Preparation and Characterization of Stimuli-Responsive Magnetic Nanoparticles
© to the authors 2008
Received: 24 May 2008
Accepted: 11 July 2008
Published: 7 August 2008
In this work, the main attention was focused on the synthesis of stimuli-responsive magnetic nanoparticles (SR-MNPs) and the influence of glutathione concentration on its cleavage efficiency. Magnetic nanoparticles (MNPs) were first modified with activated pyridyldithio. Then, MNPs modified with activated pyridyldithio (MNPs-PDT) were conjugated with 2, 4-diamino-6-mercaptopyrimidine (DMP) to form SR-MNPs via stimuli-responsive disulfide linkage. Fourier transform infrared spectra (FTIR), transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS) were used to characterize MNPs-PDT. The disulfide linkage can be cleaved by reduced glutathione (GHS). The concentration of glutathione plays an important role in controlling the cleaved efficiency. The optimum concentration of GHS to release DMP is in the millimolar range. These results had provided an important insight into the design of new MNPs for biomedicine applications, such as drug delivery and bio-separation.
Recently, stimuli-responsive magnetic nanoparticles (SR-MNPs) have attracted considerable attention in the field of nanotechnology as candidates for drug delivery and bio-separation [1–4]. Owing to their ability to switch on and switch off when necessary, particularly under the action of local stimuli characteristic of the target zone, they have been employed as the carriers of drug in biomedical and bioengineering field. Advantage may also be taken for the magnetic property of MNPs to be attracted toward the target zone in the presence of an external magnetic field. This magnetic motor effect is also attractive for the development of site-specific drug delivery and bio-separation systems. Most of these bio-systems are based on stimuli-responsive polymer, for example, temperature responsive poly(N-isopropylacrylamide) [5, 6].
It is well known that pyridyl disulfide conjugates readily with a thiol-containing drug, protein, or other functional molecule, forming a disulfide linkages with such molecules, and simultaneously releasing the byproduct, thiopyridone, under mild reaction conditions [7–9]. The disulfide linkages are chemically labile in nature and have the ability to dissociate in response to various disulfide-reducing agents, especially, glutathione [10–12]. Wang et al. [13, 14] designed a kind of nanoparticles that possess the ability to respond to intracellular glutathione concentrations.
Therefore, the combination of the activated disulfide with magnetic particles is an attractive strategy to improve the functions of MNPs. While the synthesis of MNPs has been well established, not much has been reported in the case of SR-MNPs. We report here the influence of glutathione concentration on the cleavage efficiency of the stimuli-responsive disulfide linkages.
Materials and Methods
Dichloromethane, ferric chloride hexahydrate, ferrous chloride tetrahydrate, ammonium hydroxide, methanol, ethanol, diethyl ether,N,N-dimethylformamide (DMF), succine anhydride, acetic acid, and toluene were obtained from China National Medicines Corporation Ltd. and used as received. 3-Aminopropyltriethoxysilane (APTES, 99%), thiopyridyl disulfide, 1, 3-dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), and 2-aminoethylthiol hydrochloride were supplied from Aldrich Chemical Co. 2, 4-Diamino-6-mercaptopyrimidine (DMP) was endowed by MERCK. Reduced glutathione (GSH) was purchased from MERCK.
Synthesis of S-(2-Aminoethylthio)-2-Thiopyridine Hydrochloride (AE-S-S-Py)
The synthesis of S-(2-aminoethylthio)-2-thiopyridine hydrochloride was described in reference . Thiopyridyl disulfide (1.1 g) was dissolved in 10-mL methanol and 0.2-mL acetic acid. To the solution, 2-aminoethylthiol hydrochloride (0.25 g) in 10-mL methanol was added. The mixture was stirred for 48 h and then evaporated under high vacuum to obtain yellow oil. The product was washed with 50-mL diethyl ether and dissolved in 10-mL methanol, and then was precipitated by addition of 400-mL diethyl ether. Finally, the deposit was collected by vacuum filtration. Yield: 0.4 g, 77%; 1H NMR: δ ppm (in d-chloroform) 8.66 (d, 1H), 7.75 (t, 1H), 7.48 (d, 1H), 7.1(t, 1H), 3.46 (t, 2H), and 2.8 (m, 2H).
MNPs were synthesized by chemical co-precipitation of Fe (II) and Fe (III) ions in alkaline medium. FeCl3 · 6H2O (0.02 M) and FeCl2 · 4H2O (0.01 M) were dissolved in 120-mL de-ionized water at pH 2 and under N2protection. Sixty-milliliter ammonia aqueous solution (28 wt%) was added into this mixture with violently stirring. After 30 min the reaction mixture was heated to 70 °C and kept at this temperature for about 30 min. The obtained MNPs were washed immediately with de-ionized water, ethanol, and toluene five times in turn. Then MNPs were dispersed in 100-mL toluene.
MNPs Functioned with Activated Pyridyldithio
The surface of MNPs was first coated with 3-aminopropyltriethoxysilane by a silanization reaction. 5-mL APTES was added into the MNPs solution and the reaction mixture was kept at 80 °C for 10 h under nitrogen atmosphere with vigorous mechanical stirring. Then, the obtained APTES-immobilized MNPs, defined as MNPs-NH2, were washed with DMF (5 × 50 mL2) and dispersed in 100-mL DMF.
Then, MNPs-NH2reacted with succine anhydride. Briefly, 50-mL MNPs-NH2and 2 g (excessive) of succine anhydride were added to 50-mL DMF. The mixture was kept at 80 °C for 10 h under nitrogen atmosphere with stirring. After reaction, the mixture was separated under magnetic field. The modified MNPs, defined as MNPs-COOH, were washed with dichloromethane five times and dispersed in dichloromethane. The weight percent of solid was about 3.28%.
Finally, MNPs-COOH reacted with AE-S-S-Py. Five-gram suspension of MNPs-COOH, 150 mg DCC, 150 mg DMAP, and 50 mg AE-S-S-Py were added into 40-mL dichloromethane. After stirring the mixture for 48 h, MNPs modified with pyridyldithio, defined as MNPs-PDT, were washed with methanol and phosphate buffer solution (PBS: 150 mmol/L NaCl, 1.9 mmol/L NaH2PO4, 8.1 mmol/L Na2HPO4, pH 7.4, 0.01 M) five times, respectively. The concentration of MNPs-PDT dispersed in PBS was 4 mg/mL.
Preparation of SR-MNPs
To form SR-MNPs, 100-μL DMP solutions (3.84 × 10−3 mmol/L) were added to 100-μL MNPs-PDT. The mixture was kept at room temperature for 20 h. After magnetic separation, the obtained nanoparticles were washed five times with PBS.
Hundred-microliter GHS solutions (195.0 mM) were added to SR-MNPs. The total volume was 200 μL. The mixture was shaken for 20 h and separated under magnetic field. UV–Vis spectrometer was adopted to monitor the supernatant.
FTIR was recorded in the transmission mode on a Perkin Elmer 2000 FTIR instrument (USA). XPS analyses were performed with an ESCALAB220 i-XL electron spectrometer from VG Company. To determine absorbency spectra of the supernatant Lambda 950 spectrometer (Perkin Elmer) was used. The magnetic properties of nanoparticles were measured with a vibrating sample magnetometer (VSM, Lakeshore 7307) at room temperature. The size and morphology of the particles were examined by a Philips Tecnai 20 transmission electron microscopy (TEM).
Results and Discussion
MNPs Modified with an Activated Pyridyldithio
Preparation of SR-MNPs
Drug Release Behavior
It is clear that SR-MNPs can be synthesized by modifying MNPs with activated pyridyldithio and sequently conjugating thiol-containing molecule (4-diamino-6-mercaptopyrimidine) via stimuli-responsive disulfide linkage. SR-MNPs are sensitive to glutathione concentration: the increase of glutathione concentration is likely to raise the cleavage efficiency of the stimuli-responsive disulfide linkages. The optimum concentration of GHS to cleave the disulfide linkage (smart linkage) is in the millimolar. The synthesized SR-MNPs have the advantages of magnetic motor and selective release for versatile applications, such as drug delivery and bio-separation.
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