Carbohydrate Research 402 (2015) 208–214
Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
A supramolecular vesicle of camptothecin for its water dispersion and controllable releasing Mingfang Ma a, , Wenqing Shang b, , Pengyao Xing a, Shangyang Li a, Xiaoxiao Chu a, Aiyou Hao a,⇑, Guangcun Liu c,⇑, Yimeng Zhang a a b c
School of Chemistry and Chemical Engineering and Key Laboratory of Colloid and Interface Chemistry of Ministry of Education, Shandong University, Jinan 250100, PR China Wuxi Medical School of Jiangnan University, Wuxi 214122, PR China Qianfoshan Hospital Affiliated to Shandong University, Jinan 250018, PR China
a r t i c l e
i n f o
Article history: Received 22 June 2014 Received in revised form 3 September 2014 Accepted 25 September 2014 Available online 2 October 2014 Keywords: Camptothecin Vesicles Cyclodextrin Drug release Anticancer activity
a b s t r a c t Camptothecin, as an antitumor drug, has shown significant antitumor activity against various cancers through the inhibition of topoisomerase I. However, its poor solubility severely limits the clinical applications. Here, we report a camptothecin supramolecular vesicle based on the host–guest interactions, which can uniformly disperse camptothecin into water and greatly enhance camptothecin aqueous solubility. The camptothecin vesicles were identified by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and dynamic light scattering (DLS). X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), UV–vis spectrum, 1H NMR and 2D NMR ROESY were further employed to study the formation mechanism of the vesicles. Furthermore, camptothecin could be controllably released when the competitive guests were added into the vesicles system. Finally, the camptothecin vesicles in aqueous solution exhibited comparable antitumor activity in vitro as natural camptothecin in DMSO to HeLa cells under the same conditions. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Supramolecular vesicle, which encloses a volume with membrane consisting of bilayer, can be constructed through non-covalent interactions, including host–guest recognition,1 p–p stacking,2 electrostatic forces,3 and charge transfer.4 The supramolecular vesicle based on host–guest recognition, which can be fabricated more effectively than the other non-covalent interactions in aqueous solution, has attracted much interest in supramolecular chemistry over the past decade.5 Cyclodextrin,6 calixarene,7 cucurbituril,8 and pillararene9 can all act as the macrocyclic host molecules in the host–guest supramolecular vesicles. Meanwhile, many specially designed compounds, including ferrocene derivatives,10 asymmetric viologen,11 azo-benzene compounds,12 and anthraquinone derivates13 can all play the role of guest molecules to construct the host–guest supramolecular vesicles. The host–guest supramolecular vesicle is regarded as a sensitive external-stimuli responsive system. The morphologies of this vesicle can be tuned through responding to the external stimuli, including temperature,14 pH,15 ions,16 electron,17 enzyme18 and so on, which endows this ⇑ Corresponding authors. Tel.: +86 531 88363306; fax: +86 531 88564464.
E-mail address: [email protected] (A. Hao). These authors contributed equally.
http://dx.doi.org/10.1016/j.carres.2014.09.008 0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.
soft material with promising applications in controllable selfassembly and targeted release. This advantage makes them a unique material in supramolecular chemistry, especially in the field of drug delivery. The host–guest supramolecular vesicles can not only encapsulate hydrophilic drugs in the cavity of vesicle,19 but also encapsulate hydrophobic drugs in the membrane of vesicle.20 Last year, our team21 found that cyclodextrins can include hydrophobic drugs to form particular amphiphiles, which can then form host–guest supramolecular vesicles in aqueous solution, paving a new avenue for drug formulation, and the membrane or the cavity of the vesicle can still load other drugs. Camptothecin (CPT, Scheme 1), is a plant alkaloid derived from Chinese tree Camptotheca acuminata, has shown significant antitumor activity against various cancers through the inhibition of topoisomerase I.22 However, its poor solubility in aqueous solution has greatly limited its widespread clinical applications. Different CPT drug delivery approaches, including polymer conjugates,23 nanosponges,24 nanoparticles25 and hydrogel,26 were reported to increase its solubility. Those approaches can enhance the solubility of CPT, but the tedious synthesis and preparation process increase the difficulty for practical applications. The insolubility should be improved remarkably before CPT is applied as an efficient antitumor drug. The strategy of loading CPT on the host–guest supramolecular vesicles may be a good choice to solve
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1-hydroxyadmantane. Finally, the cytotoxicity assay of Hela cells was performed to study whether b-CD/CPT vesicles can be good drug delivery vehicles, since the vesicles with nanoscale diameters are believed to possess effective enhanced permeability and retention (EPR) effect and cross the membranes of tumor cells more easily.30 It was found that the b-CD/CPT vesicles in aqueous solution exhibited comparable antitumor activity in vitro as natural CPT in DMSO to HeLa cells under the same conditions. We believe the b-CD/CPT host–guest supramolecular vesicles may have potential clinical applications in drug delivery. 2. Results and discussions 2.1. Morphologies and sizes Scheme 1. Structural illustration and sizes of b-cyclodextrin (b-CD) and camptothecin (CPT).
this problem, since our team27 has previously reported lots of works on the topic of host–guest supramolecular vesicles loaded with drugs, which can enhance hydrophobic drugs’ solubility greatly. Herein, we designed a CPT drug delivery vehicle prepared by CPT with b-cyclodextrin (b-CD, Scheme 1) directly. CD28 is a class of a-1,4-linked cyclic oligosaccharides, can be divided into a-, b-, and c-CD, according to the repeating glucose units number, six, seven, and eight respectively. b-CD,29 with a hydrophobic cavity and hydrophilic outside surface, is widely used as the host molecule in supramolecular chemistry for its low price, water-solubility, and biocompatibility. In this work, b-CD can encapsulate CPT to form supramolecular amphiphiles, which can further selfassemble into host–guest supramolecular vesicles under mild conditions. This strategy can uniformly disperse CPT into water and increase the aqueous solubility greatly. Besides, CPT can be controllably released by responding to a competitive guest
Figure 1. TEM and SEM images of b-CD/CPT vesicles (1 10 bar = 200 nm; (d) SEM, scale bar = 100 nm.
4
As shown in Figure 1(a) and (b), the b-CD/CPT vesicles, which have the hollow spherical characteristic with obvious contrast between the bright center and dark periphery, can be observed clearly under TEM.31 The diameters of the vesicles mainly range from 90 to 190 nm. The morphologies of the vesicles can be further verified by the SEM detection. SEM with gold-sputter-coating process to enhance the electro-conductivity of the samples for better observation is a significant method to observe the surface topography of vesicles.32 From the SEM images of Figure. 1(c) and (d), we could observe the spherical morphologies of the vesicles. Meanwhile, the diameter distribution of the vesicles in SEM images is consistent with that in TEM images. The solution of b-CD/CPT vesicles exhibits a typical Tyndall effect, indicating the abundance of nanometer particles in the solution.33 As shown in Figure 2, the diameters of the b-CD/CPT vesicles, which have a relatively concentrated diameter distribution, are around 200 nm and slightly larger than the results of TEM and SEM. It is reasonable that the samples of TEM and SEM are dried through air-drying before detection, while the DLS
mol/L) at room temperature: (a) TEM, scale bar = 200 nm; (b) TEM, scale bar = 100 nm; (c) SEM, scale
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Figure 2. DLS radius distribution of b-CD/CPT vesicles (1 10 4 mol/L) in water at room temperature. Inset: the Tyndall effect of b-CD/CPT vesicles in aqueous solution, 1: CPT (1 10 4 mol/L) aqueous solution, 2: b-CD/CPT vesicles (1 10 4 mol/L) in water.
Figure 3. XRD patterns of b-CD, CPT, their physical mixture, and inclusion complex.
sample is full of water in aqueous solution. Hence, the DLS result is consistent with the TEM and SEM results too. Meanwhile, the vesicles can exist for about six weeks in aqueous solution at around 20 °C, showing relatively good colloidal stability for potential clinical applications.
2.2.2. FT-IR characterization FT-IR, which is widely used in studying the intermolecular interactions, was used in the mechanism study.35 As shown in Figure 4, the characteristic absorption peaks (mOH = 3390 cm 1 and mC–O–C = 1037 cm 1) of b-CD can be found in the curves of physical mixture and inclusion complex, indicating the existence of b-CD in the physical mixture and the inclusion complex. Meanwhile, the characteristic absorption peaks (mC@O = 1750 cm 1, 1 1 mC@C = 1605 cm and mC–O–C = 1037 cm ) of CPT can be found in the curves of physical mixture and inclusion complex, indicating the existence of CPT in the physical mixture and the inclusion complex too. The physical mixture curve is more like a simple overlap of b-CD and CPT curves, demonstrating that b-CD and CPT molecules are relatively independent in the physical mixture. However, the curve of the inclusion complex is different from that of the physical mixture. The mOH = 3435 cm 1, mOH = 3258 cm 1, mCH3 = 2982 cm 1, mC@O = 1750 cm 1, and mC@C = 1605 cm 1 of CPT exist in physical mixture curve but almost disappear in inclusion complex, implying that the inclusion complex may be in a new form through the entrance of CPT into b-CD cavity. 2.2.3. NMR characterizations 1 H NMR is one of the most dependent tools in studying the host and guest molecule interactions in solutions.36 The interaction between b-CD and CPT was performed by 1H NMR at room temperature. As shown in Figure S2 and Table. S1, the remarkable upfield chemical shifts of CPT proton resonances Ha, He, and Hf were observed upon addition of b-CD due to the shielding effect of the cavities of b-CD, indicating that CPT molecules enter the cavities of b-CD molecules.9,19 Meanwhile, the protons on the cavities of b-CD also exhibit slight chemical shift changes due to the interactions between b-CD and CPT, as shown in Figure 5 and Table 1. 2D NMR ROSEY spectrum37, which can provide more direct evidence for the inclusion complex, was used in our study. The intermolecular correlations between Ha of CPT and H3 of b-CD inner cavity can be observed obviously from 2D NMR ROSEY result (Fig. S3 and Scheme 2), meaning that the lactone ring of CPT is recognized and included by the cavity of b-CD to form supramolecular complex. This result is in line with the 1H NMR analysis. 2.2.4. Detection of stoichiometry between b-CD and CPT The b-CD/CPT complex stoichiometry in aqueous solution was detected through Job’s plot method, as shown in Fig. 6.38 The absorption peaks at 367 nm of CPT were taken by UV–vis
2.2. Mechanism study 2.2.1. XRD characterization As shown in Figure 3, the XRD patterns of b-CD and CPT have many sharp peaks, illustrating their typical crystal molecular stacking on their initial state.34 Meanwhile, all the sharp peaks in b-CD and CPT patterns can be almost found in the physical mixture pattern. In other words, the physical mixture pattern is just like a simple overlap of b-CD and CPT patterns. This indicates that b-CD and CPT are still on their initial crystal state separately in the physical mixture rather than shaping a new form. Meanwhile, the inclusion complex patterns are different from the others. The sharp peaks of b-CD and CPT almost disappeared, which is an obviously amorphous state. This might be related to the b-CD/CPT complex. To further investigate the detailed information about the size of the b-CD/CPT complex, SAXD was used in our study. As shown in Figure S1, sharp diffraction peak appeared at 2h = 3.1° with d value of 2.85 nm based on Bragg equation. The d value is in agreement with the length of two b-CD/CPT complex molecules since the length of one b-CD/CPT complex molecule is about 1.49 nm (calculated by Material Studio 5.5), indicating that the b-CD/CPT aggregates may have bilayer structure.
Figure 4. FT-IR spectra comparison of b-CD, CPT, their physical mixture, and inclusion complex.
M. Ma et al. / Carbohydrate Research 402 (2015) 208–214
Figure 5. 1H NMR spectra of b-CD/CPT complex in comparison with b-CD at room temperature with D2O as the reference (400 MHz).
Table 1 H NMR: b-CD protons’ shifts (Dd) induced by the inclusion of b-CD and CPT
1
211
Figure 6. Job’s plot of the b-CD/CPT complex stoichiometry in water at room temperature.
than the primary side diameter of b-CD molecule, indicating that CPT molecules could enter the cavities of b-CD molecules to form an inclusion complex. The obtained amphiphiles can further selfassemble into host–guest supramolecular vesicles induced by hydrophobic-hydrophobic interaction in aqueous solution under mild conditions. The hydrophilic heads can contact with surrounding water molecules, while hydrophobic tails aggregate together to avoid exposing in water and thus are loaded in membrane of the vesicle. 2.3. Controllable release of CPT
Proton
H1
H2
H3
H4
H5
H6
db-CD dComplex Dd
4.9982 4.9935 0.0047
3.5934 3.5897 0.0037
3.8972 3.8621 0.0351
3.5068 3.5032 0.0036
3.7823 3.7463 0.0360
3.8025 3.7812 0.0213
spectrophotometer. The X axis of Job’s plot max peak is about 0.5, indicating the host and guest molecules’ stoichiometry is 1:1. This result can also verify the formation of b-CD/CPT complex. 2.2.5. The possible mechanism of the vesicular formation From the above results, we can propose the plausible mechanism of the vesicle formation, just as illustrated in Scheme 2. One b-CD molecule can form one supramolecular amphiphile with one CPT molecule based on host–guest recognition. The b-CD molecules act as the hydrophilic heads. Meanwhile, the CPT molecules act as the hydrophobic tails. Scheme 1 displays the sizes of CPT and b-CD molecules. The width of CPT lactone ring is slightly smaller
The b-CD/CPT vesicles show the ability of responding to external competitive guest molecules with CPT release. The release mechanism illustration of CPT triggered by 1-hydroxyadmantane is shown in Scheme 3. It is well-established that 1-hydroxyadmantane is easy to form stable complex with b-CD, since the binding constant of b-CD with 1-hydroxyadmantane is about 30000 M 1 and the binding constant of b-CD with CPT is only 266 M 1, so as a competitive guest molecule, it may enter the cavity of b-CD to replace CPT.39,40 As shown in Figure 7a, the b-CD/CPT vesicles disappeared after adding 1-hydroxyadmantane into the vesicles solution. Meanwhile, some crystals were found in the TEM image. The crystals were obtained by filtering the suspension through a 0.45 lm filter, which was proved to be CPT by MS characterization (Fig. S4). This demonstrates that the competitive guest molecules can destroy the vesicles attributing to the complex formation of 1-hydroxyadmantane with b-CD. Moreover, from Figure 7(b), we can find that the UV–vis absorbance intensity of b-CD/CPT vesicles decreased sharply after 1-hydroxyadmantane was added into vesicle solution. The decrease is consistent with the vesicle disassembly, reducing the number of CPT molecules in solution. This can also verify CPT release through the disassembly of vesicles.
Scheme 2. The proposed mechanism of the vesicle formation from b-CD and CPT.
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the morphologies of living cells in CPT and b-CD/CPT vesicles groups were all sparse. It should be noted that sole CPT was dissolved in DMSO since its poor water solubility. But, b-CD/CPT vesicles were in aqueous solution since b-CD can disperse CPT into water uniformly and enhance CPT solubility greatly. Hence, b-CD/ CPT vesicles can be regarded as good vehicles to deliver CPT and are more suitable for clinical application than natural CPT. Scheme 3. The illustration of mechanism of CPT release process triggered by 1hydroxyadmantane.
2.4. Application of b-CD/CPT vesicle system in anticancer effects The nanoparticles with the diameter around 200 nm are believed to be more easily uptaken by cells.30 To illustrate whether b-CD/CPT vesicles can be good vehicles to release CPT, we verified the antitumor activity difference of CPT and b-CD/CPT vesicles to cancer cells through the methyl thiazole tetrazolium (MTT) cellsurvival assay. HeLa cells in different groups were treated with nothing, natural CPT, and b-CD/CPT vesicles. Then the numbers of living cells in each group were recorded at different time. As shown in Figure 8, CPT and b-CD/CPT vesicles all showed efficient antitumor effect on HeLa cells at 48 h, since the number of living cells in CPT and b-CD/CPT vesicle groups was less than that of blank group (P
Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
A supramolecular vesicle of camptothecin for its water dispersion and controllable releasing Mingfang Ma a, , Wenqing Shang b, , Pengyao Xing a, Shangyang Li a, Xiaoxiao Chu a, Aiyou Hao a,⇑, Guangcun Liu c,⇑, Yimeng Zhang a a b c
School of Chemistry and Chemical Engineering and Key Laboratory of Colloid and Interface Chemistry of Ministry of Education, Shandong University, Jinan 250100, PR China Wuxi Medical School of Jiangnan University, Wuxi 214122, PR China Qianfoshan Hospital Affiliated to Shandong University, Jinan 250018, PR China
a r t i c l e
i n f o
Article history: Received 22 June 2014 Received in revised form 3 September 2014 Accepted 25 September 2014 Available online 2 October 2014 Keywords: Camptothecin Vesicles Cyclodextrin Drug release Anticancer activity
a b s t r a c t Camptothecin, as an antitumor drug, has shown significant antitumor activity against various cancers through the inhibition of topoisomerase I. However, its poor solubility severely limits the clinical applications. Here, we report a camptothecin supramolecular vesicle based on the host–guest interactions, which can uniformly disperse camptothecin into water and greatly enhance camptothecin aqueous solubility. The camptothecin vesicles were identified by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and dynamic light scattering (DLS). X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), UV–vis spectrum, 1H NMR and 2D NMR ROESY were further employed to study the formation mechanism of the vesicles. Furthermore, camptothecin could be controllably released when the competitive guests were added into the vesicles system. Finally, the camptothecin vesicles in aqueous solution exhibited comparable antitumor activity in vitro as natural camptothecin in DMSO to HeLa cells under the same conditions. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Supramolecular vesicle, which encloses a volume with membrane consisting of bilayer, can be constructed through non-covalent interactions, including host–guest recognition,1 p–p stacking,2 electrostatic forces,3 and charge transfer.4 The supramolecular vesicle based on host–guest recognition, which can be fabricated more effectively than the other non-covalent interactions in aqueous solution, has attracted much interest in supramolecular chemistry over the past decade.5 Cyclodextrin,6 calixarene,7 cucurbituril,8 and pillararene9 can all act as the macrocyclic host molecules in the host–guest supramolecular vesicles. Meanwhile, many specially designed compounds, including ferrocene derivatives,10 asymmetric viologen,11 azo-benzene compounds,12 and anthraquinone derivates13 can all play the role of guest molecules to construct the host–guest supramolecular vesicles. The host–guest supramolecular vesicle is regarded as a sensitive external-stimuli responsive system. The morphologies of this vesicle can be tuned through responding to the external stimuli, including temperature,14 pH,15 ions,16 electron,17 enzyme18 and so on, which endows this ⇑ Corresponding authors. Tel.: +86 531 88363306; fax: +86 531 88564464.
E-mail address: [email protected] (A. Hao). These authors contributed equally.
http://dx.doi.org/10.1016/j.carres.2014.09.008 0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.
soft material with promising applications in controllable selfassembly and targeted release. This advantage makes them a unique material in supramolecular chemistry, especially in the field of drug delivery. The host–guest supramolecular vesicles can not only encapsulate hydrophilic drugs in the cavity of vesicle,19 but also encapsulate hydrophobic drugs in the membrane of vesicle.20 Last year, our team21 found that cyclodextrins can include hydrophobic drugs to form particular amphiphiles, which can then form host–guest supramolecular vesicles in aqueous solution, paving a new avenue for drug formulation, and the membrane or the cavity of the vesicle can still load other drugs. Camptothecin (CPT, Scheme 1), is a plant alkaloid derived from Chinese tree Camptotheca acuminata, has shown significant antitumor activity against various cancers through the inhibition of topoisomerase I.22 However, its poor solubility in aqueous solution has greatly limited its widespread clinical applications. Different CPT drug delivery approaches, including polymer conjugates,23 nanosponges,24 nanoparticles25 and hydrogel,26 were reported to increase its solubility. Those approaches can enhance the solubility of CPT, but the tedious synthesis and preparation process increase the difficulty for practical applications. The insolubility should be improved remarkably before CPT is applied as an efficient antitumor drug. The strategy of loading CPT on the host–guest supramolecular vesicles may be a good choice to solve
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1-hydroxyadmantane. Finally, the cytotoxicity assay of Hela cells was performed to study whether b-CD/CPT vesicles can be good drug delivery vehicles, since the vesicles with nanoscale diameters are believed to possess effective enhanced permeability and retention (EPR) effect and cross the membranes of tumor cells more easily.30 It was found that the b-CD/CPT vesicles in aqueous solution exhibited comparable antitumor activity in vitro as natural CPT in DMSO to HeLa cells under the same conditions. We believe the b-CD/CPT host–guest supramolecular vesicles may have potential clinical applications in drug delivery. 2. Results and discussions 2.1. Morphologies and sizes Scheme 1. Structural illustration and sizes of b-cyclodextrin (b-CD) and camptothecin (CPT).
this problem, since our team27 has previously reported lots of works on the topic of host–guest supramolecular vesicles loaded with drugs, which can enhance hydrophobic drugs’ solubility greatly. Herein, we designed a CPT drug delivery vehicle prepared by CPT with b-cyclodextrin (b-CD, Scheme 1) directly. CD28 is a class of a-1,4-linked cyclic oligosaccharides, can be divided into a-, b-, and c-CD, according to the repeating glucose units number, six, seven, and eight respectively. b-CD,29 with a hydrophobic cavity and hydrophilic outside surface, is widely used as the host molecule in supramolecular chemistry for its low price, water-solubility, and biocompatibility. In this work, b-CD can encapsulate CPT to form supramolecular amphiphiles, which can further selfassemble into host–guest supramolecular vesicles under mild conditions. This strategy can uniformly disperse CPT into water and increase the aqueous solubility greatly. Besides, CPT can be controllably released by responding to a competitive guest
Figure 1. TEM and SEM images of b-CD/CPT vesicles (1 10 bar = 200 nm; (d) SEM, scale bar = 100 nm.
4
As shown in Figure 1(a) and (b), the b-CD/CPT vesicles, which have the hollow spherical characteristic with obvious contrast between the bright center and dark periphery, can be observed clearly under TEM.31 The diameters of the vesicles mainly range from 90 to 190 nm. The morphologies of the vesicles can be further verified by the SEM detection. SEM with gold-sputter-coating process to enhance the electro-conductivity of the samples for better observation is a significant method to observe the surface topography of vesicles.32 From the SEM images of Figure. 1(c) and (d), we could observe the spherical morphologies of the vesicles. Meanwhile, the diameter distribution of the vesicles in SEM images is consistent with that in TEM images. The solution of b-CD/CPT vesicles exhibits a typical Tyndall effect, indicating the abundance of nanometer particles in the solution.33 As shown in Figure 2, the diameters of the b-CD/CPT vesicles, which have a relatively concentrated diameter distribution, are around 200 nm and slightly larger than the results of TEM and SEM. It is reasonable that the samples of TEM and SEM are dried through air-drying before detection, while the DLS
mol/L) at room temperature: (a) TEM, scale bar = 200 nm; (b) TEM, scale bar = 100 nm; (c) SEM, scale
210
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Figure 2. DLS radius distribution of b-CD/CPT vesicles (1 10 4 mol/L) in water at room temperature. Inset: the Tyndall effect of b-CD/CPT vesicles in aqueous solution, 1: CPT (1 10 4 mol/L) aqueous solution, 2: b-CD/CPT vesicles (1 10 4 mol/L) in water.
Figure 3. XRD patterns of b-CD, CPT, their physical mixture, and inclusion complex.
sample is full of water in aqueous solution. Hence, the DLS result is consistent with the TEM and SEM results too. Meanwhile, the vesicles can exist for about six weeks in aqueous solution at around 20 °C, showing relatively good colloidal stability for potential clinical applications.
2.2.2. FT-IR characterization FT-IR, which is widely used in studying the intermolecular interactions, was used in the mechanism study.35 As shown in Figure 4, the characteristic absorption peaks (mOH = 3390 cm 1 and mC–O–C = 1037 cm 1) of b-CD can be found in the curves of physical mixture and inclusion complex, indicating the existence of b-CD in the physical mixture and the inclusion complex. Meanwhile, the characteristic absorption peaks (mC@O = 1750 cm 1, 1 1 mC@C = 1605 cm and mC–O–C = 1037 cm ) of CPT can be found in the curves of physical mixture and inclusion complex, indicating the existence of CPT in the physical mixture and the inclusion complex too. The physical mixture curve is more like a simple overlap of b-CD and CPT curves, demonstrating that b-CD and CPT molecules are relatively independent in the physical mixture. However, the curve of the inclusion complex is different from that of the physical mixture. The mOH = 3435 cm 1, mOH = 3258 cm 1, mCH3 = 2982 cm 1, mC@O = 1750 cm 1, and mC@C = 1605 cm 1 of CPT exist in physical mixture curve but almost disappear in inclusion complex, implying that the inclusion complex may be in a new form through the entrance of CPT into b-CD cavity. 2.2.3. NMR characterizations 1 H NMR is one of the most dependent tools in studying the host and guest molecule interactions in solutions.36 The interaction between b-CD and CPT was performed by 1H NMR at room temperature. As shown in Figure S2 and Table. S1, the remarkable upfield chemical shifts of CPT proton resonances Ha, He, and Hf were observed upon addition of b-CD due to the shielding effect of the cavities of b-CD, indicating that CPT molecules enter the cavities of b-CD molecules.9,19 Meanwhile, the protons on the cavities of b-CD also exhibit slight chemical shift changes due to the interactions between b-CD and CPT, as shown in Figure 5 and Table 1. 2D NMR ROSEY spectrum37, which can provide more direct evidence for the inclusion complex, was used in our study. The intermolecular correlations between Ha of CPT and H3 of b-CD inner cavity can be observed obviously from 2D NMR ROSEY result (Fig. S3 and Scheme 2), meaning that the lactone ring of CPT is recognized and included by the cavity of b-CD to form supramolecular complex. This result is in line with the 1H NMR analysis. 2.2.4. Detection of stoichiometry between b-CD and CPT The b-CD/CPT complex stoichiometry in aqueous solution was detected through Job’s plot method, as shown in Fig. 6.38 The absorption peaks at 367 nm of CPT were taken by UV–vis
2.2. Mechanism study 2.2.1. XRD characterization As shown in Figure 3, the XRD patterns of b-CD and CPT have many sharp peaks, illustrating their typical crystal molecular stacking on their initial state.34 Meanwhile, all the sharp peaks in b-CD and CPT patterns can be almost found in the physical mixture pattern. In other words, the physical mixture pattern is just like a simple overlap of b-CD and CPT patterns. This indicates that b-CD and CPT are still on their initial crystal state separately in the physical mixture rather than shaping a new form. Meanwhile, the inclusion complex patterns are different from the others. The sharp peaks of b-CD and CPT almost disappeared, which is an obviously amorphous state. This might be related to the b-CD/CPT complex. To further investigate the detailed information about the size of the b-CD/CPT complex, SAXD was used in our study. As shown in Figure S1, sharp diffraction peak appeared at 2h = 3.1° with d value of 2.85 nm based on Bragg equation. The d value is in agreement with the length of two b-CD/CPT complex molecules since the length of one b-CD/CPT complex molecule is about 1.49 nm (calculated by Material Studio 5.5), indicating that the b-CD/CPT aggregates may have bilayer structure.
Figure 4. FT-IR spectra comparison of b-CD, CPT, their physical mixture, and inclusion complex.
M. Ma et al. / Carbohydrate Research 402 (2015) 208–214
Figure 5. 1H NMR spectra of b-CD/CPT complex in comparison with b-CD at room temperature with D2O as the reference (400 MHz).
Table 1 H NMR: b-CD protons’ shifts (Dd) induced by the inclusion of b-CD and CPT
1
211
Figure 6. Job’s plot of the b-CD/CPT complex stoichiometry in water at room temperature.
than the primary side diameter of b-CD molecule, indicating that CPT molecules could enter the cavities of b-CD molecules to form an inclusion complex. The obtained amphiphiles can further selfassemble into host–guest supramolecular vesicles induced by hydrophobic-hydrophobic interaction in aqueous solution under mild conditions. The hydrophilic heads can contact with surrounding water molecules, while hydrophobic tails aggregate together to avoid exposing in water and thus are loaded in membrane of the vesicle. 2.3. Controllable release of CPT
Proton
H1
H2
H3
H4
H5
H6
db-CD dComplex Dd
4.9982 4.9935 0.0047
3.5934 3.5897 0.0037
3.8972 3.8621 0.0351
3.5068 3.5032 0.0036
3.7823 3.7463 0.0360
3.8025 3.7812 0.0213
spectrophotometer. The X axis of Job’s plot max peak is about 0.5, indicating the host and guest molecules’ stoichiometry is 1:1. This result can also verify the formation of b-CD/CPT complex. 2.2.5. The possible mechanism of the vesicular formation From the above results, we can propose the plausible mechanism of the vesicle formation, just as illustrated in Scheme 2. One b-CD molecule can form one supramolecular amphiphile with one CPT molecule based on host–guest recognition. The b-CD molecules act as the hydrophilic heads. Meanwhile, the CPT molecules act as the hydrophobic tails. Scheme 1 displays the sizes of CPT and b-CD molecules. The width of CPT lactone ring is slightly smaller
The b-CD/CPT vesicles show the ability of responding to external competitive guest molecules with CPT release. The release mechanism illustration of CPT triggered by 1-hydroxyadmantane is shown in Scheme 3. It is well-established that 1-hydroxyadmantane is easy to form stable complex with b-CD, since the binding constant of b-CD with 1-hydroxyadmantane is about 30000 M 1 and the binding constant of b-CD with CPT is only 266 M 1, so as a competitive guest molecule, it may enter the cavity of b-CD to replace CPT.39,40 As shown in Figure 7a, the b-CD/CPT vesicles disappeared after adding 1-hydroxyadmantane into the vesicles solution. Meanwhile, some crystals were found in the TEM image. The crystals were obtained by filtering the suspension through a 0.45 lm filter, which was proved to be CPT by MS characterization (Fig. S4). This demonstrates that the competitive guest molecules can destroy the vesicles attributing to the complex formation of 1-hydroxyadmantane with b-CD. Moreover, from Figure 7(b), we can find that the UV–vis absorbance intensity of b-CD/CPT vesicles decreased sharply after 1-hydroxyadmantane was added into vesicle solution. The decrease is consistent with the vesicle disassembly, reducing the number of CPT molecules in solution. This can also verify CPT release through the disassembly of vesicles.
Scheme 2. The proposed mechanism of the vesicle formation from b-CD and CPT.
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the morphologies of living cells in CPT and b-CD/CPT vesicles groups were all sparse. It should be noted that sole CPT was dissolved in DMSO since its poor water solubility. But, b-CD/CPT vesicles were in aqueous solution since b-CD can disperse CPT into water uniformly and enhance CPT solubility greatly. Hence, b-CD/ CPT vesicles can be regarded as good vehicles to deliver CPT and are more suitable for clinical application than natural CPT. Scheme 3. The illustration of mechanism of CPT release process triggered by 1hydroxyadmantane.
2.4. Application of b-CD/CPT vesicle system in anticancer effects The nanoparticles with the diameter around 200 nm are believed to be more easily uptaken by cells.30 To illustrate whether b-CD/CPT vesicles can be good vehicles to release CPT, we verified the antitumor activity difference of CPT and b-CD/CPT vesicles to cancer cells through the methyl thiazole tetrazolium (MTT) cellsurvival assay. HeLa cells in different groups were treated with nothing, natural CPT, and b-CD/CPT vesicles. Then the numbers of living cells in each group were recorded at different time. As shown in Figure 8, CPT and b-CD/CPT vesicles all showed efficient antitumor effect on HeLa cells at 48 h, since the number of living cells in CPT and b-CD/CPT vesicle groups was less than that of blank group (P
