Facile construction of shape-regulated β-cyclodextrin-based supramolecular self-assemblies for drug delivery

Journal Pre-proof Facile construction of shape-regulated ␤-cyclodextrin-based supramolecular self-assemblies for drug delivery Yang Bai, Na An, Di Chen, Ying-zhe Liu, Cai-ping Liu, Hao Yao, Chao Wang, Xin Song, Wei Tian

PII:

S0144-8617(19)31382-7

DOI:

https://doi.org/10.1016/j.carbpol.2019.115714

Reference:

CARP 115714

To appear in:

Carbohydrate Polymers

Received Date:

2 October 2019

Revised Date:

3 December 2019

Accepted Date:

5 December 2019

Please cite this article as: Bai Y, An N, Chen D, Liu Y-zhe, Liu C-ping, Yao H, Wang C, Song X, Tian W, Facile construction of shape-regulated ␤-cyclodextrin-based supramolecular self-assemblies for drug delivery, Carbohydrate Polymers (2019), doi: https://doi.org/10.1016/j.carbpol.2019.115714

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Facile

construction

of

β-cyclodextrin-based

shape-regulated

supramolecular self-assemblies for drug delivery Yang Bai1*, Na An1, Di Chen13, Ying-zhe Liu4, Cai-ping Liu1, Hao Yao2, Chao Wang1, Xin Song2, Wei Tian2* 1 Shaanxi Key Laboratory of Chemical Additives for Industry, College of Chemistry and Chemical Engineering, Shaanxi University of Science and Technology, Xi’an 710021 , China 2 MOE Key Laboratory of Material Physics and Chemistry under Extraordinary Conditions

ro of

and Shaanxi Key Laboratory of Macromolecular Science and Technology, School of Science, Northwestern Polytechnical University, Xi’an, 710072, China

3 Institute of Basic Medical Sciences, Xi'an Medical University, Xi'an 710021, China

-p

4 Xi’an Modern Chemistry Research Institute, Xi’an 710065, China

lP

re

Highlights：  β-CD-based SSAs exhibit the shaped-regulated morphology.  Host-guest and hydrophobic-hydrophilic interaction can be tuned in the SSAs.  The β-CD-based SSAs show pH-responsive curcumin self-release behaviors.  The shaped-regulated β-CD-based SSAs exhibit different biological performance.

Abstract: Although supramolecular prodrug self-assemblies have been proven as

practical

na

efficient nanocarriers for cancer therapy, tedious synthesis procedures have made their applications

more

difficult.

In

this

paper,

β-cyclodextrin-based

supramolecular self-assemblies (SSAs) were directly constructed by utilizing

ur

β-cyclodextrin trimer (β-CD3) as the host unit and unmodified curcumin as the guest unit. Due to the adjustment of host-guest inclusion and hydrophilic-hydrophobic

Jo

interactions occurring in the SSAs, their morphology could be readily tuned by changing the ratio of the two components. Different self-assembly morphologies, such as spherical complex micelles, spindle-like complex micelles and multi-compartment vesicles, were obtained. Furthermore, basic cell experiments were performed to study the corresponding effects of the SSA shape on their biological properties. Compared to the other micelles, the spindle-like complex micelles exhibited enhanced cellular toxicity, uptake behaviors and apoptosis rates, and the spherical complex micelles 1

exhibited poor performance. The performance of the multi-compartment vesicles was similar to that of the spindle-like complex micelles. The facile construction of these shape-regulated SSAs and their different cellular biological properties might be valuable in the controlled drug release field. Keywords：β-cyclodextrin; drug delivery; host-guest interaction 1. Introduction Supramolecular self-assemblies (SSAs) based on host-guest interactions have received particular attention due to their dynamic nature and specific external

ro of

stimuli-responsive assembly behaviors, which endow them with outstanding potential applications in biomedicine, particularly in the controlled drug release field (Sun,

Chen, Han, & Liu, 2017; Bai et al., 2019; Ma et al., 2014; Yang, Jia, Zhang, Li, & Liu, 2018; Fu, Chen, Yu, & Liu, 2019; Zhang, Liu, Yu, Wen, & Liu, 2019; Cui et al., 2019;

-p

Song, Lou, Ma, Gao, & Yang, 2019; Jiang et al., 2018; Braegelman, & Webber, 2019;

Bai et al., 2018; Liao et al., 2017; Li et al., 2019). Among the various SSAs drug

re

delivery systems, drugs are traditionally passive encapsulated in SSAs and are released through external stimuli-induced dissociation of host/guest inclusion

lP

complexes (Zhang, Zhang, Liu, & Liu, 2019; Cheng, Zhang, Liu, & Yoon, 2019; Zhang, Liu, & Liu, 2019; Han et al., 2018; Zuo et al., 2018; Wu, Chen, Yu, Li, & Liu,

na

2019; Zhang et al., 2018; Cheng et al., 2018; Yang et al., 2018). Since this passive loading process is associated with some inherent drawbacks, especially limited drug loading efficiency (usually less than 10 wt%) and premature burst release,

ur

stimuli-responsive SSAs with higher drug loading efficiencies based on the prodrug strategy have been constructed (Tong et al., 2016; Wang, Du, Wang , Jin, & Ji, 2015;

Jo

Shao et al., 2018; Guan, Chen, Wu, Li, & Liu, 2019; Zhang, Zhang, Xiao, Liu, & Yu, 2018; Yu , Yu , Mao , Gao, & Huang, 2015; Yu et al., 2018). For example, Ji and Jin et al. constructed some supramolecular prodrug self-assembly systems exhibiting the multi-stimuli-responsive drug release (Tong et al., 2016; Wang, Du, Wang , Jin, & Ji, 2015). Wang recently utilized the inclusion interaction between a water-soluble pillar[6]arene and a drug-drug prodrug to construct nano self-assemblies, realizing the codelivery of two types of hydrophobic drugs (Shao et al., 2018). These SSA drug 2

delivery systems usually exhibit delicate and great therapeutic effects; however, their tedious synthesis procedures made their practical applications more difficult. Thus, the establishment of a more efficient and viable strategy was necessary. Compared to a covalently linked prodrug, an active drug without modification that could be directly utilized to construct host-guest interaction-based SSAs seemed more attractive due to their simple strategy. Moreover, in addition to the host-guest inclusion interaction, hydrophilic-hydrophobic interactions also occurred in these systems (Cui et al., 2019; Song, Lou, Ma, Gao, & Yang, 2019; Jiang et al., 2018;

ro of

Braegelman, & Webber, 2019). Thus, through the regulation of these two noncovalent interactions, SSAs were endowed with adjustable self-assembly and adaptable morphological properties. According to the literature, curcumin (Cur), which

possesses potential antitumor activity, could form host-guest inclusions with

-p

β-cyclodextrin (β-CD) with a stoichiometry of 1:2 (Tang, Ma, Wang, & Zhang, 2002; Manchineella, Murugan, & Govindaraju, 2017; Poorghorban et al., 2015; Wanninger,

re

Lorenz, Subhan, & Edelmann, 2015; Wallace, Kee, & Huang, 2013; Shlar, Droby, & Rodov, 2018), which is responsible for regulating the self-assembly properties due to inherent

two

inclusion

interaction

lP

their

positions.

Hence,

if

host-guest

interaction-based SSAs could be constructed directly with curcumin as the guest

na

moiety, the simple preparation methods and adjustable self-assembly morphology of these drug delivery systems may provide them with great applications in biomedical science. In addition, the shape and size regulation of these supramolecular

ur

self-assemblies may reveal the relationship between self-assembly morphology and biological performance, which is meaningful for the design of more efficient drug

Jo

delivery systems but insufficiently understood in the context of traditional preparative methods (Hu et al., 2013; Wong, Mason, Stenzel, & Thordarson, 2017; Abdelmohsen et al., 2016; Zhang, Liu, Li, Wu, & Jiang, 2019). Based on the considerations above, supramolecular self-assemblies were directly constructed using β-CD trimer (β-CD3) as the host unit and curcumin as the guest unit. Through adjustment of host-guest inclusion and hydrophobic-hydrophilic interactions in the self-assembled systems, the morphology of the self-assemblies can be 3

transformed from spherical complex micelles to spindle-like complex micelles and then further to multi-compartment vesicles by increasing the molar ratio of β-CD trimer to curcumin (Scheme 1). Furthermore, drug release behavior could be realized and then accelerated by pH as a stimulus. Moreover, basic cell experiments indicated that these SSAs exhibited shape-regulated cytotoxicity, uptake behaviors and apoptosis rates and might have promising applications in cancer therapy.

2. Result and Discussion self-assembly

behaviors

of

β-cyclodextrin

ro of

2.1 Controllable

trimer/curcumin-based supramolecular self-assemblies

According to the literature, as one type of hydrophobic anticancer drug, curcumin

can be encapsulated into the cavity of β-CD with a stoichiometry of 1:2 (Tang, Ma,

-p

Wang, & Zhang, 2002; Manchineella, Murugan, & Govindaraju, 2017; Poorghorban et al., 2015; Wanninger, Lorenz, Subhan, & Edelmann, 2015; Wallace, Kee, & Huang,

re

2013; Shlar, Droby, & Rodov, 2018). Thus, to obtain supramolecular self-assemblies by directly utilizing hydrophobic drugs, carefully characterized β-CD trimer (β-CD3,

lP

Fig. S1-S3) (Dong, Liu, Zhou, Yan & Zhu, 2011; Yang et al., 2014) and curcumin (Cur) were chosen as the host and guest units, respectively, as presented in Scheme 1.

na

Supramolecular self-assemblies were first obtained by the dropwise addition of a curcumin solution in DMSO to β-CD3 aqueous solution with a certain molar ratio. To inspect the morphology and size of the supramolecular self-assemblies, transmission

ur

electron microscopy (TEM), atomic force microscopy (AFM) and dynamic light scattering (DLS) were utilized, and the results are shown in Fig. 1. Evidently, when

Jo

the stoichiometry of β-CD3 and curcumin was 2:3, the resulting supramolecular self-assemblies (SSAs-1) formed spherical complex micelles with an average diameter (Dav) of 270 nm (Fig. 1A, D), which was close to the hydrodynamic diameter (Dh) of 253 nm determined by DLS (Fig. 1J). Notably, as the concentration of β-CD3 increased, the self-assembly morphology of the supramolecular self-assemblies transformed from sphere complex to spindle-like micelles (SSAs-2, β-CD3: curcumin = 4:3) with a diameter of approximately 150 nm (Fig. 1B, 4E) and 4

then further changed into multi-compartment vesicles (SSAs-3, β-CD3: curcumin = 6:3) with approximately a diameter of 90 nm (Fig. 1C, 4F). For a clear description, the abovementioned self-assemblies were named SSAs-1, SSAs-2 and SSAs-3, respectively. Then, AFM was performed to investigate their morphology. As shown in Fig. 1G-I, the morphologies of supramolecular aggregates monitored by AFM were consistent with those observed from the TEM results. Specifically, AFM images of SSAs-1 showed a complex structure, suggesting the complex micelle morphology of SSAs-1. The AFM results provided bigger than that of TEM and DLS test results

ro of

which may be caused by the aggregation during the sample preparation process. All the self-assemblies exhibited stability in 7 days. In addition, the critical aggregation concentration (CAC) of SSAs-1, -2, -3 was determined as 0.050, 0.026 and 0.011 mg/mL using pyrene fluorescent method (Wilhelm, M., Zhao, C.L., Wang, Y., et al.,

-p

1991). So when the concentration was determined above CAC, the SSAs could be

obtained. Based on the above results, the transition process suggested that the

re

self-assembly morphology of these supramolecular self-assemblies could be readily adjusted only by changing the molar ratio of host/guest moieties.

lP

To investigate the supramolecular self-assembly mechanism at different host/guest molar ratios in detail, 1H NMR and 2D NOESY NMR experiments were performed,

na

as presented in Fig. 2. According to the molar ratio of the abovementioned SSAs-1, 2 and 3, their 1H NMR spectra are shown in Fig. 2A. As the molar ratio of β-CD3/Cur increased, the integral ratios of a in Cur to 2,4,3,5,6-H in β-CD3 for SSAs-1, 2 and 3

ur

were measured as 1: 30.86, 1: 29.49 and 1: 31.72, respectively, which were less than the corresponding theoretical values (1:11.67, 1:23.33, 1:35) due to the

Jo

hydrophilic-hydrophobic interaction-induced shielding effects, indicating that part of Cur might be encapsulated in the self-assemblies, especially when the molar ratio of β-CD3/Cur was low. However, the tendency of measured integral values simultaneously become closer to the theoretical values as the concentration of β-CD3 increased, also suggesting that the hydrophilic-hydrophobic interaction becomes weaker and less Cur molecules were entrapped in the SSAs. On the other hand, the downfield shift in the 1-H proton peak in β-CD3 indicated that host-guest interactions 5

occurred in the self-assemblies (Fig. 2A). Thus, the host-guest interaction detected in the above SSAs was studied using 2D NOESY NMR spectroscopy. As shown in Fig. 2B-D, the cross-peak interactions between β-CD3 and Cur protons increased as the ratio of β-CD3 increased, indicating enhancement in the host-guest interaction between Cur and β-CD3. Based on these results, we can further speculate that when the molar ratio

of

β-CD3/Cur

was

2:3,

host-guest

interactions

occurred,

but

hydrophilic-hydrophobic interactions were the main driving force for the self-assembly process of the SSAs into complex micelles with part of Cur

ro of

encapsulated in the self-assemblies. Then, as the molar ratio of β-CD3/Cur increased, the host-guest interaction was enhanced, while the hydrophilic-hydrophobic interaction was reduced, leading to morphological and size transitions of the SSAs.

Fluorescence emission spectroscopy was further employed to confirm the

-p

formation and inclusion ratio of host-guest inclusion complexes between β-CD3 and curcumin. The results are shown in Fig. 3. Evidently, when the concentration of Cur

re

was kept at 1×10-5 M (4-hydroxy-3-methoxyphenyl units: 2×10-5 M), as the β-CD concentration increased, the fluorescence maximum absorption peak moved from 550

lP

nm to 525 nm, and the fluorescence intensity increased markedly. Thus, it could be speculated that the benzene moiety in Cur, which acted as a fluorophore, moved into

na

the hydrophobic cavity of β-CD, thereby reducing the quenching of fluorescence and increasing the fluorescence intensity. To determine and verify the inclusion stoichiometry between Cur and β-CD3, a double reciprocal plot was utilized via the

(1)

Jo

ur

modified Benesi-Hildebrand equation as follows: 1 1 1 = + 𝐼−𝐼0 𝑘∆𝜀[𝐻][𝐺] ∆𝜀[𝐺]

*β-CD3 was denoted as three host units, while curcumin was denoted as two guest units

where I0 denotes the fluorescence intensity in pure water, I denotes the

fluorescence intensity, Δε denotes the molar extinction coefficient between the host and host-guest complex, and β-CD3 was denoted by three host CD units, while Cur was denoted by two guest units. Clearly, the plot of 1/(I−I0) versus 1/[β-CD] gave a

6

straight line, and the correlation coefficients R2 was equal to 0.992, which means an inclusion relationship with a stoichiometry of 1:2 between Cur and β-CD exists. Furthermore, the inclusion interaction between curcumin and β-CD3 was studied through MD simulation, and two inclusion orientations were also considered, namely, head-to-head and tail-to-tail, for the two CD hosts. After a 400 ps MD simulation, the equilibrium structures of inclusion for the two orientations are shown in Fig. 4. The binding energy of Cur with two β-CD3 units was estimated on the basis of the interaction energy via the following equation: ΔEbind = −ΔEint = −(Einclu − Ehost −Eguest)

ro of

(2)

where Einclu, Ehost, and Eguest are the potential energies of the inclusion complex, two β-CD3 units and drug, respectively. The larger the binding energy is, the stronger the binding affinity. Moreover, the binding energies are further decomposed into

-p

electrostatic and van der Waals interaction components: ΔEbind =ΔEelec + ΔEvdw

(3)

re

Table 1 shows the binding affinities of the two inclusion motifs. The TT orientation has a stronger inclusion interaction than the HH orientation. It is

lP

reasonable that the two terminal groups of the Cur molecule are relatively large and suitable for encapsulation in the head of CD. Furthermore, the main driving force is

na

the van der Waals interactions, suggesting that the hydrophobic interaction and size matching between CD and Cur dominated the inclusion interaction. 3.3 Drug release in vitro and cell experiments

ur

To further confirm the curcumin release behaviors of these supramolecular self-assemblies, drug release experiments were performed under different pH

Jo

conditions. The six release results shown in Fig. 5 represent the cumulative amount released from the controls (SSAs-1, SSAs-2 and SSAs-3) in PBS at pH 7.4 and pH 5.0. Interestingly, the cumulative amount of curcumin released from SSAs-1, SSAs-2 and SSAs-3 at pH 5.0 was always clearly greater than that at pH 7.4, indicating the pH-responsive drug release property of the SSAs. For instance, the cumulative amount released from SSAs-2 in PBS at pH 5.0 was 96% after 36 h, which is more than that released from SSAs-2 in PBS at pH 7.4 (36%). This is someway 7

counterintuitive, since the acid form of curcumin is expected a higher affinity towards the β-CD cavity (Tang, Ma, Wang, & Zhang, 2002). Modulation of Cur release kinetics from Cur-CD complexes as a function of pH has been previously achieved by embedding the complexes in pH-responsive hydrogels (Gerola, A.P., Silva, D.C., et al, 2015). In our system, the partial protonation of the tertiary amine nitrogen in β-CD3 might provoke coulombic repulsions (Gallego-Yerga, L., José González-álvarez, M., et al, 2014; Gallego-Yerga, L., Blanco-Fernández, L., 2015), leading to the dissociation of inclusions. The DLS and TEM were performed to testify the

ro of

hypothesize, the larger SSAs was found at pH 5.0 (Fig. S4), indicating the better diffusion environment for the release of Cur. These results indicated that the release rate and amount of curcumin released could be controlled by pH, which is beneficial

for cancer therapy at low pH conditions. In addition to the contribution of their

-p

dynamic nature to the drug release behavior of these SSA drug delivery systems, the

influence of pH was also investigated with the abovementioned fluorescence methods.

re

As shown in Fig. S5, compared to the results shown in Fig. 3, 1/(I−I0) versus 1/[β-CD] gave a straight line, and the correlation coefficients R2 were equal to 0.917 and 0.592

lP

at pH 5.0 tested after 4 h and 24 h, respectively, which means that the Cur could release from the self-assemblies due to the increased coulombic repulsion, leading to

na

pH-induced accelerated drug release.

An MTT cell proliferation assay was used to evaluate the cellular toxicity of the abovementioned SSAs-1, SSAs-2 and SSAs-3. PC3 and MCF-7 cells were incubated

ur

with the above self-assemblies at various concentrations. The in vitro cell viability of PC3 (Fig. 6A) and MCF-7 (Fig. 6B) cells cultured with free curcumin and SSAs-1,

Jo

SSAs-2 and SSAs-3 at the same drug concentration was determined. As shown in Fig. 6A, the in vitro half-maximal inhibitory concentration (IC50) values of SSAs-1, SSAs-2 and SSAs-3 were 12.709, 8.111 and 8.155 μg/mL, respectively, which could rival that of free Cur (9.742 μg/mL). However, except for SSAs-1, SSAs-2 and SSAs-3 exhibited higher cytotoxicity to PC3 cells than free Cur at the same free concentration, suggesting the good inhibitory effect of these SSAs on cell proliferation. In addition, SSAs-1, SSAs-2 and SSAs-3 were also cultured with 8

MCF-7 cells to test the differences among the three types of supramolecular self-assemblies. As shown in Fig. 6B, the in vitro half-maximal inhibitory concentration (IC50) values of SSAs-1, SSAs-2, SSAs-3 and free curcumin were 27.784, 14.180, 13.649 and 20.512 μg/mL, respectively. SSAs-1 exhibited lower cytotoxicity than curcumin may be caused by the worse uptake by cells due to their big size. However, SSAs-2 and SSAs-3 also exhibited higher cytotoxicity to MCF-7 cells than did free curcumin and SSAs-1 at the same drug concentration, indicating that spindle-like SSAs-2 and multi-compartment vesicles like SSAs-3 have greater

ro of

potential than SSA-1 in drug delivery and cell proliferation inhibition. Furthermore, the biocompatible property of β-CD3 was also tested, β-CD3 was cultured with LO2 normal cells. As shown in Fig. S6, β-CD3 did not show any obvious cytotoxicity to LO2 cells at the concentration below 0.16 mg/mL after 48 h, as reflected by the fact

-p

that the cell viability still exceeded 85%, suggesting the good biocompatible property of synthesized β-CD3.

re

According to the reported literature (Hu et al., 2013), cellular internalization can be influenced by different self-assembly morphologies. Thus, to study whether

lP

SSAs-1, SSAs-2 and SSAs-3 could be internalized by cancer cells, confocal laser scanning microscopy (CLSM) was applied to evaluate their intracellular uptake and

na

distribution with PC3 cells as the model. As shown in Fig. 7, when the incubation time was set as 6 h the green fluorescence intensities from curcumin in SSAs-1, SSAs-2 and SSAs-3 in PC3 cells were different: SSAs-1 showed worse cellular

ur

uptake than that of SSAs-2 and SSAs-3. The cellular uptake order of curcumin is Cur>SSAs-2>SSAs-3>SSAs-1 according to the Cur-FL intensity. This result also

Jo

suggested that the shape and size of SSAs could influence their cellular uptake. The spindle-shaped or multicompartment SSAs exhibited better efficacy than the nano-sized spherical aggregates due to their irregular shape which is agreed with previous reports (Wong, Mason, Stenzel, & Thordarson, 2017; Abdelmohsen et al., 2016; Zhang, Liu, Li, Wu, & Jiang, 2019). Moreover, the results for cellular uptake by MCF-7 cells was the same as those for cellular uptake by PC3 cells, as shown in Fig. S7.

All

the

results

described

above 9

suggested

that

β-cyclodextrin

trimer/curcumin-based self-assemblies could deliver curcumin to and release curcumin in cancer cells. Finally, to understand the inhibitory mechanism of the obtained SSAs on PC3 cells and MCF-7 cancer cells, PC3 and MCF-7 cells were incubated with SSAs-1, SSAs-2, SSAs-3 or curcumin with the Cur concentration as a constant (20 μg/mL) for 48 h before being subjected to FITC-Annexin V/PI staining to assess their cell cycle distribution. As shown in Fig. 7, the apoptosis rates of PC3 cells after treatment with SSAs-1, SSAs-2, SSAs-3 and curcumin were 8.4%, 30.8%, 23.6%, and 27.5%, respectively. In addition, the apoptosis rate for cell treated with

ro of

SSAs-2 is slightly higher than that for cells treated with pure curcumin. Similarly, the corresponding apoptosis rates of MCF-7 cells after treatment with SSAs-1, SSAs-2,

SSAs-3 and curcumin were 13.9%, 17.5%, 16.9%, and 17.7%, respectively (Fig. S8). These results showed that the obtained SSAs, especially spindle-like SSAs-2, could

-p

induce obvious apoptosis in PC3 and MCF-7 cells. 3. Conclusions

re

In summary, a simple curcumin delivery platform was obtained through host-guest interactions between β-CD trimer and curcumin. Multiple hierarchical nanostructures,

lP

such as complex micelles (SSAs-1), complex spindle-like micelles (SSAs-2) and multicompartment vesicles (SSAs-3), were easily constructed only by adjusting the

na

ratio of β-CD trimer to curcumin due to the regulation of host-guest interactions and host-guest inclusion interactions. Among these morphologies, the complex spindle-like micelles exhibited the best cellular toxicity, uptake behaviors and

ur

apoptosis rates, and the complex micelles exhibited poor performance. The performance of the multi-compartment vesicles was close to that of the complex

Jo

spindle-like micelles. All these results demonstrate the facile construction of curcumin delivery platforms and their potential application in the biomedical field.

Author Contribution Yang Bai, writing, review & editing, and Funding Na An, wring original draft; Di Chen, cell experiment; 10

Ying-zhe Liu, molecular dynamic simulation; Cai-ping Liu, methodology; Hao Yao, synthesis of cyclodextrin trimer; Chao Wang, methodology; Xin Song, software; Huai-tian Bu, helpful discussion in the manuscipt revised process

ro of

Wei Tian, review and editing.

Acknowledgements

This project was supported by the National Science Foundation of China (No. 21801162 and No. 21674086).

-p

References

Abdelmohsen, L. K. E. A., Williams, D. S., Pille, J., Ozel, S. G., Rikken, R. S. M.,

re

Wilson D. A., et al. (2016). Formation of Well-Defined, Functional Nanotubes via Osmotically Induced Shape Transformation of Biodegradable Polymersomes.

lP

Journal of the American Chemical Society, 138(30), 9353-9356. Bai, Y., Liu, C.P., Xie, F.Y., Ma, R., Zhuo, L.H., Li, N., Tian, W. (2019). Construction β-cyclodextrin-based

supramolecular

hyperbranched

polymers

na

of

self-assemblies using AB2-type macromonomer and their application in the drug delivery field. Carbohydrate polymers, 213, 411-418.

ur

Bai, Y., Liu, C.P., Song, X., Zhuo, L.H., Bu, H.T., Tian, W. (2018). Photo- and pH-

dually-responsive

β-cyclodextrin-based

supramolecular

prodrug

Jo

complexes self-assemblies for programmed drug delivery. Chemistry-an Asian Journal. 13, 3903-3911.

Braegelman, A. S., & Webber, M. J. (2019). Integrating Stimuli–Responsive Properties in Host–Guest Supramolecular Drug Delivery Systems, Theranostics, 9(11), 3017-3040.

11

Cheng, H. W., Fan, X. S., Wang, X. Y., Ye, E. Y., Loh, X. J., Li, Z. B., et al. (2018). Hierarchically Self-Assembled Supramolecular Host-Guest Delivery System for Drug Resistant Cancer Therapy. Biomacromolecules, 19(6), 1926-1938. Cheng, H.-B., Zhang, Y.-M., Liu, Y., & Yoon, J. Y. (2019). Turn-On Supramolecular Host-Guest Nanosystems as Theranostics for Cancer. Chem, 5(3), 553-574. Cui, Y.-H., Deng, R., Li, Z., Du, X.-S., Jia, Q., Wang, X.-H., et al. (2019). Pillar[5]arene pseudo[1]rotaxane-based redoxresponsive supramolecular vesicles for controlled drug release. Materials Chenistry Fronties, 3(7), 1427-1432.

ro of

Dong, R. J., Liu, Y., Zhou, Y. F., Yan, D. Y., & Zhu, X. Y. (2011). Photo-reversible supramolecular hyperbranched polymer based on host-guest interactions. Polymer Chemistry, 2(12), 2771-2774.

Fu, H.-G., Chen, Y., Yu, Q. L., & Liu, Y. (2019). A Tumor-targeting

-p

Ru/Polysaccharide/Protein Supramolecular Assembly with High Photodynamic Therapy Ability. Chemical Communications, 55(21), 3148-3151.

re

Guan, X. R., Chen, Y., Wu, X., Li, P. Y., & Liu, Y. (2019). Enzyme-responsive sulfatocyclodextrin/prodrug supramolecular assembly for controlled release of

lP

anti-cancer drug chlorambucil. Chemical Communications, 55(7), 953-956. Han, S. L., Chen, S. Y., Li, L. L., Li, J., An, H. J., Tao, H., et al. (2018). Multiscale Multifunctional

Emulsions

by

Host-Guest

Interaction-Mediated

na

and

Self-Assembly. ACS Central Science, 4(5), 600-605. Hu, X. L., Hu, J. M., Tian, J., Ge, Z. S., Zhang, G. Y., Luo, K. F., et al. (2013).

ur

Polyprodrug Amphiphiles: Hierarchical Assemblies for Shape-Regulated Cellular Internalization, Trafficking, and Drug Delivery. Journal of the American

Jo

Chemical Society, 135(46), 17617-17629.

Jiang, Y., Pan, X. S., Chang, J., Niu, W. J., Hou, W. J., Kuai, H. L., et al. (2018). Supramolecularly Engineered Circular Bivalent Aptamer for Enhanced Functional Protein Delivery，Journal of the American Chemical Society, 140(22), 6780−6784. Gallego-Yerga, L., Gozález-Álvarez, J.M., Mayordomo, N., Santoyo-González, F., Benito, J.M., et al. (2014). Dynamic self-assembly of polycationic 12

clusters based on cyclodextrins for ph-sensitive dna nanocondensation and delivery by component design. Chemistry - A European Journal, 20(22), 6622-6627. Gallego-Yerga, L., Blanco-Fernández, L., Urbiola, K., Carmona, T., Marcelo, G. , & Benito, J.M., et al. (2015). Host-guest-mediated DNA templation of polycationic supramolecules for hierarchical nanocondensation and the delivery of gene material. Chemistry - A European Journal, 21(34), 12093-12104.

ro of

Gerola, A.P., Silva, D.C., Jesus, S., Carvalho, R.A., Rubira, A.F., et al. (2015). Synthesis and controlled curcumin supramolecular complex release from

pH-sensitive modified gum-arabic-based hydrogels. RSC Advances., 5(115), 94519-94533.

-p

Li, F., Liu, D., Liao, X., Zhao, Y., Li, R., & Yang, B. (2019). Acid-controlled release complexes of podophyllotoxin and etoposide with acyclic for

low

Chemistry, 27(3), 525-532.

cytotoxicity.

Bioorganic

re

cucurbit[n]urils

&

Medicinal

lP

Liao, R., Lv, P., Wang, Q., Zheng, J., & Yang, B. (2017). Cyclodextrin-based biological stimuli-responsive carriers for smart and precision medicine.

na

Biomaterials Science, 5(9), 1736-1745.

Ma, M. F., Sun, T., Xing, P. Y., Li, Z. L., Li, S. Y., Su, J., et al. (2014). A supramolecular curcumin vesicle and its application in controlling curcumin

ur

release. Colloids and Surfaces A: Physicochemical and Engineeering Aspects, 459, 157-165.

Jo

Manchineella, S., Murugan, N. A., & Govindaraju, T. (2017). Cyclic Dipeptide-based Ambidextrous Supergelators: Minimalistic Rational Design, Structure-Gelation Studies and In Situ Hydrogelation. Biomacromolecules, 18(11), 3581-3590.

Poorghorban, M., Karoyo, A. H., Grochulski, P., Verrall, R. E., Wilson, L. D., & Badea, L. (2015). A 1H NMR Study of Host/Guest Supramolecular Complexes of a Curcumin Analog with #Cyclodextrin and a #CD-conjugated Gemini Surfactant. Molecular Pharmaceutics, 12(8), 2993-3006. 13

Shao, W., Liu, X., Sun, G. P., Hu, X.-Y., Zhu, J.-J., & Wang, L. Y. (2018). Construction of drug-drug conjugate supramolecular nanocarriers based on water-soluble

pillar[6]arene

for

combination

chemotherapy.

Chemical

Communications, 54(68), 9462-9465. Shlar, L., Droby, S., & Rodov, V. (2018). Antimicrobial Coatings on Polyethylene Terephthalate Based on Curcumin/Cyclodextrin Complex Embedded in a Multilayer Polyelectrolyte Architecture. Colloids and Surfaces B: Biointerfaces, 164, 379-387.

ro of

Song, N., Lou, X.-Y., Ma, L. J., Gao, H., & Yang Y.-W. (2019). Supramolecular nanotheranostics based on pillarenes. Theranostics, 9(11), 3075-3093.

Sun, H.-L., Chen, Y., Han, X., & Liu, Y. (2017). Tunable Supramolecular Assembly and Photoswitchable Conversion of Cyclodextrin/Diphenylalanine-Based 1D and Nanostructures.

Angewandte

Chemie-International

-p

2D

7062-7065.

Edition,

56(25),

re

Tang, B., Ma, L., Wang, H.-Y., & Zhang, G.-Y. (2002). Study on the Supramolecular Interaction of Curcumin and β-cyclodextrin by Spectrophotometry and Its

1355-1361.

lP

Analytical Application. Journal of Agricultural and Food Chemistry, 50(6),

na

Tong, H. X., Chen, Y. J., Li, Z. H., Li, H., Chen, T. T., Jin, Q. (2016). Glutathione Activatable Photosensitizer-Conjugated Pseudopolyrotaxane Nanocarriers for Photodynamic Theranostics. Small, 12(45), 6223-6232.

ur

Wallace, S. J., Kee, T. W., & Huang, D. M. (2013). Molecular Basis of Binding and Stability of Curcumin in Diamide-Linked γ- Cyclodextrin Dimers. Journal of

Jo

Physical Chemistry B, 117(41), 12375-12382.

Wang, Y., Du, J. W., Wang, Y. X., Jin, Q., & Ji, J. (2015). Pillar[5]arene based supramolecular prodrug micelles with pH induced aggregate behavior for intracellular drug delivery. Chemical Communications, 51(14), 2999-3002. Wanninger, S., Lorenz, V., Subhan, A., & Edelmann, F. T. (2015). Metal complexes of curcumin – synthetic strategies, structures and medicinal applications. Chemical Society Reviews, 44(15), 4986-5002. 14

Wilhelm, M., Zhao, C.L., Wang, Y., Xu, R., Winnik, M. A., Mura, J. L., et al. (1991). Poly(styrene-ethylene oxide) block copolymer micelle formation in water: a fluorescence probe study. Macromolecules, 24(5), 1033-1040. Wong, C. K., Mason, A. F., Stenzel, M. H., & Thordarson, P. (2017). Formation of non-spherical polymersomes driven by hydrophobic directional aromatic perylene interactions. Nature Communcation, 8, 1240. Wu,

X.,

Chen,

Y.,

Yu,

Q.

L.,

Li,

F.-Q.,

&

Liu,

Y.

(2019).

A

cucurbituril/polysaccharide/carbazole ternary supramolecular assembly for

ro of

targeted cell imaging. Chemical Communications, 55(30), 4343-4346. Yang, Z., Fan, X. D., Tian, W., Wang, D., Zhang, H. T., & Bai, Y. (2014). Nonionic

Cyclodextrin Based Binary System with Upper and Lower Critical Solution Temperature Transitions via Supramolecular Inclusion Interaction. Langmuir,

-p

30(25), 7319-7326.

Yang, G.-G., Hao, L., Cao, Q., Zhang, H., Yang, J., Ji, L.-N., et al. (2018).

re

Three-in-One Self-Assembled Nanocarrier for Dual-Drug Delivery, Two-Photon Imaging, and Chemo-Photodynamic Synergistic Therapy. ACS Applied Materials

lP

& Interfaces, 10(34), 28301-28313.

Yang, Y., Jia, X., Zhang, Y. M., Li N., & Liu, Y. (2018). Supramolecular Nanoparticle

na

Based on β-CD Modified Hyaluronic Acid for DNA Capsulation and Controlled Release. Chemical Communications, 54(63), 8713-8716. Yu, G. C., Yu, W., Mao, Z. W., Gao, C. Y., & Huang, F. H. (2015). A Pillararene-Based

ur

Ternary Drug-Delivery System with Photocontrolled Anticancer Drug Release. Small, 11(8), 919-925.

Jo

Yu, G. C., Zhao, X. L., Zhou, J., Mao, Z. W., Huang, X. L., Wang, Z. T., et al. (2018). Supramolecular Polymer-Based Nanomedicine: High Therapeutic Performance and Negligible Long-Term Immunotoxicity. Journal of the American Chemical Society, 140(25), 8005-8019. Zhang, Y. Y., Yang, D., Chen, H. Z., Lim, W. Q., Phua, F. S. Z., An, G. H., et al. (2018).

Reduction-sensitive

fluorescence

15

enhanced

polymeric

prodrug

nanoparticles for combinational photothermal-chemotherapy. Biomaterials, 163, 14-24. Zhang, Y.-M., Zhang, N.-Y., Xiao, K., Liu, Y., & Yu, Q. L. (2018). Photo-Controlled Reversible Microtubule Assembly Mediated by Cyclodextrin Derivative. Angewandte Chemie-International Edition, 57(28), 8649-8653. Zhang, Z. K., Liu, C. R., Li, C., Wu, W., & Jiang, X. Q. (2019). Shape Effects of Cylindrical versus Spherical Unimolecular Polymer Nanomaterials on in Vitro and in Vivo Behaviors. Research, DOI:10.34133/2019/ 2391486. Y.-M.,

Liu,

Y.-H.,

&

Liu,

Y.

(2019).

Cyclodextrin-Based

ro of

Zhang,

Multistimuli-Responsive Supramolecular Assemblies and Their Biological Functions. Advanced Materials, 1806158.

Zhang, Y.-M., Liu, J.-H., Yu, Q. L., Wen X., & Liu, Y. (2019). Targeted Polypeptide–

-p

Microtubule Aggregation with Cucurbit[8]uril for Enhanced Cell Apoptosis. Angewandte Chemie-International Edition, 58(31), 10553-10557.

re

Zhang, L., Zhang, Y.-M., Liu G. X., & Liu, Y. (2019). Redox-responsive

30(1), 120-122.

lP

diphenylalanine aggregate mediated by cyclodextrin. Chinese Chemical Letters,

Zuo, M. Z., Qian, W. R., Xu, Z. Q., Shao, W., Hu, X.-Y., Zhang, D. M., et al. (2018). Supramolecular

Theranostic

na

Multiresponsive

Nanoplatform

Based

on

Pillar[5]arene and Diphenylboronic Acid Derivatives for Integrated Glucose

Jo

ur

Sensing and Insulin Delivery. Small, 1801942.

16

ro of

Scheme 1 The supramolecular self-assemblies obtained by the curcumin and

Jo

ur

na

lP

re

-p

β-cyclodextrin trimer

17

ro of -p re lP na ur

Fig. 1 Supramolecular self-assemblies for controllable morphology transitions. (A-F) Typical

Jo

TEM images of SSAs-1, SSAs-2 and SSAs-3; (G-I) Typical AFM images of SSAs-1, SSAs-2 and SSAs-3; (J-L) DLS results of SSAs-1, SSAs-2 and SSAs-3.

18

ro of -p re lP

Fig. 2 1H NMR spectra of β-CD3, β-CD3/Cur with molar ratio of 2:3, 4:3 and 6:3 in D2O (A)

Jo

ur

and 6:3 (D).

na

and 2D NOESY NMR spectra of mixture of β-CD3/Cur with molar ratio of 2:3 (B), 4:3 (C)

Fig.3 Fluorescence emission spectroscopy of Cur upon the stepwise addition of β-CD3 to 19

ro of

determine the association constant of Cur/β-CD3 in supramolecular self-assemblies

(a) TT orientation

(b) HH orientation

ur

na

lP

re

-p

Fig.4 Snapshot of inclusion complex obtained from equilibrium simulation.

Jo

Fig. 5 Cumulative release results of curcumin from SS1, SS2 and SS3 as a function of time over a period of 36 h at pH 5.0 and pH 7.4 condition.

20

Fig. 6 In vitro cytotoxicity of free Cur, SSAs-1, SSAs-2 and SSAs-3 against PC3 (A) and

Jo

ur

na

lP

re

-p

ro of

MCF-7 (B) cells after incubation for 48 h 37 ℃.

21

ro of -p re lP na

ur

Fig. 7 CLSM images of PC3 cells incubated with the SSAs-1, SSAs-2, SSAs-3 and Cur at a concentration of 40 μg/ml after 6 h. From left to right: Cur (green), DAPI (blue) and a merge

Jo

of two images. Flow cytometric analysis of PC3 cells treated with SSAs-1, SSAs-2, SSAs-3 and curcumin at the same concentration after incubating for 48 h. Inserted numbers in the profiles indicate the percentage of the cells present in this area.

22

Table 1 The binding energies of two inclusion complexes along with electrostatic, van der Waals interaction contributions (unit: kcal/mol). ΔEbind

ΔEelec

ΔEvdw

TT inclusion

56.8

9.0

47.8

HH inclusion

50.1

5.5

44.6

Jo

ur

na

lP

re

-p

ro of

Orientation

23