Poly(ε-caprolactone)-block-polysarcosine by Ring-Opening Polymerization of Sarcosine N-Thiocarboxyanhydride: Synthesis and Thermoresponsive Self-Assembly

Article abstract of DOI:10.1021/acs.biomac.5b00930

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 toward polymersome formation using thin-film hydration and nanoprecipitation techniques. A few giant vesicles were obtained by thin-film hydration from both copolymers and visualized by confocal laser scanning microscope. Unilamellar sheets and nanofibers (with 8-10 nm thickness or diameter) were obtained by nanoprecipitation at room temperature and observed by Cryo-TEM. These lamellae and fibrous structures were transformed into worm-like cylinders and spheres (D 30-100 nm) after heating to 65°C (>T_m,PCL). Heating CS10 suspensions to 90°C led eventually to multilamellar polymersomes (D 100-500 nm). Mechanism II, where micelles expand to vesicles through water diffusion and hydrophilic core forming, was proposed for polymersome formation. A cell viability test confirmed the self-assemblies were not cytotoxic.

Full text of DOI:10.1021/acs.biomac.5b00930

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Article
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(εꢀcaprolactone)ꢀblockꢀPolysarcosine by RingꢀOpening Polymerization
of Sarcosine NꢀThiocarboxyanhydride: Synthesis and Thermoꢀresponsive
SelfꢀAssembly
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2,a
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Yangwei Deng,
Tao Zou, Xinfeng Tao, Vincent Semetey,
Sylvain
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1,*
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Trepout, Sergio Marco, Jun Ling, MinꢀHui Li
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MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of
Polymer Science and Engineering, Zhejiang University, 310027 Hangzhou, China.
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Institut Curie ꢀ CNRS ꢀ Université Pierre & Marie Curie, Laboratoire PhysicoꢀChimie Curie,
UMR168, 26 Rue d’Ulm, 75248 Paris, France.
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Institut de Recherche de Chimie Paris, UMR8247, CNRS ꢀ Chimie ParisTech (ENSCP), 11
rue Pierre et Marie Curie, 75231 Paris, France.
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Institut Curie, INSERM U1196, 91405 Orsay cedex, France.
*
Corresponding authors: lingjun@zju.edu.cn (J. Ling); minꢀhui.li@chimieꢀparistech.fr (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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ABSTRACT
Biocompatible amphiphilic block copolymers composed of polysarcosine (PSar) and
poly(εꢀcaprolactone) (PCL) were synthesized using ringꢀopening polymerization of sarcosine
Nꢀthiocarboxyanhydride initiated by oxyamineꢀended PCL, and characterized by NMR, SEC
and DSC. Selfꢀassembling of two triblock copolymers PSar ꢀbꢀPCL ꢀbꢀPSar (CS7) and
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PSar16ꢀbꢀPCL40ꢀbꢀPSar16 (CS10) in dilute solution was studied in detail towards polymersome
formation, using thinꢀfilm hydration and nanoprecipitation techniques. A few of giant vesicles
were obtained by thinꢀfilm 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 CryoꢀTEM, were obtained by nanoprecipitation at room temperature.
These lamellae and fibrous structures were transformed into wormꢀlike cylinders and spheres
(D ~ 30ꢀ100 nm) after heating to 65°C (>Tm, PCL). Heating CS10 suspensions to 90°C led
eventually multilamellar polymersomes (D ~ 100ꢀ500 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 selfꢀassemblies were not cytotoxic.
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INTRODUCTION
Amphiphilic block copolymers have been widely studied since last decades as interesting
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macromolecular analogues of natural amphiphiles.
Depending on their molecular
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composition, mainly hydrophilic/hydrophobic ratio, these copolymers can selfꢀassemble, in
dilute water solution, into various morphologies such as spherical micelles, cylindrical rods,
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lamellae and vesicles. The selfꢀassembling process is mainly driven by the minimization of
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free energy. Polymersomes, i.e., vesicles composed of amphiphilic copolymers, have
attracted special attention due to their vesicular structures, cellꢀ and virusꢀmimicking
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dimensions and functions.
Moreover, their membrane properties such as responsiveness
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can be adjusted easily by polymer chemistry.
Polymersomes have been studied as drug
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bioimaging contrast agent carriers and nanoreactors, that present potential
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applications in biomedical field and material science.
The formation of polymersome is controlled and affected by many parameters such as
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inherent characteristics of amphiphiles,
physical chemical conditions (temperature,
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pH and initial concentration etc.), as well as preparation methods.
amphiphilic block copolymers, poly(ethylene
Among various
glycol)ꢀblockꢀpoly(εꢀcaprolactone)
(PEGꢀbꢀPCL) is of great interest for preparation of polymersomes, because both PEG and
PCL have been applied in polymeric drugꢀcarrier formations which have entered clinical
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trials, 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
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stability against degradation under neutral pH environments. PEG is a waterꢀsoluble
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macromolecule with antiꢀfouling property. Nanoꢀ and giant vesicles prepared from
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PEGꢀbꢀPCL by different methods have been intensively investigated,
with
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discussion on their formation mechanism and on their biomedical applications.
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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
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copolymers. However, recent works have revealed that PEGs show oxidative activity in
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physiological cellular environment and that PEGs can generate complement activation
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products in human serum on a time scale of minutes.
Recently, a natural amino acidꢀbased
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polymer, polysarcosine (PSar), has become a competitive alternative for PEG. PSar, with
methyl substitution on the nitrogen atom of amino acid unit (sarcosine), shows excellent
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waterꢀsolubility comparable with and even better than PEG. PSar and PEG are both polar,
electrically neutral, hydrogenꢀbond acceptors and are not hydrogenꢀbond donors. These
characteristics are responsible for the similar protein resistance between oligomers of Sar and
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EG. Luxenhofer and coworkers demonstrated PSar and other polypeptoids could be
degraded in vivo by reactive oxygen species (ROS) generated by certain enzymes from the
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liver. Reports on the synthesis and selfꢀassembly behaviors of PSarꢀcontaining block
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copolymers started to appear in the literature in the last years.
Guo’s group reported the
synthesis of PSarꢀbꢀPCL diblock copolymer via the ring opening polymerization (ROP) of
sarcosine Nꢀcarboxyanhydride (SarꢀNCA) followed by tin(II) octanoateꢀcatalyzed ROP of
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εꢀcaprolactone. However, the structure of nanoparticles of diameter around 100 nm formed
by PSarꢀbꢀPCL has not been clarified. At the same time, we were interested in the
polymersome formation by PSarꢀbꢀPCL diblock and PSarꢀbꢀPCLꢀbꢀPSar triblock copolymers
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and reported their syntheses by a new method via ROP of sarcosine Nꢀthiocarboxyanhydride
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(
SarꢀNTA). In this paper, we describe in detail this work.
NCA polymerization is the most widelyꢀused method for polypeptide and polypeptoid
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syntheses,
and PSar are prepared, in most cases, by ROP of SarꢀNCA. However, the
application of NCA is seriously limited because of their high sensibility to moisture and heat
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and their instability upon storage.
In contrast, amino acid NTA, the thioꢀanalog of amino
acid NCA, is much more robust. But its relatively low reactivity restricted for a long time its
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use to stepwise synthesis.
Kricheldorf reported in 2008 ROP of D,Lꢀleucine NTA,
D,Lꢀphenylalanine NTA and SarꢀNTA initiated by primary amine and prepared poly(amino
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acids) with wellꢀdefined structures. This work was a milestone in the research of NTA
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polymerization. Recently Ling’s group used PEGꢀamine to initiate ROP of SarꢀNTA. New
hydrophilic diꢀ and triꢀblock copolymers, PEGꢀbꢀPSar and PSarꢀbꢀPEGꢀbꢀPSar, were
produced. Amineꢀinitiated ROP of SarꢀNTA 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 =
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90 (PDI = 1.14).
The aim of the present work was to synthesize amphiphilic diꢀ and triꢀ block copolymers,
PSarꢀbꢀPCL and PSarꢀbꢀPCLꢀbꢀPSar, with various MWs and various hydrophilic/hydrophobic
ratios, and to study their ability to form polymersomes. We used ROP of SarꢀNTA initiated by
polycaprolactone oxyamines (PCLꢀONH2 and H NOꢀPCLꢀONH ). Polycaprolactone
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PCLꢀOH and HOꢀPCLꢀOH were first synthesized by diphenyl phosphateꢀcatalyzed ROP of
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εꢀcaprolactone, followed by endꢀfunctionalization with amino groups. Selfꢀassemblies of two
triblock copolymers in aqueous solution were performed using thin film hydration and
nanoprecipitation methods towards mesoꢀ and nanoꢀscale vesicle formation. By thin film
hydration, a few of giant polymersomes were observed. By nanoprecipitation using
DMF/water as coꢀsolvents, lamellar sheets and nanofibers were produced at room temperature.
Multilamellar polymersomes were successfully obtained by thermal treatment of these
selfꢀassemblies. Heatꢀtriggered morphological transformations were for the first time detected
on the PSarꢀbꢀPCLꢀbꢀPSar systems. The mechanism of polymersome formation is discussed
at the end of the paper.\
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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), Nꢀhydroxyphthalimide (NHP, 99%, Shanghai
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Adamas Reagent, China), triphenyl phosphine (PPh , 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 benzophenone ketyl and
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potassium/benzophenone ketyl, respectively. εꢀCaprolactone (CL), benzyl alcohol (BO), 1,
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4ꢀbutanediol (BDO) and dimethyl formamide (DMF) were stirred over CaH and distilled.
Sarcosine NTA (SarꢀNTA). SarꢀNTA was synthesized according to Ref. 58. Typically
Sꢀethoxythiocarbonylmercaptoacetic 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
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Nꢀethoxythiocarbonylsarcosine (SarꢀXAA). PBr (1.2 times of SarꢀXAA) was utilized to
catalyze the cyclization of SarꢀXAA, and eventually SarꢀNTA was obtained after column
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chromatographic purification (ethyl acetate:petroleum ether = 1:3). H NMR (CDCl /TMS) δ:
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3.11 ppm (s, 3H), 4.21 ppm (s, 2H).
AminoꢀEnded Poly(εꢀCaprolactone). 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 PCLꢀOH was dissolved in methylene chloride with
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NHP (0.548 g, 4.18 mmol) and PPh (0.904 g, 4.18 mmol). DIAD (0.6 mL, 3.88 mmol) was
then added to the mixture cooled with iceꢀwater 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 PCLꢀOꢀNHP (14.721 g). Hydrazine (0.11 mL, 3.41 mmol) was added
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dropwise to the CH
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Cl
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solution of PCLꢀOꢀNHP and stirred further for 30 min. Some
waxꢀlike 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 oxyaminoꢀended polymer was obtained (10.654 g, 69.2%).
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PolysarcosineꢀblockꢀPoly(εꢀCaprolactone). ROPs of SarꢀNTA initiated by PCLꢀONH and
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NOꢀPCLꢀONH
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were conducted with Schlenk line. As a typical synthetic procedure, after
(0.645 g, 0.138 mmol) was completely dissolved in anhydrous THF (8.8
H
NOꢀPCL40ꢀONH
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mL), SarꢀNTA (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 PSarꢀbꢀPCLꢀbꢀPSar 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 milliꢀQ water was injected slowly to the organic solution under mild shaking (2−3
ꢁL 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.
ThinꢀFilm Hydration. The thinꢀfilm 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.
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Characterization. Molecular weights (MWs) and polydispersity indices (PDIs) were
determined by sizeꢀexclusion 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 ꢁm MIXEDꢀC. 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
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spectrometer ( H 500 MHz) with DMSOꢀd
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or CDCl
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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 UVꢀ2550
UVꢀVis spectrophotometer (Shimadzu) equipped with a TCCꢀ240A temperature controlled
cell holder (Shimadzu). UVꢀVis 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 selfꢀassemblies
was analyzed using cryoꢀtransmission electron microscopy (CryoꢀTEM). 5 µL of samples
were deposited onto a 200 mesh holey copper grid (Ted Pella Inc., USA) and flashꢀfrozen in
liquid ethane cooled down at liquid nitrogen temperature. CryoꢀTEM images were acquired
on a JEOL 2200FS energyꢀfiltered (20eV) field emission gun electron microscope operating
at 200 kV using a Gatan ssCCD 2048 × 2048 pixels. Energyꢀfiltered (Zeroꢀloss) cryoꢀelectron
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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 tiltꢀseries
5
9, 60
and reconstruction of the final 3D volume were computed using TomoJ v2.28.
Confocal
0
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0
Laser Scanning Microscope (CLSM) images were obtained on a Nikon confocal
laserꢀscanning microscope A1R. Cytotoxicity test was carried out with a sample of the
selfꢀassemblies of PSar16ꢀbꢀPCL40ꢀbꢀPSar16 (CS10) prepared by nanoprecipitation. In a 6ꢀwell
culture plate (Nunc/ThermoScientific, Roskilde, Denmark), 5000 cells/150 ꢁL 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 ꢁL of a 5 mg/mL
solution of the 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT) in PBS
(SigmaꢀAldrich, SaintꢀQuentinꢀFallavier, 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 ꢁL 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
selfꢀassemblies over the absorbance of cells incubated only with growth medium.
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Scheme 1. Preparation of diblock and triblock copolymers PCLꢀbꢀPSar and
PSarꢀbꢀPCLꢀbꢀPSar.
RESULTS AND DISCUSSION
Synthesis and characterization of PSarꢀbꢀPCL and PSarꢀbꢀPCLꢀbꢀPSar
We employed a totally metalꢀfree protocol to prepare the amphiphilic diꢀ 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 εꢀcaprolactone with
61
DPP as catalyst. BO and BDO were chosen as the initiators respectively for PCLs with one
(
PCLꢀOH) or two terminal hydroxyl group(s) (HOꢀPCLꢀOH). The transformation from
6
2ꢀ66
hydroxyl to oxyamino group was achieved based on Mitsunobu reaction.
Three samples
of PCLꢀONH and three samples of NH OꢀPCLꢀONH were synthesized with low PDIs and
2
2
2
high yields (see Table 1, PCL1 to PCL6). ROP of SarꢀNTA was then initiated by these PCL
oxyamines on Schlenk line (gloveboxꢀfree). 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
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NCA. The polymerization of NTA with high yields, high MWs and low PDIs was achieved
44, 58
with appropriate initiators and conditions.
Herein ROP of sarcosine NTA was for the first
time initiated by oxyaminoꢀended PCLs, and this initiator endowed the copolymer with a
cleavable NꢀO bond between PSar and PCL blocks (e.g., cleavage under reduction by
0
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67ꢀ69
Pdꢀcatalyzed hydrogenolysis or in acidic environment).
Diꢀ and triꢀ block copolymers,
PSarꢀbꢀPCL and PSarꢀbꢀPCLꢀbꢀPSar, with different MWs and hydrophilic/hydrophobic ratios
were obtained (see Table 1, CS1 to CS10). Typical characterization results (NMR and SEC)
1
are shown in Figure 1 and 2 for CS10 and its macroꢀinitiator PCL5. H NMR spectra in
Figures 1A, Figure S1 and 1B reveal a complete transformation of –OH endꢀgroup to –OꢀNH
2
1
group in the synthesis of macroꢀinitiator PCL5. H 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 SarꢀNTAs initiated by
oxyaminoꢀended PCL5.
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1
Figure 1. H NMR spectra of HOꢀPCL40ꢀOH (A), PCL5 (B) in CDCl
3
and CS10 (C) in
DMSOꢀd
6
.
Figure 2. SEC chromatograms (DMF/LiBr as eluent). A: PCL5 (H
CS10 (PSar16ꢀbꢀPCL40ꢀbꢀPSar16).
2 2
NOꢀPCL40ꢀONH ) and B:
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Table 1. Syntheses of macroꢀinitiators PCL and block copolymers PCLꢀbꢀPSar and
PSarꢀbꢀPCLꢀbꢀPSar.
c
d
Block
M
n NMR
M
n SEC
Yield
(%)
a
d
Sample Initiator
T (°C)
[M]/[I]
Composition
PDI
b
ratio
(kDa)
2.3
(kDa)
3.9
PCL1
PCL2
PCL3
PCL4
PCL5
PCL6
CS1
BO
50
50
50
50
50
50
60
60
60
60
60
60
60
60
60
60
60
60
60
20
35
70
28
35
70
23
19
38
30
70
11
21
20
48
22
38
62
48
PCL20ꢀONH
PCL35ꢀONH
PCL80ꢀONH
2
ꢀ
1.12
1.12
1.36
1.17
1.25
1.30
1.16
1.24
1.24
1.18
1.35
1.35
1.37
1.3
92.5
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9
0
BO
2
2
ꢀ
4.1
9.2
3.2
4.7
8.1
2.9
4.0
5.7
4.5
14.2
3.8
4.3
6.3
6.3
7.0
9.6
10.8
11.4
6.4
14.1
5.5
69.2
74.3
81.6
65.4
78.9
50.8
61.6
73.9
58.6
82.1
> 99
95.6
91.3
92.4
54.8
82.3
68.5
83.9
BO
ꢀ
BDO
BDO
BDO
PCL1
PCL1
PCL1
PCL2
PCL3
PCL4
PCL4
PCL4
PCL4
PCL5
PCL6
PCL6
PCL6
H NOꢀPCL ꢀONH
ꢀ
2
2
2
28
2
2
2
H
H
NOꢀPCL40ꢀONH
ꢀ
9.0
NOꢀPCL70ꢀONH
ꢀ
14.8
5.0
PCL20ꢀbꢀPSar
8
20/80
43/57
60/40
10/90
35/65
15/85
25/75
36/64
CS2
PCL20ꢀbꢀPSar24
PCL ꢀbꢀPSar
48
6.2
CS3
9.2
20
CS4
PCL35ꢀbꢀPSar
5
7.0
CS5
PCL80ꢀbꢀPSar70
21.5
6.8
CS6
PSar
4
8
ꢀbꢀPCL28ꢀbꢀPSar
4
CS7
PSar
ꢀbꢀPCL28ꢀbꢀPSar
8
7.9
CS8
PSar ꢀbꢀPCL ꢀbꢀPSar
8.4
13
28
13
CS9
PSar22ꢀbꢀPCL28ꢀbꢀPSar22 48/52
PSar16ꢀbꢀPCL40ꢀbꢀPSar16 34/66
PSar10ꢀbꢀPCL70ꢀbꢀPSar10 14/86
PSar19ꢀbꢀPCL70ꢀbꢀPSar19 26/74
10.7
14.5
18.9
22.8
21.8
1.41
1.24
1.37
1.36
1.35
CS10
CS11
CS12
CS13
PSar ꢀbꢀPCL ꢀbꢀPSar
23
29/71
2
3
70
a
1
b
1
Determined by H NMR. Hydrophilic/hydrophobic block weight ratio calculated from H NMR
c
1
d
results; Average MWs of PCL initiators and copolymers calculated from H NMR results;
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
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(
60 °C). Consequently the possibility of side reactions increases, e.g., the nucleophilic attack
7
0
of amino end groups on ester linkages of PCL block that makes chain scission and forms
chains of low MWs detected as tails in SEC curves. Fortunately, previous report on the
PEGꢀbꢀPCL polymersomes has demonstrated that the polydispersity has little effect on the
34
possibility of vesicle formation. In some cases polydispersity can influence the curvature of
the membrane and consequently the sizes and size distribution of polymersomes, because
71
longer chains prefer to locate in the outer leaflet and shorter chains in the inner leaflet.
Figure 3. DSC thermograms of the first heating scan of copolymer samples recorded at
0 °C/min. A: CS7 (PSar ꢀbꢀPCL28ꢀbꢀPSar ) and B: CS10 (PSar16ꢀbꢀPCL40ꢀbꢀPSar16). T is
the melting temperature of PCL block and T
1
8
8
m
g
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 T
had T = 53 °C and ꢂH = 27.5 J/g. Both melting peaks were attributed to PCL melting.
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 ꢂH
136 J/g) of PCL homopolymer with 100% crystallinity, reported in Ref. 60, suggesting the
PCL blocks are semiꢀcrystalline in the block copolymers. Higher is PSar/PCL ratio, lower is
the PCL crystallinity. A detectable glass transition temperature (T ) for PSar block is recorded
m m
= 54 °C and melting enthalpy ꢂH = 46.1 J/g, while CS10
0
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72
m
m
m
(
g
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 T
g
of the homopolymers PSar10 (T
g
g g
= 36°C), PSar14 (T = 47°C) and PSar19 (T
=
59°C) (see Figure S4) measured in the same conditions, the T
g
values found here for CS7
and CS10 are reasonable.
Selfꢀassembly of PSarꢀ
b
ꢀPCLꢀ
b
ꢀPSar in aqueous solution
The formation of polymersomes by selfꢀassembling polysarcosineꢀcontaining block
2
6, 45, 48, 73
copolymers has interested several groups.
Even though vesicular structures have
ꢀlactic acid),
been claimed for PSarꢀbꢀPCL and PSarꢀbꢀPLLA selfꢀassemblies (PLLA: polyꢀ
L
7
3
no structural evidence was shown clearly. Only in the case of an amphiphilic block
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polypeptide, vesicular structure was demonstrated by cryoꢀTEM and TEM experiments
without ambiguity. PSar27ꢀbꢀP(
PSar27ꢀbꢀP( ꢀLeuꢀAib) having a leftꢀhanded helix segment selfꢀassembled first into two
types of peptide nanotubes. These nanotubes transform into vesicles by membrane fusion due
6
LꢀLeuꢀAib) having a rightꢀhanded helix segment and
D
6
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2
6
to stereoꢀcomplex formation between the rightꢀhanded and leftꢀhanded helical segments.
We focus herein on polymersome formation by triblock copolymers PSarꢀbꢀPCLꢀbꢀPSar.
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
1
, 30
poly(butadiene) or poly(ethylethylene) as hydrophobic block.
Hence CS7 (25%) and CS10
(
34%) were first chosen for systematic studies of selfꢀassembly in order to obtain
polymersomes. Polymer selfꢀassembling by thinꢀfilm 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 T
m g
of PCL and T of PSar. Therefore chain
movement was enabled to facilitate selfꢀassembling. 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 selfꢀassemblies 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 selfꢀassembling using nanoprecipitation since this technique was a
1
8, 27, 74
common one to prepare nanoꢀsized polymersomes.
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, multiꢀmodal
distributions were detected (Figure S5). CryoꢀTEM was then utilized to visualize the
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morphologies. Large lamellar structures were observed for selfꢀassemblies 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
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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 cryoꢀTEM, 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 (PSar16ꢀbꢀPCL40ꢀbꢀPSar16), the measured thickness is in
good agreement with the value reported for vesicles of PEO23ꢀbꢀPCL40 where a bilayer
2
8
membrane of PCL40 is around 15 nm. The interdigitation and entanglement of the polymer
chains cause partially blending of hydrophobic layers of diblock copolymers in the
7
5, 76
unilamellar organization,
while triblock copolymers are considered to have a mixed
conformation of the bent Uꢀshape and the stretched Iꢀshape in the selfꢀassembled membrane,
76
with less diffusivities. That is the reason why the bilayer thickness of PEO23ꢀbꢀPCL40 is
slightly smaller than the twice of that of PSar16ꢀbꢀPCL40ꢀbꢀPSar16. 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 wrappingꢀup
of lamellar sheets to form the final vesicles by minimizing the sum of curvature energy and
4, 77, 78
interfacial energy (Mechanism I).
Butler’s group studied the mechanism of vesicle
formation of PEGꢀbꢀPCL in THF upon water addition, and found that vesicles formed at room
temperature through wormꢀlike micelles and sheetꢀlike intermediates as water content
28
increased. They proposed a slightly different pathway where the two ‘‘foldedꢀover’’
biꢀlayers were in close contact, thus bringing the edges together for ‘‘zippingꢀup’’. In our case,
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neither “wrappingꢀup” nor “zippingꢀup” of lamellar sheets took place during the water
addition to polymer solution in DMF, probably because of the lack of sufficient mobility of
PSarꢀbꢀPCLꢀbꢀPSar structures. Some assistance in mobility would be helpful to the membrane
enclosure. Then we performed a thermal treatment on the selfꢀassemblies.
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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. CryoꢀTEM images of polymer selfꢀassemblies in water: (1) CS7 (A) and CS10 (B)
as prepared by nanoprecipitation at room temperature; (2) CS7 (C) and CS10 (D) after
30ꢀminutes heating at 65 °C; (3) CS7 (E) and CS10 (F) after 24ꢀhours 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 T of PCL block and the T of PSar
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block (Figure 3). Major changes were indeed observed by CryoꢀTEM in the samples after the
heating treatment. Spheres with diameters ranging from 30 to 100 nm, together with
wormꢀlike cylinders having similar diameters and a few nanofibers were generated in CS7
(Figure 6C). The solid spherical nature rather than diskꢀlike one for the observed
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roundꢀparticles was proved by cryoꢀtomography made on CS7 sample (Figure 7). In Figure 7
different morphologies are represented in different colors: full spheres in blue, wormꢀlike
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
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65 °C (Figure 6D), even though the majority of morphologies remained spheres and
wormꢀlike 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 wormꢀlike 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 coꢀexistence of small solid spheres. Some vesiclesꢀinꢀvesicles
structures, onionꢀlike vesicles and small spheres (diameter typically smaller than 50 nm) were
also recorded (Figure S8). The hydrodynamic diameter measurement by DLS on the
postꢀheating samples (Figure S5) was in good agreement with the observation of cryoꢀTEM,
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 50ꢀ55 °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, wormꢀlike cylinders
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and vesicles (D < 1000 nm). This temperature range around 50ꢀ55 °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.
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Figure 7. Diversity of CS7 polymer nanoparticles obtained after heating treatment at 65°C
(same sample as Figure 6C), unveiled by cryoꢀelectron 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
selfꢀassemblies 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 nanoꢀsized polymersomes were not only dependent on polymer
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natures but also strongly affected by the preparative conditions.
For example,
poly(ethylene oxide)ꢀblockꢀpoly(butylene oxide) (PEOꢀbꢀPBO), a rather lipidꢀlike copolymer
in term of solubility, could form first lamellar phase in concentrated aqueous solution at room
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temperature. 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, sheetꢀlike lamellar structures
formed at room temperature from the stereoꢀcomplex of two polysarcosineꢀcontaining
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copolypeptides, (Sar)27ꢀbꢀ(LꢀLeuꢀAib) and (Sar)27ꢀbꢀ(DꢀLeuꢀAib) , were transformed into
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vesicles only after heating at 90 °C. Nevertheless, in the present work the formation of
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vesicles did not seem to proceed via lamellar sheet enclosing. A detailed analysis of
polymersomes of the sample CS10 (PSar16ꢀbꢀPCL40ꢀbꢀPSar16) revealed that the vesicles were
not unilamellar but multilamellar. The unilamellar thickness of CS10 at room temperature
was 9ꢀ10 nm (Figure 6B), while the vesicles detected after annealing at 90 °C had membrane
thickness ranging from 20ꢀ50 nm and the multilayers were visible in the cryoꢀTEM images
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(
Figure S8). Moreover, the selfꢀassemblies treated at 65 °C are mainly spheres, wormꢀlike
cylinders with a few of vesicles, but without any lamellar sheets remaining. After heating at
0 °C, vesicles were observed only for CS10, while CS7 (PSar ꢀbꢀPCL28ꢀbꢀPSar ) concluded
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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 selfꢀassembling behavior. More studies on the
selfꢀassemblies 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 selfꢀassemble into small
spherical micelles, which then slowly evolve into cylindrical micelles and open discꢀlike
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micelles by collision. The large discꢀlike micelles then slowly close up to form vesicles. In
mechanism II, the initial spherical micelles, rather than evolving toward cylindrical micelles
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and openꢀdisk 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
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curvature, leading to vesicles. 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
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polymersomes. 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 selfꢀassembly of
poly(ethylene oxide)ꢀblockꢀpoly(N,Nꢀdiethylaminoethyl methacrylate) (PEOꢀbꢀPDEAMA) by
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a pH switch. 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
selfꢀassemblies of CS10 in a concentration of 0.69 mg/mL, which confirmed reliable
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biocompatibility of PSarꢀbꢀPCL copolymers and their selfꢀassembly structures.
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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 ꢁg/mL.
CONCLUSION
We reported on a simple and totally metalꢀfree method for the synthesis of a series of
PSarꢀbꢀPCL and PSarꢀbꢀPCLꢀbꢀPSar block copolymers based on ROP of SarꢀNTA using PCL
ended with oxyamine group(s) as initiators. The selfꢀassembling behaviors of two triblock
copolymers PSar ꢀbꢀPCL ꢀbꢀPSar (CS7) and PSar ꢀbꢀPCL ꢀbꢀPSar (CS10) in water were
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studied in detail using thinꢀfilm hydration and nanoprecipitation methods. Rodꢀlike particles
and a few of giant polymersomes were obtained by thin film hydration at 65 °C. Large
unilamellar sheets and nanofibers (of 8ꢀ10 nm in thickness and diameters) were formed by
nanoprecipitation at room temperature using DMF/water as coꢀsolvents. A thermal treatment
by heating the aqueous suspensions to 65 °C transformed the lamellae and fibrous structures
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into wormꢀlike cylinders and spheres (of 30ꢀ100 nm in diameter). Heating the initial aqueous
suspension of nanoprecipitation to 90 °C led multilamellar polymersomes (of 100ꢀ500 nm in
diameter and 20ꢀ50 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 selfꢀassemblies of CS10 confirmed that the PSarꢀbꢀPCL systems
developed in this work were not cytotoxic and suitable for possible applications in biomedical
domains.
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Supporting Information Available
Supplemental NMR spectrum, SEC curves, DSC thermograms, phase transition data, DLS
plots, cryoꢀTEM images and photographs of selfꢀassemblies’ 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
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(
21174122, 21528402) and from the program “PIC targeted therapy” of Institut Curie. We
acknowledge the PICTꢀIBiSA for providing access to chemical imaging equipment, and the
Nikon Imaging Center@Institut Curie for confocal microscopy.
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