poly(ethylene glycol) supramolecular diblock copolymers based on three-fold complementary hydrogen bonds: Synthesis, micellization, and stimuli responsivity

Accepted Manuscript Poly(lactic acid)/poly(ethylene glycol) supramolecular diblock copolymers based on three-fold complementary hydrogen bonds: synthesis, micellization, and stimuli responsivity Xiaohua Chang, Chenlei Ma, Guorong Shan, Yongzhong Bao, Pengju Pan PII:

S0032-3861(16)30155-0

DOI:

10.1016/j.polymer.2016.03.015

Reference:

JPOL 18507

To appear in:

Polymer

Received Date: 8 January 2016 Revised Date:

1 March 2016

Accepted Date: 4 March 2016

Please cite this article as: Chang X, Ma C, Shan G, Bao Y, Pan P, Poly(lactic acid)/poly(ethylene glycol) supramolecular diblock copolymers based on three-fold complementary hydrogen bonds: synthesis, micellization, and stimuli responsivity, Polymer (2016), doi: 10.1016/j.polymer.2016.03.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphic abstract

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Poly(lactic

acid)/poly(ethylene

glycol)

supramolecular

diblock

copolymers based on three-fold complementary hydrogen bonds:

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synthesis, micellization, and stimuli responsivity

Xiaohua Chang, Chenlei Ma, Guorong Shan, Yongzhong Bao, Pengju Pan*

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State Key Laboratory of Chemical Engineering, College of Chemical and Biological

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Engineering, Zhejiang University, 38 Zheda Road, Hangzhou 310027, China

*Correspondence author. Tel.: +86-571-87951334, email: [email protected]

Abstract: Thymine-terminated poly(lactic acid) (PLA-THY) and diaminotriazine-

supramolecular

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terminated poly(ethylene glycol) (PEG-DAT) are synthesized and they can form the amphiphilic

diblock

copolymers

in

solution

through

the

thymine/diaminotriazine complementary hydrogen bonding interactions. Such

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supramolecular copolymers self-assemble into the stimuli-responsive micelles in aqueous solution. Micelle size, crystalline state, stimuli responsibility, drug loading

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and release behavior of the supramolecular micelles can be readily tuned from the block length, stereostructure, homo and stereocomplex crystallizations of hydrophobic PLA blocks. The enantiomerically-mixed supramolecular copolymers with both poly(L-lactic acid) (PLLA) and poly(D-lactic acid) (PDLA) blocks assemble into the stereocomplexed micelles, while those having the isotactic PLLA (or PDLA) and atactic poly(D,L-lactic acid) (PDLLA) blocks form the homocrystalline and amorphous micelles, respectively. The stereocomplexed and amorphous micelles

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exhibit smaller size than their homocrystalline analogs, all of which are much larger than the micelles formed from the conventional covalently-bonded diblock copolymers. The supramolecular micelles are sensitive to the external stimuli such as

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pH and ion. The homocrystalline and stereocomplexed micelles undergo fast aggregation in the acidic and salted solutions due to the disassociation of

complementary hydrogen bonds. The stereocomplexed micelles exhibit larger drug

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loading content and slower drug release rate than the amorphous and homocrystalline

ones, because of the larger polymer/drug interactions and tighter chain packing inside

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the micelle cores.

Keywords: supramolecular micelle, poly(lactic acid), stimuli-responsive

1. Introduction

Amphiphilic block copolymers that have a large solubility difference between the hydrophilic and hydrophobic segments can assemble into the nano-scaled core-

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shell micelles in an aqueous solution. These micelles have been drawn great interests because of their great potentials for biomedical applications [1]. In such core-shell micelles, the hydrophobic moieties are segregated from the aqueous exterior to form

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an inner core surrounded by the hydrophilic segments. The inner hydrophobic core of micelles serves as a microenvironment for encapsulating or solubilizing the

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hydrophobic drugs and the outer hydrophilic coronas maintains a hydration barrier to stabilize the micelles. Recently, the stimuli-responsive amphiphilic copolymers and micelles have gained considerable attentions for the programmable delivery systems, in which the drug release can be readily controlled by exerting an appropriate stimulus (e.g., pH, temperature, ion) [2]. A typical method to prepare the stimuli-

responsive amphiphilic copolymers is the use of polymer segments with stimuli responsivity.

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In contrast to conventional covalent interactions, noncovalent interactions such as metal-ligand interaction, host-guest recognition, and hydrogen bonding are reversible and more sensitive to the external stimuli [3]. Therefore, self-assembly of

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supramolecular amphiphilic copolymers based on noncovalent bonds offers a new route for designing the stimuli-responsive copolymer micelles. Compared to the

conventional covalently-bonded copolymer micelles, the supramolecular copolymer

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micelles can be simultaneously sensitive to the multiple external stimuli such as temperature, acid, base, and ions [4−6]. Among a variety of noncovalent interactions,

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hydrogen bonding interactions have been frequently employed to prepare the supramolecular polymers because they are flexible, dynamic, directional, selective, and reversible [7,8]. Multiple hydrogen bonds combine several hydrogen bonds in a single functional unit, which, as a consequence, enhances the bonding strength in comparison with the single hydrogen bonding system [9].

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The multiple (e.g., two [4,10,11], three [12−14], four-fold [15−17], etc.) complementary hydrogen bonding interactions have been widely used to prepare the supramolecular or pseudo block copolymers. For example, Zhu et al. have prepared

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the supramolecular amphiphilic diblock copolymers of poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) based on the uracil/adenine two-fold hydrogen bonds,

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which can form the stimuli-responsive core-shell micelles in aqueous media [4]. Such micelles exhibit higher drug efficacy and low cytotoxicity, enabling them promising candidates for the controlled release of drugs. On the other hand, the strength of multiple hydrogen bonds is strongly influenced by the donor and acceptor groups and it enhances remarkably with the number of formed hydrogen bonds. For example, the quadruple hydrogen bonding unit, i.e., 2-ureido-4[1H]-pyrimidinone (UPy), has a strong dimerization constant (Kdim = 6 × 107 M−1 in chloroform and 6 × 108 M−1 in

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toluene at 25 °C), which is close to the noncovalent bonds [18]. It has been reported that the inter-association equilibrium constant of three-fold complementary hydrogen bonds formed between thymine and diaminotriazine is ca. 890 M−1 [19], stronger than

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that of thymine/adenine two-fold hydrogen bonds (ca. 530 M−1) [20]. In the preparation of supramolecular stimuli-responsive copolymers and micelles, too strong

bonding would lower the stimuli responsivity and otherwise too weak bonding would

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decrease the stability. Therefore, it is envisioned that the three-fold hydrogen bonding system would be a favorable choice to design the supramolecular stimuli-responsive

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copolymers and micelles compared to their two and four-fond analogs, because of the balanced stability and stimuli responsivity.

Diaminotriazine and thymine (or uracil) are a representative pair to form the complementary three-fold hydrogen bonds [21], which have been used to prepare a variety of supramolecular (co)polymers [12,13,22,23] , In this work, we synthesized

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the thymine-terminated poly(lactic acid) (PLA-THY) as the hydrophobic block and diaminotriazine end-functionalized PEG (PEG-DAT) as the hydrophilic block to construct the supramolecular amphiphilic diblock copolymers based on the

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thymine/diaminotriazine complementary three-fold hydrogen bonds. The micellation, micelle structure, crystalline state of micelle core, stimuli-responsive and drug release

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behavior of PLA/PEG supramolecular copolymers and their micelles were investigated. Because the structure and physical properties of copolymer micelles are strongly influenced by the crystallization of hydrophobic segments inside the micelle cores [24−29], the crystallizability and crystalline polymorph of micelle cores were

controlled by varying the tacticity, stereostructure of PLA blocks and by mixing the enantiomeric supramolecular copolymers. Effects of crystallization and crystalline structure of hydrophobic PLA blocks on the microstructure and physical properties of

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PLA/PEG supramolecular micelles were also investigated. To our knowledge, this represents the first report for the supramolecular diblock copolymer micelles based on the thymine/diaminotriazine complementary hydrogen bonding interactions and also

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the first investigation on the effects of crystallization of hydrophobic blocks on the microstructure and physical properties of supramolecular copolymer micelles.

2. Experimental

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2.1. Materials

Monomethoxy PEG (mPEG, Mn = 5000 g/mol) was purchased from Sigma-

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Aldrich. L- and D-lactides (> 99.9%) were purchased from Purac Co. (Gorinchem, the Netherlands) and recrystallized from ethyl acetate before use. meso-Lactide (Jinan Daigang Biomaterial Co., Ltd), thymine (99%, J&K Chemical Ltd.), ethylene carbonate (99%, J&K Chemical Ltd.), 4-dimethylamino-pyridine (DMAP, 99%, J&K Chemical Ltd.), dicyclohexyl carbodiimide (DCC, 99%, Aladdin Reagent Co.), and

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doxorubicin hydrochloride (DOX·HCl) were used as received. Tin(II) 2ethylhexanoate [Sn(Oct)2, Sigma-Aldrich] was purified by distillation under reduced pressure. 1-(2-Hydroxyethyl)thymine was prepared according to the previous

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literatures (see supporting information) [30]. The covalently-bonded PLA-b-PEG diblock copolymers with different PLA stereostructures were synthesized according

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to a published method [27]. N,N-Dimethylformamide (DMF) was dried over CaH2 for

48 h and then distilled before use. Toluene was dried by sodium and distilled after being refluxed for 48 h. All other reagents and solvents were of analytical grade and used as received unless otherwise specified. 2.2. Sample preparation 2.2.1. Synthesis of thymine-terminated PLAs (PLA-THY)

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PLA-THY was synthesized via the ring-opening polymerization (ROP) of lactide using 1-(2-hydroxyethyl)thymine as the initiator and Sn(Oct)2 as the catalyst. A typical procedure to synthesize the thymine-terminated poly(L-lactic acid) (PLLA-

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THY) with an expected Mn of 5000 g/mol is described as follows. L-lactide (4.0 g, 27.8 mmol) and 1-(2-hydroxyethyl)thymine (0.14 g, 0.83 mmol) were added into a

Schlenk tube, which was then dried on a vacuum line at 50 °C for 0.5 h. The flask was

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then purged with dry argon and 10 mL of anhydrous DMF, 24.0 mg (0.059 mmol) of

Sn(Oct)2 were added. The polymerization was allowed to proceed at 115 °C for 15 h

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under an argon atmosphere. After the reaction, DMF was evaporated under reduced pressure and the residue was precipitated into cold diethyl ether twice. The product was finally dried in vacuum at 70 °C to a constant weight (3.3 g, yield: 80 %). 1H NMR (400 MHz, CDCl3, 298 K) δ ppm: 8.40 (1H, −CONHCO−), 6.98 (1H, −C(CH3)=CH−), 5.1-5.2 (PLLA, 1H repeating units, −CH(CH3)−), 4.1-4.4 (2H,

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−CH2CH2O−PLLA), 3.99 (2H, −NCH2CH2), 1.83 (3H, −C(CH3)=CH−), 1.4-1.6 (PLLA, 3H repeating units, −CH(CH3)−). 13C NMR (400MHz, CDCl3, 298K) δ ppm: 12.2, 16.7, 20.5, 62.9, 66.8, 69.1, 111.2, 141.1, 164.0, 169.8, 175.2. The thymine-

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terminated poly(D-lactic acid) (PDLA) and poly(D,L-lactic acid) (PDLLA) were synthesized by a similar method. meso-Lactide was used as the monomer for

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synthesizing PDLLA. The thymine-terminated isotactic PLLA, PDLA, and atactic PDLLA are abbreviated as Lx-THY, Dx-THY, and DLx-THY, respectively, in which

the subscripts represent the degree of polymerizations of corresponding lactic acid unit, as derived from the 1H NMR data. 2.2.2. Synthesis of diaminotriazine-terminated PEG (PEG-DAT) PEG-DAT was prepared in two steps. In the first step, mPEG (10.0 g, 2.0 mmol), cyanoacetic acid (0.17 g, 4.0 mmol), DMAP (10.0 mg, 0.082 mmol), and DCC (0.49

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g, 2.4 mmol) were dissolved in CH2Cl2 (50 mL) and stirred at 0 °C for 24 h. The reaction mixture was then filtered to remove the urea and the filtrate was concentrated by rotary evaporation. The remained filtrate was precipitated into cold diethyl ether

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twice. The product, PEG cyanoacetic ester, was isolated and dried in vacuum to a constant weight (9.1 g, yield: 90%). 1H NMR (400 MHz, CDCl3 , 298 K) δ ppm: 4.30 (2H, −CH2OC=O−), 3.42−3.70 (4H per PEG repeated unit, −CH2CH2O−), 3.32 (3H,

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PEG terminal, −OCH3), 2.72 (2H, −CH2CN).

In the second step, PEG cyanoacetic ester (5.0 g, 1.0 mmol), dicyanodiamine

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(0.17 g, 2.0 mmol), KOH (56.0 mg, 1.0 mmol), and anhydrous DMF (10 mL) were added into a Schlenk flask and the mixture was reacted at 80 °C for 24 h under an argon atmosphere. After the reaction, the reaction mixture was precipitated into cold diethyl ether twice. The precipitate was isolated and dried under vacuum to a constant weight (3.6 g, yield: 72%) to obtain the product.

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2.2.3. Preparation of PLA-THY/DAT-PEG micelles The micelles of PLA-THY/DAT-PEG supramolecular diblock copolymers were prepared by a dialysis method. Typically, 10.0 mg of PEG-DAT (2.0 mmol) and 2.0

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mmol of PLA-THY were dissolved in 10 mL of THF at room temperature. The equalmolar mixture of PLLA-THY and PDLA-THY is used in the preparation of

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enantiomerically-mixed PLA/PEG supramolecular micelles. The polymer solution was added dropwise into 30 ml of deionized water under continuous stirring. The micelle solution was dialyzed against deionized water for 24 h (MWCO = 3500), during which the water was changed every 4 h. The PLA/PEG supramolecular diblock copolymers having the PDLLA, PLLA, PDLA, and enantiomerically-mixed PLLA/PDLA hydrophobic blocks are denoted as E/DLx, E/Lx, E/Dx, and E/Lx-Dy, respectively, in which E, DL, L, and D, respectively, represent the PEG, PDLLA,

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PLLA, PDLA blocks and the subscripts x and y describe the degree of polymerizations of corresponding blocks. The molecular weight of PEG block is maintained as 5000 g/mol in all the supramolecular copolymers.

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2.2.4. Preparation of drug-loaded micelles DOX-loaded micelles were prepared according to a published method [31]. DOX·HCl (10 mg) and a trace of triethylamine (20 µL) were dissolved in 3 mL of

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DMSO. This drug solution was then added into a mixed solution of PLA-THY and PEG-DAT, in which the moles of two blocks are equivalent and the total weight of

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PLA-THY and PEG-DAT is 100 mg. The solution was dialyzed against deionized water for 24 h (MWCO = 3500), during which the water was changed every 2 h. The solution was finally lyophilized to attain the DOX-loaded micelles. To determine the drug loading content (DLC), the lyophilized micelles (5.0 mg) were dissolved in 2 mL of DMSO. UV-Vis absorbance (at 485 nm) of this micelle solution was measured to

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determine the DOX concentration. DLC was calculated by comparing the weights of loaded drug and supramolecular micelles, i.e., DLC (wt%) = (weight of loaded drug/weight of micelle) × 100%.

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2.2.5. In Vitro Release of Drug-Loaded Micelles Lyophilized micelles (5.0 mg) were dispersed in 2 ml of deionized water and the

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solution was then transferred to a dialysis bag (MWCO = 3500). It was then immersed in 10 mL of phosphate buffer (PBS, pH 7.4, 10 mM) in a shaking water bath at 37 °C. After a predetermined time interval, The PBS solution outside dialysis tube was removed for UV-Vis measurement and also replaced by the fresh buffer. The amount of released DOX was determined based on the absorbance at 485 nm. The release experiments were conducted in triplicate and the average results were used. 2.3. Characterization

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1

H and

13

C NMR spectra were measured on a 400 MHz Bruker AVANCE II

NMR spectrometer (Bruker BioSpin Co., Switzerland). Molecular weights were measured on a gel permeation chromatography (GPC, Waters Co., Milford, MA, USA)

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consisting of a Waters degasser, a Waters 1515 isocratic HPLC pump, a Waters 2414 RI detector, and two PL-gel mix C columns at 30 °C. THF was used as the eluent at a

flow rate of 1.0 mL/min. The molecular weight was calibrated based on the

polystyrene standards. Surface tensions of copolymer solutions with various

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concentrations (1×10−4~1.0 mg/mL) were measured on a DataPhysics OCA 20

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instrument (DataPhysics Instrument, Filderstadt, Germany) at 25 °C. Dynamic light scattering (DLS) analysis was performed on a Nano ZS 90 instrument (Malvern Instruments, Malvern, UK) with a scattering angle of 90°. Micelle morphology was observed on a JEM-1230 transmission electron microscope (TEM, JEOL, Tokyo, Japan) operated at an acceleration voltage of 80 kV. To prepare the TEM sample, the

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micelle solution (0.2 wt%) was dropped on a carbon-coated copper grid and the solvent was removed by a filter paper.

Wide angle X-ray diffraction (WAXD) patterns of lyophilized micelles were

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measured on a Rigaku RU-200 (Rigaku Co., Tokyo, Japan) instrument with a Nifiltered Cu Kα radiation (λ = 0.154 nm), working at 40 kV and 200 mA. WAXD

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patterns of micelle solutions (1 wt%) were measured on the beamline BL16B1 of

Shanghai Synchrotron Radiation Facility (SSRF) with a X-ray wavelength of 0.124 nm and a sample-to-detector distance of 130 mm. Scattering data were acquired by a Rayonix SX-165 CCD detector (Rayonix, Evanston, IL) with a resolution of 2048 × 2048 pixels and a pixel size of 80 × 80 µm2. The acquisition time of each pattern was

90s.

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Thermal properties of lyophilized micelles and PLA-THY homopolymers were measured on a NETZSCH 214 Polyma DSC (NETZSCH, Germany) equipped with an IC70 intracooler. The samples were heated from −70 to 180 (or 230 °C) at a heating

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rate of 10 °C/min. Melting temperature (Tc) and enthalpy (∆Hm) of PEG and PLA blocks were calculated on basis of the DSC results. Degree of crystallinity (Xc) of

PLA in the cores of lyophilized micelles and PLA-THY were estimated by comparing

crystallites are 93 and 142 J/g, respectively [32,33].

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3. Results and discussion

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the ∆Hm of PLA to that of an infinitely large crystal (∆Hm0). ∆Hm0 of PLA hc and sc

3.1 Synthesis of PLA-THY and PEG-DAT

Scheme 1 illustrates the synthetic route of PLA-THY and PEG-DAT. First, 1-(2hydroxyethyl)thymine was prepared by the reaction of thymine and ethylene carbonate [30], which was confirmed by the 1H NMR spectrum (Fig. S1). PLA-THY

synthesized

via

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with different stereostructures, i.e., PLLA-THY, PDLA-THY, and PDLLA-THY, was the

ROP

of

corresponding

lactide

by

using

1-(2-

hydroxyethyl)thymine as the initiator (Scheme 1a). The feed ratio and molecular

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weight of synthesized PLA-THY are listed in Table 1. Molecular weight of PLATHY was controlled by the monomer/initiator feeding ratio. Macromolecular structure

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of synthesized PLA-THY was verified by 1H, 13C NMR, GPC, and FTIR (Fig.s 1, S2,

S3).

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(b)

O

O

O

N H + O n

DCC , DMAP

C

0 oC

OH

O

N C

O n

mPEG

PEG-CN NH2

HN

CN

O

N H2N

N

O

H

N O n

PEG-DAT

N

o

KOH, 90 C

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NH2

Scheme 1. Synthetic route of (a) PLA-THY and (b) PEG-DAT. (c) Formation of

f

(a)

c

b

O

N O

O

d

N H a

e O

L40-THY

g O O

i

L19-THY L40-THY

f

L64-THY

n

e

c

a 8

4.2

d

6

4

(b)

b

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4.4

10

i OH

g CDCl3

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three-fold complementary hydrogen bonds between PLA-THY and PEG-DAT.

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Chemical shift (ppm)

16

20

24

28

Elution time (min)

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Fig. 1. (a) 1H NMR spectrum and (b) GPC curves of PLLA-THY. Table 1. Feed ratio and molecular weight of thymine-end functionalized PLAs [lactide]/[initiator] yield Mn,tha Mn,NMRb Mn,GPCc PDIc molar feed ratio (%) (g/mol) (g/mol) (g/mol) L19-THY 9.7/1 64 1060 1540 2700 1.08 L40-THY 19.7/1 86 2610 3050 4040 1.13 33.7/1 83 4200 4780 6600 1.35 L64-THY D38-THY 17.4/1 90 2430 2910 3580 1.28 DL38-THY 17.4/1 90 2430 2910 3710 1.17 a Theoretical molecular weight (Mn,th), Mn,th = [lactide]/[initiator]×yield×144+Mn,initiator,

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PLA-THY

in which Mn,initiator is the molecular weight of 1-(2-hydroxyethyl)thymine initiator (170 g/mol). bMn derived from 1H NMR. cMn and polydispersity index (PDI) derived from GPC.

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PEG-DAT was synthesized in two steps (Scheme 1b). In the first step, the terminal hydroxyl group of mPEG was converted to cyano through an esterification reaction with cyanoacetic acid [34]. After esterification, the resonance of terminal

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methylene protons in mPEG is shown at 4.30 ppm (peak c, Fig. S4). In the second step, the cyano-terminated PEG is reacted with dicyanodiamine to attain the PEG-

DAT. The resonance of NH2 protons for diaminotriazine is shown at 6.1 ppm in the

H NMR spectrum of PEG-DAT (Fig. S5). The conversion of mPEG terminal to

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diaminotriazine is nearly quantitative through comparing the resonance peak area of

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diaminotriazine NH2 protons to the CH2 protons of PEG backbone. In the FTIR spectra (Fig. S6), PEG-DAT exhibits the vibration peaks at 3404 (symmetric NH stretching), 3210 (asymmetric NH stretching) [19], 1636 (phenyl breathing), and 1566 cm−1 (C=N stretching) [35], as compared to the mPEG and PEG cyanoacetic ester. 3.2 Formation of PLA/PEG supramolecular diblock copolymers

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The formation of supramolecular diblock copolymers between PLA-THY and PEG-DAT via the complementary multiple hydrogen bonds is proved by variabletemperature 1H NMR spectroscopy [4,5,10,36]. PLA-THY and PEG-DAT with

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equivalent molar quantities of thymine and diaminotriazine end groups were dissolved in 1,1,2,2-tetrachloroethane-d2. Their 1H NMR spectra were analyzed at different

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temperatures (25~80 °C), as shown in Fig. 2a. The changes of chemical shifts for terminal NH proton of PLA-THY (11.0~11.2 ppm) and terminal NH2 protons of PEG-

DAT (6.4~6.6 ppm) with temperature are shown in Fig. 2b. As shown in Fig. 2, the resonance peaks for terminal NH proton of PLA-THY and terminal NH2 protons of PEG-DAT gradually shift upfield from 11.2 to 11.0, 6.6 to 6.4 as the temperature is raised from 25 to 80 °C, respectively. This can be attributed to the dissociation of the complementary hydrogen bonds [4,36]. As the temperature is cooled from 80 to 25 °C

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again, these resonance peaks recover to the initial positions. The intensities of NH and NH2 protons decrease as the temperature increases, because of the enhanced exchange of NH, NH2 protons with solvent at high temperatures. These results demonstrate the

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formation of complementary hydrogen bonds and supramolecular diblock copolymers between PLA-THY and PEG-DAT, as illustrated in Scheme 1c. The formed complementary hydrogen bonds and supramolecular diblock copolymers are

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Chemical shift (ppm)

(a) 80 °C 60 °C 40 °C 25 °C 12

11

10

9

8

Chemical shift (ppm)

7

11

(b)

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re-cooled to 25 °C

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thermally reversible in 1,1,2,2-tetrachloroethane-d2.

thymine NH diaminotriazine NH2

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6 20

6

40

60

Temperature (°C)

80

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Fig. 2. (a) Temperature-dependent 1H NMR spectra of PLA-THY/PEG-DAT 1:1 mixture in 1,1,2,2-tetrachloroethane-d2. (b) Changes of chemical shifts with temperature for thymine NH and diaminotriazine NH2 protons. Sample was equilibrated for 10 min at each temperature before measurement.

copolymers

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3.3 Micelle formation and micelle crystalline state of supramolecular diblock

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The micelles of PLA/PEG supramolecular diblock copolymers were prepared by

a solvent evaporation method [37]. 1H NMR spectrum of lyophilized micelles dispersed in D2O was used to confirm the micelle formation of supramolecular

copolymers (Fig. 3a). The lyophilized micelles of supramolecular copolymers can be well dispersed in D2O and water to a transparent solution or dispersion, although the

PLA-THY is insoluble in water. Compared to the 1H NMR spectrum measured in CDCl3, the NMR peaks of PEG block (δ = 3.7 ppm) can be clearly seen while those 13

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of PLA block cannot be observed in the NMR spectra collected in D2O (Fig. 3a). This verifies that the supramolecular copolymers assemble into the micelles comprised of hydrophobic PLA core and hydrophilic PEG shell in aqueous solution [38].

E/L40

E/L40-D38 in CDCl3

E/L40-D38

75

E/DL38

70

E/L40-D38 in D2O E/L40 in CDCl3

65

E/L40 in D2O

4 3 2 Chemical shift (ppm)

CMC

0.0

0.4 0.8 1.2 Concentration (g/L)

1.6

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5

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PLA

PLA

γ (dyn/cm)

PEG

D2O

(b)

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80

(a)

Fig. 3. (a) 1H NMR spectra of lyophilized supramolecular micelles in different deuterated solvents. (b) Plot of surface tension versus concentration for the supramolecular copolymers with different stereostructures.

Critical micelle concentration (CMC) of PLA/PEG supramolecular copolymers

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was investigated by the surface tension method [39,40]. The slope of surface tension versus concentration plot changes abruptly as the concentration of supramolecular copolymer is approaching CMC (Fig. 3b), which also demonstrates the micellation of

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supramolecular copolymers in aqueous solution. As shown in Table 2, CMCs of PLLA/PEG supramolecular copolymers are comparable to those of the covalently-

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bonded PLA-b-PEG with the similar compositions [27] and they decrease with the increase of PLLA block length. At the similar composition, the enantiomericallymixed supramolecular copolymers show smaller CMCs than those having the isotactic PLLA or PDLA blocks, coinciding with the results reported in literatures [24,27]. The lower CMC usually means higher micellar stability [26]. Therefore, the stereocomplexation of PLLA and PDLA (as described below) increases the stability of supramolecular copolymer micelles [24,41]. The supramolecular copolymers with

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isotactic PLLA or PDLA blocks (e.g., E/L40 and E/D38) show smaller CMCs that those having the atactic PDLLA block (e.g., E/DL38), attributable to the crystallization of micelle core (as described below).

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Crystalline state and thermal properties of the supramolecular copolymer micelles were investigated by WAXD, FTIR and DSC. Melting temperature and enthalpy of PEG and PLA blocks derived from the DSC curves are summarized in

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Table 2. WAXD patterns of supramolecular micelles were measured in both the

lyophilized powder and solution states. As shown in Fig. 4, the WAXD peaks of PEG

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blocks are observed in the lyophilized micelles but not in the micelle solution, indicating that the PEG segments are solubilized in the micelle solution. The supramolecular micelles with atactic PDLLA block (e.g., E/DL38) do not show discernible diffractions of PLA crystals in the WAXD patterns of both lyophilized micelles (Fig. 4a) and micelle solutions (Fig. 4b), and also do not show the melting

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peaks of PLA in the DSC heating curve of lyophilized micelles, except for the melting peak of PEG blocks at ~60 °C (Fig. S7a). These micelles exhibit broad FTIR absorption peak in the carbonyl stretching [ν(C=O)] region at 1758 cm−1 (Fig. S7b),

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characteristic of the amorphous PLA [42]. These results demonstrate the amorphous core structure for the supramolecular micelles having atactic PDLLA blocks.

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Table 2. Physical and thermal properties of PLA/PEG supramolecular diblock

copolymer micellesa Sample

E/L19 E/L40 E/L64 E/D38 E/L40-D38 E/DL38

CMC (mg/L) 15.3 13.8 7.6 12.9 5.7 20.0

Tm,PEG (°C) 63.8 62.7 63.2 63.1 66.5 57.3

∆Hm,PEG (J/g) 187.0 129.7 127.0 134.2 147.1 129.0

Tm,PLA (°C) 108.8 144.4 155.6 139.0 208.6 N.P.

∆Hm,PLA (J/g) 1.5 14.3 21.1 7.8 25.6 0

Xc,PLA (%) 7.0 41.4 47.1 23.2 50.1 0

Dh (nm) 115.6 144.7 199.2 173.2 111.9 105.5

DLC (%) 3.5 4.7 6.5 4.8 6.0 5.1

Tm,PEG, melting temperature of PEG; ∆Hm,PEG, melting enthalpy of PEG; Tm,PLA, melting temperature of PLA; ∆Hm,PLA, melting enthalpy of PLA; Xc,PLA, degree of crystallinity of PLA. a

15

E/L40-D38 E/D38

E/DL38 E/L40-D38 E/D38 E/L40

E/L64

H2O

E/L40

15

20 2θ (°)

25

10

30

15

20

25

30

2θ (°)

SC

10

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E/DL38

hcPLA200/110

scPLA110

(b)

PEG032/112

PEG120

scPLA300/030

scPLA110

(a)

hcPLA200/110

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Fig. 4. WAXD patterns of (a) lyophilized micelles and (b) micelle solutions (1.0 wt%)

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for PLA/PEG supramolecular copolymers with different stereostructures. The wavelengths of X-ray are 0.154 and 0.124 nm in panels a and b, respectively. Supramolecular micelles with isotactic PLLA or PDLA blocks (e.g., E/L40, E/L64, and E/D38) all show a diffraction peak at 2θ = 16.7° in the WAXD patterns of lyophilized micelles (Fig. 4a) and at 2θ = 13.4° in the synchrotron-radiation WAXD

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patterns of micelle solutions (Fig. 4b), which are characteristic of the (200)/(110) diffraction of α-form homocrystalline PLA [43]. These micelles show a melting endotherm at 130~160 °C in the DSC heating curves of lyophilized micelles (Fig. S7a)

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and a sharp FTIR absorption in the ν(C=O) band at 1758 cm−1 (Fig. S7b). These

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suggest that the supramolecular micelles having isotactic PLLA or PDLA blocks are comprised of the homocrystalline core. As shown in Table 2, the melting temperature and enthalpy of PLLA core in the homocrystalline supramolecular micelles enhance with increasing the PLLA block length, due to the increased crystalline perfection. When the PLLA/PEG and PDLA/PEG supramolecular copolymers are mixed, new diffractions at 2θ = 12.1 and 20.9° are observed in the WAXD patterns of lyophilized micelles (Fig. 4a) and a diffraction at 2θ = 9.6° is detected in the

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synchrotron-radiation WAXD profiles of micelle solution (Fig. 4b). These diffractions are characteristic of the stereocomplexed PLLA and PDLA [44]. Stereocomplex formation in the micelles of enantiomerically-mixed supramolecular copolymers is

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further confirmed by DSC and FTIR (Fig. S7). The micelles of enantiomericallymixed supramolecular copolymers exhibit higher melting point (~200 °C) and lower wavenumber (1750 cm−1) for ν(C=O) band of PLA blocks than the other two types of

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micelles. The lower frequency of PLA ν(C=O) vibration in enantiomerically-mixed

supramolecular micelles is due to the PLLA/PDLA hydrogen bonding interactions in

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stereocomplex [42]. On basis of the DSC results, melting enthalpy (∆Hm) and degree of crystallinity (Xc) of PLA blocks in the cores of lyophilized micelles were calculated and compared with those of PLA-THY. As shown in Table 2, Xcs of PLLA in the cores of lyophilized micelles increase as the PLLA block length increases. As derived from the DSC results shown in Fig. S8, Xcs of L19-THY, L40-THY, L64-THY, D38-THY

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homopolymers and L40-THY/D38-THY 1/1 mixture are 45.9%, 74.3%, 79.6%, 76.6%, and 58.3%, respectively; they are much larger than the Xcs of PLA blocks in the cores of lyophilized micelles. This indicates that the confinement effect of nanoscaled

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micelle cores retards the crystallization of PLA blocks. The size and morphology of PLA/PEG supramolecular micelles were studied by

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DLS and TEM. As shown in Figs. 5a and S9, all the supramolecular micelles with different PLA stereostructures show single peak in the size distribution curves. The hydrodynamic diameter (Dh) of supramolecular micelles measured by DLS are ranging in 100~200 nm, depending on the length and stereostructure of PLA block. As shown in the TEM image (Fig. 5b), the supramolecular micelles are approximately spherical and have the relatively broad size distribution in 100~200 nm, in accordance with the DLS result. Dhs of supramolecular micelles are similar to those of the

17

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PCL/PEG supramolecular micelles based on uracil/adenine complementary hydrogen bonds (142~172 nm) [4]. As shown in Table 2, Dh of supramolecular micelles containing PLLA blocks increases with increasing the PLLA block length, because

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the micelles with larger cores would be formed to include more insoluble chains for the supramolecular copolymers containing longer hydrophobic blocks. Meanwhile,

the micelle solution of PLLA/PEG supramolecular copolymers becomes less

micelle size. 16

E/L40-D38

8

E/DL38

4 0 0 10

10

1

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Intensity (%)

)

E/L64

12

(b)

(a)

E/L40

10

2

10

3

10

4

E/L64 micelles

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)

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Dh (nm)

(c)

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transparency with increasing the PLLA block length (Fig. 5c), due to the increased

E/L40

E/L64

E/L40-D38

E/DL38

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Fig. 5. (a) Intensity size distribution, (b) TEM image, and (c) photographs of micelle solution (1 wt%) for PLA/PEG supramolecular copolymers At the similar copolymer compositions, the supramolecular copolymers having

enantiomerically-mixed PLLA/PDLA blocks (e.g., E/L40-D38) and atactic PDLLA

blocks (e.g., E/DL38) form the smaller micelles (Figs. 5a, S9, Table 2) and those micelle solutions are also more transparent (Fig. 5c), as compared to the copolymers containing isotactic PLLA or PDLA blocks. The smaller micelle size of

18

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enantiomerically-mixed supramolecular copolymers may be ascribed to the more compact chain packing in stereocomplex [42], which would shrink the micelle cores. Even though the origin for the smaller micelles formed from the supramolecular

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copolymers with atactic PDLLA blocks is still unclear, a possible reason may be the relatively higher solubility and hydrophilicity of amorphous PDLLA segments than their homocrystalline and stereocomplexed counterparts.

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Interestingly, the micelles of PLA/PEG supramolecular copolymers (Dh = 100~200 nm) are much larger than those formed from the conventional covalently-

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bonded diblock copolymers having similar composition and stereostructure (typically Dh = 20~60 nm) [24,27,45,46]. It has also been reported that the size of PCL/PEG supramolecular micelles (Dh = 142~172 nm) [4] based on uracil/adenine complementary hydrogen bonds is much larger size than that of the conventional diblock copolymers (Dh = 30~60 nm) [39]. It is conjectured that the larger Dh of

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supramolecular micelles may be ascribed to the larger size of terminal thymine, diaminotriazine groups and the longer bond length of complementary hydrogen bonds. Also, the inclusion of unpaired PLA-THY in the micelle cores could also enlarge the

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micelle particles.

3.4 Stimuli responsivity of supramolecular micelles

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Because the complementary hydrogen bonds are reversible and sensitive to the external stimuli such as temperature, pH, and ions, the stimuli responsivity of PLA/PEG supramolecular micelles are investigated by measuring the Dh under different conditions. As shown in Fig. S10, the effect of temperature on supramolecular micelles is not so significant at the temperature below 70 °C and their Dhs only increase slightly with increasing the temperature from 20 to 70 °C. Therefore, it is concluded that the dissociation of thymine/diaminotriazine

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complementary hydrogen bonds inside the micelles is not drastic in this temperature range and the PLA/PEG supramolecular micelles are stable in a wide temperature range around the physiologic temperature.

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To investigate the stimuli responsivity, the supramolecular micelles were treated by the solution with different pH and NaCl concentrations. The micelle sizes were then measured by DLS (Figs. 6, S11, S12). Effects of pH and salt concentration on the

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size of covalently-bonded PLA-b-PEG were also investigated for comparison. The size of supramolecular micelles changes little at pH = 7~11. It was found that the PLA

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blocks in supramolecular micelles underwent significant degradation at pH = 13, so we did not investigate the pH responsivity of supramolecular micelles at pH > 11. The supramolecular micelles are unstable in the acidic and salted solutions. As shown in Figs. 6, Dhs of supramolecular micelles at pH = 5 are almost the same as those measured at pH = 7. Dhs of homocrystalline (i.e., E/L40) and stereocomplexed (i.e.,

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E/L40-D38) supramolecular micelles increase significantly with decreasing pH from 5 to 3 (Fig. 6). The solutions of homocrystalline and stereocomplexed supramolecular micelles become turbid with decreasing pH to 3 or 1 (Fig. S13), indicating the

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destabilization and aggregation of micelles. However, the sizes of covalently-bonded PLA-b-PEG is almost independent of pH at pH = 1~11. (Fig. S14a). The pH

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responsivity of supramolecular micelles is attributable to the protonation of amine in diaminotriazine under acidic conditions (Fig. 7a), which lead to the disassociation of complementary hydrogen bonds and the large-scale aggregation of hydrophobic PLA blocks.

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E/L 40

pH=7 pH=5 pH=3 pH=1

40

40

Intensity (%)

Intensity (%)

(a)

20

0 10

2

10

3

10

4

30 20 10 0 1 10

(c)

E/DL 38 pH=7 pH=5 pH=3 pH=1

10

0 1 10

10

2

2

10

3

10

4

Dh (nm)

D h (nm)

20

10

SC

Intensity (%)

30

1

E/L40-D38 pH=7 pH=5 pH=3 pH=1

10

D h(nm)

3

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10

(b)

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60

10

4

Fig. 6. Change of micelle size and its distribution with pH in acidic solution for (a)

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E/L40 , (b) E/L40-D38, and (c) E/DL38 supramolecular copolymers.

Fig. 7. Structural changes of thymine, diaminotriazine groups and disassociation of

thymine/diaminotriazine complementary hydrogen bonds in the supramolecular copolymers in (a) acidic and (b) salted aqueous solutions. Similar remarkable increases of Dh are also observed for the homocrystalline and stereocomplexed supramolecular micelles with increasing the salt concentration from 21

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0.01 to 0.1 M. However, Dh of covalently-bonded PLA-b-PEG keeps nearly constant with varying the salt concentration (Fig. S14b). The disassociation of hydrogen bonds in presence of salt ions would be attributable to the binding of anions to the amide

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groups of thymine terminals (Fig. 7c) [47]. It is notable that the low stability of supramolecular micelles in salted solution would restrict its application as a drug carrier, because the biological environment is salted.

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It is notable that the stimuli responsivity of supramolecular micelles depends strongly on the micelle size and crystalline structure of PLA core. As shown in Figs. 6

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and S12, the size increase of amorphous supramolecular micelles induced by the change of pH and salt concentration is not so significant as that observed for the homocrystalline and stereocomplexed supramolecular micelles. Even at pH = 1 and a high salt concentration (0.5 M), the measured Dhs of amorphous supramolecular micelles are all less than 500 nm, which, however, are larger than 1000 nm for those

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having the homocrystalline and stereocomplexed PLA cores. The solution of amorphous supramolecular micelles remains transparent at pH = 1 (Fig. S13). All these results demonstrate that the amorphous supramolecular micelles are more stable

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to the external stimuli. Generally, the aggregation of micelles is caused by the van der walls interactions, which depend on the particle weight and inter-particle distance.

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Previous study has found that the chain packing in amorphous PDLLA-b-PEG micelles is looser than that in the homocrystalline PLLA-b-PEG and stereocomplexed

PLLA-b-PEG/PDLA-b-PEG micelles [27]. Therefore, the smaller size and looser chain packing inside the cores of amorphous supramolecular micelles would decrease the particle weight, which thus lowers the van der walls attractions between micelles and enhances the micelle colloid stability. Besides, the higher solubility and hydrophilicity of amorphous PDLLA segments than the crystalline ones may also

22

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depress the aggregation between micelles under external stimuli. Because of the better stability of amorphous supramolecular micelles in salted solution, they would be more suitable to be the drug carrier than the homocrystalline and stereocomplexed ones.

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3.5 In vitro drug release of supramolecular micelles. A typical anticancer drug, DOX, was used to evaluate the drug loading and

release behavior of PLA/PEG supramolecular micelles. As shown in Table 2, DLCs

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of supramolecular micelles are ranging in 3.5~6.5%, larger than those of the

covalently-bonded diblock copolymers having the similar compositions (typically <

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2.0%) [35]. Larger DLCs of supramolecular micelles would be due to the larger core size and looser chain packing inside the micelle core. DLCs of supramolecular micelles improve with increasing the PLLA block length, because of the increased micelle size. DLCs of supramolecular micelles are also influenced by the crystalline state of inner core. The stereocomplexed supramolecular micelles (e.g., E/L40-D38)

of the former.

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have larger DLC than their homocrystalline counterparts, even though the smaller size

In vitro release behavior of DOX-loaded supramolecular micelles was

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investigated in PBS buffer (pH = 7.4, 10 mM) at 37 °C. As illustrated in Fig. 8, the release rate of supramolecular micelles changes in the order of amorphous >

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homocrystalline > stereocomplexed micelles. The different release kinetics and DLCs of supramolecular micelles can be correlated to the polymer/drug interactions and chain packing inside the micelle cores. Because of the more compact chain packing in stereocomplexed PLA, the larger polymer/drug interactions may exist in the stereocomplexed micelles. This would prevent the penetration and diffusion of drugs and may result in the higher DLC and slower release of encapsulated drug. Meanwhile, the fast drug release rate of amorphous supramolecular micelles would be

23

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ascribed to the looser chain packing of PLA inside the micelle cores than that of the

100

60 40 20

E/L40

E/L40-D38

E/D38

E/DL38

0 0

20

40

Time (h)

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80

SC

Cummulative release (%)

crystalline ones.

60

80

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Fig. 8. In vitro release profiles of DOX from supramolecular micelles.

4. Conclusions

PLA/PEG supramolecular amphiphilic diblock copolymers were prepared based on the thymine/diaminotriazine complementary hydrogen bonding interactions

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between PLA-THY and PEG-DAT. These supramolecular copolymers self-assemble into the stimuli-responsive micelles in aqueous solution, which exhibit much larger size than the conventional covalently-bonded diblock copolymer micelles. The

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amorphous, homocrystalline, and stereocomplexed supramolecular micelles are obtained from the copolymers having the atactic PDLLA, isotactic PLLA (or PDLA),

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and enantiomerically-mixed PLLA/PDLA hydrophobic blocks, respectively. The stereocomplexed and amorphous supramolecular micelles have smaller size than their homocrystalline counterparts. The homocrystalline and stereocomplexed micelles are more sensitive to the external stimuli such as pH and ions than the amorphous ones and they undergo fast aggregation in the acidic and salted solutions due to the disassociation of complementary hydrogen bonds. The stereocomplexed micelles exhibit larger DLC and slower drug release rate than the amorphous and homocrystalline ones. This study has prepared a novel type of stimuli-responsive 24

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supramolecular micelles with the tunable microstructures and properties by combining the reversibility of multiple hydrogen bonds and the control over crystalline state of micelle core. These materials have the advantages of both

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traditional covalently-linked copolymer micelles and the dynamic supramolecular properties, and may find potential applications in the controlled drug delivery systems.

Supplementary information

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Synthesis of 1-(2-hydroxyethyl)thymine, FTIR and DSC analysis methods, NMR, FTIR data of PLA-THY and PEG-DAT, DSC and FTIR data of supramolecular

Acknowledgements

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micelles, Dh, and transparency of supramolecular copolymers and micelles.

We acknowledge the financial supports of Natural Science Foundation of China (21274128, 21422406), Skate Key Laboratory of Chemical Engineering (SKL-ChE12D06),

and

Natural

Science

Foundation

of

Zhejiang

Province,

China

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(LR16E030003). Synchrotron radiation WAXD of micelle solutions was measured on beamline BL16B1 of SSRF, China.

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Supramolecular PLA/PEG copolymers were prepared based on complementary three-fold hydrogen bonds.




These copolymers self-assemble into stimuli-responsive micelles in aqueous




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solution. Stability, stimuli responsivity, and drug delivery property of supramolecular

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micelles can be tuned by the crystallization of PLA block.

1