Letter pubs.acs.org/Langmuir
Simple, Clean Preparation Method for Cross-Linked α‑Cyclodextrin Nanoparticles via Inclusion Complexation Wen Zhu, Ke Zhang, Yongming Chen,* and Fu Xi Laboratory of Polymer Physics and Chemistry, Institute of Chemistry, The Chinese Academy of Sciences, Beijing 100190, China S Supporting Information *
ABSTRACT: A simple, clean method was presented in this letter to prepare cross-linked α-cyclodextrin (α-CD) nanoparticles with a low dispersion. The nanoparticles were synthesized in water by cross-linking the inclusion complex of α-CDs and poly(ethylene glycol) (PEG). The structure of the nanoparticles was characterized by 1H NMR, nuclear overhauser enhancement spectroscopy (NOESY), and wideangle X-ray diffraction (XRD). Spherical morphology was observed by scanning electron microscopy (SEM) for these nanoparticles. Their average hydrodynamic radius was determined to be 67 nm by dynamic light scattering (DLS). Small guest molecules could be included in the cross-linked α-CD nanoparticles, and anticancer drug cisplatin was used to evaluate the drug release behavior.
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INTRODUCTION Cyclodextrins (CDs) are a family of cyclic oligosaccharides composed of six, seven, or eight D(+)-glucose units linked by α-1,4-linkages, which are named α-, β-, and γ-CD, respectively. Because the CD molecule has a hydrophobic cavity that can form an inclusion complex with a variety of guest molecules, CDs have played a crucial role in various fields, such as catalysis, medicine, bionics, separation processes, and environmental protection.1 Especially in the pharmaceutical industry, CDs are a kind of promising excipient because of their biocompatibility and low toxicity in animals and humans. The poor physicochemical properties of the guest drug molecules, such as insolubility in water, instability, and toxic side effects, could be masked by forming inclusion complexes with CDs.2 A CD-based supramolecular system has been developed for gene delivery.3 There are dozens of marketed products based on CD inclusion complexes worldwide with different forms of administration routes such as oral, intravenous, dermal, ophthalmic, rectal, nasal, and buccal.4 Although the use of CDs in the medical field has been documented for decades, more operative and precise CDcontaining drug delivery systems (DDSs) still need to be developed beyond the solubilization and stabilization of small molecules. Because of the prolonged blood circulation time and nonspecific accumulation in tumors through the enhanced permeability and retention (EPR) effect, nanosized DDSs (nanoparticles) have been regarded as a competitive candidate in the medical and pharmaceutical fields, especially for cancer therapy. As a biocompatible material, CD-based nanoparticles are becoming a growing concern. Presently, two approaches have been developed to prepare these novel DDSs. One is through modifying CD molecules with a hydrophobic moiety to produce an amphiphilic CD derivative. The nanoparticles were then prepared by a nanoprecipitation technique.5−7 © XXXX American Chemical Society
However, this technique with chemical modification followed by nanoprecipitation is complicated. Another approach is based on the assembly of inclusion complexes formed by CDs and polymers. The obtained supramolecular nanoparticles might be used as DDSs.8−10 However, these assemblies usually are unstable. CDs can assemble spontaneously in water to form microgels with an uncontrollable size distribution by cross-linking directly.11,12 The heterogeneity and uncontrollability of the resultant cross-linked CD products limited their applications, especially in DDSs for blood circulatory systems. If the CD nanoparticles with controllable size could be prepared simply by cross-linking the CD molecules, then a very useful DDS would be built. CDs could be selectively threaded onto some linear polymer chains such as poly(ethylene glycol) (PEG) to form polyrotaxanes.13,14 Cross-linking the inclusion complexes to fix their morphologies gives novel nanostructures including CD tubes15,16 and CD nanocapsules,17 which greatly changed the physical and chemical properties of CDs and expanded their application range. In this work, cross-linked α-CD nanoparticles with a relatively low dispersion were synthesized via the inclusion complex of α-CD and PEG in water, as shown in Scheme 1. The incomplete inclusion complex of α-CD and PEG can self-assemble into nanoparticles that are stabilized by the PEG segments with no threaded α-CD. The nanoparticles were obtained by cross-linking the hydroxyl groups of the αCDs with epichlorohydrin and then extracting the PEG chains that penetrated them. The reaction was performed in water at ambient temperature, and the final products were purified Received: February 4, 2013 Revised: March 6, 2013
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Scheme 1. Structure and Preparation Process of the Cross-Linked α-CD Nanoparticles via Inclusion Complexation
Figure 1. Rh distributions of (a) α-CD, (b) the inclusion complex of PEG45 and α-CD, (c) cross-linked inclusion complex of PEG45 and α-CD, and (d) cross-linked α-CD nanoparticles in aqueous solution, as determined by dynamic light scattering.
simply by dialysis and a washing process. It is quite a simple, clean preparation method. The CD cavities of the nanoparticles could be used for drug molecule inclusion. Cisplatin, an anticancer drug, was used here to evaluate the drug release behavior of the nanoparticles.
Under an appropriate feed ratio and concentration, an incomplete inclusion complex of α-CD and PEG can selfassemble into nanoparticles in water. The inclusion complex with poor solubility formed the particle core, and the PEG chains with no α-CD threaded onto them formed the corona that stabilizes the particle. The isolated PEG segments formed hydrogen bonds with water molecules, which prevented the effective aggregation of nanoparticles. It is reasonable that the CD-based nanoparticles with relatively low dispersion would be produced by cross-linking these self-assemblies. In aqueous solution, α-CDs may aggregate, and the aggregation size was strongly influenced by the concentration.19 The general phenomenon is that the aggregation size grows with increasing CD concentration.20 When using a concentration of 18 mg/mL (18.5 mM), the hydrodynamic radius (Rh) of α-CD aggregation was measured as 81 nm (Figure 1a). This is reasonable when compared to the data previously reported (Rh = 100 ± 5 nm for a 25.7 mM α-CD solution; Rh = 68 ± 20 nm for a 12 mM α-CD solution).19,21 Then PEG45 (molecular weight 2000 Da) was added in a molar ratio of 1:2 α-CD/EG. The concentrations of PEG45 and α-CD were 1.6 and 18 mg/ mL respectively, which were far less than their saturated
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RESULTS AND DISCUSSION It is known that the complete inclusion of α-CD and PEG (average molecular weight >200) leads to the precipitation of supramolecular complexes because of their poor solubility.18 Using these complete inclusion complexes as building blocks, the cross-linked α-CD molecular tubes15 and nanocapsules17 have been prepared. The detailed preparation process includes a cumbersome three steps: (1) obtaining the inclusion complex in a concentrated CD solution, terminating the ends of the PEG chain with bulky stoppers, and then removing free CDs; (2) cross-linking the adjacent CDs in relatively dilute solution; and (3) removing the bulky stoppers and PEG chains. In this work, we creatively presented novel cross-linked α-CD nanoparticles by only a one-step synthesis process in which the incomplete inclusion complex self-assembled into nanoparticles and the cross-linking reaction was carried out in situ. B
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Figure 2. (A) 1H NMR spectrum of cross-linked α-CD nanoparticles in DMSO-d6. (a) PEG-stabilized cross-linked α-CD nanoparticles; (b) crosslinked α-CD nanoparticles. (B) 2D-NOESY spectrum for PEG-stabilized cross-linked α-CD nanoparticles in DMSO-d6.
Figure 3. SEM images of (a) PEG-stabilized cross-linked α-CD nanoparticles, (b) cross-linked α-CD nanoparticles, and (c) cross-linked α-CD without PEG during the preparation.
calculated as 20%, corresponding to an average number of bridging groups of 3.6 for each CD molecule. The result of nuclear Overhauser effect spectroscopy (NOESY) is shown in Figure 2B because it is a useful technique that allows the correlation of nuclei through space. The cross-peak signals of 3.51 ppm (−CH2CH2−, PEG) and 3.57−3.77 ppm (H(3), H(5), α-CD) suggested a close distance of PEG and the internal H of the CD cavity, indicating the supramolecular interactions between PEG chains and CDs. The wide-angle Xray diffraction (XRD) analysis of the freeze-dried powder of the cross-linked complex showed broad peaks at 2θ = 19.4° (signal of the inclusion complex) and 23° (signal of PEG), indicating the existence of both the inclusion complex and unthreaded PEG segments in the incomplete inclusion system (Figure S1). The cross-linked structure hindered the formation of microcrystals and broadened the peaks. The Rh of PEG-stabilized cross-linked α-CD nanoparticles was 88 nm (Figure 1c), and few free CD was left after dialysis. However, the PEG cannot be removed simply by dialysis possibly because the supramolecular interaction prevented the PEG chain from slipping out of the CD cavity. To remove PEG, we dispersed the cross-linked particle complex into chloroform. Because of the poor solubility of cross-linked CD particles and the good solubility of PEG, the free cross-linked CD nanoparticles were obtained by centrifugation. As shown in Figure 2Ab, only a small resonance was left at 3.51 ppm
concentrations at room temperature. Although the feed ratio of PEG45 and α-CD was stoichiometric as the complete inclusion, the low concentration may lead to an incomplete inclusion, which was reflected by the colloid solution without precipitation.18 The Rh of the inclusion complex particle was 80 nm (Figure 1b). There were small particles in the solutions (Figure 1a and 1b) with an Rh of 2 to 3 nm, which were the oligo-aggregates of free CDs. The hydroxyl groups of α-CDs of the inclusion nanoparticles were then cross-linked by epichlorohydrin in alkaline solution. After the reaction, the solution was neutralized, and small molecules were easily removed by dialysis against water. The result of 1H NMR was shown in Figure 2A. The cross-linking reaction cannot be proven by the change in the resonance signal position because the newly formed peaks of the crosslinker overlapped with the peaks of α-CD at δ 3.57−3.63 ppm (H(5) H(6), α-CD; CH2CH(OH)CH2, cross-linker) and 4.48 ppm (O(6)H, α-CD; CH2CH(OH)CH2, cross-linker). However, the peak area of 3.57−3.77 ppm, which contained H(5), H(6), and H(3) of α-CD (24H in total) and CH2CH(OH)CH2 of the newly formed cross-linker (methylene and methine groups, 5H), was larger than 4 times the peak area of 5.43 ppm that contained O(3)H of α-CD (6H). The extra area was the resonance signal of the newly formed cross-linker, which suggested the cross-linking reaction. Moreover, according to the increased area, the average cross-linking density was C
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Figure 4. (a) UV curves of the inclusion complex of α-CD and I3¯. (b) Drug release curves of cisplatin-loaded cross-linked α-CD nanoparticles in 0.01 M PBS (pH 7.4) at 25 °C (●) and 37 °C (□).
(−CH2CH2−, PEG), indicating that the majority of PEG was removed by this method. The Rh of the free cross-linked CD nanoparticles was 67 nm (Figure 1d), less than that of PEGstabilized ones, which supported the elimination of PEG chains as well. The scanning electron microscopy (SEM) images are shown in Figure 3. The average radius of PEG-stabilized cross-linked α-CD nanoparticles was 60 ± 8 nm (Figure 3a), and that of cross-linked α-CD nanoparticles was 45 ± 14 nm (Figure 3b). The particle size of the dry sample used in electron microscopy is normally smaller than that in solution as determined by dynamic light scattering. To explore the key role of PEG in the inclusion system, α-CD without PEG inclusion was cross-linked by adding epichlorohydrin and NaOH directly to water. Although the α-CD solution showed two sharp peaks (Figure 1a), the aggregates of free CD molecules are unstable and will change their size upon changing the solution condition by the addition of the epichlorohydrin and NaOH. Moreover, the interparticle cross-linking reaction occurred randomly with a high probability because no stabilizer of the CD aggregates existed. These factors led to a large particle size (dozens of micrometers) and a broad size distribution of the cross-linked product (Figure 3c). The inclusion property of the obtained CD nanoparticles was evaluated by small guest molecules. Using the cavities that were liberated from the inclusion with PEG, the CD nanoparticles could be complexed with I3¯ ions. Similar to the inclusion complex formed by free α-CDs and I3¯ ions, the mixed solution of CD nanoparticles and I3¯ ions showed a greatly enhanced absorption peak at 350 nm compared to that of free I3¯ ions (Figure 4a), indicating the formation of the inclusion complex.15 This supports the fact that the novel CD nanoparticle system is a suitable drug carrier. Cisplatin and its analogues, such as oxaliplatin, carboplatin, and nedaplatin, were notable antineoplastic agents, and their inclusion complexes with α-CDs were proven through theoretical analysis.22 The complex stability was dictated by host−guest hydrogen bonds, mainly with the amine groups. The leaving groups (Cl¯) of cisplatin were kept free to undergo ligand exchange processes necessary to achieve the biological response. Moreover, it has been reported that the inclusion complex of carboplatin and hydroxypropyl α-CD improved the survival of animals treated for experimental brain tumors.23
Therefore, cisplatin was used to evaluate the drug loading and release behaviors of the CD nanoparticles in this work. First, free α-CD was used to prove the inclusion of cisplatin. As shown in Figure S2, the chemical shift of NH3 of free cisplatin moved from 3.97 and 4.7624 to 4.50 ppm after complexation with free α-CD, indicating the formation of the inclusion complex. The XRD result of the inclusion complex of cisplatin and free α-CD showed the disappearance of peaks at 2θ = 12.0 and 21.6°, which existed in their physical mixture (Figure S3), providing further evidence of the complexation. Cisplatin was then loaded into the CD nanoparticles. Using our method in this work (experimental section in Supporting Information), the drug-loading content (the proportion of loaded cisplatin to the drug-loaded particles) was 13 wt % and the loading efficiency (the ratio of loaded cisplatin to the used drug in the preparation) was 37.5%. The in vitro release of cisplatin exhibited different trends at 25 and 37 °C in PBS (pH 7.4), as shown in Figure 4b. Cisplatin was released constantly and completely for 28 h at 25 °C. In contrast, a faster release took place at 37 °C. Especially within the beginning 10 h, more than 85% of the cisplatin was released. This indicated that the inclusion complex of cisplatin and cross-linked α-CD nanoparticles had a lower stability at higher temperature, which caused a quicker drug release.
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CONCLUSIONS
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ASSOCIATED CONTENT
A simple, clean method was reported for preparing cross-linked α-CD nanoparticles with a relatively low dispersion. The resultant CD nanoparticles were nanosized, biocompatible, and full of reactive hydroxyl groups. Anticancer drug cisplatin can be incorporated into the nanoparticles and shows temperaturedependent release behavior, which has potential utility in drug delivery system. Subsequent work will focus on the functionalization of CD particles and the control of the drug release process.
S Supporting Information *
Information concerning the experimental details and Figures S1−S3. This material is available free of charge via the Internet at http://pubs.acs.org. D
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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REFERENCES
Financial support by the National Natural Science Foundation of China (51203171, 21090350, and 21090353) and the Knowledge Innovation Program of the Chinese Academy of Sciences (grant no. KJCX2-YW-H19) is greatly acknowledged.
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