNT (4 mg, 0.44 μmol; molar quantity of nitroxide radicals = 7.04 μmol) and hydrazine anhydride (137 mg, 4.27 mmol) were dissolved in N,N-dimethylformamide (DMF) (1 mL), and the polymer solution was transferred into a membrane tube (molecular-weight cutoff size: 3,500; Spectra/Por; Spectrum, USA) and then dialyzed for 24 h against 2 L of water, which was changed after 2, 5, 8, and 20 h. Dynamic light scattering (DLS) measurements were carried out to determine the diameter of the obtained HANP after dialysis.
2.3 Synthesis of 4-Hydroxy-2,2,6,6-Tetramethylpiperidine-1-Hydroxyl (TEMPOL-H)
TEMPOL-H was prepared using a method described in a previous paper by Henry-Riyad et al. . 4-Hydroxy-TEMPO (TEMPOL) (110 mg, 0.63 mmol) in an aqueous solution (1 mL) in the presence of sodium ascorbate (210 mg, 2.56 mmol) was stirred vigorously for 5 min, resulting in complete decolorization and the appearance of a white precipitate. The resulting suspension was extracted using diethyl ether, and the ether extracts were washed with water and brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to provide TEMPOL-H (70 mg, 63%). The obtained product was used as a control after no ESR signal of TEMPOL-H was confirmed.
2.4 DLS Measurement as a Function of pH
Light scattering intensities of the pH-sensitive HANP were measured as a function of pH using a light scattering spectrometer (Nano ZS, ZEN3600, Malvern Instruments, Ltd., UK) equipped with a He–Ne laser that produces vertically polarized incident beams at a detection angle of 173° at 25°C. First, 3.5 mg/mL of the HANP solution was prepared as stock solution A after the formation of the HANP using a dialysis method. Britton–Robinson buffers (100 μL each) with various pH values, which were prepared from a stock solution containing 1 M phosphoric acid, 1 M boric acid, and 1 M acetic acid and by adjusting the pH value using NaOH, were added to stock solution A (400 μL). The mixtures with various pH values were immediately transferred to the cells and measured using DLS.
2.5 Reaction of HANP with HRP/H2O2 Mixture
HRP and H2O2 were used as models of in vivo oxidants. HANP solution (23 μg/mL, 2.3 μM) was prepared as stock solution B after formation of HANP using a dialysis method in the presence of hydrazine. H2O2 solution (500 mM, 30 μL) and HRP solution (375 U/mL, 240 μL) in 100 mM Britton–Robinson buffers at pH 5.6 or 7.4 were added to stock solution B (30 μL). After HANP was mixed with HRP/H2O2 mixture, the samples were immediately transferred to a capillary tube and measured using ESR. The TEMPOL-H solution (46 mM) was used as a control to confirm the enzymatic activity of HRP.
2.6 ESR Measurements
The ESR spectra were recorded at room temperature using a Bruker EMX-T ESR spectrometer operating at 9.7 GHz with a 100-kHz magnetic field modulation. Spectra were collected with the following parameters: sweep width, 500 G; microwave power, 0.633 mW; receiver gain, 5.02 × 104; time constant, 5.120 ms; and conversion time, 10.240 ms.
3 Results and Discussion
3.1 Preparation and Characterization of HANP
3.2 pH Response of HANP
3.3 Detection of Oxidation Reaction Using X-Band ESR
In this paper, the design and preparation of an ESR nanoprobe for imaging inflammation sites are described. This nanoprobe is based on a pH-sensitive hydroxylamine-containing-nanoparticle (HANP) consisting of a PEG-b-PMNT-H block copolymer. Disintegration of HANP was observed at pH values below 7.0; this change was caused by protonation of amino groups on the PMNT-H segments in response to the acidic pH environment. HANP shows not only suppression of atmospheric oxidation reaction by oxygen, but also the ability of on–off regulation of the ESR signal in response to low pH and enzymatic oxidation reaction. Based on these results, pH-sensitive HANP is anticipated as high-performance nanoprobe for inflammation sites imaging.
A part of this work was supported by Grant-in-Aid for Scientific Research A (21240050) and Grant-in-Aid for Research Activity Start-up (22800004) and the World Premier International Research Center Initiative (WPI Initiative) on Materials Nanoarchitronics of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.
This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited.
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