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Cite this: DOI: 10.1039/c4cc09274k Received 20th November 2014, Accepted 2nd January 2015

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Pillar[5]arene based supramolecular prodrug micelles with pH induced aggregate behavior for intracellular drug delivery† Yin Wang, Jianwei Du, Youxiang Wang, Qiao Jin* and Jian Ji*

DOI: 10.1039/c4cc09274k www.rsc.org/chemcomm

A novel type of dual pH-responsive supramolecular prodrug micelles based on host–guest interactions of water-soluble pillar[5]arene (WP5) and methyl viologen functioned doxorubicin (MV-DOX) was prepared. It was found that the prodrug micelles could be aggregated upon acidic condition, which led to enhanced accumulation and better therapy effect.

Nowadays, cancer has emerged as one of the leading causes of human death, which motivates extensive research interests.1 One of the widely investigated areas in recent years is the usage of nanocarriers formed by noncovalent polymers for drug delivery.2 Compared with their covalent counterparts, noncovalent ones have many outstanding advantages such as dynamically controllable sizes and easy implementation of functional groups. In the past few decades, various types of macrocyclic hosts have been successfully applied as assembly units to construct supramolecular drug delivery platforms.2b,c,3 For example, nanocarriers with different morphologies, such as micelles4 or vesicles,5 could be regulated by the amount of cyclodextrin, and moreover, the integration of drug molecules could further endow them with anticancer ability.4,6 As a new class of macrocyclic hosts in supramolecular chemistry, pillar[n]arenes consist of hydroquinone units linked by methylene (–CH2–) bridges at their 2,5-positions.7 Due to their high solubility, low toxicity and specific recognition towards many model substrates, pillar[n]arenes have been widely used for the development of various interesting supramolecular systems, including nanomaterials,8 chemosensors,7c,d,9 transmembrane channels,10 and supramolecular polymers.11 There are also some pioneering works on drug delivery.7b,12 For instance, pH responsive vesicles and multiwalled microtubes have been prepared from pillar[5]arene to control release of calcein.7b Very recently, there was also a report on in vitro testing of water-soluble pillar[6]arene (WP6) for anticancer behavior.12a MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China. E-mail: [email protected], [email protected]; Fax: +86-571-87953729; Tel: +86-571-87953729 † Electronic supplementary information (ESI) available: Details of experimental section. See DOI: 10.1039/c4cc09274k

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Surprisingly, in addition to the fact that considerable attention has been paid to stimuli induced reversible/irreversible changes of nanocarriers or stimuli induced release of payload to date,1 few efforts has been made to understand the accumulation of nanocarriers in the tumor site, which is extremely important.13 It is well proved that the size of the nanocarriers during the entire drug delivery process is not a constant value. Specifically, on one hand, small sized nanocarriers can penetrate more effectively inside tumor tissues and have a possibility of re-entering the bloodstream.14 On the other hand, relatively large ones favor being trapped for a long time due to the lack of functional lymphatic drainage in tumor tissue.15 Our and other groups have demonstrated that the increase in the size of nanoparticles could be achieved by simply decreasing pH, leading to the enhanced accumulation of nanoparticles in tumors.13 Thus, it is desirable to construct nanocarriers with variable diameters to meet the demands of different sizes at different stages of the drug delivery process. Herein, we report the preparation of pH responsive supramolecular prodrug micelles by host–guest interactions between water soluble pillar[5]arene (WP5) and methyl viologen functioned doxorubicin (MV-DOX) (Scheme 1). The resultant supraassemblies are expected to aggregate upon the extracellular pH of the tumor tissues but could still be internalized into the cancer cells. The aggregation would lead to the enhanced accumulation and better therapy effect. Moreover, following cell uptake, DOX is expected to be cleaved from the backbone at endo-/lysosomal pH, further inhibiting the proliferation of the cancer cells. To prepare dual pH-responsive supramolecular prodrug micelles, water soluble pillar[5]arene (WP5)7c,16 and MV-DOX17 were synthesized. The detailed synthesis procedures are shown in the ESI† (Scheme S1). Then, 1H NMR spectrometry was employed to elucidate the formation of the host–guest complex (Fig. S4, ESI†).18 Before the preparation of supramolecular micelles, the necessity of DOX for the formation of micelles was tested. We found that MV-SH and the complex without DOX (WP5@MV-SH) cannot form micelles, except WP5, which is amphiphilic itself (Fig. S5–S7, ESI†). Therefore, DOX as the hydrophobic block is essential for the prodrug micellar formation. Consequently, as the complex has the hydrophilic

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Scheme 1 Schematic illustration of the preparation of supramolecular prodrug micelles and their pH responsive aggregate behaviour upon extracellular pH stimulus of tumor tissues.

segment of WP5 and hydrophobic segment of DOX, it should be amphiphilic and able to self-assemble into nanoassemblies in water. To confirm this process, dynamic light scattering (DLS) was applied. It can be seen from Fig. 1 that the micelles have a Z average diameter of 94.3 nm and the PDI is 0.191 when the pH is 7.4. Because ammonium carboxylate groups are decorated on the rim of WP5, the variation of pH could have a direct influence on the protonation of these groups. As revealed in Fig. 1, when the pH drops to 6.5, which mimics the pH of the tumor extracellular environment, the size of the micelles shifts to around 200 nm with a broader size distribution. The pH variation induced aggregate was also confirmed by transmission electron microscopy (TEM) (Fig. S8, ESI†). As we know, sophisticated nanocarriers for cancer diagnosis and therapy could not realize their functions until they effectively accumulated at the malignant tumor sites, which is not an easy task due to the complexity of biological systems. To date, accumulation is generally mediated by the EPR effect, which is highly relied on the physicochemical characteristics of the nanocarriers such as size.19 It also should be noted that effective accumulation could be achieved

Fig. 1 DLS plots of the supramolecular micelles at different pH values. The concentration was 2 mg mL 1.

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by the aggregation of the nanocarriers.13 Although it is not easy to achieve, it is worth the effort considering the following advantages. First, at tumor tissue level, accumulation leads to the enhancement of the local concentration of nanoplatforms and increases the possibility of cell uptake. Second and more important, accumulation occurring in the cancer cells results in the less amount of eliminated payload encapsulated in the nanocarriers. It also prolongs the time for payload to exert their functions, particularly, achieving better anticancer effect. Next, whether the supramolecular micelles in the aggregation state could be internalized into cancer cells was tested. As the model drug DOX was conjugated to MV through an acid-labile hydrazone bond, the slight acidic pH (6.5) would not cause cleavage of DOX.20 To mimic the pH of the tumor extracellular environment and induce the aggregation, we adjusted the pH of the culture media to 6.5 and incubated the supramolecular micelles with human hepatocellular carcinoma cells (HepG2 cells) for different times. Because the hydrazone bond is not stable at pH 5.0, it should show endo/lysosomal pH-sensitive DOX release after the micelles were internalized into the cancer cells. As it can be found in Fig. 2, the intensity of red fluorescence increased with longer incubation time, indicating that the prodrug micelles could be internalized into HepG2 cells effectively. Furthermore, after 3 hours of incubation, almost all of the red fluorescence is accumulated in the nucleus, suggesting that DOX has been cleaved from the backbone of prodrug micelles and escaped from the endosome. Moreover, results obtained from flow cytometry are also consistent with the abovementioned results (Fig. S9, ESI†). Therefore, these results confirm that supramolecular prodrug micelles are able to deliver and release DOX into the nuclei of cancer cells.

Fig. 2 Fluorescence microscopy images of HepG2 incubated with the prodrug (10 mg mL 1). From left to right: DAPI (blue), DOX (red) and a merge of the two images. (A) 0.5 h; (B) 1 h; (C) 3 h; (D) 5 h.

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After being internalized into the cancer cells and the pH triggered the release, another key point associated with supramolecular prodrug micelles is whether the DOX molecules could inhibit the proliferation of cancer cells. Thus, the MTT assay was performed to illustrate this. To mimic the pH of the tumor extracellular environment, we continued to use the culture media with the pH of 6.5. Considering that a slight change in pH may influence the cells, the incubation in low pH only lasted for 6 h. Before testing the anticancer ability of supramolecular micelles, the biocompatibility of the precursors for preparation of micelles was assessed. As shown in Fig. 3A, MV-SH is toxic to the cells but becomes biocompatible after forming host–guest inclusions with WP5, probably because the formation of the host–guest complex shields the electropositivity of the MV segment. Notably, after conjugation with DOX, the supramolecular micelles induce remarkable cytotoxicity to the cells. For example, when the drug dosage is lower than 0.5 mg L 1, cell viability is over 80%. However, the increase of drug dose leads to the concomitant decrease of the cell viability. It is calculated from Fig. 3B that the IC50 (i.e., inhibitory concentration to produce 50% cell death) is 3.75 mg L 1. Moreover, when the concentration of drug is elevated to a higher level such as 10 mg L 1, they achieve a better antitumor activity, which inhibits the cell viability to about 30%. These results indicate that the supramolecular micelles could be utilized as a promising platform for tumor-targeting drug delivery. As discussed above, retention of nanocarriers in cancer cells is an important issue, which determines the fate of nanoplatforms in cells. Therefore, we study whether the prodrug micelles could be well trapped in cancer cells after internalization. In this case, we used one of our previous prodrug systems based on cucurbit[8]uril (CB[8]) as control because they had the same hydrophobic segment (MV-DOX) and the CB[8] system did not show pH aggregation behavior.17 Specifically, the HepG2 cells were incubated with two types of supramolecular prodrug micelles at the same concentration for 2 h before medium replacement. It should be noted that the amount of these two types of supramolecular prodrug micelles by cell uptake is not the same due to the different outer layer and size of micelles. But we normalized the percentage of fluorescence to 100% after 2 hours incubation with supramolecular prodrug micelles for the purpose of comparison. As shown in Fig. 4, in the first 2 h following medium replacement, about 90% of the fluorescence (derived from DOX) in cells treated with WP5@MV-DOX

Fig. 3 Cell viability of HepG2 cells incubated with various concentrations of MV-SH, WP5@MV-SH (A) or prodrug micelles (B). The concentration of WP5 and MV-SH used were the same for these three experiments.

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Fig. 4 The histogram of the residual fluorescence percentage in cells after incubation with fresh media for 2 and 4 h. The concentration of prodrug micelles was 10 mg mL 1.

prodrug micelles is preserved, whereas for cells treated with CB[8] prodrug micelles, the fluorescence decreased to 80%. More importantly, in the next 2 h, the fluorescence does not show significant decrease for the cells incubated with WP5@MV-DOX prodrug micelles. In contrast, the fluorescence in the cells for the control reduced to 78%, which is 12% less than that of cells treated with WP5@MV-DOX prodrug micelles. These results confirm that the WP5@MV-DOX prodrug micelles could accumulate in cancer cells for a long time, because the micelles aggregate upon acidic condition. Last, we wonder whether this aggregate behavior would have an influence on the anticancer ability of supramolecular prodrug micelles. Therefore, the MTT assay was carried out against HepG2 cells again. It can be found from Fig. S10 (ESI†) that the cell viability is lower than that of CB prodrug micelles treated cells. This is possibly due to the accumulation of nanocarriers in cancer cells, which prolongs the time for DOX to exert its function. These results indicate that WP5@MV-DOX supramolecular prodrug micelles would have a promising future in cancer therapy such as treatment of drug resistant cancer. In summary, we have illustrated a facile strategy to construct pH responsive prodrug micelles based on water-soluble pillar[5]arene mediated host–guest interactions. The supramolecular complex could self-assemble into micelles, and the size could be tuned by pH variation. When incubated with cancer cells, the internalized micelles showed endo/lysosomal pH-sensitive DOX release and prolonged accumulation in cells, which further inhibited their proliferation. These properties indicated their promising future in cancer therapy. Moreover, because pillar[5]arenes can be easily functionalized with different groups such as target ligands, it provides us an opportunity to integrate different functional groups into the complex, which holds potential for different applications. Financial support from the National Natural Science Foundation of China (NSFC-21174126, NSFC-51333005, NSFC-51303154, NSFC51273177), the National Science Fund for Distinguished Young Scholars (51025312), the National Basic Research Program of China (2011CB606203) and the Research Fund for the Doctoral Program of

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Higher Education of China (20110101110037 and 20120101130013) and the Open Project of State Key Laboratory of Supramolecular Structure and Materials (sklssm201316) are gratefully acknowledged. We greatly thank Prof. Feihe Huang and Dr Yong Yao (Department of Chemistry, Zhejiang University) for the kind donation of WP5 and for the valuable suggestions during manuscript preparation. We also thank Qing Ye for 3D drawing, Tian Jiang for the helpful discussion of manuscript preparation and Fan Jia for the fluorescence microscopy measurements.

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Pillar[5]arene based supramolecular prodrug micelles with pH induced aggregate behavior for intracellular drug delivery.

A novel type of dual pH-responsive supramolecular prodrug micelles based on host-guest interactions of water-soluble pillar[5]arene (WP5) and methyl v...
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