Supramolecular Nanoparticles Constructed by DOX-Based Prodrug

Jan 14, 2015 - pillar[6]arene (WP6) and novel doxorubicin (DOX)-based prodrugs ... ditions, whereas the cumulative release of DOX is approximate to...
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Supramolecular Nanoparticles Constructed by DOX-Based Prodrug with Water-Soluble Pillar[6]arene for Self-Catalyzed Rapid Drug Release Yu Cao,†,§ Yan Li,‡,§ Xiao-Yu Hu,*,† Xiaochun Zou,† Shuhan Xiong,† Chen Lin,† and Leyong Wang*,† †

Key Laboratory of Mesoscopic Chemistry of MOE, Center for Multimolecular Organic Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China ‡ State Key Laboratory of Bioelectronics, Jiangsu Key Laboratory of Biomaterials and Devices, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China S Supporting Information *

ABSTRACT: The constructing of novel supramolecular prodrug nanoparticles based on the host−guest interaction of water-soluble pillar[6]arene (WP6) and novel doxorubicin (DOX)-based prodrugs (G1 or G2) is reported, in which these two kinds of prodrugs are synthesized by conjugating DOX with a flexible alkyl chain or a short EGn chain via an acid-cleavable hydrazone bond. The obtained supramolecular nanoparticles are stable under physiological conditions, whereas the cumulative release of DOX is approximate to 100% within 30 min at pH 5.5 by simulating the endolysosomal environment at 37 °C. It is noteworthy that WP6 can efficiently catalyze the cleavage of hydrazone bond of the prodrug G1 or G2 via a favored intramolecular process under acidic conditions. Moreover, intracellular localization experiments demonstrated that these two nanoparticles, taken up by cancer cells via endocytosis, can lead to efficient DOX accumulation in SKOV3 cancer cells. Cytotoxicity experiments further suggest that these nanoparticles can efficiently inhibit the proliferation of cancer cells and exhibit potent antitumor activity. nitions,28−31 have been applied to construct supramolecular micelles and vesicles for drug/DNA delivery;32−34 however, most of them are mainly based on the drug/DNA-loaded strategy through the rapidly responsive assembly/disassembly processes under external stimuli. On the other hand, in the field of prodrug nanocarrier systems for drug delivery, drugs are conjugated with polymers or dendrimers generating the socalled “polymeric prodrug,” but research focusing on the construction of supramolecular prodrug nanoparticles for drug delivery based on the host−guest interaction of the macrocycle with small molecular prodrugs is very limited.35,36 On the basis of our previous work on pillararene-based supramolecular vesicles for drug delivery,37−39 we note that water-soluble pillar[6]arenes (WP6) show good biocompatibility and acidresponsive properties40,41 in aqueous media; simultaneously, WP6 has been demonstrated to have a strong binding affinity with pyridinium salt in water driven by hydrophobic and electrostatic interactions.38,42 Therefore, we envision that we can design novel acid-responsive WP6-based supramolecular prodrug nanoparticles based on the host−guest complexation

1. INTRODUCTION In the past few years, research concerning drug-conjugated prodrug nanocarriers, especially polymeric drug delivery systems,1−10 has shown exciting efficacy for cancer treatments due to their improved pharmacokinetics11,12 and biodistribution13 profiles via the enhanced permeability and retention (EPR) effects.2,14,15 Accordingly, various kinds of smart prodrug nanocarriers including micelles,1,16,17 vesicles,1 nanorods,18 and nanocapsules19 are designed, which can not only increase their stability and reduce their side effects to normal tissue but also preferentially deliver drugs to tumor tissues via the above-mentioned EPR effect.2,20−24 However, problems still exist for those polymeric nanocarriers, one of which is the inefficient drug release rate in tumor cells to achieve a sufficient intracellular drug concentration for cancer therapy. Therefore, to address this problem, it is highly desirable to develop efficient drug delivery systems which can avoid drug premature burst release under physiological conditions21 but also can importantly achieve a rapid and almost complete release in the acidic tumor extracellular fluid or intracellular lysosomes. One effective strategy for solving the above problem is to construct supramolecular prodrug nanoparticles based on the weak and reversible noncovalent interactions.25−27 Recently, such noncovalent interactions, especially host−guest recog© XXXX American Chemical Society

Received: December 3, 2014 Revised: January 12, 2015

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particles enter cancer cells mainly via endocytosis with significant drug accumulation in SKOV3 cancer cells. And further cytotoxicity experiments demonstrate that this supramolecular prodrug nanocarrier system exhibits multiple advantages and a comparable therapeutic effect for cancer cells relative to free DOX, implying its promising future for cancer therapy.

of WP6 and a pyridinium-based prodrug which could be synthesized by conjugating a hydrophobic drug with pyridinium derivative via an acid-cleavable hydrazone bond to achieve the acid-responsiveness for drug release.43−46 Furthermore, under acidic conditions, WP6 can be protonated to their acid form, and the generated corresponding carboxylic acid derivative is a Brønsted acid which can catalyze and accelerate the cleavage of a hydrazone bond.47,48 Therefore, we speculated that it may be a good way to accelerate the drug release rate of prodrug nanocarriers and thereby achieve efficient drug accumulation in tumor cells by using the above-mentioned supramolecular strategy based on the catalytic ability of WP6. Herein, we report for the first time the construction of acidresponsive supramolecular prodrug nanoparticles based on the host−guest interaction between WP6 and a doxorubicin (DOX)-based prodrug, where the DOX-based prodrug is synthesized by directly conjugating hydrophobic DOX with a pyridinium-modified flexible alkyl chain (G1) or a short EGn (ethylene glycol) chain49,50 (G2) via an acid-cleavable hydrazone bond. Supramolecular nanoparticles formed from WP6⊃G1 and WP6⊃G2 show good stability and extremely high drug loading content under physiological and weakly alkaline conditions, whereas in the acidic environment (e.g., endolysosomes),51,52 they show rapid and almost complete release of DOX within 30 min due to the acid-sensitive property of the hydrazone bond of the prodrug, where, especially and importantly, we find that WP6 of the host−guest complexes can catalytically accelerate the cleavage of the hydrazone bond of a prodrug guest molecule through a favored intramolecular process53 under acidic conditions (Scheme 1).

2. RESULTS AND DISCUSSION Host−Guest Complexation Study. WP6 and DOX-based prodrugs G1 and G2 were synthesized according to the established procedures (for details of the synthesis and characterization of G1 and G2, see the Supporting Information). The host−guest interaction between WP6 and G1 (or G2) was investigated by 1H NMR experiments (see the Supporting Information, Figures S17 and S18), which clearly showed that WP6 could form an inclusion complex (WP6⊃G1 or WP6⊃G2) with G1 (or G2), and the binding site was in the hydrophilic pyridinium section. Construction of Supramolecular Prodrug Nanoparticles Based on the WP6⊃G1 and WP6⊃G2. The above WP6⊃G1 and WP6⊃G2 supramolecular complexes have the ability to form higher-order aggregates in a weak alkaline phosphate buffer solution (PBS) based on their amphiphilic property. As shown in Figure 1b and e, with the addition of WP6, a light opalescence and obvious Tyndall effect could be observed from the aqueous solution of WP6⊃G1 or WP6⊃G2, and the intensity of opalescence changed gradually upon adding a different amount of WP6. The morphology and size of these resulting WP6⊃G1 and WP6⊃G2 nanoparticles were further characterized by transmission electron microscopy (TEM) and dynamic laser scattering (DLS) experiments. The TEM images of amphiphilic WP6⊃G1 and WP6⊃G2 complexes showed spherical morphology with a dark core, indicating that they formed spherical nanoparticles with a well-defined smooth surface in a weak alkaline PBS buffer solution (Figure 1a and d), and the DLS results showed that these nanoparticles had an average diameter of 278 and 366 nm, respectively (Figure 1c and f), which made these supramolecular nanoparticles able to passively target tumor tissues via the EPR effect. Besides the above Tyndall effects and DLS and TEM results, another proof to confirm that the self-assembly process occurred between WP6 and the DOX-based prodrug was the obvious quench of the fluorescence intensity of the DOX-based prodrug G1 or G2 upon the addition of WP6. As shown in Figure 2a, when WP6 was added to the G1 solution, a remarkable decrease of the fluorescence intensity of G1 could be observed. A reasonable explanation was that the WP6⊃G1 complex self-assembled into supramolecular nanoparticles where the fluorescent hydrophobic DOX section existed in the interior hydrophobic space, resulting in the significant decrease of its fluorescence intensity. Moreover, the formed WP6⊃G1 supramolecular nanoparticles had an extremely high drug loading content (about 85 wt %). With respect to the other DOX-based prodrug G2, similar fluorescence changes could be observed upon the addition of WP6 (Figure S19a), and the obtained WP6⊃G2 nanoparticles also had a very high drug loading content (about 70 wt %). According to our previous work,38,39 it was found that the ζ-potential of the formed nanoparticles was a very important parameter to evaluate the stability of the obtained supramolecular aggregates, which was then applied for investigating the stability of the obtained nanoparticles. And it was found that the ζ potentials

Scheme 1. Schematic Illustration of the Formation of Supramolecular Prodrug Nanoparticles Based on WP6 and DOX-Based Prodrugs (G1 and G2) for WP6-Catalyzed Rapid Drug Release

To the best of our knowledge, the drug release rate of this system is significantly faster than that of other previously reported drug-conjugated polymeric nanocarriers. Moreover, the biological effects of these WP6⊃G1 and WP6⊃G2 supramolecular prodrug nanocarriers are also evaluated by a series of in vitro assays. Cellular uptaking and intracellular localization experiments suggest that these prodrug nanoB

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Figure 1. TEM images: (a) WP6⊃G1 and (d) WP6⊃G2 aggregates in PBS buffers (pH 8.0). (b and e) Tyndall effect of the WP6⊃G1 and WP6⊃G2 aggregates, respectively (note: right is the G1 or G2 solution (PBS, pH 8.0) and left is the G1 or G2 solution in the presence of WP6). (c and f) DLS data of the WP6⊃G1 and WP6⊃G2 aggregates, respectively.

Figure 2. (a) Fluorescence spectra of G1 (0.08 mM) in PBS buffer (pH 8.0) at 25 °C with the presence of different amounts of WP6: 0.15, 0.25, 0.28, 0.33, and 0.36 equiv, respectively; (b) the ζ potential of the aggregates formed by WP6⊃G1 ([G1]/[WP6] = 4.5:1).

Figure 3. (a) Drug release profile of G1 under acid conditions (pH 5.5) detected by HPLC under the absorption wavelength of 254 nm; (b) DOX release profile of the WP6⊃G1 nanoparticles at pH 8.0, 7.4, and 5.5, respectively (37 °C).

different pH values are shown in Figure 3b and Figure S20b, respectively. It was found that the above nanoparticles were very stable at pH 8.0, and the cumulative release of DOX was less than 3% within 6 h. Meanwhile, under simulated physiological conditions (PBS, pH 7.4), the cumulative release of DOX was less than 12% within 6 h. However, under acidic conditions (PBS, pH 5.5) by simulating the endolysosomal environment at 37 °C, a very quick release of DOX from WP6⊃G1 and WP6⊃G2 nanoparticles could be observed due to the favored acid-promoted hydrolysis of hydrazone bonds, and the cumulative release of DOX was approximately 100% within 30 min, which was not achieved in the previously reported polymeric systems.35,44 Thus, one of the advantages of these acid-responsive supramolecular nanoparticles is that they are stable under physiological conditions but can achieve a

of the nanoparticles gradually changed from positive to negative upon the addition of WP6 in the titration experiments (Figure S19b). Therefore, considering the repulsive-force-induced increasing stability of nanoparticles, the molar ratios of 4.5:1 [G1]/[WP6] (ζ potential = −29 mV, Figure 2b) and 5:1 [G2]/[WP6] (ζ potential = −38.3 mV, Figure S19c) were used for further investigation of their stimuli-responsive behavior as well as their application in controllable drug release. Drug Release Profile of Nanoparticles Based on the WP6⊃G1 and WP6⊃G2. The obtained WP6⊃G1 or WP6⊃G2 nanoparticles showed a characteristic acid-responsive behavior to release DOX due to the cleavage of the hydrazone bond of prodrug G1 or G2 under acid stimulus as detected by HPLC (Figure 3a and Figure S20a), and DOX release profiles of WP6⊃G1 and WP6⊃G2 nanoparticles in the solutions of C

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a hydrazone bond. But, to determine why the monomer of WP6 could not catalyze the cleavage of hydrazone bonds of these prodrugs, we speculated that the host−guest interaction between WP6 and the prodrug (G1 or G2) may play an important role in facilitating the cleavage of the hydrazone bond. Along this line, we further performed 1H NMR experiments with different pH solutions by using a model compound G′ (1-ethylpyridinium bromide; Figure S24 in the Supporting Information), which provided convincing evidence for the host−guest complexation effect under acidic condition. Subsequently, based on the different binding affinities between WP6⊃pyridinium [(3.26 ± 0.28) × 105 M−1]42 and WP6⊃paraquat [(1.02 ± 0.1) × 108 M−1]40 at pH 7.0 aqueous solution, control experiments by using competitive guest paraquat (G″) were carried out to further investigate the effect of host−guest interaction on the cleavage of a hydrazone bond. As shown in Figure 5A, when 4 equiv of G″ was added into the WP6⊃G′ solution, the protons on G′ which showed a remarkable upfield shift compared with the individual G′ solution now shifted downfield and returned to their original position. The above facts suggested that the host−guest complexation between WP6 and G′ disappeared. What’s more, the chemical shifts of protons Hf, Hg, and Hh on the competitive guest G″ shifted upfield, indicating the formation of a WP6⊃G″ inclusion complex. The above results clearly indicated that the complexation between WP6 and G′ could be destroyed after the addition of competitive guest paraquat, and a more stable WP6⊃G″ inclusion complex was formed, which is shown in Figure 5B. And then, the effect of G″ on the DOX release rate of a WP6⊃G1 solution under physiological conditions (pH 7.4 buffers) was investigated by HPLC. As shown in Figure 6a, with the presence of competitive guest G″, the degradation rate of prodrug G1 was obviously reduced and almost consistent with that of individual G1 at pH 7.4 buffers. Similarly, the degradation rate of G1 was also reduced with the presence of competitive guest G″ under acidic conditions (Figure 6b). This result can be explained by a rational reason that, after the addition of the competitive guest G″, a more stable inclusion complex WP6⊃G″ was formed, which led to the disassembly of the WP6⊃G1 complex under acidic conditions. Therefore, such WP6-catalyzed cleavage of a hydrazone bond could not proceed through a favored intramolecular process53,54 but became an unfavorable intermolecular process with the presence of G″ and resulted in the decreasing of the degradation rate (Scheme 2). On the basis of the above observations, we could draw the following conclusions: (1) these two types of novel supramolecular nanoparticles (WP6⊃G1 and WP6⊃G2) were stable in a weakly basic environment (pH 7.4 and 8.0), whereas rapid and effective release of DOX from its DOX-based prodrug G1 or G2 could be achieved in the target sites (e.g., the acidic microenvironment of tumor tissue, endolysosomes) due to its acid-sensitive property; (2) WP6 in this system plays a dual role, acting not only as the host molecule to construct the WP6⊃G1 and WP6⊃G2 supramolecular nanoparticles but also as a catalyst to promote the cleavage of the hydrazone bond through a favored intramolecular process under acidic conditions (Scheme 2). Therefore, compared with conventional DOX-conjugated prodrugs, which have a high stability and yet limited and very slow drug release under acidic conditions, such supramolecular nanoparticles, together with the catalytic capability of WP6 to promote the cleavage of hydrazone

quick and almost complete release of DOX in the acidic tumor tissue within a very short period of time. WP6-Catalyzed Cleavage of Hydrazone Bond of the Prodrug G1 or G2 via Intramolecular Process. In order to know whether the rapid DOX release of WP6⊃G1 and WP6⊃G2 is related only with the cleavage of the hydrazone bond of prodrug G1 or G2 under acidic conditions (pH 5.5), further investigation of the cleavage of only G1 or G2 under acidic conditions was performed. The degradation rate profile of G2 under different conditions (Figure S21 in Supporting Information) suggested that after G2 was incubated under physiological conditions (PBS, pH 7.4) for 9 h, the degradation percentage was less than 5%. Whereas, upon incubation for 9 h under acidic conditions (pH 5.5), the cumulative release of DOX was approximate to 85%. Subsequently, a control experiment by using sodium 2,2′-(1, 4-phenylenebis(oxy)) diacetate (monomer of WP6) was carried out, where 6 equiv of this monomer was added to the G2 solution at pH 5.5. It was found that the degradation percentage of the above G2 solution was almost the same as the individual G2 solution at pH 5.5 (Figure S21 in Supporting Information). However, in the other control experiment where different ratios of WP6 were added into the individual G2 solution at pH 5.5, the degradation rate of G2 was obviously accelerated with the presence of WP6 under the same conditions (Figure 4). Particularly, when 0.5

Figure 4. Degradation profile of G2 (0.4 mM, 0.7 mL) with the presence of 50 μL, 200 μL, and 400 μL of WP6 (0.28 mM), in pH 5.5 buffer at 25 °C, and degradation profile of G2 (0.4 mM, 0.7 mL) with the presence of 200 μL of WP6 (0.28 mM) in pH 8.0 buffer at 25 °C.

equiv of WP6 was added to the G2 solution, the complete degradation of G2 could be achieved only within 30 min. Whereas, under weakly alkaline conditions (pH 8.0), the degradation percentage was less than 5% with the presence of WP6 within 10 h. With respect to G1, similar degradation profiles could be obtained, as shown in Figure S22. Therefore, based on the above experiments, we could undoubtedly deduce that WP6 had an exciting and efficient capability to catalyze the cleavage of hydrazone bonds under acidic conditions. This is just the reason why the release of DOX from such DOXconjugated supramolecular nanoparticles is so much faster than other previously reported examples under the corresponding acid stimulation. In the following study, we further investigated why WP6 could remarkably catalyze the cleavage of hydrazone bonds under acidic conditions. Initially, UV−vis absorption spectra of WP6 in different pH buffers suggested that different amounts of carboxylate groups of WP6 were protonated to their acid form under different pH solutions (Figure S23 in the Supporting Information), and the generated carboxylic acid derivative is a Brønsted acid which can catalyze and accelerate the cleavage of D

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Figure 5. (A) 1H NMR spectra (400 MHz, D2O, 298 K): (a) only G′ (10.6 mM), (b) WP6 (5.3 mM) and G′ (10.6 mM), (c) when competitive guest paraquat G″ (42.4 mM) was added to the solution of b, (d) only G″ (10.6 mM). (B) Schematic illustration of adding competitive guest paraquat G″ to the solution of WP6⊃G1 or WP6⊃G2.

In Vitro Antitumor Activity. Following the above acidinduced DOX release, another key point is whether these supramolecular nanoparticles could effectively kill cancer cells, so an MTT assay was performed to investigate the anticancer efficiency of these nanoparticles. As shown in Figure 7, it was found that SKOV3 cells (human ovarian cancer cell) were more sensitive to free DOX, WP6⊃G1, and WP6⊃G2 nanoparticles than 4T1 (murine breast cancer cell) and HeLa cells (human cervical cancer cell). Meanwhile, WP6⊃G1 and WP6⊃G2 nanoparticles exhibited decreased toxicity compared with free DOX, but these two kinds of nanoparticles still showed significant in vitro antitumor activity. Moreover, from the morphology of living cells we also found that the viability of cells incubated with the WP6⊃G1 and WP6⊃G2 nanoparticles group were better than that of the free DOX group even after 32 h (Figure 8). As is well-known, free DOX permeates cellular and nuclear membranes mainly by passive diffusion.55 However, WP6⊃G1 and WP6⊃G2 nanoparticles are considered to be taken up by tumor cells via endocytosis, followed by endolysosomal escape and subsequent drug distribution in the cytosol and nucleus, which is much slower than passive diffusion, resulting in a relatively low concentration of the

Figure 6. (a) Degradation profile of G1 (0.11 mM) in pH 7.4 buffer, and G1 with the presence of WP6 (0.0275 mM), as well as the G1 (0.11 mM) solution with the presence of WP6 (0.0275 mM) and competitive guest G″ (0.55 mM) in pH 7.4 buffer at 25 °C. (b) Degradation profile of G1 (0.11 mM) in a pH 5.5 buffer and G1 with the presence of WP6 (0.0275 mM), as well as the G1 (0.11 mM) solution with the presence of WP6 (0.0275 mM) and different amounts of competitive guest G″ in pH 5.5 buffer at 25 °C.

bonds, make these DOX-based prodrugs capable of achieving rapid drug release under acidic conditions.

Scheme 2. Schematic Illustration of WP6-Catalyzed Drug Release

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Figure 7. Cytotoxicities of free DOX, WP6⊃G1, and WP6⊃G2 against 4T1 cells (a), HeLa cells (b), and SKOV3 cells (c), respectively, as determined by MTT assay. Cells were treated with designated regimes for 32 h followed by 24 h incubation with a fresh medium.

Figure 8. Images of living Hela cells in (a) blank, (b) free DOX (10 μg·mL−1), (c) WP6⊃G1 nanoparticles with an equivalent dosage of DOX (10 μg·mL−1), and (d) WP6⊃G2 nanoparticles with an equivalent dosage of DOX (10 μg·mL−1) after incubating for 32 h. The images of living 4T1 cells in (e) blank, (f) free DOX (10 μg· mL−1), (g) WP6⊃G1 nanoparticles with an equivalent dosage of DOX (10 μg·mL−1), and (h) WP6⊃G2 nanoparticles with an equivalent dosage of DOX (10 μg·mL−1) after incubating for 32 h.

released DOX from WP6⊃G1 and WP6⊃G2 nanoparticles. In addition, the cell viability also showed a dosage-dependent effect: at low DOX concentration (a drug equivalent dosage of 0.1 μg·mL−1), the cell viability was more than 80% after incubating for 32 h; when the concentration of WP6⊃G1 and WP6⊃G2 nanoparticles increased (DOX equivalent dosage reached 10.0 μg·mL−1), the cell viability decreased significantly. The above results indicated that DOX delivered by these nanoparticles usually exhibited relatively lower in vitro antitumor activity than the free DOX molecule, but such a rapid and efficient delivery system could be utilized as a promising platform for nanoprodrug delivery. Cellular Uptake and Intracellular Localization. The cellular internalization of WP6⊃G1 and WP6⊃G2 supramolecular nanoparticles and free DOX were then examined against SKOV3 cells using fluorescence microscopy (Figure 9). As mentioned above, free DOX and prodrugs G1 and G2 have inherent fluorescence, so they can be directly observed with a fluorescence microscope (shown in green), and the fluorescence intensity detected in cells treated with WP6⊃G1 or WP6⊃G2 nanoparticles or free DOX can be considered consistent with the concentration of DOX internalized into the cells. Thus, the fluorescence intensity detected in cells can clearly indicate the concentration of these systems internalized into the cells. Meanwhile, LysoTracker Red was used to label lysosomes for colocalizing these self-assembled nanostructures (shown in red). Initially, SKOV3 cells were incubated with WP6⊃G1 and WP6⊃G2 nanoparticles and free DOX at 37 °C for 1 h and 5 h, respectively. As shown in Figure 9A, after incubation with free DOX for 1 h or 5 h, the red and green

Figure 9. Cellular uptake and intracellular localization of free DOX (A), WP6⊃G1 aggregates (B), and WP6⊃G2 aggregates (C) in tumor cells observed by fluorescence microscope. SKOV3 cells were incubated with free DOX (a1−a6), WP6⊃G1 aggregates (b1−b6), and WP6⊃G2 aggregates (c1−c6) at 37 °C for 1 and 5 h, respectively; a1, a4, b1, b4, c1, and c4 are the Lyso-Tracker channel (red); a2, a5, b2, b5, c2, and c5 are the DOX channel (green); a3, a6, b3, b6, c3, and c6 are the overlap of DOX, Lyso-Tracker, and light field channels.

fluorescence in SKOV3 cells did not colocalize with each other, which is consistent with the diffusion pathway by which free DOX enters cells.55 In contrast, these two nanoparticles entered cells mainly through endocytosis, and the red and green fluorescence could colocalize very well after incubating for 5 h, as demonstrated by the colocalization of DOX and lysosomes (Figure 9B and C, yellow spots indicate the overlap F

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Figure 10. Flow cytometric profiles of SKOV3 cells incubated with PBS, DOX·HCl, WP6⊃G1, and WP6⊃G2 nanoparticles at 37 °C for 1 h (a) and 5 h (b).

of red and green fluorescence). More importantly, it was found that the relative cell uptake rates of WP6⊃G1 and WP6⊃G2 nanoparticles were different from each other. Cell images indicated that the red and green fluorescence began to colocalize with each other after incubating with WP6⊃G1 nanoparticles only for 1 h (Figure 9b3), whereas, with respect to WP6⊃G2 nanoparticles, such overlaps could not be observed within the same time (Figure 9c3). This result suggested that WP6⊃G1 nanoparticles exhibited faster internalization than WP6⊃G2 nanoparticles, probably because the size of WP6⊃G2 nanoparticles (∼350 nm) is larger than that of WP6⊃G1 nanoparticles (∼200 nm),56−58 and the surfaces of WP6⊃G2 nanoparticles were functionalized with EG, which can form an inert and hydrophilic surface,50,59,60 resulting in the obvious increase of the steric hindrance of these nanoparticles, which will affect the interactions between nanoparticles and cell membranes and consequently affect the cellular uptake. Therefore, WP6⊃G2 nanoparticles showed slower internalization than WP6⊃G1 nanoparticles. Moreover, the relative cellular uptake rates of the WP6⊃G1 group, the WP6⊃G2 group, and the DOX·HCl group were further confirmed by flow cytometric analysis (Figure 10). As shown in the flow cytometric profiles, the intensity of the WP6⊃G1 group surpassed the intensity of the WP6⊃G2 group after 5 h of incubation at the same DOX concentration (5 μg/mL) but was slower than that of the DOX·HCl group. We speculated that the surface charge of nanoparticles (negative charge) might influence the cell uptake and result in the relatively slower cellular uptake rates of WP6⊃G1 and WP6⊃G2 groups than that of the DOX·HCl group. Meanwhile, after incubating for 5 h, the green fluorescence was not only accumulated in the lysosomes but also in the nuclei in the WP6⊃G1 and WP6⊃G2 groups (Figure 9b6 and c6), suggesting that DOX had been effectively cleaved from the prodrug backbone and escaped from the endosomes. Thus, all above results clearly confirmed that these DOX-conjugated supramolecular nanoparticles were able to efficiently deliver and release DOX into the nuclei of cancer cells.

pH 5.5 due to the WP6-catalyzed cleavage of the hydrazone bond through a favored intramolecular process, which is very significant for providing a sufficient concentration of DOX in tumor cells within a short period of time. Such catalytic ability of WP6 might be particularly attractive in biological systems such as live cell imaging, reactive probes in cells, and drug release.61,62 Furthermore, cellular internalization and localization assay experiments suggested that these DOX-based supramolecular prodrug nanoparticles entered cancer cells mainly via endocytosis and could lead to significant drug accumulation in SKOV3 cancer cells as demonstrated by the colocalization of DOX and lysosomes. Cytotoxicity experiments demonstrated that these DOX-based prodrug nanoparticles exhibited excellent antitumor activities in vitro. This work thus provides a novel example of rational design for effective nanoprodrug delivery systems. Further studies on the active targeting effects of this promising prodrug platform and the application of the WP6-catalyzed cleavage of the hydrazone bond in biology systems are ongoing in our lab.

4. EXPERIMENTAL SECTION Materials Preparation. Pyridine, 11-bromoundecanoic acid, thionyl chloride, hydrazine hydrate, EG6, ethyl bromoacetate, DOX·HCl, and other reagents are commercially available and were used as received. Solvents were either employed as purchased or dried according to procedures described in the literature. WP642 was synthesized and purified according to previously reported procedures. These compounds were identified by 1H NMR, 13C NMR, HRMS, and HPLC. 1H NMR and 13C NMR spectra were recorded on a Bruker Advance DMX 300 MHz spectrometer (or Bruker DPX 400 MHz spectrometer). Low-resolution electrospray ionization mass spectra (LR-ESI-MS) were obtained on Finnigan Mat TSQ 7000 instrument. High-resolution electrospray ionization mass spectra (HR-ESI-MS) were recorded on an Agilent 6540Q-TOF LCMS equipped with an electrospray ionization (ESI) probe operating in positive-ion mode with direct infusion. The results of purity were obtained on a Shimadzu HPLC (type: UFLC), whose control system is Lab Solution, and the detector is PDA. The G1 and G2 were purified via preparative high performance liquid chromatography (PHPLC), which was obtained on a Waters 2998 instruments. Preparation and Observation of Nanoparticles Formed from WP6⊃G1 and WP6⊃G2. WP6⊃G1 and WP6⊃G2 nanoparticles were prepared using the injection method. Typically, G1 (1.4 mg) was dissolved in a weak

3. CONCLUSIONS In conclusion, we have successfully designed and synthesized two kinds of novel DOX-based prodrugs (G1 and G2), which would form WP6⊃G1 and WP6⊃G2 supramolecular nanoparticles with the presence of WP6. They are both stable under physiological conditions, whereas rapid and almost complete release of DOX from them could be achieved within 30 min at G

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penicillin and 50 U·mL−1 streptomycin, and cultured in 5% CO2 at 37 °C for 24 h. Then the three kinds of cells were exposed to serial dilutions of DOX, WP6⊃G1, or WP6⊃G2 nanoparticles and further incubated for 32 h. The cells were washed and replenished with fresh culture medium and further incubated for 2 h. Subsequently, 20 μL of MTT solution was added into each cell and incubated for another 4 h. After that, the medium containing MTT was removed, and dimethyl sulfoxide (DMSO, 150 μL) was added to each well to dissolve the MTT formazan crystals. Finally, the plates were shaken for 10 min, and the absorbance of formazan product was measured at 490 nm by a microplate reader (BioTek ELx808). Untreated cells in media were used as the blank control. All experiments were carried out with six replicates. The cytotoxicity was expressed as the percentage of the cell viability as compared with the blank control. Cellular Uptake and Intracellular Localization Observed by Fluorescence Microscope. The cellular uptake and intracellular localization of DOX was examined in SKOV3 cell lines. Briefly, tumor cells were plated onto glass-bottomed Petri dishes in 1.5 mL of complete culture medium for 24 h before treatment. Then cells were treated with free DOX solution (10.0 μg·mL−1) or WP6⊃G1 or WP6⊃G2 nanoparticles (equivalent to 10.0 μg·mL−1 DOX) for designated time periods. For SKOV3 cells, LysoTracker (Molecular Probes, USA) was directly added to the medium at a final concentration of 100 nM for 1 h to label lysosomes. The tumor cells were washed three times with fresh medium and investigated by fluorescence microscopy (IX-81, Olympus). The fluorescence characteristics of DOX, WP6⊃G1, and WP6⊃G2 nanoparticles were used to directly monitor the localization of these drugs without utilizing additional dye. Flow Cytometric Analysis. SKOV3 cells were seeded in six-well plates at a density of 5 × 105 cells per well in 3 mL of complete DMEM and cultured at 37 °C in a 5% CO2 humidified atmosphere for 24 h. After treating with DOX, WP6⊃G1, or WP6⊃G2 nanoparticles for determined intervals at 37 °C, the cells were then rinsed three times with cold PBS. After trypsinizing, the cells were washed with cold PBS, centrifuged, and dispersed in cold PBS. And then, the cells were subjected to flow cytometric analysis using a BD FACSCalibur flow cytometer, and 105 cells were tested for each sample. Statistical Analysis. Differences between treatment groups were statistically analyzed using the paired Student’s t-test. A statistically significant difference was reported if p < 0.05 or less.

alkaline PBS buffer (5 mL, pH 8.0) in the dark; then a WP6 solution (0.6 mL, C = 0.605 mM) dissolved in a weak alkaline PBS buffer (pH 8.0) was injected. Similarly, G2 (2.25 mg) was dissolved in a weak alkaline PBS buffer (3 mL, pH 8.0) in the dark; then a WP6 solution (0.73 mL, C = 0.605 mM) dissolved in a weak alkaline PBS buffer (pH 8.0) was injected. Thereafter, these WP6⊃G1 and WP6⊃G2 nanoparticles were obtained and characterized by Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM) experiments. For TEM observation, typically, one drop of the nanoparticle solution was placed onto a carbon-coated copper grid. Finally, the solution was air-dried in the dark. Observations were carried out using a JEM-2100 instrument. DLS measurements were performed under a Brookhaven BI-9000AT system (Brookhaven Instruments Corporation, USA) equipped with a 200 mW laser light and operating at λ = 514 nm. ζ-Potential Measurement. ζ-potential measurement was performed at 20 °C on a Zeta sizer-Nano Z (Malvern Instruments Ltd., Worcestershire, U.K.) using the Smoluchowski model for the calculation of the ζ potential from the measured electrophoretic mobility. UV−Vis Absorption and Fluorescence Emission Spectra. UV−vis spectra were recorded in a quartz cell (light path 10 mm) on a PerkinElmer Lambda 35 UV−vis spectrometer. Steady-state fluorescence spectra were recorded in a conventional quartz cell (light path 10 mm) on a PerkinElmer LS55 Fluorescence Spectrometer. Determination of Drug Release Properties from WP6⊃G1 and WP6⊃G2 Nanoparticles.63 In a typical release experiment, WP6⊃G1 and WP6⊃G2 nanoparticles with a concentration of 0.25 and 0.6 mg·mL−1, respectively, were prepared as described above and incubated at 37 °C in different buffers (pH 5.5, 7.4, and 8.0, respectively). At specific time intervals, samples (0.5 mL) were withdrawn and analyzed by HPLC using an AKZONOBEL KR100-5C18 reverse phase column (4.6 × 250 mm) with UV detection at 480 nm at 30 °C. A gradient elution of 30−100% acetonitrile−water both containing 0.05% TFA was applied at a flow rate of 1 mL· min−1. During assays, 20 μL of each sample was injected into the analytic column. The release of DOX was detected by UV at 480 nm, and the hydrolysis degree was determined on the basis of the ratio of free DOX peak areas to the corresponding G1 or G2 peak areas. Determination of the Degradation Rates of Prodrugs G1 and G2. In a typical experiment, G1 or G2 prodrug was dissolved at 25 °C (nanoparticles could not form at this concentration) in different buffers (pH 5.5, 7.4, and 8.0, respectively). At specific time intervals, samples (0.5 mL) were withdrawn and analyzed by HPLC using an AKZONOBEL KR100-5C18 reverse phase column (4.6 × 250 mm) with UV detection at 480 nm at 30 °C. A gradient elution of 30−100% acetonitrile−water both containing 0.05% TFA was applied at a flow rate of 1 mL·min−1. During assays, 20 μL of each sample was injected into the analytic column. The release of DOX was detected by UV at 480 nm, and the hydrolysis degree was determined based on the ratio of free DOX peak areas to the corresponding G1 or G2 peak areas. In Vitro Cell Assay. The relative in vitro cytotoxicities of DOX, WP6⊃G1, and WP6⊃G2 nanoparticles against SKOV3 cell, 4T1 cell, and HeLa cell were assessed using the MTT assay. Briefly, the cells were seeded in 96-well plates at a density of 104 cells per well in 200 uL of complete DMEM containing 10% fetal bovine serum, supplemented with 50 U·mL−1

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ASSOCIATED CONTENT

S Supporting Information *

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

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AUTHOR INFORMATION

Corresponding Authors

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

Yu Cao and Yan Li contributed equally.

Notes

The authors declare no competing financial interest. H

DOI: 10.1021/cm504445r Chem. Mater. XXXX, XXX, XXX−XXX

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (2011CB808600, 2014CB846000), National Natural Science Foundation of China (No. 91227106, 21202083), and the National Science Foundation of Jiangsu (No. BK20130608, BK20140595). We wish to thank Prof. Jeffery T. Davis from the University of Maryland for fruitful discussions and suggestions.

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DOI: 10.1021/cm504445r Chem. Mater. XXXX, XXX, XXX−XXX