Poly(Îµ-caprolactone)-block-polysarcosine by Ring-Opening

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Poly(#-caprolactone)-block-Polysarcosine by Ring-Opening Polymerization of Sarcosine N-Thiocarboxyanhydride: Synthesis and Thermo-responsive Self-Assembly Deng Yangwei, Tao Zou, Xinfeng Tao, Vincent Semetey, Sylvain Trepout, Sergio Marco, Jun Ling, and Min-Hui Li Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b00930 • Publication Date (Web): 21 Sep 2015 Downloaded from http://pubs.acs.org on September 23, 2015

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Poly(ε-caprolactone)-block-Polysarcosine by Ring-Opening Polymerization of Sarcosine N-Thiocarboxyanhydride: Synthesis and Thermo-responsive Self-Assembly Yangwei Deng,1,2 Tao Zou,2,a Xinfeng Tao,1 Vincent Semetey,2,3 Sylvain Trepout,4 Sergio Marco,4 Jun Ling,1,* Min-Hui Li 2,3,* 1

MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of

Polymer Science and Engineering, Zhejiang University, 310027 Hangzhou, China. 2

Institut Curie - CNRS - Université Pierre & Marie Curie, Laboratoire Physico-Chimie Curie,

UMR168, 26 Rue d’Ulm, 75248 Paris, France. 3

Institut de Recherche de Chimie Paris, UMR8247, CNRS - Chimie ParisTech (ENSCP), 11

rue Pierre et Marie Curie, 75231 Paris, France. 4

Institut Curie, INSERM U1196, 91405 Orsay cedex, France.

*Corresponding authors: [email protected] (J. Ling); [email protected] (M.-H. Li) a

Present address: School of Chemical Engineering and Technology, Wuhan University of

Science and Technology, 430081, Wuhan, China.

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ACS Paragon Plus Environment

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ABSTRACT

Biocompatible amphiphilic block copolymers composed of polysarcosine (PSar) and poly(ε-caprolactone) (PCL) were synthesized using ring-opening polymerization of sarcosine N-thiocarboxyanhydride initiated by oxyamine-ended PCL, and characterized by NMR, SEC and DSC. Self-assembling of two triblock copolymers PSar8-b-PCL28-b-PSar8 (CS7) and PSar16-b-PCL40-b-PSar16 (CS10) in dilute solution was studied in detail towards polymersome formation, using thin-film hydration and nanoprecipitation techniques. A few of giant vesicles were obtained by thin-film hydration from both copolymers and visualized by confocal laser scanning microscope. Unilamellar sheets and nanofibers (with 8 to 10 nm thickness or diameter), observed by Cryo-TEM, were obtained by nanoprecipitation at room temperature. These lamellae and fibrous structures were transformed into worm-like cylinders and spheres (D ~ 30-100 nm) after heating to 65°C (>Tm, PCL). Heating CS10 suspensions to 90°C led eventually multilamellar polymersomes (D ~ 100-500 nm). Mechanism II, where micelles expand to vesicles through water diffusion and forming hydrophilic cores, was proposed for polymersome formation. Cell viability test confirmed the self-assemblies were not cytotoxic.

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INTRODUCTION

Amphiphilic block copolymers have been widely studied since last decades as interesting macromolecular analogues of natural amphiphiles.1,

2

Depending on their molecular

composition, mainly hydrophilic/hydrophobic ratio, these copolymers can self-assemble, in dilute water solution, into various morphologies such as spherical micelles, cylindrical rods, lamellae and vesicles.2-4 The self-assembling process is mainly driven by the minimization of free energy.5-8 Polymersomes, i.e., vesicles composed of amphiphilic copolymers, have attracted special attention due to their vesicular structures, cell- and virus-mimicking dimensions and functions.9, 10 Moreover, their membrane properties such as responsiveness can be adjusted easily by polymer chemistry.11-15 Polymersomes have been studied as drug carriers,16-19 bioimaging contrast agent carriers20 and nanoreactors,21 that present potential applications in biomedical field and material science.10, 22-24

The formation of polymersome is controlled and affected by many parameters such as inherent characteristics of amphiphiles,18, 25, 26 physical chemical conditions (temperature,27, 28 pH17 and initial concentration29 etc.), as well as preparation methods.16, 30 Among various amphiphilic

block

copolymers,

poly(ethylene

glycol)-block-poly(ε-caprolactone)

(PEG-b-PCL) is of great interest for preparation of polymersomes, because both PEG and PCL have been applied in polymeric drug-carrier formations which have entered clinical trials,31 and some drugs containing them have been approved by FDA. PCL is a biocompatible and biodegradable hydrophobic polymer which shows slow erosion kinetics and a certain stability against degradation under neutral pH environments.32 PEG is a water-soluble

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macromolecule with anti-fouling property.33 Nano- and giant vesicles prepared from PEG-b-PCL by different methods have been intensively investigated,

18, 28, 30, 34, 35

with

discussion on their formation mechanism 28 and on their biomedical applications.16, 36

The synthetic polymer PEG has often been considered as a relatively benign and immunologically safe material, and has been a common choice for the hydrophilic block of copolymers.30 However, recent works have revealed that PEGs show oxidative activity in physiological cellular environment19 and that PEGs can generate complement activation products in human serum on a time scale of minutes.37, 38 Recently, a natural amino acid-based polymer, polysarcosine (PSar), has become a competitive alternative for PEG.39 PSar, with methyl substitution on the nitrogen atom of amino acid unit (sarcosine), shows excellent water-solubility comparable with and even better than PEG.40 PSar and PEG are both polar, electrically neutral, hydrogen-bond acceptors and are not hydrogen-bond donors. These characteristics are responsible for the similar protein resistance between oligomers of Sar and EG.41 Luxenhofer and coworkers demonstrated PSar and other polypeptoids could be degraded in vivo by reactive oxygen species (ROS) generated by certain enzymes from the liver.42 Reports on the synthesis and self-assembly behaviors of PSar-containing block copolymers started to appear in the literature in the last years.26, 43-48 Guo’s group reported the synthesis of PSar-b-PCL diblock copolymer via the ring opening polymerization (ROP) of sarcosine N-carboxyanhydride (Sar-NCA) followed by tin(II) octanoate-catalyzed ROP of ε-caprolactone.45 However, the structure of nanoparticles of diameter around 100 nm formed by PSar-b-PCL has not been clarified. At the same time, we were interested in the polymersome formation by PSar-b-PCL diblock and PSar-b-PCL-b-PSar triblock copolymers 4


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and reported their syntheses by a new method via ROP of sarcosine N-thiocarboxyanhydride (Sar-NTA).49 In this paper, we describe in detail this work.

NCA polymerization is the most widely-used method for polypeptide and polypeptoid syntheses,50-52 and PSar are prepared, in most cases, by ROP of Sar-NCA. However, the application of NCA is seriously limited because of their high sensibility to moisture and heat and their instability upon storage.53, 54 In contrast, amino acid NTA, the thio-analog of amino acid NCA, is much more robust. But its relatively low reactivity restricted for a long time its use to stepwise synthesis.55,

56

Kricheldorf reported in 2008 ROP of D,L-leucine NTA,

D,L-phenylalanine NTA and Sar-NTA initiated by primary amine and prepared poly(amino acids) with well-defined structures.57 This work was a milestone in the research of NTA polymerization. Recently Ling’s group used PEG-amine to initiate ROP of Sar-NTA.44 New hydrophilic di- and tri-block copolymers, PEG-b-PSar and PSar-b-PEG-b-PSar, were produced. Amine-initiated ROP of Sar-NTA generally resulted in PSar with moderate molecular weight MWs (DP < 100). To obtain PSar of higher MWs (DP > 100), Ling’s group employed rare earth borohydrides as initiator, and synthesized successfully PSar with DP = 390 (PDI = 1.14).58

The aim of the present work was to synthesize amphiphilic di- and triblock copolymers, PSar-b-PCL and PSar-b-PCL-b-PSar, with various MWs and various hydrophilic/hydrophobic ratios, and to study their ability to form polymersomes. We used ROP of Sar-NTA initiated by polycaprolactone

oxyamines

(PCL-ONH2

and

H2NO-PCL-ONH2).

Polycaprolactone

PCL-OH and HO-PCL-OH were first synthesized by diphenyl phosphate-catalyzed ROP of

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ε-caprolactone, followed by end-functionalization with amino groups. Self-assemblies of two triblock copolymers in aqueous solution were performed using thin film hydration and nanoprecipitation methods towards meso- and nano-scale vesicle formation. By thin film hydration, a few of giant polymersomes were observed. By nanoprecipitation using DMF/water as co-solvents, lamellar sheets and nanofibers were produced at room temperature. Multilamellar polymersomes were successfully obtained by thermal treatment of these self-assemblies. Heat-triggered morphological transformations were for the first time detected on the PSar-b-PCL-b-PSar systems. The mechanism of polymersome formation is discussed at the end of the paper.\

EXPERIMENTAL SECTION

Materials. Sarcosine (98%, Shanghai Adamas Reagent, China), carbon disulfide (99%, Sinopharm Chemical Reagent, China), sodium chloroacetate (98%, Shanghai Jingchun, China), and phosphorus tribromide (98.5%, Sinopharm Chemical Reagent, China), diphenyl phosphate (DPP, 99%, Acros Organics), N-hydroxyphthalimide (NHP, 99%, Shanghai Adamas Reagent, China), triphenyl phosphine (PPh3, 99%, Shanghai Adamas Reagent, China), diisopropyl azodicarboxylate (DIAD, 98%, J&K Chemical), methanol (99%, Sinopharm Chemical Reagent, China), diethyl ether (99%, Sinopharm Chemical Reagent, China), methylene chloride (99%, Sinopharm Chemical Reagent, China) and hydrazine (Sinopharm Chemical Reagent, China) were used as received. Tetrahydrofuran (THF) and toluene

were

refluxed

before

use

over

sodium

6


benzophenone

ketyl

and

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potassium/benzophenone ketyl, respectively. ε-Caprolactone (CL), benzyl alcohol (BO), 1, 4-butanediol (BDO) and dimethyl formamide (DMF) were stirred over CaH2 and distilled.

Sarcosine NTA (Sar-NTA). Sar-NTA was synthesized according to Ref. 58. Typically S-ethoxythiocarbonylmercaptoacetic Acid (XAA) was firstly prepared through the reaction of NaOH, ethanol, carbon disulfide and the following addition of sodium chloroacetate (molar ratio = 1:1.5:1.5:1), and then extracted by chloroform after acidification. Equivalent amounts of

sarcosine

and

XAA were

dissolved

in

basic

aqueous

solution

to

obtain

N-ethoxythiocarbonylsarcosine (Sar-XAA). PBr3 (1.2 times of Sar-XAA) was utilized to catalyze the cyclization of Sar-XAA, and eventually Sar-NTA was obtained after column chromatographic purification (ethyl acetate:petroleum ether = 1:3). 1H NMR (CDCl3/TMS) δ: 3.11 ppm (s, 3H), 4.21 ppm (s, 2H).

Amino-Ended Poly(ε-Caprolactone). All polymerizations were performed using Schlenk technique, and all polymerization tubes were predried and purged with argon. A typical synthesis procedure is described as follows. CL (15.0 mL, 140 mmol), DPP (0.528 g, 2.11 mmol) and BO (0.31 mL, 2.84 mmol) were dissolved in toluene (39 mL) with stirring for 7 h at 25 °C. The polymer was isolated by precipitation from cold methanol and dried in a vacuum (15.347 g, 91.0%). The obtained PCL-OH was dissolved in methylene chloride with NHP (0.548 g, 4.18 mmol) and PPh3 (0.904 g, 4.18 mmol). DIAD (0.6 mL, 3.88 mmol) was then added to the mixture cooled with ice-water bath. The reaction mixture was stirred for 18h, and then precipitated in cold methanol. This reaction was repeated twice to fulfill the conversion to obtain PCL-O-NHP (14.721 g). Hydrazine (0.11 mL, 3.41 mmol) was added

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dropwise to the CH2Cl2 solution of PCL-O-NHP and stirred further for 30 min. Some wax-like precipitates appeared in the solution and were removed by filtration. The purified polymer was isolated by dropping the filtrate into cold methanol. After being dried in vacuum, the oxyamino-ended polymer was obtained (10.654 g, 69.2%).

Polysarcosine-block-Poly(ε-Caprolactone). ROPs of Sar-NTA initiated by PCL-ONH2 and H2NO-PCL-ONH2 were conducted with Schlenk line. As a typical synthetic procedure, after H2NO-PCL40-ONH2 (0.645 g, 0.138 mmol) was completely dissolved in anhydrous THF (8.8 mL), Sar-NTA (0.429 g, 3.27 mmol) was added with stirring at 60 °C. The polymerization was quenched after 24 h and precipitated from diethyl ether. After being dried in vacuum, the triblock copolymer PSar-b-PCL-b-PSar was obtained (0.466 g, 54.8%).

Nanoprecipitation. The nanoprecipitation procedure was conducted according to Ref. 25. Typically the copolymer was dissolved in 1 mL of DMF (at 0.5 or 0.1 wt %). 1 mL of deionized milli-Q water was injected slowly to the organic solution under mild shaking (2−3 µL of water per minute to 1mL of polymer solution). The process of nanoprecipitation was carried out at 25 °C. The turbid mixtures were then dialyzed against water for 3 days to remove DMF using a Spectra/Por regenerated cellulose membrane with a molecular weight cutoff (MWCO) of 3500.

Thin-Film Hydration. The thin-film hydration procedure was employed to assemble the copolymers in a similar approach to Ref. 30. The copolymers were dissolved in chloroform (1 wt%) with 0.01 wt% Nile Red, and deposited on the surface of roughened Teflon. The polymer films were then dried for >12 h under vacuum and rehydrated in a 250 mmol/L

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sucrose aqueous solution. The samples were subsequently sealed and heated at 65 °C for 24 to 48 h. Each aqueous sample was then diluted in 250 mmol/L glucose aqueous solution at the ratio 30:70 for the following microscopic observation.

Characterization. Molecular weights (MWs) and polydispersity indices (PDIs) were determined by size-exclusion chromatography (SEC) which was consisted of a Waters 1515 isocratic high performance liquid chromatograph pump, a Wyatt DAWN DSP MALLS detector, and a column of PLgel 5 µm MIXED-C. DMF containing 0.05 mol/L LiBr was used as the eluent with a flow rate of 1.0 mL/min at 60 °C. Commercial monodispersed poly(methyl methacrylate)s (PMMA) were used as the calibration standards. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance DMX 500 spectrometer (1H 500 MHz) with DMSO-d6 or CDCl3 as solvent and tetramethylsilane (TMS) as internal reference. Differential scanning calorimetry (DSC) analyses were performed on a TA Q200 instrument. The synthesized copolymers were heated from 0 to 130 °C at a rate of 10 °C/min under a nitrogen purge. Turbidity measurements were performed using a UV-2550 UV-Vis spectrophotometer (Shimadzu) equipped with a TCC-240A temperature controlled cell holder (Shimadzu). UV-Vis optical density (O.D.) of the samples was recorded at a wavelength of 600 nm with a slit width of 2 nm. The morphology of polymer self-assemblies was analyzed using cryo-transmission electron microscopy (Cryo-TEM). 5 µL of samples were deposited onto a 200 mesh holey copper grid (Ted Pella Inc., USA) and flash-frozen in liquid ethane cooled down at liquid nitrogen temperature. Cryo-TEM images were acquired on a JEOL 2200FS energy-filtered (20eV) field emission gun electron microscope operating at 200 kV using a Gatan ssCCD 2048 × 2048 pixels. Energy-filtered (Zero-loss) cryo-electron 9


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tomography images were collected at 12000x (corresponding pixel size 0.8 nm) from -64° up to 64° using a Saxton scheme (1° at high tilts and 2° at low tilts). Alignment of the tilt-series and reconstruction of the final 3D volume were computed using TomoJ v2.28.59, 60 Confocal Laser Scanning Microscope (CLSM) images were obtained on a Nikon confocal laser-scanning microscope A1R. Cytotoxicity test was carried out with a sample of the self-assemblies of PSar16-b-PCL40-b-PSar16 (CS10) prepared by nanoprecipitation. In a 6-well culture plate (Nunc/ThermoScientific, Roskilde, Denmark), 5000 cells/150 µL of cell suspension (L929 cell line) were used to seed each well. The cells were incubated overnight to allow for cell attachment, recovery and growth. CS10 suspensions were then added to the wells in order to obtain 6.9, 34.5, 69, 345, 690 µg/mL as final concentration of CS10, respectively. Cells were incubated with CS10 samples for 4 hours at 37 °C. The medium was then removed and replaced by 1.8 mL of fresh culture medium, and 200 µL of a 5 mg/mL solution of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in PBS (Sigma-Aldrich, Saint-Quentin-Fallavier, France) was added to each well. Cells were incubated further at 37 °C for 2 h. The resulting violet formazan precipitate was solubilized by the addition of 500 µL DMSO and was allowed to incubate at 37 °C for additional 10 min. The samples were then analyzed on UV/Vis spectrophotometer (Lambda 800, Perkin Elmer) at 540 nm to determine the absorbance. Experiments were performed in triplicate. Relative cell viability was defined as the ratio of the absorbance of cells incubated with CS10 self-assemblies over the absorbance of cells incubated only with growth medium.

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Scheme 1. Preparation of diblock and triblock copolymers PCL-b-PSar and PSar-b-PCL-b-PSar.

RESULTS AND DISCUSSION

Synthesis and characterization of PSar-b-PCL and PSar-b-PCL-b-PSar

We employed a totally metal-free protocol to prepare the amphiphilic di- and triblock copolymers. The synthesis was conducted in a sequential approach (Scheme 1). Firstly we prepared PCLs with different degrees of polymerization (DP) by ROP of ε-caprolactone with DPP as catalyst.61 BO and BDO were chosen as the initiators respectively for PCLs with one (PCL-OH) or two terminal hydroxyl group(s) (HO-PCL-OH). The transformation from hydroxyl to oxyamino group was achieved based on Mitsunobu reaction.62-66 Three samples of PCL-ONH2 and three samples of NH2O-PCL-ONH2 were synthesized with low PDIs and high yields (see Table 1, PCL1 to PCL6). ROP of Sar-NTA was then initiated by these PCL oxyamines on Schlenk line (glovebox-free). In previous works we showed that NTA was a promising monomer that could be synthesized easily and be kept stable for a longer time than 11


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NCA. The polymerization of NTA with high yields, high MWs and low PDIs was achieved with appropriate initiators and conditions.44, 58 Herein ROP of sarcosine NTA was for the first time initiated by oxyamino-ended PCLs, and this initiator endowed the copolymer with a cleavable N-O bond between PSar and PCL blocks (e.g., cleavage under reduction by Pd-catalyzed hydrogenolysis or in acidic environment).67-69 Di- and triblock copolymers, PSar-b-PCL and PSar-b-PCL-b-PSar, with different MWs and hydrophilic/hydrophobic ratios were obtained (see Table 1, CS1 to CS10). Typical characterization results (NMR and SEC) are shown in Figure 1 and 2 for CS10 and its macro-initiator PCL5. 1H NMR spectra in Figures 1A, Figure S1 and 1B reveal a complete transformation of –OH end-group to –O-NH2 group in the synthesis of macro-initiator PCL5. 1H NMR spectrum of CS10 (Figure 1C) shows legible signals from both PCL and PSar blocks, while SEC chromatogram of CS10 together with that of its initiator PCL5 (Figure 2) show clearly a MW increase from 9.0 kDa for PCL5 to 14.5 kDa for CS10 without distribution broadening (PDI from1.25 to 1.24). These

results

indicate

the

successful polymerization

of

oxyamino-ended PCL5.

12


Sar-NTAs

initiated

by

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Figure 1. 1H NMR spectra of HO-PCL40-OH (A), PCL5 (B) in CDCl3 and CS10 (C) in DMSO-d6.

Figure 2. SEC chromatograms (DMF/LiBr as eluent). A: PCL5 (H2NO-PCL40-ONH2) and B: CS10 (PSar16-b-PCL40-b-PSar16).

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Table 1. Syntheses of macro-initiators PCL and block copolymers PCL-b-PSar and PSar-b-PCL-b-PSar.

a

Mn NMR c

Mn SEC d

(kDa)

(kDa)

-

2.3

3.9

1.12

92.5

PCL35-ONH2

-

4.1

6.4

1.12

69.2

70

PCL80-ONH2

-

9.2

14.1

1.36

74.3

50

28

H2NO-PCL28-ONH2

-

3.2

5.5

1.17

81.6

BDO

50

35

H2NO-PCL40-ONH2

-

4.7

9.0

1.25

65.4

PCL6

BDO

50

70

H2NO-PCL70-ONH2

-

8.1

14.8

1.30

78.9

CS1

PCL1

60

23

PCL20-b-PSar8

20/80

2.9

5.0

1.16

50.8

CS2

PCL1

60

19

PCL20-b-PSar24

43/57

4.0

6.2

1.24

61.6

CS3

PCL1

60

38

PCL20-b-PSar48

60/40

5.7

9.2

1.24

73.9

CS4

PCL2

60

30

PCL35-b-PSar5

10/90

4.5

7.0

1.18

58.6

CS5

PCL3

60

70

PCL80-b-PSar70

35/65

14.2

21.5

1.35

82.1

CS6

PCL4

60

11

PSar4-b-PCL28-b-PSar4

15/85

3.8

6.8

1.35

> 99

CS7

PCL4

60

21


25/75

4.3

7.9

1.37

95.6

CS8

PCL4

60

20


36/64

6.3

8.4

1.3

91.3

CS9

PCL4

60

48


48/52

6.3

10.7

1.41

92.4

CS10

PCL5

60

22


34/66

7.0

14.5

1.24

54.8

CS11

PCL6

60

38


14/86

9.6

18.9

1.37

82.3

CS12

PCL6

60

62


26/74

10.8

22.8

1.36

68.5

CS13

PCL6

60

48


29/71

11.4

21.8

1.35

83.9

Sample

Initiator

T (°C)

[M]/[I]

Composition a

PCL1

BO

50

20

PCL20-ONH2

PCL2

BO

50

35

PCL3

BO

50

PCL4

BDO

PCL5

Determined by 1H NMR.

results;

c

b

Block ratio

b

PDI d

Yield (%)

Hydrophilic/hydrophobic block weight ratio calculated from 1H NMR

Average MWs of PCL initiators and copolymers calculated from 1H NMR results;

d

Determined by SEC (DMF/LiBr as eluent) with PMMA as standard.

Nevertheless, DPs of PSar blocks could not be accurately controlled by feeding ratio, PDIs of copolymers were not always below 1.2, and tails of SEC peaks were recorded for some copolymer samples (Figure S2). These observations can be explained by two factors.

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On one hand the oxyamino group is less nucleophilic than alkylamino group and has a relatively low initiation activity. Thus the initiation reaction is slow, which may results in relatively high PDI. On the other hand the relatively low reactivity of NTA makes the time necessary for the polymerization longer (up to 48 h) and the reaction temperature higher (60 °C). Consequently the possibility of side reactions increases, e.g., the nucleophilic attack of amino end groups on ester linkages of PCL block 70 that makes chain scission and forms chains of low MWs detected as tails in SEC curves. Fortunately, previous report on the PEG-b-PCL polymersomes has demonstrated that the polydispersity has little effect on the possibility of vesicle formation.34 In some cases polydispersity can influence the curvature of the membrane and consequently the sizes and size distribution of polymersomes, because longer chains prefer to locate in the outer leaflet and shorter chains in the inner leaflet.71

Figure 3. DSC thermograms of the first heating scan of copolymer samples recorded at 10 °C/min. A: CS7 (PSar8-b-PCL28-b-PSar8) and B: CS10 (PSar16-b-PCL40-b-PSar16). Tm is the melting temperature of PCL block and Tg the glass transition of PSar block.

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The thermal behaviors of copolymers were analyzed by DSC. Figure 3 shows typical DSC thermograms of CS7 and CS10 at the first heating scan at 10 °C/min. CS7 exhibited a melting peak with peak value Tm = 54 °C and melting enthalpy ∆Hm = 46.1 J/g, while CS10 had Tm = 53 °C and ∆Hm = 27.5 J/g. Both melting peaks were attributed to PCL melting.72 When weighted by the weight ratio of PCL part, the melting enthalpy equals to 61.5 J/g for CS7 (75% PCL) and 41.7 J/g for CS10 (66% PCL). These values are smaller than that of ∆Hm (136 J/g) of PCL homopolymer with 100% crystallinity, reported in Ref. 60, suggesting the PCL blocks are semi-crystalline in the block copolymers. Higher is PSar/PCL ratio, lower is the PCL crystallinity. A detectable glass transition temperature (Tg) for PSar block is recorded at 40 °C for CS7. For CS10, we attempt to assign the signal around 62 °C as the glass transition of PSar block, however this assignment need to be further confirmed because it was not observable either in first cooling scan or in the second heating scan (see Figure S3). Comparing to Tg of the homopolymers PSar10 (Tg = 36°C), PSar14 (Tg = 47°C) and PSar19 (Tg = 59°C) (see Figure S4) measured in the same conditions, the Tg values found here for CS7 and CS10 are reasonable.

Self-assembly of PSar-b-PCL-b-PSar in aqueous solution

The formation of polymersomes by self-assembling polysarcosine-containing block copolymers has interested several groups.26, 45, 48, 73 Even though vesicular structures have been claimed for PSar-b-PCL and PSar-b-PLLA self-assemblies (PLLA: poly-L-lactic acid), no structural evidence was shown clearly.73 Only in the case of an amphiphilic block

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polypeptide, vesicular structure was demonstrated by cryo-TEM and TEM experiments without ambiguity. PSar27-b-P(L-Leu-Aib)6 having a right-handed helix segment and PSar27-b-P(D-Leu-Aib)6 having a left-handed helix segment self-assembled first into two types of peptide nanotubes. These nanotubes transform into vesicles by membrane fusion due to stereo-complex formation between the right-handed and left-handed helical segments.26

We focus herein on polymersome formation by triblock copolymers PSar-b-PCL-b-PSar. Vesicular morphologies are generally considered to be generated from the block copolymers possessing a hydrophilic weight fraction of 20%~40% based on diblock copolymers with poly(butadiene) or poly(ethylethylene) as hydrophobic block.1, 30 Hence CS7 (25%) and CS10 (34%) were first chosen for systematic studies of self-assembly in order to obtain polymersomes. Polymer self-assembling by thin-film hydration method was first carried out. Polymer thin films were prepared with mixtures of copolymers and Nile Red (100:1 in weight) as described in the experimental section, followed by hydration at 65 °C for 24 h. The temperature of 65 °C was higher than the Tm of PCL and Tg of PSar. Therefore chain movement was enabled to facilitate self-assembling. Rods (Figures 4A and 4B) and giant vesicles (Figures 4C and 4D) were observed for both copolymers CS7 and CS10. The observation of giant polymersomes is encouraging because it indicates the membrane structure can be formed by both copolymers.

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Figure 4. Fluorescence confocal micrograph of self-assemblies obtained from CS7 (A, C) and CS10 (B, D). Nile Red was added in the preparation at a weight ratio of 1:100 (Nile Red to copolymer) for observation. Rods and giant vesicles were observed. Scale bar = 10 µm.

Then we performed self-assembling using nanoprecipitation since this technique was a common one to prepare nano-sized polymersomes.18,

27, 74

Water was dropped into a

copolymer solution in DMF, and PCL blocks tended to assemble together by hydrophobic interaction as water content increased. Turbidity measurement was conducted during water addition until water content reached 50% (see Figure 5). For both copolymers the turbidity of solutions increased sharply at water content around 5%, revealing the appearance of nanoparticles. The turbid mixtures were then dialyzed against water for 3 days to remove DMF. After dialysis, the mixtures remained turbid and visible precipitates appeared at bottom of flask after hours of standing (data not shown). Dynamic laser scattering (DLS) was used to try to measure the hydrodynamic diameters of nanoparticles. Unfortunately, multi-modal distributions were detected (Figure S5). Cryo-TEM was then utilized to visualize the

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morphologies. Large lamellar structures were observed for self-assemblies of CS7 (Figure 6A and Figure S6), while lamellae together with long nanofibers were observed for CS10 (Figure 6B and Figure S6). The membrane thickness can be measured in the place where the sheet folds up. It is 8 - 9 nm for CS7 and 9 - 10 nm for CS10, and the diameter of nanofibers of CS10 is of 9 - 10 nm. As only hydrophobic part is measured in cryo-TEM, the values of 8 - 9 nm and 9 - 10 nm correspond to the PCL thickness of the unilamellar organization of triblock copolymers. In the case of CS10 (PSar16-b-PCL40-b-PSar16), the measured thickness is in good agreement with the value reported for vesicles of PEO23-b-PCL40 where a bilayer membrane of PCL40 is around 15 nm.28 The interdigitation and entanglement of the polymer chains cause partially blending of hydrophobic layers of diblock copolymers in the unilamellar organization,75,

76

while triblock copolymers are considered to have a mixed

conformation of the bent U-shape and the stretched I-shape in the self-assembled membrane, with less diffusivities.76 That is the reason why the bilayer thickness of PEO23-b-PCL40 is slightly smaller than the twice of that of PSar16-b-PCL40-b-PSar16. The lamellar structure is a good indication of the possibility of vesicle formation, because the generally assumed pathway in lipids or block copolymer amphiphiles in water concludes with the wrapping-up of lamellar sheets to form the final vesicles by minimizing the sum of curvature energy and interfacial energy (Mechanism I).4,

77, 78

Butler’s group studied the mechanism of vesicle

formation of PEG-b-PCL in THF upon water addition, and found that vesicles formed at room temperature through worm-like micelles and sheet-like intermediates as water content increased.28 They proposed a slightly different pathway where the two ‘‘folded-over’’ bi-layers were in close contact, thus bringing the edges together for ‘‘zipping-up’’. In our case,

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neither “wrapping-up” nor “zipping-up” of lamellar sheets took place during the water addition to polymer solution in DMF, probably because of the lack of sufficient mobility of PSar-b-PCL-b-PSar structures. Some assistance in mobility would be helpful to the membrane enclosure. Then we performed a thermal treatment on the self-assemblies.

Figure 5. Turbidity versus water content measured at wavelength of 600 nm for CS7 (○) and CS10 (■) at 25 °C. Water was added dropwise to a solution of polymer in DMF at an initial concentration of 5 mg/mL.

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Figure 6. Cryo-TEM images of polymer self-assemblies in water: (1) CS7 (A) and CS10 (B) as prepared by nanoprecipitation at room temperature; (2) CS7 (C) and CS10 (D) after 30-minutes heating at 65 °C; (3) CS7 (E) and CS10 (F) after 24-hours heating at 90 °C. Scale bar = 300 nm in A, B, E and F. Scale bar = 100 nm in C and D. For the lamellar sheets in A and B, the membrane thickness can be measured in the place where the sheet folds up, indicated by the two pairs of arrows.

The aqueous suspensions of CS7 and CS10 nanoparticles were first heated at 65 °C for 30 min followed by a slow cooling to room temperature at about 0.2 °C/min. The temperature of 65 °C was chosen since it was higher than both the Tm of PCL block and the Tg of PSar block (Figure 3). Major changes were indeed observed by Cryo-TEM in the samples after the heating treatment. Spheres with diameters ranging from 30 to 100 nm, together with worm-like cylinders having similar diameters and a few nanofibers were generated in CS7 (Figure 6C). The solid spherical nature rather than disk-like one for the observed

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round-particles was proved by cryo-tomography made on CS7 sample (Figure 7). In Figure 7 different morphologies are represented in different colors: full spheres in blue, worm-like cylinders in violet, thin nanofibers in orange; we even observed one vesicle with empty core near the center of image (in yellow). More vesicles were observed in CS10 after heating at 65 °C (Figure 6D), even though the majority of morphologies remained spheres and worm-like cylinders (Figure 6D and Figure S7). We noticed also that solid spheres seemed to bud from the tip of cylinders (Figure 6C, 6D and Figure S7). To try to complete the morphological transformation a reinforced thermal treatment was then carried out for both samples by heating at 90 °C for 24 h. In the case of CS7, only increases in size and in amount of the spheres occurred accompanied by the disappearance of worm-like cylinders (Figure 6E). However, abundant vesicular structures with diameters ranging mainly from 100 to 500 nm and with membrane thickness from 20 - 50 nm were indeed generated in CS10 sample (Figure 6F and Figure S8), with the co-existence of small solid spheres. Some vesicles-in-vesicles structures, onion-like vesicles and small spheres (diameter typically smaller than 50 nm) were also recorded (Figure S8). The hydrodynamic diameter measurement by DLS on the post-heating samples (Figure S5) was in good agreement with the observation of cryo-TEM, nearly monomodal size distribution below 1000 nm being detected. Furthermore, dynamic turbidity measurements were performed during the thermal treatment of CS7 and CS10 suspensions at a rate of 1 °C/min. Figure 8 shows the optical densities of the samples recorded as a function of temperature. Dramatic declines of turbidities happened around 50-55 °C for both samples, corresponding to the size decrease of the particles in the suspensions from larges sheets (width > 1000 nm) and long nanofibers to smaller spheres, worm-like cylinders

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and vesicles (D < 1000 nm). This temperature range around 50-55 °C falls into the melting peak of PCL block recorded in DSC analysis. Therefore, these dynamic turbidity measurements suggest that the chain mobility improvement upon melting is responsible for the morphological change. Direct observation with naked eyes showed the turbid suspensions transformed to translucent ones when heating from 25 to 80 °C (Figure S9). The transformation was kept unchanged as sample cooling back to room temperature, and stable even for months of conservation at room temperature. This morphological preservation suggests that the transitions are irreversible and the resulted morphologies are probably frozen by PCL partial crystallization.

Figure 7. Diversity of CS7 polymer nanoparticles obtained after heating treatment at 65°C (same sample as Figure 6C), unveiled by cryo-electron tomography. Solid spheres of various diameters in blue; cylindrical structures in purple; nanofibers in orange; a vesicle in yellow.

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Figure 8. Turbidity evolution as a function of temperature for aqueous dispersions of self-assemblies of CS7 (○) and CS10 (■) prepared by nanoprecipitation, measured at wavelength of 600 nm with temperature interval of 1 °C at the rate of 1 °C/min from 25 to 80 °C.

The morphological transformation upon thermal treatment as aforementioned shows how the preparation method can influence the vesicle formation. Other works in the literature also reported that the formation of nano-sized polymersomes were not only dependent on polymer natures but also strongly affected by the preparative conditions.27-29,

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For example,

poly(ethylene oxide)-block-poly(butylene oxide) (PEO-b-PBO), a rather lipid-like copolymer in term of solubility, could form first lamellar phase in concentrated aqueous solution at room temperature.29 A transformation from this lamellar phase to dispersed vesicles was obtained by a simple diluting process, and membrane undulation and membrane unbinding were proposed to be responsible for the vesicle formation. In contrast, sheet-like lamellar structures formed at room temperature from the stereo-complex of two polysarcosine-containing copolypeptides, (Sar)27-b-(L-Leu-Aib)6 and (Sar)27-b-(D-Leu-Aib)6, were transformed into vesicles only after heating at 90 °C.26 Nevertheless, in the present work the formation of 24


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vesicles did not seem to proceed via lamellar sheet enclosing. A detailed analysis of polymersomes of the sample CS10 (PSar16-b-PCL40-b-PSar16) revealed that the vesicles were not unilamellar but multilamellar. The unilamellar thickness of CS10 at room temperature was 9-10 nm (Figure 6B), while the vesicles detected after annealing at 90 °C had membrane thickness ranging from 20-50 nm and the multilayers were visible in the cryo-TEM images (Figure S8). Moreover, the self-assemblies treated at 65 °C are mainly spheres, worm-like cylinders with a few of vesicles, but without any lamellar sheets remaining. After heating at 90 °C, vesicles were observed only for CS10, while CS7 (PSar8-b-PCL28-b-PSar8) concluded with spheres. Therefore, we propose a different scenario for the vesicle formation here. Upon heating from room temperature to 65 °C, lamellar sheets reorganized into cylindrical and spherical polymer nanoparticles; upon further heating to 90 °C, cylindrical structures were transformed completely into spheres (by budding) and water diffused then into the spherical structures. In the case of CS10, multilamellar vesicles were eventually formed. As for CS7 the final morphology was mainly spherical polymer nanoparticles; we have no explanation for the difference between CS7 and CS10 self-assembling behavior. More studies on the self-assemblies of copolymers with extended composition ranges are necessary. This work is in progress.

Two mechanisms I and II have been proposed for the polymer vesicle formation in the literature. In mechanism I, amphiphilic block copolymers rapidly self-assemble into small spherical micelles, which then slowly evolve into cylindrical micelles and open disc-like micelles by collision. The large disc-like micelles then slowly close up to form vesicles.79 In mechanism II, the initial spherical micelles, rather than evolving toward cylindrical micelles 25


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and open-disk micelles, regroup the copolymer chains remaining in solution to increase their size, forming bigger and complex spherical micelles with a hydrophilic core. The solvent then diffuses into the bigger micelles to lower their bending energy by increasing the radius of curvature, leading to vesicles.80 The scenario of vesicle formation for the CS10 proposed in this work corresponds to the mechanism II rather than the mechanism I of polymersome formation, even though the pathway to achieve the bigger and complex micelles (called here spherical particles) is different from that described theoretically. Such a mechanism that polymersomes are formed by water diffusion into bigger and complex micelles upon thermal treatment, if being confirmed, will have considerable impact on the encapsulation in polymersomes.81 Their ability to trap hydrophilic molecules should be extremely reduced. The loading efficiency for hydrophilic molecules into the aqueous interior of polymersomes should be consequently low, as already reported on vesicles made from the self-assembly of poly(ethylene oxide)-block-poly(N,N-diethylaminoethyl methacrylate) (PEO-b-PDEAMA) by a pH switch.81 However, hydrophobic drugs or contrast agents can still be incorporated efficiently in the thick membrane of polymersomes.

Cell viability test

To assess the biocompatibility, the dialyzed aqueous solution of CS10 was employed in cell viability test with MTT as the indicator. The histogram presented in Figure 9 declared that the relative viability of the tested cells sustained to be over 80% even with the self-assemblies of CS10 in a concentration of 0.69 mg/mL, which confirmed reliable

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biocompatibility of PSar-b-PCL copolymers and their self-assembly structures.

Figure 9. Relative cell viability of CS10 for L929 cells after 4 h incubation at 37 °C and at concentration of 6.9, 34.5, 69.0, 345 and 690 µg/mL.

CONCLUSION

We reported on a simple and totally metal-free method for the synthesis of a series of PSar-b-PCL and PSar-b-PCL-b-PSar block copolymers based on ROP of Sar-NTA using PCL ended with oxyamine group(s) as initiators. The self-assembling behaviors of two triblock copolymers PSar8-b-PCL28-b-PSar8 (CS7) and PSar16-b-PCL40-b-PSar16 (CS10) in water were studied in detail using thin-film hydration and nanoprecipitation methods. Rod-like particles and a few of giant polymersomes were obtained by thin film hydration at 65 °C. Large unilamellar sheets and nanofibers (of 8-10 nm in thickness and diameters) were formed by nanoprecipitation at room temperature using DMF/water as co-solvents. A thermal treatment by heating the aqueous suspensions to 65 °C transformed the lamellae and fibrous structures 27


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into worm-like cylinders and spheres (of 30-100 nm in diameter). Heating the initial aqueous suspension of nanoprecipitation to 90 °C led multilamellar polymersomes (of 100-500 nm in diameter and 20-50 nm in thickness) coexisting with a few polymer spheres for CS10, while only polymer spheres were obtained for CS7 after heating to 90 °C. The turbidity measurement as a function of temperature showed the morphological transformation started around the melting point of PCL blocks, suggesting a close connection with the crystalline behavior of PCL and its mobility. A pathway close to mechanism II of polymersome formation was then proposed, where multilamellar polymersomes are formed by water diffusion into polymer spheres (i.e., bigger and complex micelles) upon thermal treatment. The cell viability test on the self-assemblies of CS10 confirmed that the PSar-b-PCL systems developed in this work were not cytotoxic and suitable for possible applications in biomedical domains.

Supporting Information Available

Supplemental NMR spectrum, SEC curves, DSC thermograms, phase transition data, DLS plots, cryo-TEM images and photographs of self-assemblies’ dispersions. This material is available at free of charge via the Internet at http://pubs.acs.org

ACKNOWLEDGEMENTS

This work received financial support from the National Natural Science Foundation of China 28


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(21174122, 21528402) and from the program “PIC targeted therapy” of Institut Curie. We acknowledge the PICT-IBiSA for providing access to chemical imaging equipment, and the Nikon Imaging Center@Institut Curie for confocal microscopy.

REFERENCES 1. Discher, D. E.; Eisenberg, A. Science 2002, 297 (5583), 967-973. 2. Blanazs, A.; Armes, S. P.; Ryan, A. J. Macromol. Rapid. Comm. 2009, 30 (4-5), 267-277. 3. Jain, S.; Bates, F. S. Science 2003, 300 (5618), 460-464. 4. Antonietti, M.; Forster, S. Adv. Mater. 2003, 15 (16), 1323-1333. 5. Gennes, P.-G. D. Solid State Physics 1978, Suppl. 14, 1-18. 6. Zhang, L. F.; Eisenberg, A. Polym. Advan. Technol. 1998, 9 (10-11), 677-699. 7. Halperin, A.; Tirrell, M.; Lodge, T. P. Adv. Polym. Sci. 1992, 100, 31-71. 8. Zhulina, E. B.; Borisov, O. V. Macromolecules 2012, 45 (11), 4429-4440. 9. Discher, D. E.; Ahmed, F., Polymersomes. In Annu. Rev. Biomed. Eng., 2006; Vol. 8, pp 323-341. 10. LoPresti, C.; Lomas, H.; Massignani, M.; Smart, T.; Battaglia, G. J. Mater. Chem. 2009, 19 (22), 3576-3590. 11. Le Meins, J. F.; Sandre, O.; Lecommandoux, S. Eur. Phys. J. E 2011, 34 (2), 14. 12. Li, M.-H.; Keller, P. Soft Matter 2009, 5 (5), 927-937. 13. Mabrouk, E.; Cuvelier, D.; Brochard-Wyart, F.; Nassoy, P.; Li, M.-H. P. Natl. Acad. Sci. U.S.A. 2009, 106 (18), 7294-7298. 14. Hocine, S.; Brulet, A.; Jia, L.; Yang, J.; Di Cicco, A.; Bouteiller, L.; Li, M.-H. Soft Matter 2011, 7 (6), 2613-2623. 15. Jia, L.; Cui, D.; Bignon, J.; Di Cicco, A.; Wdzieczak-Bakala, J.; Liu, J.; Li, M. H. Biomacromolecules 2014, 15 (6), 2206-2217. 16. Lee, J. S.; Feijen, J. J. Control. Release 2012, 158 (2), 312-318. 17. Yassin, M. A.; Appelhans, D.; Wiedemuth, R.; Formanek, P.; Boye, S.; Lederer, A.; 29


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Temme, A.; Voit, B. Small 2015, 11 (13), 1580-1591. 18. Yang, X.; Grailer, J. J.; Rowland, I. J.; Javadi, A.; Hurley, S. A.; Steeber, D. A.; Gong, S. Biomaterials 2010, 31 (34), 9065-9073. 19. Meng, F.; Zhong, Z.; Feijen, J. Biomacromolecules 2009, 10 (2), 197-209. 20. Ghoroghchian, P. P.; Frail, P. R.; Susumu, K.; Blessington, D.; Brannan, A. K.; Bates, F. S.; Chance, B.; Hammer, D. A.; Therien, M. J. P. Natl. Acad. Sci. U.S.A. 2005, 102 (8), 2922-2927. 21. Grafe, D.; Gaitzsch, J.; Appelhans, D.; Voit, B. Nanoscale 2014, 6 (18), 10752-10761. 22. Du, J.; O'Reilly, R. K. Soft Matter 2009, 5 (19), 3544-3561. 23. Christian, N. A.; Benencia, F.; Milone, M. C.; Li, G.; Frail, P. R.; Therien, M. J.; Coukos, G.; Hammer, D. A. Mol. Imaging Biol. 2009, 11 (3), 167-177. 24. Carlsen, A.; Lecommandoux, S. Curr. Opin. Colloid In 2009, 14 (5), 329-339. 25. Jia, L.; Cao, A.; Lévy, D.; Xu, B.; Albouy, P.-A.; Xing, X.; Bowick, M. J.; Li, M.-H. Soft Matter 2009, 5 (18), 3446-3451. 26. Ueda, M.; Makino, A.; Imai, T.; Sugiyama, J.; Kimura, S. Chem. Commun. 2011, 47 (11), 3204-3206. 27. Jia, L.; Lévy, D.; Durand, D.; Impéror-Clerc, M.; Cao, A.; Li, M.-H. Soft Matter 2011, 7 (16), 7395-7403. 28. Adams, D. J.; Kitchen, C.; Adams, S.; Furzeland, S.; Atkins, D.; Schuetz, P.; Fernyhough, C. M.; Tzokova, N.; Ryan, A. J.; Butler, M. F. Soft Matter 2009, 5 (16), 3086-3096. 29. Battaglia, G.; Ryan, A. J. Macromolecules 2006, 39 (2), 798-805. 30. Ghoroghchian, P. P.; Li, G. Z.; Levine, D. H.; Davis, K. P.; Bates, F. S.; Hammer, D. A.; Therien, M. J. Macromolecules 2006, 39 (5), 1673-1675. 31. Talelli, M.; Barz, M.; Rijcken, C. J. F.; Kiessling, F.; Hennink, W. E.; Lammers, T. Nano Today 2015, 10 (1), 93-117. 32. Sinha, V. R.; Bansal, K.; Kaushik, R.; Kumria, R.; Trehan, A. Int. J. Pharm. 2004, 278 (1), 1-23. 33. Balakrishnan, B.; Kumar, D. S.; Yoshida, Y.; Jayakrishnan, A. Biomaterials 2005, 26 (17), 3495-3502. 34. Qi, W.; Ghoroghchian, P. P.; Li, G.; Hammer, D. A.; Therien, M. J. Nanoscale 2013, 5 30


Page 30 of 34

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(22), 10908-10915. 35. Sui, X.; Kujala, P.; Janssen, G.-J.; de Jong, E.; Zuhorn, I. S.; van Hest, J. C. M. Polym. Chem. 2015, 6 (5), 691-696. 36. Lee, J. S.; Feijen, J. J. Control. Release 2010, 148 (1), E15-E16. 37. Hamada, I.; Hunter, A. C.; Szebeni, J.; Moghimi, S. M. Mol. Immunol. 2008, 46 (2), 225-232. 38. Sung, H.-J.; Luk, A.; Murthy, N. S.; Liu, E.; Jois, M.; Joy, A.; Bushman, J.; Moghe, P. V.; Kohn, J. Soft Matter 2010, 6 (20), 5196-5205. 39. Barz, M.; Luxenhofer, R.; Zentel, R.; Vicent, M. J. Polym. Chem. 2011, 2 (9), 1900-1918. 40. Zhang, D.; Lahasky, S. H.; Guo, L.; Lee, C.-U.; Lavan, M. Macromolecules 2012, 45 (15), 5833-5841. 41. Ostuni, E.; Chapman, R. G.; Holmlin, R. E.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17 (18), 5605-5620. 42. Ulbricht, J.; Jordan, R.; Luxenhofer, R. Biomaterials 2014, 35 (17), 4848-4861. 43. Birke, A.; Huesmann, D.; Kelsch, A.; Weilbacher, M.; Xie, J.; Bros, M.; Bopp, T.; Becker, C.; Landfester, K.; Barz, M. Biomacromolecules 2014, 15 (2), 548-557. 44. Tao, X.; Deng, C.; Ling, J. Macromol. Rapid. Comm. 2014, 35 (9), 875-881. 45. Cui, S.; Wang, X.; Li, Z.; Zhang, Q.; Wu, W.; Liu, J.; Wu, H.; Chen, C.; Guo, K. Macromol. Rapid. Comm. 2014, 35 (22), 1954-1959. 46. Heller, P.; Birke, A.; Huesmann, D.; Weber, B.; Fischer, K.; Reske-Kunz, A.; Bros, M.; Barz, M. Macromol. Biosci. 2014, 14 (10), 1380-1395. 47. Hara, E.; Ueda, M.; Kim, C. J.; Makino, A.; Hara, I.; Ozeki, E.; Kimura, S. J. Pept. Sci. 2014, 20 (7), 570-577. 48. Huesmann, D.; Sevenich, A.; Weber, B.; Barz, M. Polymer 2015, 67, 240-248. 49. Deng, Y.; Zou, T.; Tao, X.; Dembele, F.; Ling, J.; Li, M.-H., Polymersomes of biodegradable polysarcosine-block-poly(ε-caprolactone). In The Third Symposium on Innovative Polymers for Controlled Delivery Suzhou, China, 2014. 50. Kricheldorf, H. R. Angew. Chem. Int. Edit. 2006, 45 (35), 5752-5784. 51. Deming, T. J., Polypeptide and polypeptide hybrid copolymer synthesis via NCA polymerization. In Peptide Hybrid Polymers, Klok, H. A.; Schlaad, H., Eds. 2006; Vol. 202, 31


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pp 1-18. 52. Luxenhofer, R.; Fetsch, C.; Grossmann, A. J. Polym. Sci. Pol. Chem 2013, 51 (13), 2731-2752. 53. Aliferis, T.; Iatrou, H.; Hadjichristidis, N. Biomacromolecules 2004, 5 (5), 1653-1656. 54. Kramer, J. R.; Deming, T. J. Biomacromolecules 2010, 11 (12), 3668-3672. 55. Kato, H.; Higashim.T; Okamura, S. Makromol. Chem. 1967, 109 (Nov), 9-21. 56. Dewey, R. S.; Schoenew.Ef; Joshua, H.; Paleveda, W. J.; Schwam, H.; Barkemey.H; Arison, B. H.; Veber, D. F.; Denkewal.Rg; Hirschma.R. J. Am. Chem. Soc. 1968, 90 (12), 3254-3255. 57. Kricheldorf, H. R.; Sell, M.; Schwarz, G. J. Macromol. Sci., Part A: Pure Appl. Chem. 2008, 45 (6), 425-430. 58. Tao, X.; Deng, Y.; Shen, Z.; Ling, J. Macromolecules 2014, 47 (18), 6173-6180. 59. Sanchez Sorzano, C. O.; Messaoudi, C.; Eibauer, M.; Bilbao-Castro, J. R.; Hegerl, R.; Nickell, S.; Marco, S.; Carazo, J. M. BMC Bioinformatics 2009, 10, 124. 60. Messaoudil, C.; Boudier, T.; Oscar Sanchez Sorzano, C.; Marco, S. BMC Bioinformatics 2007, 8, 288. 61. Makiguchi, K.; Satoh, T.; Kakuchi, T. Macromolecules 2011, 44 (7), 1999-2005. 62. Thygesen, M. B.; Sorensen, K. K.; Clo, E.; Jensen, K. J. Chem. Commun. 2009,

(42),

6367-6369. 63. Novoa-Carballal, R.; Muller, A. H. Chem. Commun. 2012, 48 (31), 3781-3783. 64. Holder, P. G.; Francis, M. B. Angew. Chem. Int. Edit. 2007, 46 (23), 4370-4373. 65. Novoa-Carballal, R.; Pfaff, A.; Müller, A. H. E. Polym. Chem. 2013, 4 (7), 2278. 66. Schlick, T. L.; Ding, Z. B.; Kovacs, E. W.; Francis, M. B. J. Am. Chem. Soc. 2005, 127 (11), 3718-3723. 67. Frankel, M.; Knobler, Y.; Bonni, E.; Bittner, S.; Zvilicho.G. J. Chem. Soc. C -Org. 1969, (13), 1746-1749. 68. Krogsgaardlarsen, P.; Nielsen, L.; Falch, E.; Curtis, D. R. J. Med. Chem. 1985, 28 (11), 1612-1617. 69. Wathen, S. P.; Czarnik, A. W. Bioorg. Med. Chem. Lett. 1993, 3 (6), 1245-1246. 70. Koide, A.; Kishimura, A.; Osada, K.; Jang, W. D.; Yamasaki, Y.; Kataoka, K. J. Am. 32


Page 33 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Chem. Soc. 2006, 128 (18), 5988-5989. 71. Terreau, O.; Luo, L. B.; Eisenberg, A. Langmuir 2003, 19 (14), 5601-5607. 72. Perez, R. A.; Cordova, M. E.; Lopez, J. V.; Hoskins, J. N.; Zhang, B.; Grayson, S. M.; Mueller, A. J. React. Funct. Polym. 2014, 80, 71-82. 73. Makino, A.; Yamahara, R.; Ozeki, E.; Kimura, S. Chem. Lett. 2007, 36 (10), 1220-1221. 74. Yang, J.; Levy, D.; Deng, W.; Keller, P.; Li, M. H. Chem. Commun. 2005,

(34),

4345-4347. 75. Battaglia, G.; Ryan, A. J. J. Am. Chem. Soc. 2005, 127 (24), 8757-8764. 76. Itel, F.; Chami, M.; Najer, A.; Lörcher, S.; Wu, D.; Dinu, I. A.; Meier, W. Macromolecules 2014, 47 (21), 7588-7596. 77. Leng, J.; Egelhaaf, S. U.; Cates, M. E. Biophys. J. 2003, 85 (3), 1624-1646. 78. Goltsov, A. N.; Barsukov, L. I. J. Biol. Phys. 2000, 26 (1), 27-41. 79. Uneyama, T. J. Chem. Phys. 2007, 126 (11), 114902. 80. He, X. H.; Schmid, F. Macromolecules 2006, 39 (7), 2654-2662. 81. Adams, D. J.; Adams, S.; Atkins, D.; Butler, M. F.; Furzeland, S. J. Control. Release 2008, 128 (2), 165-170.

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Biomacromolecules

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Graphical Abstract for Table of Contents

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Poly(Îµ-caprolactone)-block-polysarcosine by Ring-Opening

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