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Supramolecular Amphiphilic Polymer-Based Micelles with 7-Armed Polyoxazoline Coating for Drug Delivery Peng Zhang, Xiaoping Qian, Zhengkui Zhang, Cheng Li, Chen Xie, Wei Wu, and Xiqun Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14464 • Publication Date (Web): 26 Jan 2017 Downloaded from http://pubs.acs.org on January 27, 2017

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ACS Applied Materials & Interfaces

Supramolecular Amphiphilic Polymer-Based Micelles with 7-Armed Polyoxazoline Coating for Drug Delivery

Peng Zhang, Xiaoping Qian, Zhengkui Zhang, Cheng Li, Chen Xie, Wei Wu, Xiqun Jiang* MOE Key Laboratory of High Performance Polymer Materials and Technology, Department of Polymer Science & Engineering, College of Chemistry & Chemical Engineering, and Jiangsu Key Laboratory for Nanotechnology, Nanjing University, Nanjing, 210093, P. R. China.

* To whom correspondence should be addressed Email: [email protected]

Keywords: poly(2-methy-2-oxazoline), pseudo block copolymers, supramolecular polymer micelles, drug delivery, antitumor activity.

ACS Paragon Plus Environment

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Abstract

Supramolecular polymer micelles comprised of 7-armed poly(2-methy-2-oxazoline) as the coating and linear poly(DL-lactide) as the core were prepared through synthesizing βcyclodextrin-termineated poly(2-methy-2-oxazoline) and adamantine-terminated linear poly(DLlactide), following by host–guest interaction between β-cyclodextrin and adamantine groups in two polymers and self-assembly in aqueous solution. Dynamic light scattering measurement showed that the micelles based on supramolecular amphiphilic polymers have the size of 120 nm and were highly stable in salt solution. When the micelles were used as the carrier of cabazitaxel, an antitumor agent for drug-resistant cancers, satisfactory drug loading content and encapsulation efficacy were obtained. In vitro cellular cytotoxicity assays found that cabazitaxel-loaded micelles presented obvious cytotoxicity against taxane-sensitive and -resistant cancer cells. Further in vivo antitumor activity evaluation showed that cabazitaxel-loaded micelles have signiﬁcantly superior eﬃcacy in inhibiting tumor growth and prolonging survival in tumorbearing mice than that of free paclitaxel and free cabazitaxel.


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Introduction Nanoscale polymer vehicles are very promising transport systems for delivering chemotherapeutic and biological drugs.1,2,3,4 On the basis of the enhanced permeability and retention (EPR) effect, the nanoscale polymer vehicles are easier to gather and retain in the tumor sites for a longer time, compared with small molecules.5 Among the nanoscale polymeric vehicles such as micelles and nanoparticles, amphiphilic block copolymers are basic assembly blocks. They are formed by covalently linked hydrophobic and hydrophilic polymer blocks. Nonetheless, the preparation condition of block copolymers is greatly rigorous, and preparation processes are usually quite complicated and time consuming. On the other hand, by using intermolecular bonds instead of covalent linkages, supramolecular polymer chemistry provides an alternative strategy to link different polymers.6 Hydrophobic block A and hydrophilic block B can be linked, based on intermolecular interactions, such as host–guest recognition,7 hydrogen bonds8,9 and ligand-metal coordination,10 to generate so-called pseudo block copolymers. These pseudo block copolymers can further assemble into micelles and nanoparticles. Among the supramolecular interactions, β-cyclodextrin-based inclusion complexation is often employed due to high association constant with guest molecules. For example, the association constant reaches 1×105 M-1 when β-cyclodextrin (β-CD) coordinated with adamantane (Ad) in water.11 Also, based on host-guest interaction, various stimuli-responsive moieties can be incorporated into supramolecular micelles,12 endowing these micelles to be responsive for environmental stimuli, such as pH,13,14 redox,15, enzymes16 and temperature.17 Compared with common covalently linked block copolymers, preparing amphiphilic pseudo block copolymers based on supramolecular interactions offers several advantages: (1) the using of intermolecular bonds instead of covalent linkages significantly reduce the complexity of synthesis; (2) the


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noncovalently linking among different blocks provides great flexibility and linkage among copolymer building blocks on demand like the plug-in units; (3) separate syntheses of different blocks may simplify purification and separation procedures; (4) supramolecular chemistry is often conducted under mild and environmental friendly condition, which is very suitable for biorelated materials; (5) various functional moieties can be incorporated into individual block easily; (6) the blocks with various topologies can be linked on demand.18,19 Up to now, although numerous pseudo block copolymers and their micelles have been reported, rarely of them addressed the drug delivery ability, biologic effects, and antitumor activity in vitro and in vivo for supramolecular drug delivery systems.20 The behaviors of nanoscale supramolecular polymer vehicles in vivo are still poorly understood. Poly(2-oxazoline)s (POx), also called as pseudo-polypeptides, are a class of hydrophilic and biocompatible polymers.21,22 They are polymerized by living cationic ring-opening polymerization (LCRP), which yields low polydispersity index and versatile end-active polymers.23 The most typical hydrophilic POx are poly(2-ethyl-2-oxazoline) (PEtOx) and poly(2-methyl-2-oxazoline) (PMeOx). Similar to poly(ethylene glycol) (PEG), they have an immunological stealth property and present finite interactions with human serum proteins.24-26 Although they are non-biodegradable polymers, PMeOx and PEtOx with low molecular weight are excreted easily via the kidneys, and show no significant nonspecific uptake in vivo after intravenous administration.27 In contrast of PEG, which shows an accelerated blood clearance (ABC) from circulation when it is repeatedly injected into the body, POx shows the same pharmacokinetic behavior over multiple injections. Thus, POx is considered as a potential alternative to PEG.


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As a new taxane, cabazitaxel (CBZ) has shown a broad spectrum of antitumor activities28 and can overcome P-glycoprotein (Pgp)-mediated drug resistance.29 CBZ is also clinically used in treatment of metastatic breast cancer with taxane-resistant and docetaxel previously treated metastatic castration-resistant prostate cancer.30,31 Nonetheless, similar to that reported for taxanes, CBZ show the side effects such as neutropenia and neurosensory disturbances,32 and a poor water solubility. In this work, we report a novel kind of supramolecular pseudo block copolymer micelles with linear poly(DL-lactide) (PDLLA) as hydrophobic micellar core and 7-armed star PMeOx as the hydrophilic micellar shell. By living cationic ring-opening polymerization of 2-methyl-2oxazoline (MeOx), 7-armed star PMeOx was prepared from β-CD core. Also, adamantine capped linear PDLLA was synthesized by ring-opening polymerization of DL-lactide. Thank to non-covalently host-guest interaction between β-CD and adamantine moieties, 7-armed PMeOxPDLLA pseudo block copolymers were initially generated, and then formed supramolecular micelles by self-assembly in water. The biological performance and antitumor efficiency of these supramolecular micelles loaded with CBZ were evaluated both in cultured cells and tumorbearing mice. Results and discussion Synthesis of 7-armed star PMeOx 7-Armed star PMeOx was prepared by LCRP of 2-methyl-2-oxazoline (MeOx) using heptaiodide-substituted-β-cyclodextrin as the initiator (Scheme 1).33-36 First, the primary hydroxyl groups in β-cyclodextrin were selectively replaced by iodine atoms, affording iodidesubstituted-β-cyclodextrin (CD-I) (Scheme S1).37 The remaining hydroxyl groups of iodide-


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substituted-β-cyclodextrin were then acetylated in order to eliminate possible side reaction induced by the hydroxyl groups in the polymerization of MeOx (Scheme S1). Figure 1 shows 1

H NMR spectra of iodide-substituted-β-cyclodextrin, and acetylation of iodide-substituted-β-

cyclodextrin. All the peaks are clearly seen as marked in Figure 1, and the degree of iodidesubstituted-β-cyclodextrin was calculated as 7. The polymerization of MeOx monomer was performed by living cationic ring opening reaction of MeOx with CD-I as the initiator. Although iodine can accelerate the polymerization of MeOx in some cases, the polymerization reaction still proceeded for several days. After precipitated into excess diethyl ether and purified, 7-armd star PMeOx (β-CD-PMeOx) was prepared (Figure S1). The molecular weight and polydispersity index of 7-armd star PMeOx were measured by gel permeation chromatography (GPC) and determined to be 22000 Da and 1.30, respectively (Figure S2). Next adamantane-capped poly(DL-lactide) (Ad-PDLLA) was synthesized through ring opening polymerization of DL-lactide using adamantine as the initiator and stannous octoate as the catalyst. The 1H NMR spectrum of Ad-PDLLA obtained is shown in Figure S3. All the peaks are clearly seen, demonstrating the successful synthesis of Ad-PDLLA. The molecular weight of Ad-PDLLA was calculated to be 16000 Da based on the areas of peak 2 at 5.18 ppm (methyne protons linked to O atom) and peak 3 at 4.37 ppm (methyne protons linked to hydroxyl group at the terminal of PDLLA). Thus, we successfully obtained β-CD terminated 7 armed star PMeOx and adamantane-terminated linear Ad-PDLLA. Preparation of supramolecular polymer micelles Supramolecular 7 armed star PMeOx and linear PDLLA micelles were constructed by the inclusion complexation between β-CD and Ad ends in PMeOx and PDLLA, respectively. β-CD


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terminated 7 armed star PMeOx and adamantane-terminated linear Ad-PDLLA were dissolved in water and acetone, respectively. With slow dropping Ad-PDLLA solution into β-CD-PMeOx aqueous solution, the micelles were formed spontaneously. As a result, hydrophobic linear PDLLA constituted the core of micelles while hydrophilic 7-armed PMeOx consisted of the corona. The interface linkage between the core and the corona was made up of complexation of β-CD and adamantane. Two-dimensional nuclear overhauser effect spectroscopy of NMR (2D NOESY) was used to confirm the formation of the inclusion complexation between two polymers and the spectra are presented in Figure 2. It can be seen that the supramolecular polymer micelles show well correlation of protons between Ad group (2.0 ppm) and of β-CD moiety (3.5 ppm) respectively, indicating the successful inclusion complexation between β-CD end of 7 armed PMeOx and adamantane end in linear PDLLA. The result is well consistent with previous studies on the preparation of pseudo block copolymers through the host-guest interaction of β-CD and adamantine.38 We designed these supramolecular polymer micelles obtained as 7PMOxLA micelles. The size of 7PMOxLA micelles was investigated by a dynamic light scattering (DLS) method. The size distribution of the micelles was divided into two groups with a dominant peak at 119 nm and a minor peak at 250 nm (Figure 3A). A small amount of larger micelles arises probably from the aggregation of the micelles. The zeta potential of 7PMOxLA micelles was measured to be around -4.1 mV, suggesting that the micelles have a neutral surface. Further the size and morphology of supramolecular polymer micelles were evaluated by a transmission electron microscopy (TEM). It is seen that the micelles distribute uniformly and have spherical morphology (Figure 3B). The average size of the micelles is about 58 nm at dry state, about half


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of its diameter obtained from the DLS measurement. Obviously, the micellar exterior, PMeOx is highly swelled by water molecules when 7PMOxLA micelles are in aqueous solution. It has been reported that the aggregation of poly(ethylene glycol)-b-poly(ε-caprolactone) micelles and PEGylated mesoporous silica nanoparticles occurs in a salt aqueous solution.39,40 To investigate the stability of 7PMOxLA micelles, the hydrodynamic diameter of 7PMOxLA micelles as a function of time in saline and PBS was monitored by DLS (Figure 3c). It can be seen that the hydrodynamic diameter of 7PMOxLA micelles is invariant in PBS (pH = 7.4, 10 mM) and saline (0.9% w/w) as a time function up to 57 hours. This result suggests that 7PMOxLA micelles have good stability in saline and PBS. Drug loading of 7PMOxLA micelles To evaluate the drug delivery ability of 7PMOxLA micelles as the drug vehicles, cabazitaxel (CBZ) was loaded into 7PMOxLA micelles. The CBZ loading content was 17.5% (per 100 mg of CBZ-loaded micelles contains 17.5 mg of CBZ) and the loading efficiency was 65%. The DLS measurement shows that the CBZ-loaded micelles are still a bimodal distribution: a major peak at 89 nm and a weak peak around 190 nm (Figure 4A). In contrast to empty micelles, the drug loaded 7PMOxLA micelles are more compact and their size decreases after drug loading.41,42 Drug encapsulation is supposed to have two contrary effects on particle size. One effect is that drug may improve the compact arrangement among chains through hydrophobic interactions. The other effect is the enlargement of core bulk due to the drug incorporation. Hydrophobic CBZ might induce the compactness of the hydrophobic core and smaller micelles are formed. Subsequently, the diameter of CBZ-loaded micelles in PBS and saline were measured for stability evaluation as a time function (Figure 4B). Within 57 hours, no


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significant change in hydrodynamic diameter of the micelles is found, indicating the great stability in salt solution of CBZ-loaded micelles as empty micelles do. In vitro drug release and cytotoxicity of the micelles CBZ release from CBZ-loaded 7PMOxLA micelles in vitro was examined in phosphatebuffered saline (PBS, 0.1 M, pH = 7.4, 0.1% v/v Tween 80) at 37 oC. The released CBZ from the micelles was analyzed by using high performance liquid chromatography (HPLC), as done in detection of paclitaxel release from micelles.43 The release profile was determined up to 264 hour (11 days). Two stages of release profile are observed: a relatively fast release rate at first stage, followed by a slow release at second stage (Figure 4C). At 24 hours, about 40% of loaded CBZ is release from the micelles and no initial burst is observed. At the end of release profile (264 hours), this value reaches 74.1%, indicating release of most of loaded drugs. There are two reasons which may cause two stages of release profile of drugs in 7PMOxLA micelles. One is that some CBZ molecules are loaded at the hydrophobic-hydrophilic interface of the micelles, leading to initial CBZ diffusion and relatively fast release from the micelles. Another reason is that the release speed of drug is dependent on the drug payload and drug density in the micelles. The higher CBZ loading leads to faster release.44,45,46 The pharmacological activity and the potential toxicity were evaluated by the cytotoxicity of CBZ-loaded micelles against A549 cell line (human lung adenocarcinoma cell line) through MTT assay. Figure 5A shows the cell viabilities after 48 hour of incubation with free PTX, CBZ , and 7PMOxLA micelles loading with CBZ, respectively. Both samples display obvious dose dependent cytotoxicity. The cytotoxicity of free CBZ is higher than CBZ-loaded micelles. The IC50 value is 5.2, 8.1 and 23.2 ng/mL for free CBZ and CBZ-loaded micelles, respectively.


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This might be resulted from the supramolecular polymer micelles which have not release CBZ completely. Multidrug resistance (MDR) is a common problem which we observed in clinic chemotherapy failure. Compared with docetaxel and paclitaxel, cabazitaxel which shows weak affinity with P-glycoprotein is considered to be a potential candidate to overcome MDR.29 To estimate the ability of CBZ-loaded 7PMOxLA micelles to overcome drug resistance in cellular level, A2780/T, a kind of paclitaxel (PTX)-resistant human ovarian cell line, was used as a drug resistant cell model.47 The cytotoxicity of paclitaxel was also investigated to evaluate the drug resistance ability of A2780/T cells to PTX. It can be seen that no obvious cytotoxicity of PTX is observed after 48 hour of incubation except the highest concentration (Figure 5B), confirming the drug resistance of A2780/T cell to PTX. In contrast, CBZ treated A2780/T cells present a dose dependent cytotoxicity. The significantly higher cytotoxicity of CBZ than PTX at each concentration indicates the ability of breaking through the multidrug resistance of cancer cells. Figure 5B also displays the cytotoxic effect of CBZ-loaded 7PMOxLA micelles on the A2780/T cells in vitro. The inhibition effect of CBZ-loaded micelles on A2780/T cells is also dose-dependent, similar to the A549 cells, suggesting that A2780/T cell proliferation is effectively inhibited. Free CBZ shows a little higher cytotoxicity than CBZ-loaded 7PMOxLA micelles against A2780/T cells. Similar results are also observed in A2780 cells without PTX resistant (Figure 5C) and H22 cells (Figure 5D). The IC50 values of CBZ-loaded 7PMOxLA micelle against different types of cells are presented in Table S1. Additionally, no significant inhibition effect of empty 7PMOxLA micelles is observed even at high concentration on all the four cells (Figure S5), demonstrating the great cytocompatibility of these supramolecular polymer micelles.


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To investigate the cellular uptake and intracellular distribution, 7PMOxLA micelles were labeled with rhodamine B and co-incubated with all four kinds of cancer cells, A549 cells, PTXresistant A2780/T cells, A2780 cells and H22 cells. Confocal laser scanning microscopy (CLSM) images of A549 cells, PTX-resistant A2780/T cells, A2780 cells and H22 cells after coincubation with the micelles for 4 hour at 37 oC are shown in Figure 6. For A549 cells, A2780 cells and H22 cells, it can be seen that 7PMOxLA micelles with punctulate red fluorescence are localized in cytoplasm region and accumulated around the nucleus (Figure 6A, 6C, 6D), indicating the effectively internalization of 7PMOxLA micelles by A549 cells through endocytosis pathway. Similarly, co-incubated with A2780/T cells, a great amount of 7PMOxLA micelles are distributed in the cytoplasm region (Figure 6B), indicating that the 7PMOxLA micelles can bring more CBZ into A2780/T cells with the potential to overcome multidrug resistant. The flow cytrometry analysis was also good agreement with CLSM observation above (Figure S6). The three-dimensional multicellular spheroids (MCS) represent the avascular areas in tumor tissues. Unlike conventional two-dimensional cell cultures, MCS can mimic the 3D cellcell interactions and cellular environment of solid tumors in vivo, and are an appropriate model to evaluate the penetration and accumulation profiles of 7PMOxLA micelles.48-51 In present work, multicellular tumor spheroids (MCTS) of SH-SY5Y cells were established and used to study the penetration behavior of 7PMOxLA micelles. After co-incubation with MCTS for 4 h, 8 h and 24 h respectively, a time-dependent penetration and accumulation of the rhodamine Blabeled 7PMOxLA micelles are presented in Figure 7A. At the initial 4 h and 8 h after coincubation, the fluorescent signal is weak and only accumulates in the periphery of MCTS. Nonetheless, 24 hours later, the fluorescent intensity of MCTS becomes stronger than that at 4 h


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and 8 h. Also, the micelles penetrate deeper into the MCTS at 24 h after co-incubation. The mean fluorescent intensity within MCTS at different times based on semi-quantitative evaluation presents a time-dependent cell uptake and penetration (Figure 7B). The fluorescent intensity of MCTS at 24 h is 14- and 7- fold higher than that at 4 h and 8 h, respectively. This result suggests that more 7PMOxLA micelles are taken up by the MCTS and penetrate into MCTS to a deeper extent with time. Biodistribition of 7PMOxLA micelles In order to estimate the delivery efficiency of CBZ-loaded 7PMEOxLA micelles in vivo, the biodistribution of cabazitaxel in vivo is necessary to be determined. The mice were fed with food and water for 7 days after tumor inoculation. After that, 15 H22 tumor bearing ICR mice were injected via tail vein with CBZ loaded micelles at an equivalent dose of 10 mg/kg body weight. Free CBZ which dissolved in 0.1% tween 80 was also injected in the same number of mice as the control. The mice were sacrificed at 1 h, 4 h, 8 h, 12 h and 24 h after i.v. administration. Plasma samples were mixed with methanol (plasma: methanol, 1:2.5, v/v) and centrifuged at 14,500 r.p.m. for 15 min. Supernatants were collected for HPLC quantification. Subsequently, organs were collected carefully and then washed with water. Tissue samples were homogenized in water and the homogenates were mixed with acetonitrile (DDB as internal standard) (1:1, v/v),52

centrifuged and supernatants were transferred to clean tubes. Equal

volumes of methanol were added to precipitate proteins and supernatants were collected following centrifugation. The supernatants were flowed dried by N2 and 1 mL acetonitrile was added. HPLC was used to measure the containing of CBZ. The mobile phase was composed by double distilled water and acetonitrile (HPLC grade) with the ratio of 52/48 in the flow rate of 1mL/min. The CBZ contents in plasma were presented in Figure S8. Very low CBZ


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concentration in plasma is observed for both free CBZ and CBZ-loaded 7PMEOxLA micelles in the time range from 2min to 24 h. However, the blood circulation time of micelle formulation is much longer than free CBZ. The half-life time of CBZ in blood circulation for free CBZ and CBZ-loaded micelles is calculated to be around 1.4 h and 12 h, respectively. The CBZ contents in heart, liver, spleen, lung, kidney and tumor for two groups of free CBZ and CBZ-loaded 7PMEOxLA micelles are presented in Figure 8. The content of CBZ for CBZ-loaded micelles in tumor is higher than that of free CBZ and reaches around 3.9% injection dose per gram of tumor (ID %/g) at the first hour after injection and decreases few in the next 24 hours (Figure 8A), suggesting that the micelles can maintain high drug concentration in tumor for longer time. For free CBZ, the accumulation of CBZ in H22 tumor is 2.5% ID%/g at 1 hour and decreases dramatically with time (Figure 8B). This result indicates that the tumor-targeting drug delivery can be improved by 7PMEOxLA micelles. It is also noteworthy that the accumulation of CBZ enwrapped in micelles is higher than that of free CBZ complex in liver and spleen. In vivo NIRF imaging of 7PMOxLA micelles Optical NIRF imaging was carried out at different time points after intravenous injection of NIR-797 labeled 7PMOxLA micelles. The fate of the micelles in H22 tumor bearing mice was shown in Figure S7 by time. Initially, strong NIR fluorescence signal occurs in liver and intestine, while weak signal in tumor site. Gradually, the fluorescence signal in tumor became stronger and stronger, indicating the successive accumulation of 7PMOxLA micelles in tumor, which favors the enhanced antitumor efficacy of supramolecular micelles. In contrast, the signal in liver decreases with time. Antitumor activity of CBZ-loaded 7PMOxLA micelles


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Next antitumor efficacy in vivo was evaluated with different CBZ and PTX formulations in the mice bearing subcutaneous H22 tumor. When the tumor volume grew up to approximately 50 mm3 on average, the mice were treated with saline, empty 7PMEOxLA micelles, free paclitaxel (10 mg/kg), free CBZ (10 mg/kg) and CBZ-loaded 7PMEOxLA micelles (10 mg/kg based on cabazitaxel) by single intravenous (i.v.) injection. Time dependent tumor volumes and survival rates were then measured and used to evaluate the antitumor effect. The growth curves of H22 tumors of mice with time (day) are shown in Figure 9A. We can see that tumor volumes of the control groups treated with saline and blank micelles increase rapidly and reach 2010 ± 439 mm3 and 2425 ± 1336 mm3 on day 15, respectively. Compared with the initial volume of 50 mm3 at day 1, the tumor volumes grow up about 50 folds. However, in the initial 7 days the tumors grow relatively slowly in the free PTX and CBZ treatment groups, but accelerate later follow in a similar pace of the control groups (saline and blank micelles). At day 15, tumor volume increases 21 and 22 folds in PTX and CBZ groups, respectively, compared to day 1. The tumor growth inhibition (TGI) are just about 56.5% and 53.9%, respectively. In contrast, the group received CBZ-loaded micelles has smallest tumor volume (CBZ micelles in the Figure 9A). The tumor growth almost stops in the first 11 days. At day 15, the tumor volume of the group treated by CBZ-loaded micelles increases 13 folds compared to that on day 1. The TGI of the group treated by CBZ-loaded micelles is 78.8%. This is the highest value among the five groups. Thus, CBZ-loaded 7PMEOxLA micelles have a significant higher antitumor activity against H22 tumor compared to both free PTX and CBZ (P < 0.01). The survival rate of each treated group of the mice was recorded and presents in Figure 9B. All mice die in the control groups during 35 days after treatment. Meanwhile about half of mice are alive for free PTX and CBZ treated groups during 35 days, and the remaining mice in


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both groups die completely during 52 day. By comparison, the mice received CBZ-loaded 7PMEOxLA micelles show the most survival number after treatment among five groups. Two third of the mice survive at day 45, and 3 mice still keep alive at day 60 when the experiment is ended. This result indicates that CBZ-loaded micelles can significantly prolong the survival time of H22 tumor-bearing mice. The body weights of mice show similar change tendency in different groups after treatment (Figure 9C). Because of the remarkable increase of tumor volumes, the body weights of control groups are higher than those of other groups. Also, no significant decrease in body weight for all groups is observed, indicating the doses of PTX and CBZ we used are appropriate. Conclusions A novel type of supramolecular polymer micelles was successfully prepared through host-guest interaction of β-CD-terminated 7-armed star PMeOx and adamantine-terminated linear PDLLA. It was found that these supramolecular polymer micelles with multi-armed polyoxazoline as the shell were highly stable in saline and PBS. When CBZ, an antitumor agent that does not succumb to drug resistance, was encapsulated into the micelles, the satisfied drug loading content (17.5%) was achieved. CBZ-loaded micelles could effectively be internalized by drug-sensitive A549 and drug-resistant A2780/T cancer cells and had much larger ability to inhibit the growth of PTX-resistant A2780/T cancer cells than free PTX. In the examination of antitumor activity in vivo, CBZ-loaded supramolecular polymer micelles showed much higher antitumor efficiency than free CBZ and PTX, and the survival time of tumor-bearing mice was significantly prolonged after treating H22 tumor-bearing mice. Moreover, the biodistribution analysis in vivo demonstrated that the supramolecular polymer micelles could transport more drugs into tumor than free drug, indicating a promising drug delivery system for cancer therapy.


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Supporting Information: Additional experimental methods, polymer characterization data. This material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGMENT

This work was supported by the Natural Science Foundation of China (No. 51690153, 21474045, 51273090 and 51422303), Specialized Research Fund for the Doctoral Program of Higher Education, and the Program for Changjiang Scholars and Innovative Research Team in University.


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Scheme 1 Schematically showing the preparation of 7PMOxLA micelles and CBZ-loaded 7PMOxLA micelles.


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Figure 1. (A) 1H NMR (DMSO-d6, 400 MHz) spectra of iodide-substituted-β-cyclodextrin; (B) 1

H NMR (DMSO-d6, 400 MHz) and (C)

13

C NMR (DMSO-d6, 400 MHz) spectra of the

acetylation of iodide-substituted-β-cyclodextrin.


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Figure 2. 2D NOESY NMR spectra of 7PMOxLA micelles in D2O/D6-acetone (8/3).


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Figure 3. (A) Size distribution of 7PMOxLA micelles in PBS determined by DLS; (B) TEM image of 7PMOxLA micelles; (C) Changes in the diameter of 7PMOxLA micelles in PBS (pH = 7.4, 10 mM) and saline (0.9% w/w).


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Figure 4. (A) Size distribution of CBZ-loaded 7PMOxLA micelles in PBS determined by DLS; (B) Changes of diameters of CBZ-loaded 7PMOxLA micelles in PBS (pH = 7.4, 10 mM) and saline (0.9% w/w); (C) CBZ release from CBZ-loaded 7PMOxLA micelles in vitro (PBS, 0.1 M, pH = 7.4, 0.1% v/v Tween 80) at 37 oC.


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Figure 5. (A) Cell viabilities of free CBZ, CBZ-loaded 7PMOxLA micelles, PTX against A549 cells ; (B) A2780/T cells (PTX resistant); (C) A2780 cells (PTX non- resistant) ; (D) H22 cells for 48 hour; Data are presented as mean ± SD (n = 3).


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Figure 6. CLSM images of A549 cells (A), A2780/T cells (B), A2780 cells (C), and H22 cells (D) incubated with RB-labeled 7PMOxLA micelles. All the scale bars are 20 µm. Red: RBlabeled 7PMOxLA micelles. Blue: Nuclear. The micelles concentration = 200 µg/mL).


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Figure 7. (A) CLSM images of SH-SY5Y MCTS incubated with RB-labeled 7PMOxLA micelles for 4 h, 8 h and 24 h at 37 oC, respectively. Images were taken every 15 µm from equatorial plane to top. All the scale bars are 100 µm; (B) Fluorescent mean intensity in SHSY5Y MCTS incubated with RB-labeled 7PMOxLA micelles (200 µg/mL) for 4 h, 8 h and 24 h. Data are shown as mean ± SD (n = 3).


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Figure 8. Biodistribution of CBZ-loaded 7PMOxLA micelles (A) and free CBZ (B) in H22 tumor bearing mice at various time points after i.v. injection. The values are shown as the percentage of injection dose per gram of organs, mean ± SD (n = 3).


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Figure 9. (A) In vivo tumor volume curves of H22 tumor bearing mice with different treatments. Data are shown as mean ± SD (n = 9). ** represent P < 0.01 since the 9th day; (B) Kaplan-Meier curves presenting survival of H22 tumor bearing mice in different groups; (C) Body weight changes with different treatments. The treatment doses of both PTX and CBZ are 10 mg/kg per mice. Empty micelles (the same amount polymers as CBZ loaded micelles).


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