Introduction

Most of brain cancer is glioblastoma multiforme (GBM), while temozolomide (TMZ, Scheme 1) is one of the most effective drugs in GBM treatment [1]. As illustrated in Fig. 1, under physiological conditions, TMZ can hydrolyze into 5-(3-methyl-triazen-1-yl)imidazole-4-carboxamide (MTIC) [2]. Only TMZ molecules cross the blood–brain barrier, enter into GBM cells, and hydrolyze into MTIC; it can induce DNA damage of GBM cells. MTIC is inactive when it is outside of GBM cells. Unfortunately, half-life period of TMZ is very short in plasma, only about 1.8 h [3], since TMZ can degrade into MTIC in the blood. Besides, the solubility of TMZ in water is very low [4]. Those above deficiencies all limit TMZ clinical applications. Chemists have tried their best to develop drug carriers to solve those deficiencies in the past decade. Polycefin, prodrug, microemulsion, nanoparticles, liposome, and so on can all be used as drug carriers to deliver TMZ [1,2,3,4,5,6,7,8]. For example, Scherman et al. has encapsulated TMZ into the cavity of cucurbit[7]uril, prolonging the lifetime of the TMZ since encapsulation can decrease TMZ degradation under physiological conditions [9]. Moreover, encapsulation can improve TMZ activity compared to natural TMZ. Recently, Golomb et al. has successfully encapsulated TMZ into PEGylated liposome, leading longer survival of rats treated with TMZ liposomes compared to treatment with TMZ solution [10].

Scheme 1
scheme 1

Structures of hydroxypropyl-β-cyclodextrin (HP-β-CD) and temozolomide (TMZ)

Full size image
Fig. 1
figure 1

Chemical structures of TMZ and MTIC

Full size image

However, as far as we know, the supramolecular vesicle based on hydroxypropyl-β-cyclodextrin (HP-β-CD, Scheme 1) encapsulated with TMZ has not been reported yet. The vesicle containing bilayer membrane [11,12,13,14], which is similar with liposome, has been applied widely in nanoreactors, template synthesis, cell membrane mimicking, and gene or drug delivery [15,16,17,18,19]. The supramolecular vesicle is an important branch of vesicle, which can be constructed by non-covalent interactions, consisting of π–π stacking, electrostatic forces, host–guest recognition, and charge transfer [20,21,22,23,24]. Supramolecular vesicle is sensitive to external stimuli, including pH, ions, electrons, enzymes, and light [25,26,27,28,29,30], endowing this colloid material with a potential application in drug delivery and release. As a drug delivery soft material, supramolecular vesicle can deliver hydrophilic drug. Liu et al. has constructed a supramolecular vesicle by host–guest recognition between p-sulfonatocalixarene and asymmetric viologen [31]. After ultracentrifugation and dialysis, doxorubicin can be successfully loaded into the interior of the vesicle. The doxorubicin-loaded vesicles show lower damage for normal cells while same antitumor activity to cancer cells in contrast with doxorubicin itself. Supramolecular vesicle can deliver hydrophobic drug as well. Our team has reported a supramolecular vesicle based on amphiphilic β-cyclodextrin [32]. Paclitaxel can be loaded into the vesicle’s membrane. Paclitaxel-loaded vesicles exhibit remarkable anticancer activity compared to natural paclitaxel. Afterwards, we find that paclitaxel can form amphiphile with ethanediamine-arm modified β-cyclodextrin; the supramolecular amphiphile can assemble into supramolecular vesicles spontaneously [33], paving a new avenue to construct drug-loaded vesicle.

Herein, a novel stimulus-responsive TMZ supramolecular vesicle is firstly reported. β-cyclodextrin (β-CD) is a cyclic oligosaccharide, containing seven glucose units connected by α-1,4-glucosidic bond [34, 35]. β-CD has hydrophobic cavity and hydrophilic outside the surface, which can encapsulate hydrophobic molecules [36, 37]. β-CD is used widely in supramolecular chemistry because of its high water solubility, favorable biocompatibility, and easy functionalization [38, 39]. HP-β-CD is one of the most significant β-CD derivatives. The water solubility of HP-β-CD is higher than β-CD [40]. As shown in Scheme 1, the narrow diameter of HP-β-CD cavity is 0.43 nm, while the size of TMZ is 0.41 nm calculated by Materials Studio 5.5; hence, HP-β-CD can easily encapsulate TMZ. In this study, HP-β-CD can encapsulate TMZ to form supramolecular amphiphile through host–guest recognition. The obtained supramolecular amphiphile can further self-assemble into a vesicle. The diameters of HP-β-CD/TMZ vesicles mainly distribute from 200 to 240 nm in aqueous solution. Nanoparticles including vesicles can selectively accumulate in tumor by enhanced permeability and retention (EPR) effect [32]. So, our HP-β-CD/TMZ vesicles may be preferentially delivered to tumor through this passive targeting mechanism. Moreover, HP-β-CD/TMZ vesicles exhibit sensitive stimulus responsiveness to copper ions. HP-β-CD/TMZ vesicles will change to irregular aggregates when copper ions are added into this vesicle system. Hence, our HP-β-CD/TMZ vesicle is a smart drug delivery system, which may have potential application in GBM treatment.

Experimental section

Materials

HP-β-CD was bought from Binzhou Zhiyuan Biotechnology Co. Ltd., China. TMZ and all other chemical reagents were all purchased from Sinopharm Chemical Reagent Co. Ltd., China.

Analytical instruments and methods

Transmission electron microscope (TEM) pictures were obtained through a transmission electron microscope (JEM-100CX, JEOL Ltd). Scanning electron microscope (SEM) images were carried out on a scanning electron microscope (Hitachi S-4800). Phosphotungstic acid was used to stain TEM samples for detection. TEM samples were then dried by an infrared lamp after staining. SEM samples for measurement were sprayed using gold. Ultraviolet-visible (UV-vis) spectroscopy was used to obtain UV-vis curves by a TU-1800pc UV-vis spectrophotometer (Purkinje General Co. Ltd. Beijing, China). Dynamic light scattering (DLS, Wyatt QELS Technology DAWN HELEOS instrument) was used to measure hydration radius of vesicles. Water was filtered by a 0.45-μm filter before preparation of DLS sample. A German Bruker D8ADVANCE diffractometer was used to get X-ray diffraction (XRD) spectrum. An Avatar 370 FT-IR Spectrometer was used to obtain Fourier transform infrared (FT-IR) spectrum by KBr pellet method. 1H nuclear magnetic resonance (1H NMR) measurement was on API Bruker Avance 300 M NMR. 2D NMR ROESY experiments were recorded by an API Bruker Avance 400M NMR. Materials Studio 5.5 by Accelrys was used to calculate the sizes of HP-β-CD and TMZ molecules.

Preparation of HP-β-CD/TMZ solid complex and their physical mixture

Through freeze-drying HP-β-CD/TMZ complex aqueous solutions, HP-β-CD/TMZ solid complex was prepared. By mixing HP-β-CD and TMZ powders directly, HP-β-CD/TMZ physical mixture was prepared. For HP-β-CD/TMZ physical mixture sample preparation of FT-IR detection, 1:1 ratio of HP-β-CD and TMZ is ground with KBr firstly, then HP-β-CD/KBr and TMZ/KBr are ground immediately.

Detection of the stoichiometries of HP-β-CD/TMZ

HP-β-CD (10−3 mol/L) and TMZ (10−3 mol/L) aqueous solutions were prepared firstly. Then different HP-β-CD/TMZ solutions with different molar ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10 were prepared. UV-vis spectrums of above solutions were detected; the max absorption peaks were picked. Finally, Job’s plot was obtained.

Preparation of the vesicles

HP-β-CD (2 × 10−4 mol/L) and TMZ (2 × 10−4 mol/L) solutions were prepared firstly. Then HP-β-CD/TMZ (1 × 10−4 mol/L) solution was prepared by mixing HP-β-CD and TMZ solution in a centrifuge tube. Finally, the vesicle samples were prepared by sonicating HP-β-CD/TMZ solution for 30 min and placing for about one day. HP-β-CD/TMZ vesicles can be obtained by dialyzing HP-β-CD/TMZ solution through dialysis membrane.

Stimulus responsiveness of vesicle system

TMZ contains an amido group, so it may coordinate with copper ions. Hence, the effect of copper ions on HP-β-CD/TMZ vesicles was studied. CuCl2 aqueous solution (1 × 10−4 mol/L) was prepared firstly. Then CuCl2 aqueous solution was dropped into HP-β-CD/TMZ vesicles’ solution. Finally, the mixed solution was sat a day at ambient temperature.

Results and discussions

Morphologies and sizes

The UV-vis absorbance of different concentrations of HP-β-CD/TMZ sample solutions was obtained by UV-vis detection as shown in Fig. S1. UV-vis absorption intensity increased along with HP-β-CD/TMZ sample’s increasing concentration. The maximum absorption intensity was picked. Interestingly, the slope changed obviously when HP-β-CD/TMZ sample’s concentration was above 1 × 10−4 mol/L, illustrating critical aggregate concentration of HP-β-CD/TMZ sample should be about 1 × 10−4 mol/L. So the morphology of HP-β-CD/TMZ sample at critical aggregate concentration was observed by electron microscopy. From Fig. 2a and b, we can see the obtained HP-β-CD/TMZ vesicles clearly under TEM [41]. The diameters of spherical vesicles are about 170 to 200 nm. SEM detection was further used to confirm the vesicle’s existence since it can provide surface topography of aggregates. For organic material such as vesicle, it is better to be coated with gold to improve the electroconductivity for clear observation. As shown in Fig. 2c and d, aggregates with a spherical shape can be seen under SEM, which can further confirm HP-β-CD/TMZ vesicle’s existence. Moreover, the diameters of vesicles under SEM are almost the same as those in TEM images.

Fig. 2
figure 2

HP-β-CD/TMZ vesicles’ TEM and SEM images (1 × 10−4 mol/L) at room temperature, a TEM, scale bar = 500 nm; b TEM, scale bar = 200 nm; c SEM, scale bar = 200 nm; d SEM, scale bar = 200 nm

Full size image

As shown in the inserted image in Fig. 3, a typical Tyndall effect can be seen clearly under laser in HP-β-CD/TMZ vesicles’ solution. This phenomenon illustrated that there were abundant nanoparticles in HP-β-CD/TMZ sample solution [42]. Moreover, HP-β-CD/TMZ vesicles’ diameters were mainly from 200 to 240 nm in DLS result. The vesicles’ diameters in DLS were a little larger than the vesicles’ diameters in TEM and SEM detection. This may attribute to the reason that DLS detected vesicles’ hydration radius while vesicular samples in TEM and SEM were dried. Hence, DLS detection confirmed HP-β-CD/TMZ vesicles’ existence as well. Those above results all give us a strong signal that HP-β-CD/TMZ vesicles can be constructed by reasonable design and preparation.

Fig. 3
figure 3

HP-β-CD/TMZ vesicles’ (1 × 10−4 mol/L) DLS radius distribution in water at room temperature. Inset: image of HP-β-CD/TMZ vesicles’ Tyndall effect in aqueous solution

Full size image

Mechanism study

XRD characterization

As shown in Fig. 4, TMZ is a typical crystal since there are many sharp peaks at 2θ including 10.8°, 14.6°, 17.9°, 19.1°, 21.5°, 26.5°, 28.9°, and 29.7° in its XRD pattern [43]. While for HP-β-CD’s XRD pattern, almost no sharp peak can be seen, there are two broad peaks in the range of 5°–25°, showing an amorphous state obviously [44]. Furthermore, there are many sharp peaks in HP-β-CD/TMZ physical mixture as well, which is similar to TMZ itself. Almost all of the sharp peaks in TMZ pattern including 10.8°, 14.6°, 17.9°, 19.1°, 26.5°, 28.9°, and 29.7° and two broad peaks in the range of 5°–25° in HP-β-CD’s XRD pattern existed in HP-β-CD/TMZ physical mixture, indicating that HP-β-CD/TMZ physical mixture is just a mixture of HP-β-CD and TMZ. In other words, HP-β-CD and TMZ were all in their separate initial crystal states in the physical mixture. But, compared to HP-β-CD/TMZ physical mixture, HP-β-CD/TMZ inclusion complex is very different; it shows an amorphous state since only two broad peaks in the range of 5°–25° of HP-β-CD exist while TMZ’s sharp peaks almost disappear. This may be attributed to HP-β-CD/TMZ complex formation. TMZ molecule enters into the cavity of HP-β-CD molecule, leading to the disappearance of TMZ’s sharp peaks.

Fig. 4
figure 4

XRD patterns of HP-β-CD, TMZ, their physical mixture, and inclusion complex

Full size image

FT-IR characterization

FT-IR, which can provide intermolecular interaction information, was used in vesicle formation mechanism exploration [45]. As shown in Fig. 5, HP-β-CD’s characteristic absorption peaks (νOH = 3426 cm−1, νC-H = 2920 cm−1, and νC-O-C = 1032 cm−1) can be found in FT-IR curves of HP-β-CD/TMZ physical mixture and HP-β-CD/TMZ inclusion complex. This illustrated that HP-β-CD existed in HP-β-CD/TMZ physical mixture and their inclusion complex. Meanwhile, TMZ’s characteristic absorption peaks (νNH = 3382 cm−1 and νC=O of TMZ = 1745 cm−1) can be found in FT-IR curves of HP-β-CD/TMZ physical mixture and HP-β-CD/TMZ inclusion complex as well. This indicated that TMZ existed in HP-β-CD/TMZ physical mixture and their inclusion complex. However, HP-β-CD/TMZ inclusion complex pattern was different from HP-β-CD/TMZ physical mixture pattern clearly. As for HP-β-CD/TMZ physical mixture pattern, it was just like a simple overlap between HP-β-CD curve and TMZ curve. This demonstrated that HP-β-CD and TMZ molecules are relatively independent in HP-β-CD/TMZ physical mixture. However, νNH of amido = 3116 cm−1 and νC=O of TMZ = 1745 cm−1 of TMZ existed in HP-β-CD/TMZ physical mixture curve but almost disappeared in HP-β-CD/TMZ inclusion complex curve. This implied that HP-β-CD/TMZ inclusion complex may be in a new form by entrance of TMZ into HP-β-CD’s cavity.

Fig. 5
figure 5

FT-IR spectra comparison of HP-β-CD, TMZ, their physical mixture, and inclusion complex

Full size image

NMR characterizations

1H NMR characterization

1H NMR is a significant tool to study interaction between different molecules [46]. The interaction of HP-β-CD and CPT was performed through 300 M 1H NMR. As shown in Fig. 6 and Table 1, compared to H2 and H4, H3 and H5 protons of HP-β-CD all shifted to a higher field. Meanwhile, H5 proton shift is highest than the other HP-β-CD protons. This suggested that TMZ molecules entered the cavities of HP-β-CD molecules under shielding effect. Meanwhile, TMZ molecule should enter the cavity of HP-β-CD molecule from the primary side rather than the second side because H5 proton’s shift is higher than H3 proton’s shift.

Fig. 6
figure 6

1H NMR spectra comparison of HP-β-CD and HP-β-CD/TMZ sample with D2O as the reference

Full size image
Table 1 HP-β-CD protons’ shifts (Δδ) induced by the inclusion between HP-β-CD and TMZ in 1H NMR
Full size table

2D NMR characterization

2D NMR ROSEY detection was used in our study since it can give accurate evidence for inclusion complex formation between host and guest molecules [47]. This detection was performed by 400 M 1H NMR. As shown in Fig. 7, clear correlations between Ha of TMZ and H3 and H5 of HP-β-CD’s inner cavity can be observed from 2D NMR ROSEY spectrum. This indicated that HP-β-CD and TMZ can form supramolecular complex through host–guest recognition. Just like 1H NMR analysis, this result further confirms HP-β-CD/TMZ complex formation. The simulated diagram of HP-β-CD/TMZ complex is inserted in Fig. 7.

Fig. 7
figure 7

2D NMR ROSEY (400 MHz) spectrum of HP-β-CD/TMZ sample with D2O as the reference at room temperature

Full size image

Detection of stoichiometry between HP-β-CD and TMZ

The stoichiometry of HP-β-CD/TMZ complex in aqueous solution can be detected through Job’s plot. UV-vis spectrophotometer was used to record TMZ’s absorption peaks at 266 nm with different HP-β-CD/TMZ ratios. The horizontal ordinate of Job’s plot max peak was 0.5 [48] (Fig. 8). This indicated that TMZ molecule can be included by HP-β-CD molecule with 1:1 stoichiometry. Meanwhile, this result also verified that HP-β-CD can form complex with TMZ. The stoichiometry of HP-β-CD/TMZ complex can be verified by NMR detection as shown in Fig. S2. A slight upfield shift can be observed clearly for TMZ’s aromatic proton with the addition of one molar HP-β-CD. However, aromatic proton of TMZ has no change with more HP-β-CD addition, illustrating HP-β-CD/TMZ complex formation with 1:1 stoichiometry. Moreover, the equilibrium binding constant of HP-β-CD/TMZ complex was determined about 0.893 × 103 M−1 (Fig. S3) according to the reported method [49].

Fig. 8
figure 8

Job’s plot of HP-β-CD/TMZ inclusion complex in water by UV-vis detection

Full size image

The possible mechanism of the vesicular formation

From the above analysis, a reasonable mechanism of HP-β-CD/TMZ vesicle formation was proposed, as shown in Scheme 2. One HP-β-CD molecule can recognize and encapsulate one TMZ molecule to form one supramolecular amphiphile through host–guest recognition. In one HP-β-CD/TMZ supramolecular amphiphile, HP-β-CD molecule is the hydrophilic, while TMZ molecule is hydrophobic. The obtained HP-β-CD/TMZ supramolecular amphiphiles can further self-assemble into vesicles by non-covalent interactions. The hydrophilic part of HP-β-CD/TMZ supramolecular amphiphile exposes to water, while the hydrophobic part of HP-β-CD/TMZ supramolecular amphiphile buries into the membrane of the vesicle to avoid water. In this way, anticancer drug TMZ can be loaded into the membranous layer of HP-β-CD/TMZ vesicles successfully.

Scheme 2
scheme 2

The proposed mechanism of HP-β-CD/TMZ vesicle formation and its stimulus responsiveness

Full size image

Stimulus responsiveness of HP-β-CD/TMZ vesicles

It is reported that copper ions play an important role in cancer cell proliferation, leading to that copper ions accumulated in cancer cells are higher than those in normal cells [50]. Hence, it is necessary to study copper ions’ responsiveness of HP-β-CD/TMZ vesicles. As shown in Fig. 9b, HP-β-CD/TMZ vesicle disappeared while a large number of irregular aggregates emerged when copper ions were dropped into this vesicle solution, indicating disassembly of HP-β-CD/TMZ vesicle. As shown in Scheme 2, disassembly of HP-β-CD/TMZ vesicle may be induced by coordination between copper ions and amido group of TMZ. Moreover, UV-vis detection [51] can further verify disassembly of HP-β-CD/TMZ vesicle since the UV-vis absorbance intensity of HP-β-CD/TMZ vesicle treated with copper ions decreased obviously than that of HP-β-CD/TMZ vesicle. So, HP-β-CD/TMZ vesicle is a smart vesicle, which can respond to copper ions sensitively, as illustrated in Scheme 2. Besides, HP-β-CD/TMZ vesicle is very sensitive to zinc ions, HP-β-CD/TMZ vesicles changed to irregular aggregates, and the UV-vis absorbance intensity of HP-β-CD/TMZ vesicles decreased clearly as well when zinc ions were added into the HP-β-CD/TMZ vesicle system (Fig. S4). But HP-β-CD/TMZ vesicle cannot respond to lithium ions since the addition of lithium ions almost did not change the morphologies and UV-vis absorbance intensity of HP-β-CD/TMZ vesicles (Fig. S5). This may be attributed to that copper ions and zinc ions have more strong complex ability with an amido group of TMZ than lithium ions. For TMZ itself, when copper ions or zinc ions were added, its UV-vis absorbance intensity reduced clearly (Fig. S6 and Fig. S7). Copper ions or zinc ions can coordinate with an amido group of TMZ, which can further decrease TMZ’s UV-vis absorbance intensity.

Fig. 9
figure 9

(a) TEM image of the HP-β-CD/TMZ vesicle (1 × 10−4 mol/L), scale bar = 200 nm; (b) TEM image of the HP-β-CD/TMZ vesicle (1 × 10−4 mol/L) treated with copper ions (5 × 10−5 mol/L), scale bar = 300 nm; (c) UV-vis spectra comparison of HP-β-CD/TMZ vesicle and HP-β-CD/TMZ vesicle treated with different molar ratio copper ions at room temperature

Full size image

Conclusions

In conclusion, we have successfully fabricated a supramolecular amphiphile vesicle based on HP-β-CD and TMZ. TMZ, which is the primary drug for GBM treatment, can be loaded into the membranous layer of HP-β-CD/TMZ vesicle effectively. The morphology of this vesicle was confirmed by electron microscopy. Moreover, the mechanism of vesicle formation was studied by XRD, FT-IR, UV-vis spectrum, 1H NMR, and 2D NMR ROSEY. Finally, we find that HP-β-CD/TMZ vesicle can respond to copper ions sensitively. Hence, our HP-β-CD/TMZ vesicle as a smart drug delivery material may have great application potential in GBM treatment since copper ions in cancer cells are higher than those in normal cells.