Supramolecular Chemotherapy: Cooperative Enhancement of

Feb 14, 2017 - chemotherapy by utilizing the clinical antitumor drug, oxaliplatin ..... 0.05 compared to the nontreated cell control group, one way AN...
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Supramolecular Chemotherapy: Cooperative Enhancement of Antitumor Activity by Combining Controlled Release of Oxaliplatin and Consuming of Spermine by Cucurbit[7]uril Yueyue Chen,†,‡,§ Zehuan Huang,†,⊥ Hanyang Zhao,⊥ Jiang-Fei Xu,⊥ Zhiwei Sun,*,‡,§ and Xi Zhang*,⊥ ‡

Department of Toxicology and Sanitary Chemistry, School of Public Health, and §Beijing Key Laboratory of Environmental Toxicology, Capital Medical University, Beijing 100069, P. R. China ⊥ Key Lab of Organic Optoelectronics & Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084, P. R. China S Supporting Information *

ABSTRACT: Supramolecular chemotherapy is aimed to employ supramolecular approach for regulating the cytotoxicity and improving the efficiency of antitumor drugs. In this paper, we demonstrated a new example of supramolecular chemotherapy by utilizing the clinical antitumor drug, oxaliplatin, which is the specific drug for colorectal cancer treatment. Cytotoxicity of oxaliplatin to the colorectal normal cell could be significantly decreased by host−guest complexation between oxaliplatin and cucurbit[7]uril (CB[7]). More importantly, oxaliplatin-CB[7] exhibited cooperatively enhanced antitumor activity than oxaliplatin itself. On the one hand, the antitumor activity of oxaliplatin can reappear by competitive replacement of spermine from oxaliplatin-CB[7]; on the other hand, CB[7] can consume the overexpressed spermine in tumor environments, which is essential for tumor cell growth. These two events can lead to the cooperatively enhanced antitumor performance. Supramolecular chemotherapy can be applied to treat with spermine-overexpressed tumors. It is highly anticipated that this strategy may be employed in many other clinical antitumor drugs, which opens a new horizon of supramolecular chemotherapy for potential applications in clinical antitumor treatments. KEYWORDS: supramolecular chemotherapy, host−guest chemistry, antitumor drugs, cucurbiturils chemistry



INTRODUCTION Cisplatin has been widely used as the first line antitumor drugs for more than three decades to help millions of tumor patients extend their survival time.1 Lots of cisplatin derivatives, such as oxaliplatin, carboplatin, and nedaplatin, have been developed for trying to achieve higher antitumor activity, better watersolubility, and fewer side effects.2 Most of the side effects of these cisplatin-based drugs are caused by low specificity among normal cells and tumor cells, which can make tumor patients suffer a lot during their treatments.3−5 Many efforts have been taken for trying to solve these problems by encapsulating these cisplatin-based drugs into micelles, liposomes, or protein selfassemblies to decrease their cytotoxicity on the delivery. In our previous communication, we have presented a new strategy of supramolecular chemotherapy to face the above challenges.6 Supramolecular chemotherapy is aimed to decrease the cytotoxicity of antitumor drugs by host−guest chemistry and recover their antitumor activity by the competitive replacement of overexpressed tumor biomarkers. As a proof of concept, we employed dimethyl viologen (MV) as a model antitumor agent encapsulated by cucurbit[7]uril (CB[7]) to form the host−guest complex of MV-CB[7]. In this way, we © XXXX American Chemical Society

successfully regulated the cytotoxicity of MV and also released its antitumor activity in the spermine-overexpressed tumor environments. Interestingly, MV-CB[7] even exhibited higher antitumor activity than MV itself. Two reasons behind this effect can be described: on the one hand, the antitumor activity of MV can be recovered by competitive replacement of spermine from MV-CB[7]; on the other hand, CB[7] can consume the spermine in tumor environments, which is essential for tumor cell growth, therefore decreasing the cancer cell viability furthermore. This cooperative enhancement caused by host−guest complexation between the antitumor drug and macrocyclic host may benefit significantly to antitumor treatment. In this article, we wondered if this strategy could work with clinical antitumor drugs, such as cisplatin derivatives. To answer this question, we introduced the oxaliplatin (OxPt) as the clinical antitumor drug (Scheme 1).2 By using CB[7], oxaliplatin can be incorporated into its hydrophobic cavity. Received: January 23, 2017 Accepted: February 14, 2017 Published: February 14, 2017 A

DOI: 10.1021/acsami.7b01157 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Scheme 1. Schematic Presentation of Supramolecular Chemotherapy Using Oxaliplatin and CB[7] To Form the Host−Guest Complex of Oxaliplatin-CB[7] and Its Competitive Release and Replacement by Spermine

UV/Vis Spectroscopy (UV). UV−vis spectra were obtained using a HITACHI U-3010 spectrophotometer. Scan range was 200−340 nm and scan speed was 600 nm/min. The concentration of oxaliplatin and oxaliplatin-CB[7] was chosen to be 0.40 mM. And the solution of spermine in high concentration (100.0 mM) was added into oxaliplatin-CB[7] to replace oxaliplatin from oxaliplatin-CB[7] in different ratios. The conversion rate of the competitive process was calculated by absorption at 248 nm, which is the typical absorption peak of oxaliplatin. Cell Viability Assay. NCM460 cells and HCT116 cells were cultured in RPMI-1640 and HT29 were cultured in DMEM/F-12 supplemented with 10% fetal bovine serum and 1% antibiotics in a humidified incubator at 37.0 °C (95% room air, 5% CO2). For viability assay, the cells were grown in 96-well plates, with 3000 cells seeded into each well. When cells reached 60−70% confluency, drugs were added at appropriate concentrations (12.5, 25, and 50 μM oxaliplatin/ CB[7]/oxaliplatin-CB[7] concentration). Cells were incubated in the presence of drugs at 37.0 °C for 24, 48, and 72 h, respectively. Cells were then washed with culture media. Cell proliferation was measured using the cell counting kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. Absorbance of the bioreduced soluble formazan product was measured at 450 nm using a Versamax microplate reader. Results were quantified by manually subtracting the blank value from each value then normalizing against the control values. Human Spermine Synthase Elisa Kit. Specimen requirement: HCT116 and HT29 cells treated with 50 μL of CB[7], 50 μL of oxaliplatin, and 50 μL of oxaliplatin-CB[7] for 24 h. The concentration of intracellular spermine synthase was detected according to the manufacturer’s instructions. Cell Cycle Analysis. Cells were grown in 6-well plates, 1 × 106 cells per well. When cells were 60−70% confluent, drugs were added in appropriate concentrations (50 μM oxaliplatin/CB[7]/oxaliplatinCB[7] concentration) and incubated for 12 h. They were then collected, treated with cold 70% ethanol, and incubated at 20 °C for 30 min to permeabilize the cell membrane. The cells were then centrifuged, washed with PBS, and resuspended in propidium iodide (PI) solution. The cell suspensions were then transferred to fluorescence-activated cell sorting (FACS) tubes and analyzed for PI staining on a BD FACS Calibur instrument. The data were analyzed using a FlowJo software. Statistics Analysis. All experiments were repeated independently at least thrice, and data were expressed as mean ± SE. Data were subjected to ANOVA followed by an appropriate post hoc test to measure statistical significance. P < 0.05 was set as the level of significance.

CB[7] belongs to a family of water-soluble macrocycles of cucurbit[n]urils and has good solubility in water and strong affinity with many kinds of guest molecules.7−18 Oxaliplatin, the clinical antitumor drugs, has been proved to be capable of binding to CB[7] with considerable binding affinity.19,20 Oxaliplatin has good antitumor activity but high cytotoxicity to normal cells; however, the host−guest complex of oxaliplatin-CB[7] could exhibit low cytotoxicity and this disguised antitumor drugs would not cause any harm to normal cells during the delivery process. Many research works have revealed that spermine is an overexpressed tumor biomarker in some tumor environments, such as colorectal tumors and lung tumors.21 Thus, spermine may competitively bind with CB[7] to release oxaliplatin from its disguised form of oxaliplatin-CB[7] and regain its antitumor activity. We envisioned that the cooperative enhancement of antitumor activity by combining controlled release of oxaliplatin and consuming of spermine by CB[7] may be achieved in this way.



EXPERIMENTAL SECTION

Materials. All the materials as following were of analytical purity grade and used without further purification and all of them were obtained from commercial suppliers: oxaliplatin, CB[7], monopotassium phosphate, hydrochloride acid, human spermine synthase Elisa Kit (R&D Systems Inc., USA) and deuterium oxide (D, 99.8%). Human colorectal normal cell line NCM460, tumor cell line HCT116 and HT29 were purchased from American Type Culture Collection (ATCC, Rockeville, MD). Fetal bovine serum (FBS, Gibco), Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F12 medium (DMEM/F-12, Gibco), and Roswell Park Memorial Institute 1640 medium (RPMI-1640, Corning) were purchased from Invitrogen, USA. Water was obtained from a Milli-Q Integral Water Purification System (18.2 MΩ cm−1). Considering that the pH condition in the tumor cell environment is mild acidic (5.5−6.5), all of our experiments were performed in the pH of 6.0. The phosphorus buffer (PBS) was prepared by mixing monopotassium phosphate and water then adjusted to pH 6.0 by hydrochloric acid and the PBS D2O buffer was prepared by the same way. Isothermal Titration Calorimetry (ITC). ITC experiments were carried out with a Microcal VP-ITC apparatus. All the sample solutions for titration were prepared in 20 mM phosphorus buffer (pH = 6.0). In a typical titration experiment, CB[7] was in the injection syringe at a concentration of 0.5 mM, whereas the guest was in the sample cell at a concentration in the range of 0.05 mM. All the titration schedules consisted of 1 injection of 5 μL for eliminating initial errors and 28 consecutive injections of 10 μL with a 300 s interval between injections. All solutions were degassed prior to titration. The temperature during titration was set at 37.0 °C. The ITC titration curves were fitted by Origin 7.0 using one set of sites binding model for determining the macroscopic enthalpy change, entropy change and association constant.



RESULTS AND DISCUSSION Cytotoxicity Regulated by Encapsulating Oxaliplatin with CB[7]. Considering that oxaliplatin is the specific drug for colorectal cancer treatment, we chose colorectal normal and tumor cell lines in our studies.22 To investigate the controlled disguise of oxaliplatin by host−guest complexation with CB[7], B

DOI: 10.1021/acsami.7b01157 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

effectively improving the safety of oxaliplatin during delivery process. To understand the reversible binding of oxaliplatin-CB[7], we utilized a competitive guest, adamantylamine (ADA), with the strong affinity with CB[7] to replace the oxaliplatin from its host−guest complex.23 As shown in Figure 2, after treating the

we measured the cytotoxicity of oxaliplatin and oxaliplatinCB[7] on health colorectal cell (NCM460) with a series of concentrations (12.5, 25.0, and 50.0 μM) for different incubation time (24, 48, and 72 h). As shown in Figure 1,

Figure 2. In vitro cytotoxicity of ADA-oxaliplatin-CB[7], oxaliplatin, CB[7], ADA-CB[7], and ADA in the colorectal normal cell NCM460 after incubation for 24 h. Data shown are mean ± SD from n = 3. *p < 0.05 compared to the nontreated cell control group, one way ANOVA.

health colorectal NCM460 cells with the equal amount of oxaliplatin, CB[7] and ADA for 24 h, the cell viability returned to be about 38.2%, which is almost the same as the control group (38.5%) treated with oxaliplatin itself. These results indicate that the complexation between oxaliplatin and CB[7] is reversible and the antitumor activity of oxaliplatin can be recovered in this way. Thus, it is possible to use an intracellular tumor biomarker, such as spermine, to achieve the recovery of antitumor activity in the tumor cell environments. Competitive Replacement of Oxaliplatin from Oxaliplatin-CB[7] by Spermine in Vitro. We wondered if our strategy of supramolecular chemotherapy could work in the case of oxaliplatin. To answer this question, we investigated in vitro competitive replacement of oxaliplatin from oxaliplatinCB[7] by spermine (SPM) and studied the host−guest chemistry behind this competitive binding. At first, 1H NMR experiments were performed to confirm the formation of oxaliplatin-CB[7] and the competitive binding process in the physiological conditions. As shown in Figure 3, the protons of 1,2-diaminocyclohexane as the ligand of oxaliplatin showed significant upfield shifts after complexation with CB[7], indicating that the 1,2-diaminocyclohexane group can be incorporated into CB[7]’s cavity to form host−guest complex of oxaliplatin-CB[7]. Then, spermine was quantitatively added into the solution of oxaliplatin-CB[7] at the ratio of 1.0, 5.0, 10.0, and 30.0 equiv. With the increased amount of spermine, the signals of the protons of 1,2-diaminocyclohexane on oxaliplatin gradually decreased and finally disappeared, indicating a dynamic exchange of oxaliplatin and spermine in CB[7]’s cavity. These results suggest that the binding affinities of oxaliplatin with CB[7] and spermine with CB[7] may be close to each other. To further study the binding affinities of the complexations of oxaliplatin-CB[7] and spermine-CB[7], ITC was employed to obtain the thermodynamic information about their bindings.

Figure 1. In vitro cytotoxicity of CB[7], oxaliplatin and oxaliplatinCB[7] for the health colorectal NCM460 cell line in a series of concentrations in the incubation time of (a) 24, (b) 48, and (c) 72 h. Data shown are mean ± SD from n = 3. (*) P < 0.05 vs vehicle treated control, one way ANOVA.

the cytotoxicity was increased with the increase of concentrations of oxaliplatin and the extension of its incubation time. In case of the concentration of 50 μM, oxaliplatin killed health colorectal cell up to 93.0% (72 h) but oxaliplatin-CB[7] only up to 11.5% (72 h). This indicates that oxaliplatin has quite high cytotoxicity to health colorectal cell while oxaliplatinCB[7] has considerably less cytotoxicity. Besides, it can be observed that CB[7] has no cytotoxicity for health colorectal NCM460 cell. Therefore, the cytotoxicity of oxaliplatin can be significantly decreased by encapsulating it with CB[7], thus C

DOI: 10.1021/acsami.7b01157 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

affinities are quite close to each other, which corresponds to the H NMR results. Thus, the competitive replacement of oxaliplatin by spermine should be a concentration-dependent process. For observing the competitive binding of spermine and oxaliplatin with CB[7], UV/vis experiments were rationally performed. As shown in Figure S1, after host−guest complexation with CB[7], the absorption of the broad peak around 240−260 nm of oxaliplatin became larger than oxaliplatin itself. Considering that CB[7] has no absorption here, this should be the consequence of host−guest complexation of oxaliplatinCB[7]. By quantitatively titrating spermine into the solution of oxaliplatin-CB[7], the absorption of the broad peak gradually returned to its original level. This indicates that the sufficient amount of spermine can replace oxaliplatin from its host−guest complex with CB[7]. By calculating the conversion rate based on the absorption of the peak at 248 nm, the correlation curve of conversion rate and ratio of spermine to oxaliplatin-CB[7] were obtained. As shown in Figure 5, 2.0 equiv of spermine can replace about 50% oxaliplatin, and 25.0 equiv can replace more than 90%. Therefore, these results elucidate that spermine can replace oxaliplatin from oxaliplatin-CB[7] and such a competitive binding process is based on the sufficient amount of spermine. Antitumor Activity Enhanced by Host−Guest Complexation of Oxaliplatin and CB[7]. To study if competitive binding of spermine to replace oxaliplatin from oxaliplatinCB[7] could happen in spermine-overexpressed tumor cell environments and recover its antitumor activity, we chose two kinds of colorectal tumor cell lines (HCT116, HT29) as model tumor cell lines to be tested with oxaliplatin-CB[7]. As shown in Figure 6, in the incubation time of 24 h, the cell viability of HCT116 treated with oxaliplatin-CB[7] was lower than the cases treated with oxaliplatin only. After extension of incubation time to 48 and 72 h, such effect was much clear. And the 1

Figure 3. 1H NMR spectra of (1) oxaliplatin, (2) oxaliplatin-CB[7], and oxaliplatin-CB[7] with addition of (3) 1.0, (4) 5.0, (5) 10.0, and (6) 30.0 equiv of spermine in the 20 mM PBS D2O buffer of pD 6.0 at 37.0 °C.

By separately titrating spermine into CB[7] and titrating CB[7] into oxaliplatin in the PBS buffer of pH 6.0 at 37.0 °C, two titration curves could be observed as shown in Figure 4. As expected, both of the spermine and oxaliplatin exhibited a strong binding with CB[7] at 1:1 molar ratio. By fitting the titration curve with one set of sites binding model, the binding constants of oxaliplatin-CB[7] and spermine-CB[7] were calculated to be 2.89 × 106 M−1 (Figure 4a) and 1.18 × 106 M−1 (Figure 4b), respectively. This indicates that their binding

Figure 4. ITC titration curves and fitted data of host−guest complexation of (a) oxaliplatin-CB[7], (b) spermine-CB[7] in the 20 mM PBS buffer of pH 6.0 at 37.0 °C. D

DOI: 10.1021/acsami.7b01157 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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the decreased concentration of intracellular spermine in tumor cells may be the key point for this phenomenon. It is revealed that the binding between spermine and DNA or RNA can stabilize the highly ordered structures of chromosome and RNA complexes, which can benefit to the cell growth and cell proliferation.25−30 Especially, some tumor cells can overexpress spermine to assist their fast cell growth and proliferation. Inhibiting the biosynthetic pathway of spermine has been used for chemotherapeutic intervention,31−33 and oxaliplatin-CB[7] could significantly interfere with this pathway by directly reducing the concentration of intracellular spermine. The balance between biosynthesis and consuming of spermine of should be rebuilt to maintain the necessary level of intracellular spermine, which may result in high expression of intracellular spermine synthase.34 To prove the above hypothesis, we examined the concentrations of the intracellular spermine synthase (SPMS) after treated with oxaliplatin-CB[7] and its control groups for 24 h in the colorectal tumor HCT116 and HT29 cells by Elisa Kit. As shown in Tables 1 and 2, we obtained the expression

Figure 5. Conversion rate of the competitive process obtained by equilibrium calculation based on the absorption of the peak of oxaliplatin at 248 nm.

antitumor performance on HT29 was the same as the above results. Especially in the cases of the incubation time of 72 h and the concentration of 50 μM, the cell viabilities of HCT116 (7.1%) and HT29 (6.3%) treated with oxaliplatin-CB[7] were much lower than the cases treated with oxaliplatin only (HCT116, 16.2%; HT29, 13.3%). Considering that CB[7] itself has almost no cytotoxicity for both two kinds of colorectal tumor cells,24 these results indicate that oxaliplatin-CB[7] has higher antitumor activity on HCT116 and HT29 than oxaliplatin itself. Therefore, we have demonstrated interestingly that the antitumor activity of oxaliplatin can be cooperatively enhanced indeed by host−guest complexation between oxaliplatin and CB[7]. Mechanism Behind the Enhancement of Antitumor Activity. What is the reason behind the enhancement of the antitumor activity of oxaliplatin-CB[7]? We hypothesized that

Table 1. Expression Levels of Spermine Synthase (SPMS) in HCT116a concentration of SPMS in HCT116 (pg/mL) sample

12.5 μM

25.0 μM

50.0 μM

oxaliplatin-CB[7] oxaliplatin CB[7] control

362.4 (± 0.1)b 151.9 (± 0.3) 329.2 (± 0.2) 301.5 (± 0.2)

533.6 (± 0.2)b 273.3 (± 0.1) 435.3 (± 0.2) 301.5 (± 0.2)

899.9 (± 0.7)b 357.4 (± 0.1) 583.2 (± 0.1) 301.5 (± 0.2)

Note: Data shown are mean ± SD from n = 3. bp < 0.05 vs vehicle treated control, one way ANOVA. a

Figure 6. In vitro antitumor activity of oxaliplatin-CB[7] and comparison with oxaliplatin in HCT116 and HT29 colorectal tumor cell lines. Graphs show oxaliplatin concentration response on cell survival after treatment with free oxaliplatin and oxaliplatin-CB[7] for (a) 24, (b) 48, and (c) 72h on HCT116 and (d) 24, (e) 48, and (f) 72 h on HT29. Data shown are mean ± SD from n = 3. *P < 0.05 compared to the nontreated cell control group, one way ANOVA. E

DOI: 10.1021/acsami.7b01157 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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UV/vis spectra to observe the competitive replacement of oxaliplatin from oxaliplatin-CB[7] by spermine, 1H NMR spectra to confirm the host−guest complexation between spermine and CB[7], and cell cycle studies (PDF)

Table 2. Expression Levels of Spermine Synthase (SPMS) in HT29a concentration of SPMS in HT29 (pg/mL) sample oxaliplatin-CB[7] oxaliplatin CB[7] control

12.5 μM 322.7 213.4 226.8 210.8

(± (± (± (±

0.1)b 0.1) 0.1) 0.1)

25.0 μM 469.3 238.0 279.3 210.8

(± 0.1)b (±0.2) (±0.1) (±0.1)

50.0 μM 624.6 274.4 312.0 210.8



(± 0.1)b (±0.1) (±0.1) (±0.2)

AUTHOR INFORMATION

Corresponding Authors

Note: Data shown are mean ± SD from n = 3. bp < 0.05 vs vehicle treated control, one way ANOVA.

*E-mail: [email protected]. *E-mail: [email protected].

a

ORCID

Xi Zhang: 0000-0002-4823-9120 levels of intracellular spermine synthase in HCT116 and HT29. All of them were much higher than the expression levels after treated with oxaliplatin, CB[7] and control group in the same conditions. Especially, for HCT116 tumor cells in the concentration of 50.0 μM, the expression level was significantly increased from 301.5 pg/mL treated with oxaliplatin to 899.9 pg/mL treated with oxaliplatin-CB[7]. A similar phenomenon was observed in the experiments for HT29 tumor cells. These results indicated that the uptake of oxaliplatin-CB[7] into colorectal tumor cells (HCT116 and HT29) could significantly activate the biosynthetic path way of spermine because of the consuming of it by competitive binding with oxaliplatin. It should be mentioned that CB[7] can also slightly improve the expression levels of spermine to some extent, because CB[7] itself can bind with spermine as well. However, CB[7] does not exhibit any antitumor activity on colorectal tumor cells as shown in Figure 6. These data suggest that the tumor cells cannot be killed by simply consuming the spermine. The tumor cells can only be killed by simultaneously releasing oxaliplatin from oxaliplatin-CB[7] and consuming the spermine by CB[7]. Therefore, there exists the cooperative effect between these two events in the spermine-involved path way, resulting in the enhancement of the antitumor performance of oxaliplatinCB[7].

Author Contributions †

Y.C. and Z.H. contributed equally.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This research was supported by the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (21421064) and the National Basic Research Program of China (2013CB834502).





CONCLUSION We have developed a supramolecular chemotherapy by using the clinical antitumor drug, oxaliplatin. Cytotoxicity of oxaliplatin to the colorectal normal cell can be significantly decreased by host−guest complexation with CB[7] to form oxaliplatin-CB[7] complex, thus decreasing the cytotoxicity of oxaliplatin during drug delivery. More importantly, oxaliplatinCB[7] exhibited higher antitumor activity than oxaliplatin itself, because the releasing of oxaliplatin from oxaliplatin-CB[7] by the overexpressed spermine, and simultaneously consuming spermine by CB[7] can result in the cooperatively enhanced antitumor performance. Supramolecular chemotherapy could be employed to other clinical antitumor drugs and other macrocyclic molecules can be the candidate hosts as well. This research opens a new horizon of supramolecular chemotherapy for potential applications in clinical antitumor treatments. It is highly anticipated that supramolecular chemotherapy could be further extended to programmable and simultaneous drug delivery.



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ASSOCIATED CONTENT

S Supporting Information *

]The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b01157. F

DOI: 10.1021/acsami.7b01157 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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DOI: 10.1021/acsami.7b01157 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX