Cytotoxicity Regulated by Host–Guest Interactions: A Supramolecular

Letter www.acsami.org

Cytotoxicity Regulated by Host−Guest Interactions: A Supramolecular Strategy to Realize Controlled Disguise and Exposure Yueyue Chen,¶,‡,§ Zehuan Huang,¶,⊥ 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: This work is aimed at providing a supramolecular strategy for tuning the cytotoxicity in chemotherapy. To this end, as a proof of concept, we employed dynamic cucurbit[7]uril(CB[7])-mediated host−guest interaction to control the loading and releasing of dimethyl viologen (MV) as a model antitumor agent. MV has high cytotoxicity to both normal cells and tumor cells without specificity. By encapsulating MV into the hydrophobic cavity of CB[7], the cytotoxicity of MV to normal cells can be significantly decreased. When the host−guest complex of MV-CB[7] is added into tumor cells with overexpressed spermine, the antitumor activity of MV can be recovered in tumor cell environment. There are two reasons behind this effect: on the one hand, spermine has a high affinity to CB[7], leading to releasing of MV from MV-CB[7]; on the other hand, CB[7] can soak up spermine, which is essential for tumor cell growth, therefore decreasing the cell viability furthermore. Then, it is highly anticipated that this kind of supramolecular strategy could apply to clinical antitumor agents and provide a new approach for decreasing the cytotoxicity and increasing the antitumor activity, thus opening horizons of supramolecular chemotherapy. KEYWORDS: cytotoxicity regulation, supramolecular chemotherapy, antitumor agent, host−guest chemistry, cucurbiturils chemistry

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guest interactions, some overexpressed tumor markers in the tumor environment can competitively bind with the macrocycle, therefore releasing the antitumor agent from its disguised form and regaining its antitumor activity. This supramolecular strategy may help us realize control over cytotoxicity of the commercially available antitumor agents and develop new supramolecular antitumor agents. As a proof of concept, cucurbit[7]uril (CB[7]) was chosen as the model macrocycle (Scheme 1 bottom). Cucurbit[n]urils (CB[n]s) are a family of water-soluble macrocyclic hosts, which have a hydrophobic cavity capable of the binding one or two guest molecules depending on the size of different CB[n]s.8−16 Among them, CB[7] has good solubility in water and strong affinity with guest molecules.17−25 In addition, the dimethyl viologen (MV) was used in this work as the model antitumor agent which can form the host−guest complex of MV-CB[7] (Scheme 1 bottom).26 Although there exists a dynamic equilibrium between the host and the guest, the high binding

hemotherapy is still one of the important clinical antitumor strategies, in spite of new therapeutic development.1 Many kinds of chemotherapeutic agents have been commonly used in clinical treatment, such as oxaliplatin,2 doxorubicin,3 and paclitaxel;4 however, the main disadvantages are poor antitumor efficacy and severe side effects, because most of chemotherapeutic agents cannot distinguish tumor cells from normal cells. Several approaches have been developed to improve safety of chemotherapeutic agents on normal cells and maintain their antitumor activity.5 To solve the above problems, a widely used approach is to encapsulate chemotherapeutic agents into micelles6 or liposomes.7 The advantage of this approach is to improve the solubility of chemotherapeutic agents and decrease their cytotoxicity on the delivery. This letter is aimed at developing a new supramolecular strategy to regulate the cytotoxicity of antitumor agent by host− guest chemistry (Scheme 1 top). By introducing a water-soluble macrocycle, we can decrease the cytotoxicity by encapsulating the antitumor agents into the macrocycle. This host−guest complex can be regarded as a disguised antitumor agent which may exhibit low cytotoxicity or even noncytotoxicity in normal cell environment. On the basis of the reversible nature of host− © XXXX American Chemical Society

Received: July 7, 2016 Accepted: August 22, 2016

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DOI: 10.1021/acsami.6b08295 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Scheme 1. Schematic Representation of Supramolecular Chemotherapy: (1) Controlling Disguise and Exposure of Antitumor Agents by Host−Guest Chemistry and (2) Demonstration of This Concept Using Dimethyl Viologen, Cucurbit[7]uril, and Spermine

affinity between CB[7] and MV guarantee the stability of the host−guest complex. The MV has good antitumor activity but high cytotoxicity; however, the host−guest complex of MVCB[7] may display low cytotoxicity and this disguised antitumor agent would not kill normal cells during the drug delivery process. Lots of research works have revealed that spermine is an overexpressed tumor marker in tumor environment.27,28 Thus, in a tumor environment, spermine can competitively bind with CB[7] to release MV from the disguised antitumor agent of MVCB[7] and regain its antitumor activity.29 It is hoped that controlled disguise and release of antitumor agents can significantly decrease the cytotoxicity and increase its efficacy. To investigate the controlled disguise of MV and controlled release by spermine, we utilized BEAS-2B normal lung cells as model demonstration. By adding MV, CB[7] and MV-CB[7] into the cell incubation environment of BEAS-2B respectively, we wondered if the cytotoxicity of MV could be decreased. Cell viability was determined after the cultured cells exposed to various concentrations of MV, CB[7] and MV-CB[7] (0.625, 1.25, 2.50, and 5.00 mmol/L) for 24 h. Mimethyl thiazolyl diphenyl tetrazolium (MTT) assays were used to test the cytotoxicity in cell lines.30 As shown in Figure 1, MV exhibited significant cytotoxicity to BEAS-2B cells and CB[7] showed low cytotoxicity; however, the cytotoxicity of MV-CB[7] was dramatically decreased to almost noncytotoxicity. Because cytotoxicity is always concentration-dependent, a little bit cytotoxicity can be observed in high concentration of MVCB[7]. Therefore, these results indicate that the host−guest complexation can significantly reduce the cytotoxicity of MV. We wondered if spermine could compete with MV to release it from MV-CB[7] and regain its cytotoxicity. To answer this question, MV-CB[7]-spermine and spermine itself were added into the BEAS-2B cell medium. As shown in Figure 2, spermine itself displayed noncytotoxicity and moreover, it benefited to cell proliferation, because spermine is essential for cell growth, proliferation, differentiation and cancer progression.31 After adding both MV-CB[7] and spermine into the BEAS-2B cell medium, MV regained its cytotoxicity on accounts of the competitive complexation of spermine with CB[7]. In other

Figure 1. In vitro BEAS-2B cytotoxicity of MV-CB[7] was measured by MTT after 24 h in different concentrations, compared with MV and CB[7] (*p < 0.05).

Figure 2. In vitro BEAS-2B cytotoxicity of MV-CB[7] was measured by MTT after 24 h in different concentrations, compared with spermine and MV-CB[7]-Spermine (*p < 0.05, [MV]:[CB7]:[Spermine] was 1:1:1 in molar ratio).

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DOI: 10.1021/acsami.6b08295 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 3. ITC titration curves and fitted curves of titrating CB[7] into (a) MV and (b) spermine in PBS of pH 5.50 at 25.0 °C.

Figure 4. UV−vis spectra of gradually titrating spermine into MV-CB[7] and conversion rate of the competitive process using spermine to release MV from MV-CB[7] obtained by equilibrium calculation. ([MV] = 0.05 mM).

words, MV is released from the host−guest complex of MVCB[7] by replacing MV with spermine to form the host−guest complex of spermine-CB[7]. To study the mechanism behind this competitive host−guest complexation, we employed isothermal titration calorimetry (ITC) to obtain the thermodynamic information on the complexation of MV-CB[7] and spermine-CB[7]. As shown in Figure 3a, by adding MV dropwise into CB[7] in PBS solution, a titration curve was observed. There was a titration jump at the ratio of 1.0, suggesting that MV can form a 1:1 host−guest complex with CB[7]. By fitting this titration curve, the binding constant of MV complexed with CB[7] was calculated to be (5.25 ± 0.14) × 105 M−1. In addition, as shown in Figure 3b, spermine can also bind with CB[7] to form 1:1 host−guest complex with a binding constant of (3.26 ± 0.17) × 105 M−1. How can spermine replace the MV from the host−guest complex of MV-CB[7] since the binding constant of MV-CB[7] is a little bit higher than spermine-CB[7]? To address this question, we performed UV−vis titration experiments to investigate the concentration-dependent competitive complexation. As shown in Figure 4 (left), the typical peak of MV can be

Figure 5. In vitro A549 cytotoxicity of MV-CB[7] was measured by MTT after 24 h in different concentrations, compared with MV (*p < 0.05).

observed in 255 nm. After host−guest complexation with CB[7], the absorption of this peak showed significant decrease. By titrating spermine into MV-CB[7] from the ratio of 1.0 to 20.0, C

DOI: 10.1021/acsami.6b08295 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 6. In vitro HT-19 and HepG-2 cytotoxicity of MV-CB[7] were measured by MTT after 24 h in different concentrations, compared with MV (*p < 0.05).

clinical antitumor drugs, such as cisplatin derivatives, to confirm this strategy furthermore. It is highly anticipated that this line of research will open new horizons of supramolecular chemotherapy.

the absorption of this peak exhibited considerable increase, indicating that the addition of spermine can replace MV from the host−guest complex of MV-CB[7] indeed. By plotting the conversion of this competitive process at different ratios of spermine to MV-CB[7] (Figure 4 right), we found that the addition of spermine with 2.0 equiv could release about 50% MV from its host−guest complex. If the ratio of spermine to MVCB[7] is higher than 2.0, more MV can be released. Therefore, this competitive complexation process depends on the concentration of spermine. In order to confirm that the above strategy could work on tumor cells, we chose A549 lung cancer cell line as demonstration. As shown in Figure 5, we found interestingly that both of MV and MV-CB[7] exhibited considerable antitumor activity. Moreover, MV-CB[7] displayed even higher antitumor activity than MV. When the incubation time was extended from 24 to 48 h, it displayed the same tendency, but the antitumor effect in 48 h was better than that of in 24 h (Figure S4a−c). On the one hand, MV can be released from MV-CB[7] by the high concentration of overexpressed spermine around tumor cells. On the other hand, CB[7] can soak up spermine from the tumor environment, leading to decrease of the concentration of spermine, which can also decrease the cell viability. Therefore, this cooperative effect is responsible for the higher antitumor activity of MV-CB[7]. To understand if the above strategy could work on other kinds of tumor cells, we selected HT-19 intestinal tumor cell line and HepG-2 hepatic tumor cell line as demonstration. As shown in Figure 6, in the case of HT-19, MV-CB[7] also exhibited higher antitumor activity than MV itself, which is similar to above A549 lung tumor cell line. However, in the case of HepG-2, MV-CB[7] showed lower antitumor activity than MV itself. The plausible reason is that both HT-19 intestinal tumor cell and A549 lung tumor cell lines can overexpress spermine,32,33 whereas HepG-2 hepatic tumor cell line cannot.34 Therefore, it indicates that this strategy works well with the tumor cells, which can overexpress spermine. In summary, by marrying supramolecular chemistry with chemotherapy, we have developed a new supramolecular strategy to regulate the cytotoxicity of antitumor agents. The cytotoxicity of antitumor agents to normal cells can be significantly decreased and the efficacy of them to tumor cells can be enhanced. Besides the model antitumor agents in this study, we are employing

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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.6b08295. Experimental procedures, all ITC binding isotherms, fitted curves and experimental results, along with supporting figures (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions ¶

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

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by National Natural Science Foundation of China (21434004, 21274076) and the National Basic Research Program of China (2013CB834502).

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DOI: 10.1021/acsami.6b08295 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX