Supramolecular Vesicles Based on Complex of Trp-Modified Pillar[5

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Supramolecular Vesicles Based on Complex of Trp-modified Pillar[5]arene and Galactose Derivative for Synergistic and Targeted Drug Delivery kui Yang, Yincheng Chang, Jia Wen, Yuchao Lu, Yuxin Pei, Shoupeng Cao, Feng Wang, and Zhichao Pei Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.6b00696 • Publication Date (Web): 11 Mar 2016 Downloaded from http://pubs.acs.org on March 11, 2016

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Chemistry of Materials

Supramolecular Vesicles Based on Complex of Trp-modified Pillar[5]arene and Galactose Derivative for Synergistic and Targeted Drug Delivery Kui Yang, Yincheng Chang, Jia Wen, Yuchao Lu, Yuxin Pei,* Shoupeng Cao, Feng Wang, and Zhichao Pei* College of Science, Northwest A&F University, Yangling, Shaanxi 712100, People’s Republic of China Failure of cancer treatment is mainly caused by adverse side effects to normal cells and multidrug resistance (MDR) of cancer cells induced by chemotherapy.1, 2 Targeting and stimuli-responsive drug delivery systems (DDSs) based on nanotechnology with tailored properties have shown great potential to succeed as effective cancer treatments. This has significantly increased the interest in developing advanced drug nanocarriers3, 4 via new concepts or strategies towards enhanced cytotoxicity as well as anti-MDR effect in drug delivery in the past decade. Remarkable examples include nanocapsules formed with prodrugs leading to a synergetic cytotoxicity of the encapsulated drug5 and a more sophisticated intracellular selfassembly of taxol nanoparticles achieved via a biocompatible condensation reaction and enzymatic self-assembly for anti-MDR.6 These works indicate a paradigm shift in the strategies used to design nanocarries. Considering that many anticancer drugs, such as doxorubicin hydrochloride (DOX),7 cause cytotoxicity via their intercalation with DNA, we envision that a drug nanocarrier with the ability of cooperatively interacting with DNA intracellularly might lead to synergistically enhanced cytotoxicity. The exploration of DNA interacting nanocarriers with little or no cytotoxicity is of a great significance for opening new ways towards optimal cancer therapy and would be valuable to the design of advanced DDSs. Unfortunately, to the best of our knowledge, such drug nanocarriers have yet to be explored. Vesicles have been one of the most popular DDSs for their excellent abilities to encapsulate and controllably release drugs in response to specific stimuli.8-11 Among the various molecular building blocks for constructing vesicles, we are particularly interested in pillar[n]arenes,12 a new type of macrocyclic hosts. Pillar[n]arenes possess a hydrophobic core sandwiched between two functional rims, which endow facile modification with hydrophilic groups to obtain water soluble amphiphilic molecules. Their intrinsic symmetrical and rigid structure and excellent properties in host–guest chemistry make them an attractive candidate to construct various interesting supramolecular vesicles for DDSs.8, 10, 13 For instance, by using ferrocenium as head groups of pillar[5]arenes, our group developed the first redox-responsive cationic vesi-

cles for DOX/siRNA co-delivery, which successfully restored the drug sensitivity of SKOV-3/ADR.9

Scheme 1. Cartoon representation of the selfassembly and drug loading process of the vesicle, and their possible cellular path ways. In searching for a suitable heading group for an amphiphilic pillararene that can interact with DNA, tryptophan (Trp), an essential amino acid in the human diet, was investigated. It’s known for its interaction with DNA via indole ring in the molecule.14, 15 More importantly, it has been reported that the multiple indole rings on Trprich structures can strengthen their supramolecular interaction with DNA.15 We envisioned that modification of pillararene with Trp would not only lead to a new type of water soluble amphiphilic macrocyclic molecule resulting from its free amino groups, but the interaction between the 10 indole rings of Trp residues on the macrocyclic

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molecule and DNA may lead to synergistic effect with the drug encapsulated in their self-assembled vesicles. Herein, Trp-modified pillar[5]arene (TP5) was synthesized, and supramolecular vesicles (TP5G) based on complex of TP5 and galactose derivative (G) assembled via host-guest interaction were constructed and evaluated toward synergistically enhanced cytotoxicity by interacting with DNA in targeted drug delivery (Scheme 1), where the galactose residue on one end of G can be acted as the targeting ligand to asialoglycoprotein receptor (ASGP-R) overexpressing HepG2 cells via the carbohydrate-protein interactions.16 According to the published procedure,9, 15, 17 Trpmodified pillar[5]arene (4) was synthesized with 5 Trp residues as heading groups on each rim of the hydrophobic core. The acidification of free amino groups in 4 by HCl gave a water soluble amphiphilic TP5. A galactose derivative G was designed as the guest, where the galactose residue on one end of G was acted as the targeting ligand and the pyridinium on the other end was used for complexing with TP5. The details of the synthesis and characterization of TP5 and G can be found in Supporting Information.

Figure 1. (a) Partial 1H NMR spectra of the mixtures of TP5 and G at different molar ratios; (b) Tyndall effect of TP5G and DOX-loaded TP5G; (c) SEM image of TP5G; (d) SEM image of DOX-loaded TP5G complexes. Scale bar: 500 nm.

The host−guest complexation between TP5 and G was first investigated by 1H NMR spectroscopy, where 1H NMR titration experiment was performed by adding G into a solution of TP5 in D2O. As can be seen in Fig. 1a, the upfield chemical shifts of the pyridine protons (H1-3) of G were observed upon the addition of G due to the shielding effect of the electron-rich cavities of TP5 toward G, indicating the inclusion of the pyridine moiety of G into the hydrophobic cavity of TP5. Also, the curve of molar fraction of G vs. its UV-Vis absorption fraction at 288 nm showed that the complex formed between TP5 and G had 1:1 stoichiometry (Fig. S16). The association constant (Ka) of TP5⊃G is about 3.5 ± 0.6×105 M-1 through a non-linear curve-fitting method by fluorescence titration experiments (Fig. S17).8

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Next, the construction of supramolecular vesicles (TP5G) based on the complex between TP5 and G was investigated. By subjecting an aqueous solution of TP5 and G to sonication for 30 min, a clear Tyndall effect was observed (Fig. 1b, left), indicating the presence of abundant nanoparticles, which were determined by scanning electron microscopy (SEM, Fig. 1c) to have spherical morphology. Negative-stained transmission electron microscopy (TEM) images suggested that these nanospheres were vesicles with a wall thickness of about 4 nm (Fig. S18). Further analysis with dynamic light scattering (DLS) showed that the average diameter of the vesicles was 110 nm (Fig. S19a). The critical aggregation concentration (CAC) was found to be 30 µM by the water surface tension method (Fig. S21). The drug loading and release profiles of TP5G were studied using DOX as a model drug. Upon the load of DOX to TP5G, the solution of DOX-loaded TP5G, similar to that of TP5G, showed clear Tyndall effect (Fig. 1b, right). The SEM image of DOX-loaded TP5G complexes (Fig. 1d) showed that their average size was about 250 nm. This coincides with the result from the analysis by DLS, which showed that the average diameter of the vesicles was 274 nm (Fig. S19c). The encapsulation efficiency of DOX was calculated to be 30 wt % by UV-Vis spectroscopy, which indicated a good drug-loading capability of TP5G. On the other hand, DOX-loaded TP5G released DOX in response to pH, where more than 74 % of encapsulated DOX was released at pH no higher than 5.0 within 12 h, while only 30 % at pH 7.2 (Fig. 2a). These results suggest that DOX-loaded TP5G vesicles are relatively stable under physiological condition and can be used for controllable release. To investigate whether the Trp-rich structure of TP5 would lead to significant interaction with DNA, a series of experiments were performed. Firstly, agarose gel retention assay was used to determine the DNA-binding ability of TP5. The results showed that the mobility of negatively charged DNA was obviously retarded by positively charged TP5 at a Trp/P (Fig. S22a). Further study with fluorescence spectroscopy (Fig. 2b) showed that the fluorescence intensity at 378 nm (the charact- eristic peak corresponding to the Trp residues, light blue line) decreased with the addition of DNA, owing to the binding of the indole rings to DNA; while simultaneously a new peak at 576 nm appeared, ascribed to the light scattering of the TP5/DNA aggregates.18, 19 UV-Vis spectroscopy study disclosed that the broad absorption of TP5 at 250–300 nm increased with the addition of DNA, and the absorption peak gradually blue shifted with the increased amount of DNA, which was ascribed to the π-π interactions within the TP5/DNA aggregates (Fig. S23). In addition, a transparent solution of DNA gradually became turbid with the addition of TP5, which clearly indicated the formation of TP5/DNA aggregates (Fig 2c); this was further corroborated by SEM (Fig. 2d) and the analysis of DLS (Fig. S19b, d). The aggregates were also tested by FTIR spectra (Fig. S24). What’s more, 10 amino groups could not bind to DNA at the same amine/phosphate ratio of 20:1 (Fig.


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S22b). All the results obtained showed that TP5 can strongly interact with DNA, which are in agreement with that reported from the literature.15, 20 The cellular uptakes of DOX-loaded TP5G and TP5FITC (fluorescein isothiocyanate) were respectively investigated by confocal laser scanning microscope (CLSM). As

cence intensity of HepG2 cells incubated with DOXloaded TP5G is similar to that incubated with free DOX, which is higher than that of DOX-loaded TP5 and DOXloaded TP5G with LA preincubation. The difference was caused by the blockade of the asialoglycoprotein receptors by LA, which subsequently led to the inhibition of G’s galactose residue mediated endocytosis, and cellular uptake was thus only driven by non-specific endocytosis. The results implied that TP5G displays hepatoma- targeting ability and can be used for targeted drug delivery.

Figure 2. (a) DOX release profiles from DOX-loaded TP5G in PBS at different pH values; (b) Fluorescence emission spectra of TP5/DNA aggregates and light scattering at different Trp/P ratios (λex = 288 nm); (c) DNA aqueous solution and mixtures of DNA and TP5 at different Trp/P ratios; (d) SEM image of TP5/DNA aggregates at Trp/P = 10 : 1.

can be seen from the results shown in Fig. 3, red or green fluorescence could clearly be observed in the nuclei of HepG2 cells afterincubating with either DOX-loaded TP5G or FITC-labeled TP5 for 4 h, indicating both could be taken up by the cells efficiently. Moreover, TP5-FITC accumulated in the nuclei in a similar fashion to DOX.

Figure 4. (a) Cell viability of HepG2 cells and HepG2/ADR cells incubated with DOX-loaded TP5G and free DOX for 72 h, respectively. (b) TEM image of HepG2 in blank control. (c) An enlarged view of the marked area of (b). (d) TEM image of HepG2 incubated with DOX-loaded TP5G at 5 μM for 24 h. (e) An enlarged view of the marked area of (d).

Figure 3. CLSM images of HepG2 cells incubated with DOXloaded TP5G (a-d) and TP5-FITC (e-h) at 5 μM for 4 h: (a) and (e) are blue channel with Hoechst 33342, (b) is red channel for DOX, (f) is green channel for FITC, and (c) and (g) are bright field, and (d) and (h) are merged images of (a-c) and (e-g), respectively. Scale bar: 40 μm.

To evaluate the targeting effect of G, HepG2 cells were incubated with DOX-loaded TP5G, DOX-loaded TP5 and free DOX at 5 μM for 4 h, respectively, where the efficiency of cellular uptake was analyzed by flow cytometry. HepG2 cells pre-incubated with lactobionic acid (LA, 2 mg/mL) for 4 h were used to incubate with DOX-loaded TP5G for comparison. As shown in Fig. S25, the fluores-

The anticancer efficacy of DOX-loaded TP5G was evaluated using methyl thiazole tetrazolium (MTT) cell survival assay. HepG2 and DOX-resistance HepG2 (HepG2/ADR) cells were independently incubated with free DOX and DOX-loaded TP5G under five different concentrations for 24, 48, and 72 h, respectively (Fig. S26 and 4a). The relative viabilities after the incubation with DOX-loaded TP5G at all three tested time periods were visibly lower than those with free DOX (especially at the concentrations of 5 and 10 μM). In contrast, G and TP5 showed none or relatively low cytotoxicity (Fig. S27, S28). These results indicate that DOX-loaded TP5G offered enhanced anticancer efficacy of DOX, as was expected. To our delight, DOX-loaded TP5G also showed strongly enhanced anticancer efficacy to HepG2/ADR cells at the three tested time periods in comparison with free DOX, suggesting TP5G as a potential DDS for overcoming MDR.



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Further study of the HepG2 cells incubated with free DOX, TP5, or DOX-loaded TP5G, respectively, by TEM disclosed that there was a striking amount of microaggregates in the nuclei of the cells incubated with DOXloaded TP5G (Fig. 4d-e). In contrast, no similar phenomenon was observed either in the control cells (Fig. 4b-c), or in the cells incubated with free DOX or TP5G (Fig. S29). We deduced that these micro-aggregates composed of the complex of TP5, DOX, and DNA resulting from the strong binding ability of the indole rings in TP5 to DNA, and the intercalation of DOX in DNA. In other words, the tryptophan-rich structure of TP5 worked as multivalent ligands to cause an amplification effect or cluster effect,21 which greatly enhanced the supramolecular interactions between the Trp residues and DNA. This, in conjunction with the intercalation of DOX in DNA, induced the formation of complexes between TP5, DNA, and DOX, which led to the conformational change of DNA and thus inhibition of DNA synthesis,22 and eventually caused synergistically enhanced cytotoxicity for cell apoptosis and death. In summary, we developed a DNA interacting targeted drug delivery system based on the supramolecular vesicles that were self-assembled by the host–guest complex of TP5 and G. Drug loading experiments demonstrated that DOX was successfully encapsulated into the vesicles. DOX-loaded TP5G vesicles exhibit an excellent pH responsiveness and quick release of DOX at acidic environment, enabling them for controlled drug release. As expected, DOX-loaded TP5G not only possesses hepatoma-targeting ability to ASGP-R overexpressing HepG2 cells but also has significantly enhanced cytotoxicity compared with free DOX towards hepatoma cells and DOX-resistant hepatoma cells owing to the synergistic effect of TP5 with DOX, as a result of the significant interactions between Trp-rich structure of TP5 and DNA in cells. Our unique strategy provides insights for the design of advanced nanocarriers towards synergistically enhanced cytotoxicity for cancer chemotherapies.

ASSOCIATED CONTENT Supporting Information. Experimental procedures and supporting ﬁgures. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected]; [email protected].

Notes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENT This research work was supported by the National Natural Science Foundation of China (NSFC 21572181, 21174113 and NSFC 31270861).

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Supramolecular Vesicles Based on Complex of Trp-Modified Pillar[5

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