Chinese Journal of Chemistry, 2008, 26, 1737—1740 Note

* E-mail: mlpeng@nwu.edu.cn, chaochen@nwu.edu.cn; Tel. & Fax: 0086-029-88303551 Received March 18, 2008; revised April 17, 20087; accepted May 26, 2008.

© 2008 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Magnetic HP-β-CD Composite Nanoparticle: Synthesis, Characterization and Application as a Carrier of

Doxorubicin in vitro

ZHANG, Huaa (张华) PENG, Ming-Li*,b(彭明丽) CUI, Ya-Lia(崔亚丽) CHEN, Chao*,c(陈超)

a School of Life Sciences, Northwest University, Xi'an, Shaanxi 710069, China b Department of Chemistry, Northwest University, Xi'an, Shaanxi 710069, China

c National Engineering Research Center For Miniaturized Detection Systems, Xi'an, Shaanxi 710069, China

Hydroxylpropyl-β-cyclodextrin (HP-β-CD) was introduced to the surface of magnetic nanoparticles in the presence of NH3•H2O to synthesize magnetic HP-β-CD composite nanoparticles, which were used as a carrier in magnetic targeted drug delivery. The composite nanoparticles were characterized by FTIR, ICP-AES, TEM and VSM. The results showed that the iron content in the composite nanoparticles was 55.4%. The range of size is 10—20 nm and the magnetization is 59.9 emu/g. The capacity of composite nanoparticles for doxorubicin adsorption is 87.8 µg/mg after incubation for 72 h. The cumulative percentage of released doxorubicin in PBS buffer (pH＝7.4) in 1, 4, 10 d were 35.5%, 49.3% and 76.5%, respectively. Thus, the magnetic HP-β-CD composite nanoparticles could be a potential carrier in the magnetic targeted drug delivery.

Keywords HP-β-CD, doxorubicin, magnetic composite nanoparticle, extended release

Introduction

Based on biocompatibility/biodegradation and super- paramagnetism, magnetic particles have been an attrac-tive carrier for targeted drug delivery to improve the therapeutic index of drug molecule and to minimize side effects on healthy cells and tissues.1-3 Cyclodextrins (CD) are a series of cyclic oligosaccharides consisting of α-CD, β-CD and γ-CD glucopyranose units, respec-tively. The three-dimensional structure of the cyclodex-trin forms a truncated cone and provides a hydrophobic cavity. Some drug molecules can form reversible inclu-sion complexes with the cavity,4 which plays a role as “reservoir” of molecules without interfering with their activities since complexation is a reversible dynamic process in an aqueous solution. The cavity also ensures molecules to form the inclusion compound through host-guest interaction. Formation of the inclusion com-plex can increase the guest’s solubility and stability against hydrolysis, oxidation and dehydration.5 CD has been used as a stabilizer to dispersing magnetic nanoparticles in aqueous medium.6 α-CD had been used to “pull” the hydrophobic particles into aqueous phase by locking the oleic acid in cavity.7 A composite parti-cle has also been synthesized by combining the magnet-ite and β-CD.8 β-CD-citrate-gum arabic modified mag-netic nanoparticles were used for hydrophobic drug de-livery.9 However, α-CD is too expensive and the renal toxicity of β-CD at high concentration limits its applica-

tion as potential drug carriers.10 HP-β-CD is a β-CD derivative, which is widely used in the field of drug en-capsulation owing to its inclusion with a higher water solubility and lower toxicity compared to β-CD.11 It is safe enough to be tolerated by the human body both through intravenous and oral administrations as ap-proved by FDA.12-15 Herein, HP-β-CD was introduced to the surface of magnetic nanoparticles in the presence of NH3•H2O to synthesize magnetic HP-β-CD compos-ite nanoparticles. And the composite nanoparticles were used as a carrier of doxorubicin (Dox) in magnetic tar-geted drug delivery. The optimal conditions of Dox load and release in vitro were also discussed.

Experimental

Materials

All chemicals were of analytical grade. HP-β-CD (average Mw: 1564) was obtained from Shaanxi Institute of Microbiology. FeCl2•4H2O, FeCl3•6H2O and doxoru-bicin were purchased from Sigma.

Synthesis of magnetic composite nanoparticles

Magnetic nanoparticles have been obtained by a co-precipitation method.16 HP-β-CD composite magnetic nanoparticles was prepared according to Ref. 17. Briefly, magnetite (200 mg) and HP-β-CD (386.5 mg) were dis-solved in 2 mL of water under continuous and vigorous stirring. After the solution was heated to 40 ℃, 0.8 mL

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© 2008 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

of NH3•H2O (25%) was added, the mixture was heated to 50 ℃ and kept for 5 h, resulting in black suspension. The suspension was washed with water using magnetic separation for several times until the pH value of super-natant was neutral.

Characterization of magnetic composite nanoparti-cles

The magnetic composite nanoparticles were charac-terized by infrared spectra (Nicolet 5700 FT-IR, Thermo Electron Corporation), recording the wavenumbers in the 400—4000 cm－1, using KBr disks. The iron content of the composite nanoparticles was determined by an inductively coupled atomic emission spectrometer (ICP-AES, Thermo Elemental IRIS), after dissolving the particles in concentrated HCl. Magnetization curve was then determined by a Lake Shore 7307 Vibrating Sample Magnetometer. The TEM images were obtained by Transmission Electron Microscopy (Hitachi HT-600) by dropping the samples on a copper grid coated with a formvar film and air-dried with accelerating voltage of 200 kV.

Drug load and release in vitro

Drug load process was performed by adding differ-ent amounts of Dox into 5 mL tubes containing 5 mg of the magnetic composite nanoparticles. The mixture was incubated in 2 mL aqueous solution at room temperature for at least 72 h. After separated by the magnetic field for 5 min, the Dox-loaded magnetic composite nanopar-ticles were prepared and kept in refrigerator under 4 ℃ with aluminium film covered.

The drug release in vitro was carried out under sink condition. 5 mg Dox-loaded composite nanoparticles were added into a tube with 15 mL PBS (pH＝7.4). The mixture was placed into a shaker at 37 ℃, 180 r/min for 10 d. At specific time 0.5 mL solution was pipetted out and fresh PBS buffer was added in the same volume. The concentrations of Dox were determined by the variation of absorbance at 480 nm.

Results and discussion

Components of magnetic composite nanoparticles

The magnetic composite nanoparticles consisting of HP-β-CD and magnetite were evidented by the infrared spectra shown in Figure 1. The spectrum of HP-β-CD characterized in 3600—3200 cm－1 and 850—1650 cm－1 was due to O—H stretching vibrations and vibrations of β-CD (Figure 1a). The characteristic absorption bands at 583 cm－1 were attributed to Fe—O bond of the magnet-ite (Figure 1b). The spectrum of composite particles (Figure 1c) was similar to the superimposition of the individual spectrum of HP-β-CD and Fe3O4 with ab-sorptions at 588, 800—1630 and 3600—3200 cm－1 re-spectively, indicating that magnetic HP-β-CD composite nanparticles have been obtained.

Another evidence for formation of the composite nanoparticles is the iron content in Fe3O4 and composite

Figure 1 FTIR spectra of HP-β-CD (a), Fe3O4 (b) and magnetic composite nanoparticles (c).

nanoparticles. The contents of Fe were 72.8% and 55.4% individually determined by ICP-AES. The de-crease of Fe in composite nanoparticles implies that Fe3O4 was coated by HP-β-CD and the content of HP-β-CD was ca. 23.6% in weight.

Properties of magnetic composite nanoparticles

The magnetization curves of Fe3O4 and HP-β-CD composite nanoparticles are shown in Figure 2 with magnetization (Ms) 67.4 and 59.9 emu/g at 300 K, re-spectively. The little decrease of Ms for the composite nanoparticles can be easily understood due to the coat-ing effect of HP-β-CD.18

Figure 2 Magnetization curves at 300 K of Fe3O4 nanoparticles (a) and magnetic composite nanoparticles (b).

The diameters of the magnetic HP-β-CD composite nanoparticles before and after drug loading were deter-mined by TEM as shown in Figure 3. The magnetic composite nanoparticles have a size in range of 10—20 nm.

Drug capacity and loading efficiency

The incubation time for Dox-loading was optimized (Figure 4) by determining the absorbance of Dox in so- lution. The absorbance of Dox reached a plateau, after the mixture was rotated for 72 h, implying that the loading amount of the magnetic HP-β-CD composite nanoparticles for Dox was saturated. The drug contents and loading efficiency in nanoparticles were determined by the following equation.

Amount of Dox in nanoparticlesDrug concent 100%

Weight of nanoparticles＝ ×

Hydroxylpropyl-β-cyclodextrin Chin. J. Chem., 2008 Vol. 26 No. 9 1739

© 2008 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 3 TEM images of magnetic composite nanoparticles (a) and Dox-loaded magnetic composite nanoparticles (b).

Figure 4 Dox loading curve on magnetic composite nanopati-cles.

Loading efficiency

Residue amount of Dox in nanoparticles100%

Feeding amount of Dox

＝

×

Correlation between drug content and loading effi-ciency was shown in Figure 5. The optimal amount of Dox was 150 µg for 1 mg magnetic HP-β-CD composite nanoparticles, giving drug content of 100.8 µg/mg and 67.7% loading efficiency.

In vitro drug release

The drug release curves of the Dox-loaded magnitite and composite nanoparticles were obtained by monitor-ing the concentration of Dox in PBS buffer in 10 d

Figure 5 Correlation between drug content and loading effi-ciency.

as shown in Figure 6. An initial burst occurred in 2 h in both curves, as described in the literature.19 It was probably due to physical adsorption on the surfaces. And the composite nanoparticles showed extended re-lease sustained in the later stage. The t50 (time taken for 50% drug release) was calculated as 100 h and the re-lease continued over 10 d.20 The cumulative release percentages of Dox in vitro in 1, 4 and 10 d were 35.5%, 49.3% and 76.5%, respectively, while those of Fe3O4 nanoparticles in 1, 4 and 10 d were 61.9%, 67.0% and 67.1%, respectively, which could be attributed to the slow diffusion of drug from the cavity of HP-β-CD of the magnetic composite nanoparticles. HP-β-CD played as a “reservoir” of drugs, because complexation of Dox and HP-β-CD is a rapid reversible dynamic process in aqueous solution. Such structural modifications pro-longed the release of Dox from the magnetic composite nanoparticles, which shows promising prospects for the magnetic targeted drug delivery.

Figure 6 In vitro drug release behavior of the magnetic and composite nanoparticles.

Conclusion

In conclusion, HP-β-CD was introduced to the sur-face of magnetic nanoparticles in the presence of NH3•H2O to synthesize new magnetic composite nanoparticles. The application of the magnetic compos-

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© 2008 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

ite nanoparticles as a Dox carrier in vitro indicated that the HP-β-CD coated on the composite nanoparticles led to an extended release. These characteristics of the composite nanoparticles show promising prospects for magnetic targeted drug delivery.

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