ATP-Driven Temporal Control over Structure Switching of Polymeric

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ATP-Driven Temporal Control over Structure Switching of Polymeric Micelles Bingyang Dong, Li Liu, and Cong Hu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00769 • Publication Date (Web): 01 Aug 2018 Downloaded from http://pubs.acs.org on August 2, 2018

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ATP-Driven Temporal Control over Structure Switching of Polymeric Micelles Bingyang Dong, Li Liu,* Cong Hu Key Laboratory of Functional Polymer Materials, Ministry of Education, Institute of Polymer Chemistry, College of Chemistry, Nankai University, Tianjin 300071, P. R. China.

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ABSTRACT

An adenosine triphosphate (ATP)-fueled micellar system in the out-of-equilibrium state was constructed based on 4,5-diamino-1,3,5-triazine (DAT)-containing block copolymer. The block copolymer self-assembled into spherical micelles in equilibrium steady state at pH higher than its pKa. The pendant DAT residues in protonated form acted as ATP catchers via hydrogen-bonding and electrostatic interactions in a synergistic manner. Activated by ATP fuel, the polymeric micelles spontaneously disrupted into small aggregates of ATP/polymer hybrid complex. The consumption of ATP energy via the enzymatic hydrolysis led to dissociation of the complex and reversible formation of polymeric micelles. A transient self-assembly cycle, in which the assembly underwent autonomous division-fusion motion, was created using ATP fuel and enzyme; the switching of assembly structure was sustained by continuous supply of ATP fuel. This DAT-containing block copolymer have good biocompatibility, and drug-loaded micelles displayed ATP-responsive release behavior. It is expected that this ATP-fueled supramolecular assembly system will provide a functional platform for biomimic chemistry and therapeutic applications.


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INTRODUCTION Living systems extensively exploit the dissipative self-assembly (DSA) under nonequilibrium state to accomplish spatio-temporal regulation of various important functions such as cell division, motility and signal transduction.1-4 The dissipative self-assembly is an energetically uphill process that requires a continuous supply of energy.5,6 For example, cytoskeleton proteins self-assemble into microtubules and actin filaments that perform cell division and motility by exhibiting a transient change in length of the filament/tubule fueled by guanosine triphosphate (GTP) and adenosine triphosphate (ATP), respectively.7,8 A class of integral membrane proteins, known as ATP-binding cassette (ABC) transporters, undergo nonequilibrium conformational changes to perform active transport in cells by utilizing ATP binding and hydrolysis energy.9 The temporal control over structure transformations obtained by these natural systems is a consequence of their fuel-driven functioning. In contrast, the self-assembly of the synthetic materials is an energetically downhill process driven by thermodynamics to reach a global or a local minimum in the free energy. There is a strong drive to develop intelligent materials and devices with life-like properties, such as autoconfiguration, self-healing and adaptability, based on stimuli provided in the form of energy. It is important to add time as an extra dimension for the design of artificial systems with spatiotemporal control over their structures and functions. Recently reported studies on the supramolecular systems that require energy to make self-assembly, shed light on the fabrication of transient nanostructures.10,11,12 Various forms of energies have been utilized to realize dissipative self-assembly, such as light,13 ultrasound,14 chemical oscillations15 and electric fields.16 Inspired by the autonomous oscillatory phenomena in living systems, different selfoscillating polymeric systems have been developed by coupling with chemical oscillating


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reactions, including gels,17 polymer brushes,18 colloidsomes,19 and block copolymers that are able to undergo cyclical change between unimer/micelle or unimer/vesicle structures.20-23 These materials exhibit a spontaneous and periodical response under constant conditions without “ON/OFF” switching through external stimuli. The natural scenario has been the inspiration for researchers to construct supramolecular assemblies mimicking biological structures and functions. Known as energy currency of the cell,24 ATP plays a crucial role in cell physiology as part of signal transduction cascades involving phosphorylation of proteins. The cleavage of the ‘‘high-energy (phosphate–phosphate) bond’’ of ATP (or GTP) is utilized to fuel dissipative processes in the biological system. ATP contains triphosphate ion, nucleotide group and 1,2-cis-diols, which can provide effective recognition sites for building supramolecular assemblies applicable in drug delivery and molecular glues.25-27 ATP can also be employed as a chemical fuel to drive dissipative supramolecular assembly of synthetic molecules. Prins and co-workers displayed transient signal generation of a self-assembled nanosystem fueled by ATP,28,29 and fabricated vesicular nanoreactors showing temporal control over the reaction kinetics through regulating the concentration of ATP.30 George and co-workers developed adenosine-phosphate-driven chiral supramolecular polymerization of zinc-receptors-functionalized naphthalenediimide (NDI) chromophores, and temporally programmed the polymers with multiple transient helical conformations by various combinations of fuels and enzymes.31-33 More recently, they reported an ATP-selective and ATP-fuelled controlled supramolecular polymerization of a π-conjugated monomer which could undergo time-dependent nucleation-growth and seeded growth on interaction with ATP, and achieved a transient self-assembly in the presence of ATP-hydrolysing enzyme.34 Using a symmetric peptide derivative of 3,4,9,10-perylenediimide as a building block,


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Hermans and co-workers35 investigated ATP-fueled supramolecular polymers in sustained nonequilibrium steady states by a continuous influx of fuel and outflux of waste inside a membrane reactor, and demonstrated the transient change in the polymer structure and chirality. Based on an amphiphilic block copolymer appended with biguanidine-cyclodextrin units as specific catchers for ATP,36 Yan and coworkers37 reported a pulsatile micellar system undergoing autonomous expansion-contraction cycles through continuous supply of ATP fuel. As an energy provider of living cells, exploiting ATP as a source of fuel represents a promising strategy to achieve the active synthetic assemblies for biological motion mimics. In this paper, we describe a transient micellar system in which the polymeric micelles can undergo reciprocating division-fusion motion driven by biofuel, ATP. To realize the capture of ATP, 4,6-diamino-1,3,5-triazine (DAT) moieties were anchored to the side chains of a block copolymer. DAT can function like melamine in molecular recognition via multiple hydrogen bonds, coordination bonds and π-π stacking.38 Melamine in its protonated form can self-assemble into hydrogels triggered by oxoanions, such as PO43-, NO3-, SO42- and ATP, due to the cooperative effects of hydrogen bonding, electrostatic and π-π stacking interactions.39 We envision that the obtained DAT-containing block polymer, PDAT, is able to recognize ATP and construct ATP-fueled polymeric assemblies. As shown in Scheme 1, we established a transient self-assembly cycle based on competition of binding (activation) and enzymatic dissociation (deactivation) between ATP and PDAT micelles. In a cycle, PDAT micelles spontaneously divided into small aggregates of ATP/PDAT hybrid complex by capturing ATP, and the enzymatic consumption of ATP induced the fusion of these aggregates to reversibly form the PDAT micelles. The cyclical switch of assembly structure was sustained by continuous input of


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ATP fuel. It is expected that this ATP-fueled self-assembly system will provide a multifunctional platform for mimicking activities of biological system and therapeutic applications.

Scheme 1. Illustration of ATP-driven transient change in structure of a micellar system based on the DAT-containing block copolymer. EXPERIMENTAL SECTION Materials. Poly(ethylene glycol) methyl ether methacrylate (PEGMA, Mn = 500 g/mol, Sigma-Aldrich, 99%) was purified through a basic aluminum oxide column before use. Nhydroxysuccinimide acrylate (NHSA, Energy Chemical, 98%), 6-chloro-1,3,5-triazine-2,4diamine (Energy Chemical, 98%), adenosine-5’-triphosphate (ATP, HEOWNS, 98%), adenosine-5’-diphosphate (ADP, HEOWNS, 98%) and adenosine-5’-monophosphate (AMP, Energy Chemical, 98%) were used as received. 4,4′-Azobis (4-cyanopentanoic acid) (ACPA, Acros, 97%) was dried under vacuum before use. All other reagents were commercial chemicals and used directly. Aminoethylmelamine was synthesized by the reaction of ethylenediamine and


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6-chloro-1,3,5-triazine-2,4-diamine.40 4-Cyanopentanoic acid dithiobenzoate (CPADB) was synthesized according to the previous paper.41 All solvents were distilled before use. Characterizations. 1H NMR and

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P NMR measurements were performed on a Varian

UNITY-plus 400 M nuclear magnetic resonance spectrometer using CDCl3, DMSO-d6 or D2O as the solvent. The number-average molecular weights (Mn), weight-average molecular weights (Mw), and polydispersities (Mw/Mn) of the polymers were determined by gel permeation chromatography (GPC) at 40 °C with a Hitachi L-2130 HPLC pump, three Varian PL columns with 1000–100 K (100, 000Å), 100-10 K(10, 000 Å), and 100-10 K (1000 Å) molecular ranges, and a Hitachi L-2490 refractive index detector. N, N-Dimethylformamide (DMF) with 0.01 M LiBr was used as the mobile phase at a flow rate of 1.0 mL min−1. The apparent molecular weights of the polymers were calibrated on poly(methyl methacrylate) (PMMA) standards. Dynamic light scattering (DLS) measurements and ξ-potentials of the micelles were conducted on a Malvern Zetasizer Nano-S90 and a Zetasizer Nano-ZS respectively. Malvern Instruments equipped with a 10 mW He-Ne laser at a wavelength of 633 nm. UV-vis spectroscopy was performed on a Shimadzu UV-2450 UV-visible spectrophotometer. Transmission electron microscopy (TEM) observations were carried out on a Tecnai G2 20 S-TWIN electron microscope equipped with a Model 794 CCD camera. The aqueous solution of the PDAT micelles was deposited on a carbon-coated copper grid, and water was evaporated in air. To enhance the contrast, the TEM sample was stained under hydrazine hydrate (80%) vapor for 8 h and OsO4 vapor for 2 h. Synthesis of P(PEGMA) homopolymer by RAFT polymerization. P(PEGMA) was synthesized as the following procedure. PEGMA (3.20 g, 6.40 mmol), CPADB (0.089 g, 0.32 mmol) and ACPA (0.0042g, 0.015 mmol) were dissolved in 4 mL of anhydrous DMF in a flask,


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and the solution was degassed by three freeze- vacuum-thaw cycles. The polymerization was carried out at 70 °C for 6 h, and quenched by cooling the flask in ice water and exposing the solution to air. P(PEGMA) hompolymer was precipitated into cold ethyl ether, centrifuged and dried overnight under vacuum at room temperature. Synthesis of P(PEGMA)-b-P(NHSA) diblock copolymer. NHSA (0.44 g, 2.6 mmol) was dissolved in anhydrous DMF (5 mL), followed by addition of P(PEGMA) (0.50 g, 0.044 mmol) and ACPA (1.4 mg, 0.005mmol). After degassed by three freeze-vacuum-thaw cycles, the block copolymerization was carried out at 70 °C for 24 h. The reaction was stopped by cooling in ice water and exposing the solution to air. The solution was concentrated under vacuum, and P(PEGMA)-b-P(NHSA) block copolymer was precipitated into cold ethyl ether, centrifuged and dried overnight under vacuum at room temperature. Synthesis of DAT-containing block copolymer copolymer PDAT. Typically, P(PEGMA)-bP(NHSA) (0.27 g, 0.18 mmol NHS functional group) was dissolved in 2 mL of anhydrous DMSO, aminoethylmelamine (0.26 g, 1.5 mmol) and trimethylamine (0.15 g, 1.5 mmol) were added. After degassed by two freeze-vacuum-thaw cycles, the solution was heated to 50 °C and stirred overnight. The product was precipitated in ice diethyl ether, then dissolved in 5 mL of water at pH 4.3 and dialyzed (MWCO 3500) against deionized H2O (pH=4.3) for 48 hours. The DAT-containing block copolymer PDAT was recovered by lyophilization. Determination of pKa value. The aqueous solution of PDAT (3 mg/mL, 10 mL) was first adjusted with 0.1M NaOH to a pH value of 11, and subsequently titrated with 0.1M HCl at 25°C while being monitored using a pH-meter (Mettler Toledo FE 20). The pKa value corresponded to the midpoint between two pH jumps.


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Measurement of critical micelle concentration (CMC) by fluorescence spectroscopy. Pyrene was used as a fluorescence probe to determine the CMC of the block copolymer PDAT. A predetermined amount of pyrene solution in acetone was added into a series of volumetric flasks, and acetone was then evaporated completely. A series of copolymer solutions at different concentrations were added to the flask, while the concentration of pyrene in each flask was fixed at a constant value (6.0×10-7 mol L-1). The solution was in equilibrium for 30 min at room temperature. The excitation spectra were recorded at 25°C on a Shimadzu RF-5301PC spectrofluorophotometer with λem=390 nm. Isothermal titration calorimetry (ITC) assays. All ITC measurements were carried out on a MicroCal Auto-ITC200 titration calorimeter at 25°C with 307 rpm stirring. An injection volume of 10 µL was introduced into the cell with 20 s duration. In a typical measurement, the aqueous solution of ATP (0.7 mM) in acetate buffer (pH 5.0, 50 mM) was titrated into the reservoir cell filled with the PDAT solution (0.02 mM) in the same buffer. In the case of ADP or AMP, the concentration was fixed at 1.0 mM. In vitro cytotoxicity assays. In vitro cell viabilities of the block polymer PDAT were evaluated on MCF-7 cells Cells were seeded in 96-well plates at a density of 5×103 cells/well, and cultured in 100 µL of Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% FBS, penicillin (100 U mL-1), and streptomycin (100 mg mL-1) for 24 h in a humidified incubator (37 °C, 5% CO2). Then the culture medium was replaced with 100 µL of fresh medium containing the block polymer at 20, 50, 80, 100, 150 and 200 µg/mL, respectively, and the cells were incubated for 24h. Thereafter, the medium was removed, and 10 µL of CCK-8 solution and 90 µL of fresh medium were added into each well. After 2 h, the plate was gently shaken for 2 min to dissolve formazan crystals. The absorbance was measured at 450 nm using a


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multifunctional ELISA plate reader (Thermo Varioskan Flash). All experiments were carried out in triplicate. The cell viability (%) was calculated using the following formula: ஺ೞೌ೘೛೗೐ ି஺್೗ೌ೙ೖ

Cell viability (%) =஺

೎೚೙೟ೝ೚೗ ି஺್೗ೌ೙ೖ

Where Asample and Acontrol represent the absorbance of CCK-8 reagents determined for cells treated with different samples and for control cells (untreated) respectively, and Ablank is the absorbance of CCK-8 reagents without cells. Preparation of DOX-loaded PDAT micelles. Doxorubicin hydrochloride (DOX·HCl) (18 mg) was dissolved in 4 mL DMSO with an excess amount of trimethylamine and the solution was stirred for 30 min. PDAT (166 mg) was added into the solution of DOX and kept stirring for another 2 h. DOX was loaded into micelles by dropwise addition of 20 mL water to this mixture under stirring, followed by dialysis against water for 24 h (MWCO 3.5 kDa). Water was changed five times. To determine the drug loading content, the lyophilized DOX-loaded micelles were dissolved in DMSO and analyzed by UV-vis spectroscopy. The drug loading content (LC, 6.0 %) and encapsulation efficiency (EE, 58%) was determined by UV-Vis absorbance at λ480 nm with a standard calibration curve prepared from a series of DOX solution with DMSO/H2O (1/9, v/v) as solvent. The sample was assayed in triplicate. In vitro release study. DOX-loaded PDAT micelles (10 mg) were dispersed into 2 mL of TrisHCl buffer solution (pH 7.4, 100 mM) without/with ATP. The solution was quickly transferred into a dialysis bag (MWCO 7 kDa), which was immersed in 10 mL Tris-HCl buffer (pH 7.4, 100 mM) or 10 mL Tris-HCl buffer (pH 7.4, 100 mM) containing ATP (4 or 10 mM). The release study was performed under constant shaking at 37 °C. At desired time intervals, 3 mL of release media was taken out and replaced with 3 mL of fresh media. DOX concentration in the release


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media was measured by UV-vis absorbance at λ480 nm. All release assays were carried out in triplicate.

Scheme 2. Outline for the synthesis of DAT-containing diblock copolymer PDAT by postpolymerization modification. RESULTS AND DISCUSSION Synthesis of DAT-containing diblock copolymer. Melamine and its derivatives are highly versatile modules for constructing fascinating nano-micro architectures and developing functional materials via non-covalent interactions.38 The H-bonded sites on melamine can modulate selectivity of the polymer chains, imitating the recognition of biological systems to the guest molecules.42-44 Herein, a melamine-derived polymer was prepared through side-chain functionalization of a block copolymer with aminoethylmelamine. First, poly(poly(ethylene glycol) methyl ether methacrylate) (P(PEGMA)) homopolymer (Mn = 9000 g mol-1, PDI = 1.17, DPn,NMR = 23) was synthesized by RAFT polymerization (Scheme 2). Subsequently, P(PEGMA) was successfully chain-extended with an active ester monomer N-hydroxysuccinimide acrylate (NHSA), yielding the desired block copolymer P(PEGMA)-b-P(NHSA) with narrow


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polydispersity (Mn = 12000 g mol-1, PDI = 1.27), which was revealed by the shift of GPC peak to a higher molecular weight part relative to that of P(PEGMA) (Figure S1). The 1H NMR spectrum of P(PEGMA)-b-P(NHSA) block copolymer is shown in Figure 1. The characteristic signal (peak i) at δ 2.80 ppm corresponds to methylene protons in NHSA units. The numberaverage polymerization degree of NHSA units was calculated to be 33 by the integration ratio of peak i to peak d (ester methylene protons in PEGMA units) at δ 4.02 ppm. The block copolymer had an average composition of P(PEGMA)23-b-P(NHSA)33, calculated based on the 1H NMR results (Figure 1).

Figure 1. 1H NMR spectra of P(PEGMA) homopolymer in CDCl3 (A), P(PEGMA)-b-P(NHSA) block copolymer in d6-DMSO (B) and PDAT in d6-DMSO (C). P(PEGMA)-b-P(NHSA) diblock copolymer was reacted with excess aminoethylmelamine at 50 °C, and the activated ester functionalities converted to the corresponding amides, yielding


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DAT-containing diblock copolymer PDAT. 1H NMR spectrum showed the disappearance of the signal corresponding to NHSA groups at 2.80 ppm and significant increases in the intensities of the characteristic signals of melamine moieties at δ 6.0-8.0 ppm (peak j, k, l). Based on 1H NMR result, the average number of DAT moieties attached to the copolymer was calculated to be 28. Aqueous behaviors of PDAT block copolymer. For a melamine molecule, the protonacceptor sites change to proton-donor sites as the N atoms in the triazine ring are protonated at pH lower than its pKa (pKa 5.10).45 To understand the effect of protonation on the self-assembly behavior of the diblock copolymer PDAT, the pKa value of DAT on the polymer chains was determined by potentiometric titration and was found to be pH 5.4 (Figure S2). As the pH value of the medium is lower than its pKa, the PDAT block copolymer is expected to show positive surface charges due to the protonation of DAT moieties in the polymer side chain. This was studied by Zeta potential measurements. As shown in Figure S2, the ξ value gradually changed from negative charge (-15 mV) to positive (+24 mV) charge as pH changed from pH 8 to 3. The net positive ξ indicates the existence of protonated DAT moieties on the surface at pH lower than pH 5.7. Using pyrene as a fluorescence probe, the CMC values of PDAT in the buffer solutions at pH 6.0 (acetate buffer, 50 mM) and 7.4 (Tris-HCl buffer, 50 mM) were determined by fluorescence spectroscopy. It was reported that the concentration dependence of the I338/I333 ratios of the (0, 0) band of pyrene was very sensitive to CMC.46 The intensity ratio of I338/I333 was plotted against the polymer concentrations, as shown in Figure 2A. The intensity ratio I338/I333 remained almost constant at low concentrations, taking the characteristics of pyrene in a water environment. As the polymer concentration increased, the intensity ratio increased sharply, indicating the formation of polymeric micelles and encapsulation of pyrene in the hydrophobic cores. From the


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sigmoidal shape curves, the CMC values were determined to be 51 mg/L at pH 6.0 and 41 mg/L at pH 7.4, respectively.

Figure 2. (A) Dependence of the intensity ratio I338/I333 from excitation spectra of pyrene on PDAT concentrations at pH 6.0 and 7.4 (λem = 390 nm). (B) DLS curves of PDAT block copolymer in the buffer solutions at pH 7.4 (Tris-HCl buffer, 50 mM), pH 6.0 (acetate buffer, 50 mM) and pH 5.0 (acetate buffer, 50 mM). Driven by thermodynamics, the PDAT block copolymer self-assembles into micelles in the aqueous solution at pH higher than its pKa owing to the amphiphilic property of the block copolymer. The hydrophobic DTA-containing block forms the cores of micelles, and the hydrophilic PEG block forms the shells. DLS analyses show that the average hydrodynamic diameters, , of the micelles prepared at pH 7.4 and 6.0 are both 110 nm (Figure 2B). As pH changes from 6.0 to 5.0, DLS reveals a decrease in from 110 to less than 10 nm, indicating the disassembly of the micelles to unimers due to the protonation of DAT moieties at lower pH. Interaction between PDAT and ATP in aqueous solution. 31P NMR analysis was employed to elucidate the interaction between ATP and PDAT block copolymer.

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P NMR spectrum of


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ATP shows three sharp phosphorus peaks at δ -10.75 (γ-P), -11.30 (α-P), and -22.97 ppm (β-P) in D2O at pH 5.0 (Figure 3A). With an increase in the molar ratio of PDAT to ATP from 1:300 to 1:30, β and γ-P peaks gradually shift to lower field and all the three peaks became broader, indicating the formation of hydrogen-bonding interactions between ATP and DAT moieties.36 Calculated by Henderson-Hasselbalch equation (pH=pKa+log([base]/[acid]), 72% of DAT moieties in PDAT exist in the protonated form at pH 5.0, and the protonated DAT moieties can function as proton-donor in the hydrogen bonding formation with phosphates in ATP. The possible H-bonding between phosphates of ATP and DAT moiety is schematically illustrated in Figure 3B. As shown in Figure 3B, ATP exists in the form of tri-anionic ion at pH 5.0.47 After ATP (1 mM) was added to the solution of PDAT (0.5 mg/mL) at pH 5.0, the Zeta potential switched from a positive value (+6.8 mV) to a negative value (-5.3 mV), confirming the electrostatic interactions of the phosphates with protonated DAT moieties.48 The results from 13P NMR and Zeta potential measurements demonstrate the formation of ATP/PDAT hybrid complex through hydrogen bonding and electrostatic interactions.

Figure 3. (A) 31P NMR spectra of ATP in the presence of various amount of PDAT in D2O (pH 5.0). The ratios represent the molar ratios of PDAT polymer to ATP. (B) Proposed structure of ATP/PDAT hybrid complex.


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Figure 4. ITC results of ATP/PDAT (A-C) at pH 5.0 (A, acetate buffer), pH 6.0 (B, acetate buffer) and pH 7.4 (C, Tris-HCl buffer), ADP/PDAT (D) and AMP/PDAT (E) at pH 5.0 (acetate buffer). All the measurements were carried out at 25 °C. Isothermal titration calorimetry (ITC), one of the most powerful methods in anion-receptor interaction study, was utilized to study the interaction between PDAT block copolymer and ATP. ITC method provides an effective access to quantify biological intermolecular interactions and thermodynamic parameters.49 A stock solution of ATP (0.7 mM) was injected into PDAT buffer solution (0.02 mM) at different pH values. A significantly exothermic signature was clearly observed at pH 5.0 (Figure 4), and the binding affinity (KB) was calculated to be 4.33×104 M-1. As the pH value increased to 6.0 and 7.4, KB decreased to 3.31×103 and 82.4 M-1, respectively, which indicated that the block copolymer exhibited weaker interaction with ATP at pH>pKa due to less DAT moieties were protonated. The interactions of PDAT with various adenosine phosphates, ADP and AMP, were also analyzed by ITC at pH 5.0 (Figure S3). As indicated by a decrease in the binding affinity (KB = 2.72×103 M-1), ADP displayed a little weaker association with PDAT than the binding of ATP with PDAT, due to the lack of a phosphate group. Because AMP has only one phosphate group and cannot form multiple H-bonds with the protonated DAT


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moieties, it cannot even associate with the block copolymer. The ITC results demonstrate the formation of multiple H-bonds between the protonated DTA moieties and the triphosphate group which plays a key role in the formation of ATP/PDAT hybrid complex. ATP-responsive Polymeric Micelles. DLS was employed to demonstrate the effect of ATP on the hydrodynamic diameter of PDAT micelles. Figure 5 shows DLS curves of PDAT micelles before and after addition of ATP at two different pH values. Upon addition of ATP at pH 7.4 or 6.0, the of micelles decreased from 110 to 15 nm, indicating the disruption of PDAT micelles and the formation of ATP/PDAT hybrid complexes (Figure 5). The concentration of ATP required to induce the disruption of the micelles was higher at pH 7.4 than that at pH 6.0, due to the weaker hydrogen-bonding interaction between the block copolymer and ATP at pH 7.4. DLS results displayed that PDAT micelles were also disrupted by ADP at pH 7.4, however, higher concentration of ADP was required. Because of the absence of multiple H-bonds, AMP cannot induce the disruption of PDAT micelles even if the concentration of AMP is 6 times higher than that of ATP.

Figure 5. (A) DLS curves of PDAT block copolymer (0.5 mg/mL) without or with ATP in the buffer solutions at pH 7.4 and pH 6.0. (B) DLS curves of PDAT block copolymer in the presence of ADP and AMP at pH 7.4. All the measurements were carried out at 25 °C.


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It is interesting to find out that the disruption and the re-association of the block copolymer chains into micelles in the presence of ATP are thermal reversible. As shown by DLS results (Figure 6), the PDAT micelles (1 mg/mL) were disrupted upon addition of 4 mM ATP at 25 °C, however, as the temperature was raised to 37 °C, the shifted back to the initial size of the micelles, indicating the formation of the polymeric micelles at this temperature. This result demonstrates that the hydrogen-bonding between ATP and DAT moieties can be easily destroyed by heating, resulting in the dissociation of the ATP/PDAT complexes. This process can be reversibly repeated for several cycles by alternatively decreasing or raising the temperature. This phenomenon is similar to the finding reported by Komiyama’s group.48 In their study on the recognition of nucleotides by poly(vinyldiaminotriazine) through hydrogen bonding in water, it was found that the adsorbed nucleotide by poly(vinyldiaminotriazine) powder was promptly released into water on raising the temperature and the absorption was reversible. However, the complexes formed by the block copolymer chains and ATP at higher ATP concentration (6 mM) were stable at both 25 and 37 °C。

Figure 6. Hydrodynamic size measured by DLS during investigations of the influence of temperature on the formation ATP/PDAT hybrid complexes at pH 7.4.


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Transient self-assembly structure fueled by ATP. In order to switch on a backward motion from ATP/PDAT complexes to the formation of the PDAT micelles, an enzymatic hydrolysis process was introduced to release the chemical energy stored in ATP, leading to destroying the interaction between ATP and pendant DAT moieties of PDAT block copolymer. A natural occurring enzyme, calf intestinal alkaline phosphatase (CIAP), was utilized to cleave the phosphoanhydride bonds of ATP. CIAP can dissociate all three forms of adenosine phosphates to adenosine and phosphates.31 After addition of CIAP (12 U/mL) into the solution of ATP/PDAT complexes at pH 7.4, it was found that the DLS curve shifted back to the same position as the initial micelles (Figure 7A), implying the reversible formation of PDAT micelles after hydrolysis of ATP. In the presence of CIAP, the in-situ hydrolysis of ATP→ADP→AMP→adenosine+Pi resulted in the changes in the concentration and structure of ATP, and thus, the interaction between ATP and DAT residues became weaker and weaker. Therefore, the consumption of ATP fuel by CIAP led to the dissociation of ATP/PDAT complexes, and the released PDAT copolymer chains made self-assembly into micelles. The process is shown in Scheme 1. At pH 6.0, acid phosphatase was added to the solution of ATP/PDAT hybrid complexes, and the same phenomenon was observed (Figure S4). Thus, DLS results confirm that the ATP/PDAT hybrid complexes are highly dynamic and adaptive to the presence of ATP fuel. Hence, we envisage to temporally program the structure of the assembly using ATP fuel and phosphatase. TEM observations also verified the ATP-adaptive feature of the assemblies. The PDAT block copolymer self-assembles into spherical micelles with sizes in the range of 60 to100 nm at pH 7.4 (Figure 7B). Upon introduction of ATP into the micellar solution, TEM image clearly reveals the appearance of small aggregates (20 nm) and disappearance of the large micelles, demonstrating the disruption of the polymeric micelles and the formation of aggregates of


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ATP/PDAT complexes. Upon addition of CIAP, these complex aggregates undergo a spontaneous fusion stage, reverting to the spherical micelles (Figure 7D).

Figure 7. (A) DLS curves of PDAT block copolymer (a), ATP/PDAT hybrid complex before (b) and after CIAP addition (c) in Tris-HCl buffer solution at pH 7.4 and 37°C. (B-D) TEM image of PDAT micelles (B), aggregates of ATP/PDAT hybrid complex (C) and aggregates after adding CIAP to ATP/PDAT hybrid complex (D). A transient self-assembly was realized by using ATP as a chemical fuel. ATP (6 mM) was added to the solution of PDAT micelles (1 mg/mL) and CIAP (32 U/mL) at pH 7.4. A change in the size of the self-assemblies with time was tracked by DLS at 37°C (Figure S6). The large PDAT micelles first disrupted into the small aggregates of transient ATP/PDAT hybrid complexes very quickly ( dropped from 150 to 18 nm) (Figure 8). Subsequently, the small aggregates underwent a spontaneous fusion to the polymer micelles within 8 min (size up from 18 to 150 nm). A second cycle was reinitiated by addition of 0.6 mM ATP, and the process of division-fusion transition completed within 35 min. The reversible process was exhibited for additional 2 cycles by ATP fueling, and only one-tenth of the initial ATP was added for each cycle. In the presence of CIAP, the hydrolytic pathway of ATP is via ATP to ADP to AMP before converting to adenosine and phosphates.31 The data reported by George’s studies33


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showed that it took about 10 h to completely hydrolyze ATP (1 mM) by CIAP (100 U/mL) to adenosine and phosphates. In our experiment, not all the ATP (6 mM) can be hydrolyzed by CIAP (32 U/mL) within 8 min in the first cycle. One of the intermediate products, ADP, also interacts with PDAT. In addition, it was found that the pH value changed to pH 6.6 after the first cycle completed, and kept at pH 6.6 during the subsequent cycles. At pH 6.6, the association between ATP and PDAT is stronger than that at pH 7.4. Therefore, less amount of ATP is required to initiate the subsequent cycles. We also observed that the cycles lasted longer and longer, presumably because of the accumulation of chemical wastes (adenosine and Pi) in the closed system. These two different types of nanoparticles can be identified as the transient selfassembly structures controlled by the competition of ATP binding and enzymatic dissociation, and can periodically undergo the structure transition by continuous supply of ATP fuel.

Figure 8. Hydrodynamic size measured by DLS during investigations of the reversible formation of PDAT micelles and ATP/PDAT hybrid complexes in Tris-HCl buffer solution at 37°C. In vitro cytotoxicity. The biocompatibility of DAT-containing polymer is a key factor for its applications in biomedical areas. The relative cytotoxicity of the block copolymer PDAT was


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investigated in MCF-7 cells by CCK-8 assays. The cells were incubated with the PDAT block copolymer for 24 h. As shown in Figure 9A, the PDAT-treated cells exhibit high viability (>90%) at the tested concentrations up to 200 µg mL-1 of polymer. This result suggests the block copolymer PDAT has a low level of toxicity and is thus a potential candidate for biological applications.

Figure 9. (A) Cytotoxicity of the block copolymer PDAT at different concentrations by CCK-8 assays using MCF-7 cells. Data are presented as the average ± SD (n=3). (B) In vitro release of DOX from DOX-loaded PDAT micelles at pH 7.4 and 37 °C in Tris-HCl buffer (100 mM) solution without ATP (a) and with 4 mM ATP (b) and 10 mM ATP (c). In vitro release of DOX-loaded polymeric micelles. Using doxorubicin (DOX) as a drug model, we investigated the possibility of encapsulating a hydrophobic drug into the PDAT micelles and the release profile. The drug loading content and encapsulation efficiency were calculated to be 6.0 % and 58 %, respectively. The DOX-loaded micelles were dispersed in TrisHCl buffer (100 mM) at pH 7.4 and 37°C with/without ATP to evaluate the effect of ATP on the release of DOX (Figure 9(B)). Without ATP, only 8% of DOX was released from the micelles in 24 h and 11% was released in 60 h. In the presence of 4 mM ATP, about 20% of DOX was


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released in 24 h and 28.5% was released in 60 h, which is ascribed to the loose cores of micelles under this condition. However, drug release was enhanced greatly with 10 mM ATP, wherein about 34.1 and 50% of DOX was released in 24 and 60 h, respectively. Notably, DOX release amount reached to 14 % within the initial 2 h in the presence of 10 mM ATP, while the release of DOX was only 1.3% without ATP. This is because the DOX-loaded PDAT micelles were disrupted to form aggregates of ATP/PDAT complexes in the presence of 10 mM ATP, thus resulting in an abrupt release of DOX during the transition course. As the transition completed, DOX was entrapped into aggregates of ATP/PDAT complexes. The drug release from these aggregates displayed a sustained release in the following time.

CONCLUSIONS In summary, we made a transient self-assembly system using a DAT-containing block copolymer and a biofuel ATP. The protonated DAT moieties function as hydrogen-donors and interact with triphosphates of ATP via multiple hydrogen-bonding and electrostatic interactions. Without ATP, the DAT-containing block copolymer self-assembled into spherical micelles in a thermodynamically steady state at pH higher than its pKa. By binding with ATP fuel, the polymeric micelles disrupted into the aggregates of ATP/polymer hybrid complexes spontaneously. Once the energy stored in ATP was consumed through enzymatic hydrolysis, the aggregates of hybrid complex underwent an automatous fusion to the initial PDAT micelles. Transient self-assembly cycles were established by ATP fuel and phosphatase. The temporal change in the assembly structure was controlled by the ATP binding and hydrolysis energy, and the cyclical transition of polymeric micelles to hybrid complexes driven by the continuous supply of ATP fuel. The good biocompatibility of this DAT-containing polymer make it a


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promising candidate for biological application. Taking advantage of the molecular recognition of DAT-containing polymer via versatile nonconvalent interactions, we expect this study will provide a new strategy to construct active biomimetic assemblies for imitating cellular and organellar activities and applying in therapeutics.

ASSOCIATED CONTENT Supporting Information. GPC curves of polymers, pH titration curve, plot of zeta potential at diffierent pHs, ITC results of ADP/PDAT and AMP/PDAT, DLS results of PDAT block copolymer with acid phosphatase. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Notes The authors declare no competing financial interest. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT This project was financially supported by the National Natural Science Foundation of China under contract No. 21374047.


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ATP-Driven Temporal Control over Structure Switching of Polymeric Micelles Bingyang Dong, Li Liu and Cong Hu


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ATP-Driven Temporal Control over Structure Switching of Polymeric

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