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Mar 1, 2016 - ABSTRACT: Although self-assembled polymeric micelles have received significant attention as anticancer drug delivery systems, most of th...
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Organocatalytic Anticancer Drug Loading of Degradable Polymeric Mixed Micelles via a Biomimetic Mechanism Julian M. W. Chan,† Jeremy P. K. Tan,‡ Amanda C. Engler,† Xiyu Ke,‡ Shujun Gao,‡ Chuan Yang,‡ Haritz Sardon,†,§ Yi Yan Yang,*,‡ and James L. Hedrick*,† †

IBM Almaden Research Center, 650 Harry Road, San Jose, California 95120, United States Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669, Singapore § POLYMAT, University of the Basque Country UPV/EHU Joxe Mari Korta Center, Avda. Tolosa 72, 20018 Donostia-San Sebastián, Spain ‡

S Supporting Information *

ABSTRACT: Although self-assembled polymeric micelles have received significant attention as anticancer drug delivery systems, most of them suffer initial burst release of drugs after injection. Herein, a novel organocatalytic drug loading approach is reported to chemically conjugate anticancer drugs to the micellar core through an acid-labile bond that only breaks in the acidic tumor tissue and endolysosomal environments. Specifically, a degradable polymeric micelle system based on amphiphilic mPEG-b-polycarbonate block copolymers was developed. The mussel-inspired polymer design features catechol side chains to which the anticancer drug doxorubicin (DOX) can be covalently conjugated as pH-sensitive p-quinoneimines via a mechanism that mimics the Raper−Mason pathway of mammalian melanogenesis. We demonstrate that a higher drug loading is achieved when N-methylimidazole is cointroduced during self-assembly as an organocatalyst. The DOX-loaded mixed micelles formed from a catechol-functionalized polycarbonate/PEG block copolymer and a sister polymer with imidazole side chains are kinetically stable and display no signs of premature drug release, but possess comparable cytotoxicity in cancer cells to free DOX by a pH-triggered intracellular release. Moreover, we show that the nanoparticles accumulate in tumors through the enhanced permeability and retention (EPR) effect, and that the DOX-loaded mixed micelles suppress tumor growth more effectively than free DOX without causing toxicity in a mouse breast cancer model.

1. INTRODUCTION Cancer is currently the second most common cause of death in the U.S., accounting for 1 in every 4 deaths.1 Many anticancer drugs employed in chemotherapy are hydrophobic molecules that show poor water solubility and short blood circulation times in the body. Frequent dosing, however, is unfeasible due to the dose-limiting toxicities of these nonspecific drugs (e.g., doxorubicin), which are often associated with side effects in patients undergoing chemotherapy.2 In an effort to address these problems, well-defined nanostructures self-assembled from various macromolecules have been developed to function as drug delivery carriers.3 These supramolecular assemblies serve to encapsulate the anticancer drugs within their core, resulting in prolonged blood circulation lifetimes and lower overall clearance rates. In particular, micellar drug delivery systems based on synthetic polymers have received a significant amount of attention in recent years.4 These polymeric micelles are organic nanoparticles that offer several advantages as drug delivery vehicles. First, their characteristic core−shell structures allow drug cargo to be encapsulated within the hydrophobic core while the hydrophilic shell/corona, usually composed of © XXXX American Chemical Society

poly(ethylene glycol) (PEG), confers water solubility and provides a stealth effect against opsonization.5 Second, their 10−200 nm particle size range is ideal for passive targeting of tumors via the enhanced permeability and retention (EPR) effect.6 Lastly, the physical and biological properties of these self-assembled nanostructures can also be readily fine-tuned through chemical modifications of the constituent polymer structure. Thus, by manipulating key structure−propertyfunction relationships, it is possible to rationally design and synthesize polymeric micelles with a specific set of desired properties.7 For the purpose of drug delivery using polymeric micelles, there are several important parameters to be considered. These include micelle stability (thermodynamic and kinetic), drug loading capacity, nanoparticle size, size distribution, and biocompatibility.8 The stability of the micelles determines their blood circulation lifetimes, while their particle size and Received: December 28, 2015 Revised: February 21, 2016

A

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Figure 1. (a) Mixed micelle system based on mPEG-b-polycarbonate copolymers; Organocatalytic anticancer drug loading. (b) Proposed biomimetic mechanism of drug-loading onto the catechol side chains of the block copolymer (left). Raper−Mason mechanism of melanin biosynthesis (inset) and (c) UV−vis spectroscopic monitoring of a model study involving a simple aminoalcohol.

targeting via the EPR effect, and (3) acid-triggered intracellular drug release in tumors.

dispersities influence biodistribution in the body. From previous studies, it is known that micelle stability is dependent upon the noncovalent interactions between the hydrophobic components of the polymer.9 These interactions can be engineered and fine-tuned through the introduction of specific functional groups into the polymer structure. In the current work, we have utilized similar rational design principles to develop a polymeric system capable of conjugating high levels of anticancer drug via a biomimetic loading mechanism. In addition to its biocompatibility and biodegradability, this bioinspired drug delivery system features an ideal combination of high drug loading capacity, passive tumortargeting ability, and selective intracellular drug release. Herein we report an amphiphilic mPEG-b-polycarbonate diblock copolymer system that enables the following: (1) high levels of doxorubicin (DOX) to be covalently loaded through an organocatalyzed and bioinspired pathway, (2) passive tumor

2. RESULTS AND DISCUSSION 2.1. Polymer Synthesis. A series of diblock copolymers comprising a 5-kDa methoxyPEG block and an 11-repeat unit polycarbonate block bearing pendant functional groups were synthesized via organocatalytic ring-opening polymerization (OROP) of cyclic carbonate with an active pentafluorophenyl ester group (MTC-OC6F5) followed by postpolymerization functionalization (Figure 1a).10−12 Each copolymer is functionalized with either pendant catechol, imidazole, pyridine or phenol moieties, tethered to the polycarbonate block by amide linkages. Besides providing hydrogen-bonding handles for mixed micelle formation, the catechols and imidazoles or pyridines also serve as DOX conjugation sites and basic organocatalysts/promoters, respectively. The catechol unit forms the basis of many mussel-inspired functional materials,13 B

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Macromolecules Table 1. Characteristics of Blank and DOX-Loaded Micelles

PEG−imidazole PEG−pyridine PEG−phenol PEG−catechol phenol/imidazole mixed micelle phenol/pyridine mixed micelle catechol/imidazole mixed micelle catechol/pyridine mixed micelle PEG−catechol + N-methylimidazole

particle sizea (nm)

PDIb

particle sizec (nm)

PDId

20, 164 91, 294 29 25 28, 386 29 26 24 −

0.30 0.39 0.10 0.05 0.45 0.07 0.10 0.07 −

315 187 105, 793 125 429 17, 100 148 37 118

0.33 0.14 0.51 0.23 0.39 0.33 0.20 0.26 0.22

DOX loading (wt %) 3.4 1.8 0.7 20.6 3.4 3.4 19.9 12.1 31.5

± ± ± ± ± ± ± ± ±

0.3 0.3 0.1 3.2 0.3 0.3 0.1 0.2 2.4

a

Hydrodynamic diameter of micelles in DI water by dynamic light scattering. bPolydispersity index of micelles in DI water by dynamic light scattering. cHydrodynamic diameter of DOX-loaded micelles in DI water by dynamic light scattering. dPolydispersity index of DOX-loaded micelles in DI water by dynamic light scattering.

2.2. Drug Loading. As part of our preliminary studies, we prepared a mixture of the catechol and imidazole copolymers (50% w/w) in methanol, and introduced a small quantity of 2amino-1-butanol to the solution. The clear and initially colorless solution was monitored by UV−vis spectroscopy over 3 h, during which a spectrum was acquired every 30 min. Within 1 h, a perceptible color change in the solution was observed and a distinct absorption peak at 431 nm appeared (Figure 1c). This absorption band, located in a similar range as L-dopachrome (λmax 475 nm), continued to increase in intensity with time, resulting in a highly colored solution after 3 h. The observed spectral feature is likely due to the formation of a 2hydroxy-p-quinoneimine chromophore. A control solution containing an identical polymer mixture but without primary amine remained clear and colorless even upon exposure to air for 24 h. Following this, the covalent conjugation of DOX to the catechol polymer as a p-quinoneimine was attempted. In one trial, we used a single-component system featuring only the catechol-functionalized diblock copolymer, whereas in a second trial, a mixed system containing catechol and imidazole copolymers (50 wt %) was employed. Gratifyingly, drug loading proved successful and up to 20.6% by weight of DOX with respect to the polymer was achieved in the catechol-only system (Table 1). Interestingly, in the case of the mixed system, the DOX loading level was 19.9 wt %, which is surprising given that 50% dilution of the catechol copolymer with the imidazole copolymer. This observation can be rationalized through an analysis of the drug loading mechanism. Following a Raper− Mason-type pathway, it can be seen that a primary amine (e.g., DOX) becomes covalently attached to the polymer during the aza-Michael addition step. In this case, for the calculated degree of polymerization of the catechol block a theoretical value of 42% DOX loading level should be possible. A loading level of 20.6 wt % constitutes ∼49% loading efficiency/capacity. Therefore, if the yield of this step was improved, then drug loading levels would increase by the same factor. On the basis

offering numerous possibilities through its unique and versatile chemistry. The catechol group is an electron-rich polyphenol that plays a central role in many biochemical and nonbiological pathways.14 These include the chemistry of mussel adhesive proteins,15 mammalian melanogenesis (Raper−Mason pathway),14 enzymatic browning of fruits and vegetables,16 oxidation of adrenaline,17 coatings,18 delivery agents,19,20 and dopamine polymerization.21 The common mechanistic step in all the above processes is an initial oxidation of the electron-rich catechol to an o-quinone via enzyme-catalyzed oxidation (e.g., by polyphenol oxidase) or autoxidation by molecular oxygen. The highly reactive o-quinone is susceptible to multiple reaction pathways, e.g., Schiff base formation, oxidative coupling, and Michael-type 1,4-conjugate addition of thiols, amines, and DNA bases, etc.22 In view of the primary amine (NH2) present in DOX, we envisioned using the aza-Michael addition to covalently conjugate the drug to o-quinones formed in situ via autoxidation of catechol side chains. Similar 1,4additions to o-quinones occur in both melanin biosynthesis23 and dopamine polymerization,24 albeit intramolecularly, as well as mild bioconjugation reactions.25,26 In the Raper−Mason mechanism of melanin bioynthesis, the aza-Michael reaction is followed by rapid tautomerization to 1,2-dihydroxyaniline with concomitant regeneration of aromaticity. The resulting benzene ring, being even more electron-rich than the initial catechol, is readily oxidized to a 4-amino-substituted o-quinone that exists in equilibrium with its 2-hydroxy-p-quinoneimine form. In melanogenesis, the p-quinoneimine intermediate is the pigment 27 L-dopachrome (λmax = 475 nm). Attracted by the facile nature of the reactions in this biochemical pathway, we decided to explore an analogous way to load NH2-containing DOX molecules onto the catechol polymers as p-quinoneimines (Figure. 1b). Furthermore, based on the knowledge that imines are hydrolytically labile under acidic conditions,28 we also envisaged the possibility of eventual pH-triggered drug release in the endosomes of tumor cells. C

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Figure 2. In vitro drug release kinetics and cytotoxicity. (a) Release profiles of DOX from catechol/imidazole mixed micelles (covalent bond) at pH 5 and 7.4 and urea- and acid-functionalized mixed micelles (noncovalent interaction). Inset: Release profile for extended period of time. (b) Cytotoxicity of DOX-loaded catechol micelles and catechol/imidazole mixed micelles on HepG2 cells.

of the wealth of chemical literature on aza-Michael additions where mild Lewis bases are used as organocatalysts,29−33 it is unsurprising that the imidazole-functionalized polymer was able to boost DOX conjugation to the autoxidized catechols. In particular, the use of N-methylimidazole as an organocatalyst in aza-Michael additions to α,β-unsaturated ketones mirrors the chemistry in the present system. The calculated maximum capacity of DOX for the mixed micelles is 26 wt %, and a loading level of 19.9 wt % is ∼77% of the possible capacity. To further confirm the catalysis-assisted DOX loading, Nmethylimdazole was used as the catalyst during the preparation of DOX-loaded catechol micelles. The small molecular Nmethylimidazole was easily removed during the dialysis process in micelles preparation. DOX loading was increased from 20.6% to 31.5% in the presence of the catalyst (Table 1). This loading level is ∼75% of the theoretical capacity and consistent with the mixed micelle loading data. Clearly, the use of an organic catalysis has a pronounced effect on the loading capacity and efficiency. However, higher efficiencies were not observed likely due to either steric congestion or incomplete auto-oxidation.

For comparison, we also synthesized a separate pair of analogous block copolymers featuring phenol and pyridine side chains instead of catechol and imidazole. In subsequent studies using identical drug loading conditions as before, all four possible phenol/N-heterocycle combinations were examined. In addition, each individual polymer was also employed on its own as a control. The DOX loading levels obtained with each of these polymeric systems are given below in Table 1. Summarizing the results, it was found that any system containing phenol-functionalized polymer, whether used alone or in combination with an imidazole/pyridine polymer, was ineffective at loading DOX. This was expected since the oxidation potential of phenol is much higher than catechol, resulting in no quinone being available for Michael addition of the drug. A small amount of DOX was nonetheless loaded (i.e., 0.7−3.4%), likely due to encapsulation via weak hydrophobic interactions. As predicted, the controls containing only Nheterocycle-functionalized polymers were also unable to load any appreciable quantity of the drug, since only weak intermolecular binding forces are possible. In contrast, significant DOX loading levels were attained in all three D

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Figure 3. Biodistribution of catechol and catechol/imidazole mixed micelles. (a) Whole-body imaging of BLBA/c mice bearing 4T1 tumor after injection of free DiR, DiR-loaded catechol micelles and catechol/imidazole mixed micelles at various time points post i.v. injection (Arrows indicating the position of the tumor); (b) Imaging of tumor and various organs of mice treated with free DiR, DiR-loaded catechol micelles or catechol/ imidazole mixed micelles at 7 days post i.v. injection; (c) Relative tumor volume of 4T1 tumor-bearing mice as a function of time. Treatments: saline as control, free DOX (5 mg/kg), DOX-loaded catechol/imidazole mixed micelles (5 mg/kg), and blank catechol/imidazole mixed micelles (equivalent amount as DOX-loaded catechol/imidazole mixed micelles). 1, p < 0.01 vs saline or blank catechol/imidazole mixed micelles; 2, p < 0.05 vs free DOX.

catechol-containing systems. When used in conjunction with catechol polymer, pyridyl side chains were much less effective at promoting drug loading than imidazoles. This may be explained by the lower basicity of pyridines relative to imidazoles, which is an important factor in driving the aza-Michael addition forward. Thus, the optimal platform in terms of DOX conjugation was the catechol/imidazole mixed micelle system, with loading levels of 40% by weight with respect to the catechol polymer, nanosize and narrow size distribution (Table 1). Catechol/ imidazole mixed micelle system without and with DOX was observed under transmission electron microscopy (TEM) and spherical micelles were seen (Figure S1). 2.3. pH-Sensitive in Vitro Drug Release and Cell Cytotoxicity. The drug release kinetics of the DOX-loaded catechol/imidazole mixed micelles were then investigated and compared against our previously reported urea-carboxylate system (Figure 2a).34,35 In the latter platform, where the drug is encapsulated by weak noncovalent interactions, over 50% of the DOX was released in just half a day. With the catechol/ imidazole platform, however, there was negligible drug release during the first 8 h (∼1%) and only about 7% of the drug was released after 4 days. This is consistent with the drug molecules being covalently bound to the polymer side chains. The small amount of drug released may have arisen from noncovalently encapsulated DOX molecules and/or via imine hydrolysis under the test conditions. Because of the acid sensitivity of imines, a much higher percentage of the drug was released by hydrolysis at low pH (Figure 2a). This would enable the selective and pH-triggered release of DOX within the acidic environments of endosomes in cells. To investigate this, we conducted in vitro studies in which HepG2 liver carcinoma and 4T1 mouse mammary carcinoma cells were incubated with DOX-loaded catechol-containing polymers for 48 h. Two systems, the catechol/imidazole mixed micelles and pure catechol polymer, were evaluated against a control featuring

free DOX and no added polymer. Cell viability was then monitored as a function of polymer concentration, the results of which are shown in Figures 2b and S2. The experiment indicates that the drug-loaded polymer nanoparticles were almost as effective as free DOX at inducing cell death and the cell death was only due to the release of DOX from the different formulations. No cell death was induced by the polymers as seen in Figures S3−S5. Taken together, the above studies show that while DOX release from the loaded micelles was negligible under the simulated extracellular condition (pH 7.4), their cytotoxic effects nevertheless rivaled that of free DOX when they were incubated with cells. Hence, if the micelles could accumulate in solid tumors via the EPR effect, then selective eradication of tumor cells should be possible. In order to demonstrate this, the relevant animal studies were subsequently carried out. 2.4. Biodistribution of Micelles in Tumor-Bearing Mice. The tumor targeting effects of the catechol micelles and the catechol/imidazole mixed micelles were investigated on BALB/c mice bearing subcutaneous 4T1 mouse breast tumors using a near-infrared fluorescent (NIRF) dye, DiR-loaded micelles via iv injection. The 4T1 tumor-bearing mice were injected with free DiR at an equivalent dose as that in micelles were used as control. Whole body images showed that the DiR signal was seen in the tumor at 5 h post injection of both the catechol micelles and catechol/imidazole mixed micelles, suggesting accumulation of the micelles in the tumor (Figure 3a). At 24 h, the signal of DiR in the tumor was much stronger than that in the other organs of mice treated with either catechol micelles or catechol/imidazole mixed micelles. This phenomenon became more obvious at 4 and 7 days post injection. In contrast, the mice treated with free DiR showed nonspecific fluorescence distribution, and no signal or very weak signal of fluorescence was observed in the tumor at all time points. Compared to the mice treated with the micelles, E

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Figure 4. Histological analysis of 4T1 tumors and hearts at the end of antitumor efficacy study for TUNEL-positive apoptotic bodies from a representative mouse in each treatment group. Tumor or heart sections: saline (a, d), 5 mg/kg free DOX (b, e), 5 mg/kg DOX-loaded catechol/ imidazole mixed micelle (c, f). Quantitation of mean apoptotic bodies per field (×400) in tumor (g) and heart (h) sections for the ten highest densities of apoptotic bodies. All scale bars are at 100 μm.

the intensity of the fluorescence signal in the control group was weaker at all time points, implying that the free dye was quickly eliminated from the body. At 7 days post injection, the mice were sacrificed and the organs were collected and photographed (Figure 3b). Comparing to the control group treated with free DiR, the intensity of DiR signal in tumors was much stronger in the groups treated with the catechol micelles or catechol/imidazole mixed micelles. In addition, no signal of fluorescence was observed in the heart, suggesting that the cardiotoxicity of DOX might be eliminated by using the micelles as a delivery carrier. Moreover, the signal of DiR dye was stronger in the tumor than other organs, indicating that the catechol micelles or catechol/imidazole mixed micelles preferably accumulated in the tumor. These findings demonstrated that both the catechol micelles and catechol/imidazole mixed micelles effectively targeted the tumor via the EPR effect and are expected to achieve higher antitumor activity than free drug. 2.5. Antitumor Efficacy of Micelles in Tumor-Bearing Mice. The antitumor efficacy of the DOX-loaded micelles was studied on BLBA/c mice bearing 4T1 mouse breast cancer using DOX-loaded catechol/imidazole mixed micelles by measuring the tumor volume as a function of time. The mice treated with saline and blank catechol/imidazole micelles showed the highest tumor growth rates among all the groups, indicating the polymers have no effect on suppressing tumor growth, and the inhibition effects on tumor were due to DOX (Figure 3c). As compared to free DOX, the DOX-loaded catechol/imidazole mixed micelles showed significantly higher antitumor efficacy (p = 0.01). This was due to the preferable accumulation of micelles in the tumor via the EPR effect. The body weight of the mice was also monitored to evaluate the general toxicity of free DOX and DOX-loaded mixed micelles. The body weight loss in the mice treated with free DOX was

significantly higher than that in the mice treated with the DOXloaded micelles, saline or blank micelles, implying that the mixed micelles were capable of reducing the toxicity of free DOX (Figure S6). The histological analysis of tumor and heart sections of mice treated with saline, free DOX and DOX-loaded catechol/ imidazole mixed micelle was TUNEL-stained for apoptotic bodies as shown in Figure 4. Through TUNEL staining, the brown apoptotic bodies (as shown by white arrows in Figure 4) can be visualized. The number of apoptotic bodies in the tumors of untreated control mice (injected with saline) was low (Figure 4, parts a and g, 3 ± 1 per field) and there was no significant difference between the control group and the free DOX-treated group (Figure 4, parts b and g, 4 ± 2 per field). This is in agreement with the observation that no therapeutic effect of saline and free DOX treatments (Figure 3c). In contrast, a significantly higher number of apoptotic bodies was seen with the treatment of DOX-loaded catechol/imidazole mixed micelles (Figure 4, parts c and g, 10 ± 2 per field) due to the preferable accumulation of micelles at the tumor (Figure 3b). This is in agreement to their higher antitumor efficacy (Figure 3c). These results indicate that the anticancer mechanism of the DOX-loaded catechol/imidazole mixed micelles is based on DOX-induced apoptosis. DOX treatment usually leads to cardiotoxicity in clinical use.36 Few apoptotic bodies were seen in the heart tissues of mice treated with saline and DOX-loaded catechol/imidazole mixed micelles (Figure 4, parts d and f), while a high number of apoptotic bodies were found in the heart tissues of mice treated with free DOX (Figure 4e). Quantitation analysis of the apoptotic bodies showed that the mean average number of apoptotic bodies for saline, free DOX and DOX-loaded catechol/imidazole mixed micelles treatments was 1 ± 1, 10 ± 3, and 1 ± 1 per field. This indicates that the precision F

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CH2OCOO), 3.50 (s, 455H, H of PEG), 3.33 (s, 2H, C(O)NHCH2), 3.00 (s, 2H, CH2Imidazole), 1.04 (m, 3H, CH3). PEG-b-Poly(MTC-pyridine), 1H NMR (DMSO-d6, 400 MHz, 22 °C): δ 8.45 (s, 1H, H of pyridine), 7.96 (m, 1H, H of Pyridine), 7.63 (m, 1H, H of pyridine), 8.15 (m, 2H, H of pyridine and C(O)NH), 4.14 (s, 4H, CH2OCOO), 3.51 (s, 455H, H of PEG), 3.32 (s, 2H, C(O)NHCH2), 2.80 (m, 2H, CH2-pyridine), 1.07 (m, 3H, CH3). PEG-b-Poly(MTC-phenol), 1H NMR (DMSO-d6, 400 MHz, 22 °C): δ 9.17 (s, 1H, PhOH), 7.93 (s, 1H, C(O)NH), 6.90 (s, 2H, PhH), 6.69 (s, 2H, PhH), 4.18 (s, 4H, CH2OCOO), 3.51 (s, 455H, H of PEG), 3.22 (s, 2H, C(O)NHCH2), 2.51 (s, 2H, CH2Ph), 1.04 (s, 3H, CH3). PEG-b-Poly(MTC-catechol), 1H NMR (DMSO-d6, 400 MHz, 22 °C) (Figure S8): δ 8.71 (d, 2H, PhOH), 7.90 (s, 1H, C(O)NH), 6.52 (m, 3H, PhH), 4.22 (s, 4H, CH2OCOO), 3.50 (s, 455H, H of PEG), 3.18 (s, 2H, C(O)NHCH2), 2.48 (s, 2H, CH2Ph), 1.07 (s, 3H, CH3). 4.5. Formation of 2-Hydroxy-p-quinoneimine Chromophore. Equal molar ratio of PEG-imidazole and PEG-catechol mixture was dissolved in methanol and measured using the UV−vis spectrophotometer (Agilent 8453 UV−visible system) at peak wavelength of 431 nm. This would be considered as time = 0. 2Amino-1-butanol was added to the solution and monitored at a regular time interval of 30 min. 4.6. Preparation of DOX-Loaded Micelles and Measurement of DOX Loading. DOX encapsulation into various polymeric micelles was carried out through a membrane dialysis method. Briefly, 5 mg of DOX was dissolved in 1.5 mL of N,N-dimethylacetamide (DMAc) and neutralized with 3 mol excess of triethylamine (TEA). After the neutralization, DOX solution was mixed with polymer solution (10 mg in 0.5 mL DMAc) by vortexing. For mixed micelles, a molar ratio (1:1) of the different functional groups was used. Subsequently, the mixed solution was placed into a dialysis bag (MWCO of 1000 Da, Spectra/ Por 7, Spectrum Laboratories Inc.) and dialyzed against 1 L of DI water. The water was changed at 8, 24, and 32 h. After 48 h, the solution in the dialysis bag was collected and lyophilized. Each experiment was carried out in triplicates. DOX-loaded catechol micelles using N-methylimidazole as a catalyst were prepared using the same method as DOX-loaded catechol micelles. A mixture of Nmethylimidazole and catechol-functionalized polymer (1:1 molar ratio) and DOX were dissolved in DMAc, and incubated at room temperature (∼22 °C) for 30 min before performing dialysis. To determine the loading level of DOX in the micelles, a known amount of lyophilized DOX-loaded micelles was weighed and dissolved in 1 mL of dimethyl sulfoxide (DMSO). Absorbance of the solution was measured using a UV−vis spectrophotometer at 480 nm. DOX concentration was calculated based on a standard calibration curve obtained from free DOX in DMSO (1−100 mg/L), and DOX loading level was obtained using the following formula:

delivery of DOX using the catechol/imidazole mixed micelles mitigates cardiotoxicity.

3. CONCLUSION In conclusion, we have developed a polymeric drug delivery vehicle for anticancer therapy based on a bioinspired molecular design and a biomimetic DOX-conjugation mechanism. This system offers an ideal combination of biodegradability, biocompatibility, high drug-loading capacity, nanosize, passive tumor-targeting ability, and pH-triggered drug release in tumor cells, enhances in vivo anticancer activity and mitigates nonspecific toxicity of anticancer drugs. To our knowledge, this is the first example of an organocatalytic drug−polymer conjugation that provides a highly efficient drug loading capacity (75%) and mitigates initial burst release of drug. This strategy should be applicable to a number of drug substrates as many drugs contain amine functional groups. Coupled with the synthetic accessibility of the constituent polymers and the ease of nanoparticle self-assembly, this drug delivery nanoplatform holds great promise for cancer chemotherapy while mitigating the usual dose-limiting toxicity issues. 4. EXPERIMENTAL SECTION 4.1. Materials. All chemicals were purchased from Sigma-Aldrich and used as received unless mentioned otherwise. Phosphate-buffered saline (PBS) was purchased from first Base (Singapore) and diluted to the intended concentrations before use. 4.2. Synthesis of MTC−OC6F5. A 100 mL round-bottom flask was charged with 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) (3.00 g, 22 mmol), bis(pentafluorophenyl)carbonate (PFC) (21.70 g, 55 mmol), CsF (0.70 g, 4.6 mmol), and 70 mL of anhydrous THF. Initially the reaction was heterogeneous, but after 1 h, a clear homogeneous solution was formed that was allowed to stir for 20 h. The solvent was removed in vacuo. The residue was redissolved in methylene chloride, and after 10 min, a byproduct was precipitated, and filtered out. The filtrate was extracted with sodium bicarbonate and water, and dried with MgSO4. The solvent was evaporated in vacuo and the product was recrystallized from ethyl acetate/hexane mixture to give MTC−OC6F5 as a white crystalline powder. Yield: 5.50 g (75%). 1H NMR (400 MHz, CDCl3, 22 °C): δ 4.85 (d, 2H, CH2OCOO), 4.36 (d, 2H, CH2OCOO), 1.55 (s, 3H, −CH3). 4.3. Synthesis of PEG-b-Poly(MTC−OC6F5). In a nitrogenpurged glovebox, a small vial was charged with PEG5k−OH (4.0 g, 0.80 mmol), MTC−OC6F5 (3.3922 g, 10.4 mmol), and 13.83 g dichloromethane (1 M with respect to MTC−OC6F5). The solution was stirred until the PEG5k−OH was fully dissolved. The MTC− OC6F5 only partially dissolves at this concentration. Triflic acid (0.12 g, 0.8 mmol) was added to the stirring solution. As the reaction proceeded, the undissolved MTC−OC6F5 slowly went into solution. The reaction was monitored by 1H NMR. Once the reaction was complete, the polymer was precipitated into hexanes. The crude polymer was then redissolved and precipitated into diethyl ether, isolated, and dried to obtain a white solid (yield: 0.616 g, 62.1%). 1H NMR (CDCl3, 400 MHz, 22 °C): δ 4.48 (s, 4H, CH2OCOO), 3.65 (s, 455H, H of PEG), 1.51 (s, 3H, CH3). GPC (RI): Mn (PDI) = 10.1 kDa (1.10), DP MTC−OC6F5 by NMR 11.2. 4.4. General Procedure for Postpolymerization Modification. PEG-b-Poly(MTC−OC6F5) (0.60 g, 0.77 mmol/C6F5OH) and tyramine (0.127 g, 0.93 mmol) were dissolved in 2 mL of DMF. A solution containing triethylamine (0.094 g, 0.93 mmol) in 0.5 mL of DMF was added. The reaction mixture was left to react for 4 h. The reaction solution was then precipitated into diethyl ether. The crude product was further purified by dialysis in water. PEG-b-Poly(MTC-imidazole), 1H NMR (DMSO-d6, 400 MHz, 22 °C) (Figure S7): δ 7.56 (s, 1H, H of imidazole), 7.13 (m, 2H, H of imidazole and C(O)NH), 6.86 (s, 1H, H of imidazole), 4.16 (s, 4H,

DOX loading (wt %) −

mass of DOX loaded in micelles × 100% mass of DOX − loaded micelles

4.7. Particle Size Analysis. Dynamic light scattering (DLS) was performed using Zetasizer 3000 HAS (Malvern Instrument Ltd., Malvern, U.K.) to obtain particle sizes and polydispersity index (PDI) for all the samples. The equipment is equipped with a He−Ne laser beam at 658 nm. Each sample was measured 5 times, and an average particle size and PDI were obtained. 4.8. In Vitro Release Studies for DOX-Loaded Micelles. Lyophilized DOX-loaded micelles were dissolved in 150 mM PBS (pH 7.4) or acetate buffer (pH 5) at a concentration of 1 mg/mL and placed into a dialysis bag (MWCO of 1000 Da). The bag was submerged into a bottle containing 30 mL of PBS (pH 7.4) or acetate buffer (pH 5) respectively inside a water bath (Polyscience) maintained at 37 °C while being shaken at 100 rev/min. At regular interval of every 1 h, 1 mL of the release media was collected and replaced with 1 mL of fresh buffer. DOX concentration was measured using the UV−vis spectrophotometer at 480 nm using the calibration curve of DOX in PBS or acetate buffer. The release of DOX was normalized with the initial DOX loading content at a particular time point and all experiments were performed in triplicates. G

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Macromolecules 4.9. In Vitro Cytotoxicity. Cytotoxicity of free DOX, blank micelles and DOX-loaded micelles was investigated by MTT assay (3(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide). HepG2 (liver carcinoma), HEK 293 (human embryonic kidney), and 4T1 (mouse mammary carcinoma) cell lines were purchased from ATCC and cultured according to ATCC’s recommendation. HepG2 and HEK 293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and 4T1 in Roswell Park Memorial Institute 1640 medium (RPMI 1640) at 37 °C and 1% CO2. All media were supplemented with 10% fetal bovine serum (FBS) and 5% penicillin-streptomycin. The cell suspension (100 μL) was seeded in 96-well cell culture plates at a density of 10000 cells/well overnight before treatment was performed. Free DOX, blank micelles, and DOX-loaded micelles were dissolved in the corresponding cell culture medium at various concentrations. The prepared solution (100 μL) was used to replace the medium in each well and the plates were returned to the incubator and maintained at 37 °C and 5% CO2 for 48 h. Fresh growth media (100 μL) and 20 μL MTT stock solution (5 mg/mL in PBS) were used to replace the medium in each well and incubated for 4 h. To dissolve the purple formazan formed by viable cells crystals, 150 μL of DMSO was added to each well after all the MTT solution had been removed. Each sample was tested in eight replicates and the absorbance of the formazan crystals was measured at 550 nm deducted by that at 690 nm using a microplate reader (TECAN, Switzerland). Cell viability was expressed as a percentage of absorbance of the nontreated control sample. 4.10. Biodistribution of DiR-Loaded Micelles. The real-time biodistribution and tumor targeting effect of catechol micelles and catechol/imidazole micelles was studied through noninvasive bioimaging. A near-infrared fluorophore 1,1′-dioctadecyltetramethyl indotricarbocyanine iodide (DiR) was loaded into the micelles through a sonication/dialysis method. Briefly, polymer (10 mg) and DiR (0.3 mg) were dissolved in 2 mL of DMSO. The mixture was pipetted dropwise into 10 mL of DI water with sonication using a 130 W probe sonicator (Vibra Cell VCX 130) for 10 min. The resulting solution was dialyzed against 1 L of DI water using a MWCO 1000 Da membrane. Water was changed at 3, 6, and 24 h, and sample was collected and lyophilized after 48 h. The loading level of DiR was determined using the same method as DOX. The absorbance for DiR was measured at 759 nm. DiR loading level in the micelles was ∼0.2 wt %. The animal protocols were approved by Institutional Animal Care and Use Committee (IACUC), Biological Resource Center, Agency for Science, Technology and Research (A*STAR), Singapore. 4T1 cells suspended in PBS (0.5 × 106 in 200 μL) were injected subcutaneously into each female BALB/C mice (6 weeks of age, 20− 22 g). After 2 weeks post injection, when the tumor reached 200 mm3 in volume, DiR-loaded catechol micelles and DiR-loaded catechol/ imidazole mixed micelles (8 mg/kg, 0.32 μg DiR per mouse) were administrated via i. v. tail vein injection, and free DiR (0.32 μg) was used as control. Whole-body fluorescence images were taken using Xenogen IVIS 100 (Caliper Life Sciences, U.S.A.) with the ICG filter (excitation at 710−760 nm, emission at 810−875 nm) at time points of 30 min, 5 h, 24 h, 48 h, 4 days and 7 days post injection. Anesthetized animals (n = 3 for each micelle formulation and n = 2 for free DiR) were placed in one lateral position on the heated plate (37 °C) for imaging. The exposure time was set to 3 s. The tumors and major organs (heart, liver, spleen, lung and kidney) were removed from sacrificed mice at 7 days post administration and subsequently imaged. 4.11. In Vivo Therapeutic Efficacy and Histological Analysis. The antitumor efficacy of DOX-loaded catechol/imidazole mixed micelles was evaluated on BLBA/c mice bearing 4T1 tumors. The tumor model was established as described in the above section. On the 14 days post tumor inoculation (recorded as day 0), the mice were randomly divided into 4 groups (n = 7 for each group): group 1 for saline, group 2 for catechol/imidazole mixed micelles, group 3 for free DOX, and group 4 for DOX-loaded catechol/imidazole mixed micelles. The dose of DOX given was 5 mg/kg. The formulations were given to mice via iv tail vein injection at day 0, 4, 7, and 11. The tumor size and body weight of the mice were recorded regularly to

assess tumor inhibition activity and overall toxicity of each formulation, respectively. The tumor dimensions were measured with a Vernier caliper, and tumor volume was calculated using the following formula: Tumor volume = length × width2/2. At day 21, the mice were sacrificed, and the tumor and heart were excised, fixed in 4% formalin solution followed by paraffin embedding and stained with terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL). Apoptotic bodies in the tumor and heart sections were quantified by counting the number of TUNEL-positive nuclei in ten fields of x400 magnification with the highest number of apoptotic bodies and obtaining the mean of the ten fields for each sample. Embedding and staining were done by the Histopathology Unit of Biopolis Shared Facilities, Singapore. Images and counting of apoptotic bodies were performed using a Nikon AZ 100 microscope.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.5b02784. Experimental details of TEM, in vitro cytotoxicity, in vivo biodistribution, antitumor efficacy, and histological studies, TEM micrographs, 1H NMR spectra, cytotoxicity data of blank micelles/polymers in 4T1, HepG2, and HEK 293 cell lines, cytotoxicity data of free DOX and DOX-loaded mixed micelles in 4T1 cell line, and mouse body weight changes during the treatment (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(J.L.H.) E-mail: [email protected]. *(Y.Y.Y.) E-mail:[email protected]. Notes

The authors declare no competing financial interests.

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ACKNOWLEDGMENTS This work was supported by IBM Almaden Research Center, USA, and the Institute of Bioengineering and Nanotechnology (Biomedical Research Council and Joint Council Office, Agency for Science, Technology and Research, Singapore).

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DOI: 10.1021/acs.macromol.5b02784 Macromolecules XXXX, XXX, XXX−XXX