Native Chemical Ligation for Cross-Linking of Flower-Like Micelles

Article abstract of DOI:10.1021/acs.biomac.8b00908

In this study, native chemical ligation (NCL) was used as a selective cross-linking method to form core-cross-linked thermosensitive polymeric micelles for drug delivery applications. To this end, two complementary ABA triblock copolymers having polyethylene glycol (PEG) as midblock were synthesized by atom transfer radical polymerization (ATRP). The thermosensitive poly isopropylacrylamide (PNIPAM) outer blocks of the polymers were copolymerized with either N-(2-hydroxypropyl)methacrylamide-cysteine (HPMA-Cys), P(NIPAM-co-HPMA-Cys)-PEG-P(NIPAM-co-HPMA-Cys) (PNC) or N-(2-hydroxypropyl)methacrylamide-ethylthioglycolate succinic acid (HPMA-ETSA), P(NIPAM-co-HPMA-ETSA)-PEG-P(NIPAM-co-HPMA-ETSA) (PNE). Mixing of these polymers in aqueous solution followed by heating to 50 °C resulted in the formation of thermosensitive flower-like micelles. Subsequently, native chemical ligation in the core of micelles resulted in stabilization of the micelles with a Z-average of 65 nm at body temperature. Decreasing the temperature to 10 °C only affected the size of the micelles (increased to 90 nm) but hardly affected the polydispersity index (PDI) and aggregation number (N_agg) confirming covalent stabilization of the micelles by NCL. CryoTEM images showed micelles with an uniform spherical shape and dark patches close to the corona of micelles were observed in the tomographic view. The dark patches represent more dense areas in the micelles which coincide with the higher content of HPMA-Cys/ETSA close to the PEG chain revealed by the polymerization kinetics study. Notably, this cross-linking method provides the possibility for conjugation of functional molecules either by using the thiol moieties still present after NCL or by simply adjusting the molar ratio between the polymers (resulting in excess cysteine or thioester moieties) during micelle formation. Furthermore, in vitro cell experiments demonstrated that fluorescently labeled micelles were successfully taken up by HeLa cells while cell viability remained high even at high micelle concentrations. These results demonstrate the potential of these micelles for drug delivery applications.

Full text of DOI:10.1021/acs.biomac.8b00908

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Article
NATIVE CHEMICAL LIGATION FOR
CROSSLINKING OF FLOWER-LIKE MICELLES
Marzieh Najafi, Neda Kordalivand, Mohammad-Amin Moradi, Joep van den Dikkenberg, Remco
Fokkink, Heiner Friedrich, Nico A.J.M. Sommerdijk, Mathew Hembury, and Tina Vermonden
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00908 • Publication Date (Web): 13 Aug 2018
Downloaded from http://pubs.acs.org on August 14, 2018
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Native chemical ligation for crosslinking of flowerꢀ
like micelles
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Marzieh Najafi1, Neda Kordalivand1, Mohammad-Amin Moradi2,3, Joep van den Dikkenberg1, Remco
Fokkink4, Heiner Friedrich2,3, Nico A. J. M. Sommerdijk2,3, Mathew Hembury1, Tina Vermonden1
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1 Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Science for Life,
Faculty of Science, Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands.
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2 Laboratory of Materials and Interface Chemistry & Centre for Multiscale Electron Microscopy
Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, Eindhoven
5600 MB, The Netherlands
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3 Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven 5600
MB, The Netherlands
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4 Physical Chemistry and Soft Matter, Wageningen University & Research, Stippeneng 4, 6708 WE
Wageningen, The Netherlands.
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Abstract
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In this study, native chemical ligation (NCL) was used as a selective crosslinking method to form coreꢀ
crosslinked thermosensitive polymeric micelles for drug delivery applications. To this end, two
complementary ABA triblock copolymers having polyethylene glycol (PEG) as midblock were
synthesized by atom transfer radical polymerization (ATRP). The thermosensitive poly isopropyl
acrylamide (PNIPAM) outer blocks of the polymers were coꢀpolymerized with either Nꢀ(2ꢀ
hydroxypropyl)methacrylamideꢀcysteine (HPMAꢀCys), P(NIPAMꢀcoꢀHPMAꢀCys)ꢀPEGꢀP(NIPAMꢀcoꢀ
HPMAꢀCys) (PNC), or Nꢀ(2ꢀhydroxypropyl)methacrylamideꢀethylthioglycolate succinic acid (HPMAꢀ
ETSA), P(NIPAMꢀcoꢀHPMAꢀETSA)ꢀPEGꢀP(NIPAMꢀcoꢀHPMAꢀETSA) (PNE). Mixing of these
polymers in aqueous solution followed by heating to 50 °C resulted in the formation of thermosensitive
flowerꢀlike micelles. Subsequently, native chemical ligation in the core of micelles resulted in stabilization
of the micelles with a Zꢀaverage of 65 nm at body temperature. Decreasing the temperature to 10 °C only
affected the size of the micelles (increased to 90 nm) but hardly affected the polydispersity index (PDI)
and aggregation number (Nagg) confirming covalent stabilization of the micelles by NCL. CryoTEM
images showed micelles with an uniform spherical shape and dark patches close to the corona of micelles
were observed in the tomographic view. The dark patches represent more dense areas in the micelles
which coincide with the higher content of HPMAꢀCys/ETSA close to the PEG chain revealed by the
polymerization kinetics study. Notably, this crosslinking method provides the possibility for conjugation
of functional molecules either by using the thiol moieties still present after NCL or by simply adjusting the
molar ratio between the polymers (resulting in excess cysteine or thioester moieties) during micelle
formation. Furthermore, in vitro cell experiments demonstrated that fluorescently labeled micelles were
successfully taken up by HeLa cells while cell viability remained high even at high micelle
concentrations. These results demonstrate the potential of these micelles for drug delivery applications.
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Keywords
Core crosslinked micelles, ATRP polymerization, Thermosensitive polymer, CryoꢀTEM.
Introduction
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Polymeric micelles (PM) can be formed by amphiphilic block copolymers and have been studied
extensively to improve delivery of mainly hydrophobic drugs1,2. In aqueous media, at concentrations
above the critical micelle concentration (CMC), amphiphilic block copolymers (AB or ABA) selfꢀ
assemble into nanoꢀsized particles. While AB polymers selfꢀassemble into starꢀlike micelles, ABA
triblock copolymers with a hydrophilic midblock (B) are capable of selfꢀassembling into soꢀcalled flowerꢀ
like micelles3,4,5. Although flowerꢀlike micelles share many characteristics with starꢀlike micelles, they
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have been reported to have some advantageous properties regarding lower CMC and higher stability6,7,8
.
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The hydrophilic midblock (B block) in flowerꢀlike micelles is often composed of poly (ethylene glycol)
(PEG)6. Starting from this PEG midblock, there are several methods to synthesize ABA block copolymers
among which atom transfer radical polymerization (ATRP) provides good control over the structure and
sequence of monomers in a polymer9,10. ATRP conditions enable the growth of hydrophobic Aꢀblocks
with a desired length and tunable monomer composition11.
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During micellization of ABA block polymers, in aqueous solution, the PEG block forms a looped
structure of the hydrophilic corona surrounding the hydrophobic core composed of the selfꢀassembled
outer blocks3. The hydrophobic core has been used to solubilize hydrophobic drugs and the hydrophilic
shell stabilizes micelles and enhances their circulation time in body fluids12,13. However, the equilibrium
between micelles and unimers is highly affected by dilution and at concentrations below the CMC,
micelles start to dissociate. This dissociation can lead to premature release of a loaded drug from the
micelles upon administration in e.g. the circulation14. Moreover, proteins present in the body can extract
drugs from micellar formulations. To enhance the in vivo stability of micelles several methods have been
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developed, including the frequently applied method of core crosslinking and covalent linking of drugs to
the polymers present in the micelles15.
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For covalent crosslinking of the micellar core, the reactive groups should be located in the hydrophobic
block of the polymers. Of course, it is important that the chemical properties of these functional groups do
not hamper micelle formation. A commonly applied method for core crosslinking of micelles makes use
of radical polymerization. In this method, polymerizable groups such as methacrylates or acrylates are
introduced in the hydrophobic block of amphiphilic polymers, which are located in the core of the
micelles after micellization. Subsequently, by UV illumination in the presence of a photoinitiator or by
thermal radical polymerization, these functional groups can be crosslinked in the core of this type of
micelles16,8. Another common method for covalent crosslinking of micelles is introducing reactive groups,
such as, epoxy or acidic moieties in the side chain of amphiphilic polymers that are subsequently
crosslinked by a bifunctional molecule, such as a diamine17,18. Also, disulfide bridges as crosslinks can be
introduced by using cystamine in the micellar core, which gives the micelles triggered release properties
interesting for compounds that need to be delivered intracellularly. These disulfide bridges will be broken
in the intracellular reducing environment19,20. Also, free thiols in the hydrophobic block of the amphiphilic
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polymer can be used for disulfide core crosslinking, however, oxidative conditions should be provided21,22
.
Recently, ‘click’ reactions received attention for crosslinking of polymeric micelles23,24. Several studies
reported the formation of crosslinked micelles and nanoparticles using copperꢀcatalyzed alkyneꢀazide
cycloaddition reaction (CuAAC)25,26. For example, redoxꢀresponsive core crosslinked micelles were
synthesized using bisꢀ(azidoethyl) disulfide as a redoxꢀresponsive crossꢀlinker27. Other studies used Dielsꢀ
Alder reactions for crosslinking, e.g. formation of starꢀlike micelles by crosslinking block copolymers
having furan functionalities using bismaleimide crosslinkers in tetrahydrofuran (THF)28. Furthermore,
PEGꢀpolyester type polymers with pending mercapto groups were crosslinked via a thiolꢀene ‘click’
chemistry approach using a diacrylate crosslinker29.
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Besides all the developments in this field, many of the applied crosslinking methods suffer from some
limitations (e.g. exposure to radicals, oxidative conditions, use of (toxic) catalysts which mostly require an
inert environment, or use of organic solvents), that can potentially damage the cargo of the micelles.
Furthermore, the functional groups used in these methods will be consumed during crosslinking and
cannot be used for further modification steps. Therefore, introducing a crosslinking method that is not
only applicable in aqueous solutions but also retains its reactive groups in the micellar core after
crosslinking is of high interest. Among chemical conjugation methods performed in aqueous solution,
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native chemical ligation30, a chemoselective method, has been studied for crosslinking of hydrogels31,32,33
.
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However, this crosslinking method has not been applied yet for coreꢀcrosslinking of micelles. Native
chemical ligation involves nucleophilic attack of the thiol group of an Nꢀterminal cysteine to a thioester
moiety. The obtained thioester intermediate rearranges by an intramolecular S, Nꢀacyl shift that results in
the formation of an amide. Notably, after the S, Nꢀacyl shift a free thiol moiety remains available, which
offers the possibility to further conjugate desirable molecules via e.g. a disulfide bond.
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The goal of this study was to investigate native chemical ligation as a selective reaction that occurs in
water for core crosslinking of flowerꢀlike micelles34. For this purpose, an ABA triblock copolymer
consisting of polyethylene glycol (PEG) as midblock and thermosensitive poly Nꢀisopropyl acrylamide
(NIPAM)ꢀcoꢀNꢀ(2ꢀhydroxypropyl)methacrylamideꢀcysteine (HPMAꢀCys) as outer blocks P(NIPAMꢀcoꢀ
HPMAꢀCys)ꢀPEGꢀP(NIPAMꢀcoꢀHPMAꢀCys) (PNC)) was synthesized by ATRP. A complementary
polymer, P(NIPAMꢀcoꢀHPMAꢀETSA)ꢀPEGꢀP(NIPAMꢀcoꢀHPMAꢀETSA) (PNE), containing a novel
monomer of Nꢀ(2ꢀhydroxypropyl)methacrylamideꢀethylthioglycolate succinic acid (HPMAꢀETSA)
carrying a thioester moiety, was developed. In these amphiphilic polymers designed to selfꢀassemble into
micelles, the PEG block represents the hydrophilic part, PNIPAM blocks initiate micellization in aqueous
solution by increasing temperature and HPMAꢀCys/ETSA monomers provide Nꢀterminal cysteine and
thioester functionalities for NCL crossꢀlinking to stabilize the micellar structure. Upon mixing of the
polymers in aqueous solution, the presence of PNIPAM thermosensitive blocks allows micelle formation
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by increasing the temperature above the lower critical solution temperature (LCST) of these polymers35.
The stability of the micelles after covalent crossꢀlinking by NCL was investigated by lowering the
temperature of the micellar solution below the LCST of the polymers. In addition, the shape and
conformation of micelles were studied by static light scattering and CryoTEM. To investigate
cytocompatibility for potential biomedical applications of the obtained micelles, a cytotoxicity assay was
performed. Moreover, cellular uptake of labeled micelles was studied using A549 (human lung carcinoma
cell line) and HeLa (human epithelial cervix carcinoma cell line) cells.
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Materials and Methods
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All commercial chemicals were obtained from SigmaꢀAldrich (Zwijndrecht, The Netherlands) and used
as received unless indicated otherwise. Nꢀ(2ꢀhydroxypropyl)methacrylamide (HPMA) was synthesized by
a reaction of methacryloyl chloride with 1ꢀaminopropanꢀ2ꢀol in dichloromethane according to a literature
procedure36. Peptide grade dichloromethane (CH2Cl2) was obtained from Biosolve (Valkenswaard, The
Netherlands). N,N′ꢀdimethylaminopyridine (DMAP) was purchased from Fluka (Zwijndrecht, The
Netherlands). BocꢀSꢀacetamidomethylꢀLꢀcysteine (BocꢀCys(Acm)ꢀOH was purchased from Bachem
(Bubendorf,
Switzerland).
Nꢀ(2ꢀhydroxypropyl)methacrylamideꢀBocꢀSꢀacetamidomethylꢀLꢀcysteine
(HPMAꢀBocꢀCys(Acm)) was synthesized as described by Boere et al33. Ethylthioglycolate succinic acid
(ETSA) was prepared according to a literature procedure31. Phosphate buffered saline (PBS) pH 7.4 (8.2
g/L NaCl, 3.1 g/L Na2HPO4·12H2O, 0.3 g/L NaH2PO4·2H2O) was purchased from B. Braun (Melsungen,
Germany).
Celltiter
AQꢀMTS
(3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ5ꢀ(3ꢀcarboxymethoxyphenyl)ꢀ2ꢀ(4ꢀ
sulfophenyl)ꢀ2Hꢀtetrazolium) #3580 and Lysis Solution #G182A were purchased from Promega (USA).
NHSꢀAlexa Fluor® 647 and maleimideꢀAlexa flour C5 568 fluorescent dyes were obtained from
Invitrogen (Eugene, OR, USA).
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Synthesis of poly(ethylene glycol) bis(2-bromoisobutyrate)
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The synthesis of a PEG macroinitiator was achieved according to a method previously reported3 with
slight modifications. Briefly, dried PEG 6 kDa (5 gr) was dissolved in 70 mL of dry THF and purged with
nitrogen. To this solution, triethylamine (0.7 mL) and αꢀbromoisobutyryl bromide (0.6 mL) were added
and the reaction mixture was stirred overnight at room temperature. Next, the formed bromide salt was
filtered off and subsequently, THF was evaporated under reduced pressure. The crude product was
dialyzed against water for 2 days and afterwards lyophilized. The degree of functionalization was
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determined by addition of trichloroacetyl isocyanate (TAIC) reacting with unmodified OH groups37. Hꢀ
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NMR analysis confirmed the formation of a fully functionalized PEG ATRP macroinitiator. HꢀNMR
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(CDCl3): δ = 4.3 (t, 4H, OCH2), 3.85 (t, 4H, OCH2), 3.65 (t, 531H, OCH2), 3.35 (t, 4H, OCH2), 1.85 ppm
(s, 12H,CCH3).
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Synthesis of N-(2-hydroxypropyl) methacrylamide-ethylthioglycolate succinic acid (HPMA-ETSA)
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In a typical procedure, ethylthioglycolate succinic acid (ETSA) (1.54 g, 7 mmol), HPMA (1.00 g, 7
mmol), and DMAP (85 mg, 7 µmol) were dissolved in dry CH2Cl2 (7 mL). To this solution, N,N'ꢀ
dicyclohexylcarbodiimide (DCC) (1.44 g, 7 mmol) was added, and the reaction mixture was stirred for 16
h under a nitrogen atmosphere at room temperature. Subsequently, the suspension was cooled to 0 °C and
filtered, after which the filtrate was concentrated. The crude product was purified by silica gel
chromatography, using CH2Cl2/CH3OH (9:1 v/v) as eluent. The monomer HPMAꢀETSA was obtained as
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milky oil with a yield of 57%. HꢀNMR (CDCl3): δ = 6.28 (s, 1H, NH), 5.68 (s, 1H, H2C=CH), 5,31 (s,
1H, H2C=CH), 5.05 (m, 1H, CH2CH(CH3)O), 4.16 (q, 2H, CH3CH2O), 3.67 (d, 2H, C(O)SCH2C(O)), 3.56
(dd, 1H, NHCH2CH(CH3)O), 3.32 (dd, 1H, NHCH2CH(CH3)O), 2.95 (t, 2H, SC(O)CH2CH2), 2.63 (t, 2H,
SC(O)CH2CH2), 1.94 (s, 3H, C=C(CH3)), 1.24 (m, 6H, CH3CH2O) and NHCH2CH(CH3)O). 13CꢀNMR
(CDCl3): δ = 196.5, 172.3, 171.3, 168.5, 139.8, 119.5, 70.7, 61.9, 44.0, 38.2, 31.2, 29.4, 18.9, 17.5, 14.0
(SIꢀFig. 1)
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Poly(ethylene glycol) bis(2ꢀbromoisobutyrate) (50 mg, 7.9 µmol), CuBr (4.5 mg, 0.031 mmol), CuBr2
(4.7 mg, 0.021 mmol), NIPAM (268.9 mg; 2.3 mmol) and HPMAꢀETSA (55 mg, 0.16 mmol) were
dissolved in a mixture of 2.5 mL water and 2 mL ethanol. The mixture was stirred and deoxygenated by
flushing with nitrogen for 15 minutes at room temperature, then placed in an ice bath for another 15
minutes. After addition of 16 µL (0.06 mmol) of tris[2ꢀ(dimethylamino)ethyl]amine (Me6TREN) to the
solution, the color of the mixture immediately changed to blue and the reaction was left for 5 hours in an
ice bath. The final product was dialyzed (cut off 10 kDa) against water at 4 °C for one day and
lyophilized. The obtained polymer was characterized by GPC and 1HꢀNMR (SIꢀFig. 2, 4A).
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Synthesis of P(NIPAM-co-HPMA-Cys)-PEG-P(NIPAM-co-HPMA-Cys), PNC
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Poly(ethylene glycol) bis(2ꢀbromoisobutyrate) (50 mg, 7.9 µmol), CuBr (4.5 mg, 0.031 mmol), CuBr2
(4.7 mg, 0.021 mmol), NIPAM (268.9 mg; 2.4 mmol) and HPMAꢀBocꢀCys(Acm) (67 mg, 0.16 mmol)
were dissolved in a mixture of 3.5 ml of water and acetonitrile with a ratio of 3:1. The mixture was stirred
and deoxygenated by flushing with nitrogen for 15 minutes at room temperature, then placed in an ice
bath for another 15 minutes. After addition of 16 µL (0.06 mmol) of Me6TREN to the solution, the color
of the mixture immediately changed to blue and the reaction was left stirring for two hours in an ice bath.
The final product was dialyzed (cut off 10 kDa) against water at 4 °C for one day and subsequently
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lyophilized. The obtained polymer was characterized by GPC and HꢀNMR. To replace the bromide end
groups, the obtained polymer was dissolved in 5 mL CH2Cl2 and treated with an excess (500 µL) of
mercaptoethanol for 24 h. Next, the solvent was concentrated by evaporation and the pure polymer was
obtained after precipitation in cold diethyl ether. Finally, the Acm and Boc protecting groups of cysteine
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were removed as explained previously32. The obtained polymer was characterized by GPC and HꢀNMR
(SIꢀFig. 3, 4B).
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At several time points, 250 µL samples were taken from polymerization mixtures to monitor the
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conversion of polymerization by HꢀNMR and GPC. Samples of 50 µL were diluted with airꢀsaturated
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CDCl3 and analyzed by HꢀNMR. The integrals of signals at 5.5 and 5.30 ppm related to NIPAM and
HPMAꢀCys/ETSA respectively, were compared to the PEG signal at 3.65 ppm as a reference. The
remaining 200 µL was dialyzed against water for one day and then lyophilized. The dried product was
dissolved in DMF/LiCl and the molecular weight was analyzed by GPC as described below.
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NMR spectroscopy
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The obtained monomers, polymers, and micelles were studied by HꢀNMR (400 MHz) and CꢀNMR
(100 MHz) measured on an Agilent 400ꢀMR NMR spectrometer (Agilent Technologies, Santa Clara,
USA). The chemical shifts were calibrated against residual solvent peaks of CDCl3 (δ = 7.26 ppm), D2O (δ
= 4.79 ppm) for 1HꢀNMR, and δ = 77.16 ppm of CDCl3 for 13CꢀNMR. Micelle solutions were analyzed on
a Varian Inova 500 NMR instrument (Varian Inc., California, USA) in D2O.
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Gel permeation chromatography (GPC)
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To determine the molecular weight of polymers, GPC was performed on a Waters Alliance System
(Waters Corporation, Milford, MA, USA) equipped with a refractive index detector using a MixedꢀD
column (Polymer Laboratories) at a temperature of 65 °C. The eluent consisting of 10 mM LiCl in DMF
with a flow of 1 mL/min was used as mobile phase and samples were prepared in the same solvent at a
concentration of 5 mg/mL. A series of linear PEGs with narrow and defined molecular weights were used
as calibration standards.
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The cloud point (CP) of the obtained polymers was measured on a Jasco FP8300 spectrofluorometer
(Tokyo, Japan) and excitation/emission wavelength was set at 650 nm. Polymers were dissolved at a
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concentration of 1 mg/mL in PBS and heated from 10 to 50 °C with a heating rate of 1 °C/min. The CP
was defined as the onset of increasing scattering intensity.
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Micelle formation
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Micelles were formed by a fast heating method as follows: PNC and PNE were dissolved separately in
PBS at a concentration of 3 mg/mL at 4 °C. PNC and PNE polymers were mixed in a 1:1 mass ratio since
they have equal molar ratios of HPMAꢀCys and HPMAꢀETSA and similar molecular weights.
Subsequently, the mixed solution was quickly heated up to 50 °C using an oil bath and left at 50 °C for 3
hours. Resulting micellar solutions were dialyzed against water for two days at room temperature and
subsequently lyophilized. Micelles were characterized by DLS and NMR both before and after
lyophilization.
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CryoTEM
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Transmission electron microscopy at cryogenic temperatures (CryoTEM) was done using the TU/e
CryoTitan (FEI Company)38. Graphene oxide grids, prepared by adding a single layer graphene sheet to a
Quantifoil grid39, were used for sampling via an automated robot (Vitrobot™ Mark III, FEI Company),
which was kept at room temperature. The excess of liquid sample on the grid was blotted away with filter
paper to form a thin film of the dispersion. The thin layer of liquid on the grid was plunged rapidly into
liquid ethane. The vitrification was done in 100% humidity atmosphere. A tilt series of 27 cryoTEM
images from ꢀ65 to +65 degrees with 5 degrees steps were taken to reconstruct the 3D structure of the
particles using IMOD, via patch tracking alignment, and AVIZO 9.0 software40.
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Dynamic light scattering (DLS)
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The size of the micelles was measured by DLS on a Malvern CGSꢀ3 goniometer (Malvern Ltd.,
Malvern, U.K.), ALV/LSEꢀ5003 Correlator and He–Ne 633 nm laser. All measurements were carried out
at a 90° angle at temperatures of 10, 37 and 45 °C controlled by a thermostat water bath Julabo FS18. The
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solvent viscosity was corrected at each temperature by the software. The Zꢀaverage radius and
polydispersity index was calculated by ALV and DTS software, respectively.
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Static light scattering (SLS)
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Weightꢀaverage molecular weight of the micelles and the radius of gyration were determined by SLS
using an ALV7004 correlator, ALV/LSEꢀ5004 Goniometer, ALV/Dual High QE APD detector unit with
fiber splitting device with a setꢀup of 2 off detection system and a Uniphase Model 1145P HeꢀNe Laser.
The laser wavelength and power were set to 632.8 nm and 22 mW, respectively and the temperature was
controlled by a Julabo CF41 Thermostatic bath.
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Ellman assay
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Ellman’s reaction was performed on micelles to quantify the crosslink density in the micelles. Cysteine
hydrochloride monohydrate standards were prepared at concentrations ranging from 0ꢀ1.5 mM in a 0.1 M
sodium buffer/1 mM EDTA at pH 8.0. A HiTrapTM 1.5 mL, desalting column was equilibrated with the
same buffer and was used for separation of micelles from ethyl thioglycolate. A stock solution of 4
mg/mL of Ellman’s reagent was made and 50 µL was added to a mixture of 2.5 ml of buffer and 250 µL
of each standard and ethyl thioglycolate sample. The thiol contents of samples were determined by
measuring the absorbance at λ= 412 nm using a BMG spectrostar nano well plate reader.
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To fluorescently label the micelles, micelles were formed following the same procedure as described
above, then dialyzed and lyophilized. Subsequently, 6 mg of the obtained micelles was dissolved in 1 mL
DMSO and 10 µL of a 20 µg/mL NHSꢀAlexa fluor 647 or maleimideꢀAlexa fluor C5 568 stock solution in
DMSO was added and left to react overnight. Next, the labeled micelles were dialyzed against DMSO and
then water and used for the cellular uptake study.
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To make sure enough cysteine moieties were available for conjugation of NHSꢀAlexa fluor 647,
micelles with an excess of cysteine moieties (PNC/PNE ratio of 3:2) were made following the same
protocol. Conjugation of maleimideꢀAlexa fluor C5 568 was conducted on micelles formed from the 1:1
molar ratio of PNC and PNE.
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Cellular internalization study
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To investigate the cellular uptake, HeLa and A549 cells were seeded in a glassꢀbottomed 96 wellꢀplate
at a density of 104 cells/well and incubated at 37 °C for 24 h. Then, fluorescently labeled micelles were
added and incubated with cells at concentrations of 100, 200, and 400 µg/mL for 2, 4 and 24 h at 37 °C.
Subsequently, Hoechst 33430 was added to each well 30 min before imaging with a final concentration of
10 nM. The cells were washed twice with PBS and the plate was transferred into a Yokogawa CV7000
(Tokyo, Japan) spinning disk microscope with a 60x 1.2NA water objective.
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Cytocompatibility of the formed micelles was assessed by MTS assay41. HeLa cells were seeded one
day before the experiment into 96ꢀwell plates at a density of 8000 cells/well and were maintained in 200
µl DMEM Low Glucose (1g/l) medium containing 1 % antibiotics/ antimycotics and 10% FBS for 24 h, at
37 °C. A stock solution of the lyophilized micelles at a concentration of 50 mg/mL in PBS was prepared.
The micelle solution was diluted in medium to final concentrations of 1.5, 0.75, 0.375, 0.187, 0.093,
0.046, 0.023, and 0.011 mg/mL. The cells were incubated with the micelles at different concentrations for
24 h. Then, one washing step was performed using PBS and subsequently, 100 µL fresh medium and 20µl
MTS reagent was added to the cells and incubated for 2 h. As a negative control group, cells were
incubated with 100% culture medium and as a positive control group, cells were incubated in medium
containing 1% Triton Xꢀ100. The cell viability was determined by measuring the absorbance at 492 nm
using a Biochrome EZ microplate reader.
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Results and discussion
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Figure 1 shows the overall synthesis scheme of the two complementary polymers, PNC and PNE,
containing cysteine and thioester functional groups, respectively. First, the monomer HPMAꢀETSA was
designed as coꢀmonomer in the thermosensitive blocks of PNE. This monomer was synthesized by
conjugation of the hydroxyl group of HPMA to the carboxylic acid functionality of ETSA via a DCCꢀ
activated esterification method using DMAP as a catalyst. After column chromatography, the pure
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monomer was isolated in a yield of 57% and its structure was confirmed by HꢀNMR and CꢀNMR (SIꢀ
Fig. 1).
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PEG with a numberꢀaverage molecular weight (Mn) of 6 kDa was completely functionalized at both
chain ends with bromoisobutyryl bromide groups. In the next step, this PEG macroinitiator was used for
the synthesis of PNC and PNE triblock copolymers by ATRP (Fig. 1B and 1C).
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For PNE, the thermosensitive outer blocks consisted of Nꢀisopropylacrylamide (NIPAM) and the above
described novel monomer HPMAꢀETSA with a ratio of 93:7. The HPMAꢀETSA monomer was introduced
to obtain a thioester functionality in the thermosensitive domain. After polymerization, the pure polymer
was obtained by dialysis and lyophilization in a yield of 81%. The complementary polymer PNC was
designed to have a similar structure and size as PNE. Therefore, the same PEG macroinitiator was used for
polymerization and a ratio of NIPAM and HPMAꢀBocꢀCys(Acm) of 93:7 was used. Next, the bromide
end groups of PNC polymer chains were substituted by mercaptoethanol to prevent polymer selfꢀ
crosslinking after deprotection of Bocꢀcysteine (Acm)42.
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Several methods have been reported for the polymerization of NIPAM in aqueous solutions43,44, here for
the first time, we introduced an ATRP method for copolymerization of NIPAM and HPMAꢀBocꢀ
Cys(Acm) or HPMAꢀETSA in aqueous solutions. It has been reported before that the polymerization of
HPMA can proceed uncontrolled mainly due to complexation of the amine group to the transitionꢀmetal
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complex45. Using ligands with a high complexation constant, such as Me6TREN and the addition of CuBr2
to increase deactivation rate can improve control over the polymerization45. In addition, using solvents
with the ability to form hydrogen bonds with polymer and monomers reduces the risk of monomer
complexation to catalyst46.
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Figure 2A shows the conversion of monomers during the polymerization of PNE and PNC. For PNE,
the residual monomer concentration decreased exponentially over time revealing that the kinetics of
polymerization is relatively wellꢀcontrolled. Moreover, Mn evolved linearly with the conversion which
indicates that no significant termination due to recombination occurred (Fig. 2C). During the
polymerization of PNC, a very fast conversion of HPMAꢀBocꢀCys(Acm) and NIPAM was observed. The
monomer conversion displayed pseudoꢀfirstꢀorder kinetics as observed from the linear curves in figure 2B;
however, Mn as a function of conversion did not result in a linear relationship as was observed for PNE
(Fig. 2C). The higher reactivity of HPMAꢀBocꢀCys(Acm)/ETSA monomers compared to NIPAM’s
reactivity during polymerization resulted in a not completely random copolymer having a relatively high
HPMAꢀBocꢀCys(Acm)/ETSA content close to the PEG block. The outer parts of polymeric chains in PNC
and PNE are mainly formed of PNIPAM only. The PDIs of the obtained polymers (1.7ꢀ1.8) are slightly
higher than usually observed for ATRP47 (Table 1) which can be attributed to the difference in reactivity
of acrylamide and methacrylamide monomers, the high length of polymers and the composition of three
polymers blocks48. A higher Mn determined by GPC compared to NMR is often observed for these kinds
of polymers and can be explained by the use of PEGs as GPCꢀstandards, which are not perfectly
representative polymers to compare hydrodynamic volumes with the synthesized triblock copolymers in
the used eluent49,50 (Table 1).
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The cloud point (CP) of PNC prepared by ATRP was 34 °C, which was similar to the CP of a PNC
polymer synthesized by free radical polymerization in a previous study (33 °C)33. As expected, the
presence of the more hydrophobic monomer HPMAꢀETSA in PNE resulted in a slightly lower CP (29 °C)
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than the wellꢀknown value of 32 °C for homopolymers of PNIPAM51 (Table 1).
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Figure 1: Synthesis route of A) HPMAꢀETSA and BꢀC) ABA triꢀblock copolymers containing PEG as
midꢀblocks and either copolymer of (B) NIPAM and HPMAꢀBocꢀCys(Acm)(PNC) or (C) NIPAM and
HPMAꢀETSA (PNE) as outerꢀblocks.
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Table 1: Characteristics of the two ABA triblock copolymers (PNC and PNE) synthesized by ATRP.
Both polymers contain PEG mid blocks of 6 kDa with outer blocks composed of either NIPAM and
HPMAꢀBocꢀCys(Acm)(PNC) or NIPAM and HPMAꢀETSA (PNE).
Feed ratio
Obtained ratio of
a
b
[NIPAM]:[HPMAꢀ [NIPAM]:[HPMAꢀ Mn
Mn
CP
(°C)
Yield
(%)
Polymer
PDIb
BocꢀCys(Acm)]/
HPMAꢀETSA]
93:7
BocꢀCys(Acm)]/
[HPMAꢀETSA]a
93:7
(kDa)
(kDa)
PNC
PNE
42.7
42.1
83
67
1.82
1.72
34.1c
29.2
87
81
93:7
92:8
9
a Determined by 1HꢀNMR. b Determined by GPC. c Cloud point of deprotected PNC
A
B
C
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Figure 2: Kinetics of the ATRP of PNE and PNC. A: ln([M]0/[M]) as a function of time for the
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copolymerization of HPMAꢀETSA and NIPAM measured by HꢀNMR. B: ln([M]0/[M]) as a function of
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time for the copolymerization of HPMAꢀBocꢀCys(Acm) and NIPAM measured by HꢀNMR. [M]0: the
initial concentration of monomers (NIPAM/HPMAꢀBocꢀCys(Acm)/HPMAꢀETSA) and [M] concentration
of monomers in time (NIPAM/HPMAꢀBocꢀCys(Acm)/HPMAꢀETSA). Polymerization of PNC is
significantly faster than polymerization of PNE. C: molecular weight evolution of PNC and PNE as a
function of monomer conversion. Representative results from one out of three experiments are shown.
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Micelle characterization
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Both PNC and PNE exhibit an increase in solution turbidity above 34.1 and 29.2 °C respectively,
indicating lower critical solution temperature (LCST) behavior. Mixing of these polymers at 4 °C, at a
relatively low concentration of 3 mg/mL followed by fast heating (above the LCST of polymers) resulted
in dehydration of the outer Aꢀblocks of the polymers and their selfꢀassembly into flowerꢀlike micelles
(Fig. 3). The obtained micelles had a diameter of 65 nm and PDI of 0.11 at 37 °C (Fig. 4A), similar to
previously reported PNIPAMꢀPEGꢀPNIPAM triblock copolymer micelles3. Noteworthy, separate
solutions of PNC or PNE displayed formation of similar micelles at high temperature, but micelles
disappeared immediately upon cooling.
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afterwards mixed in a 1:1 ratio and immediately heated up to 50 °C using an oil bath. The micellar
solution was left at 50 °C for 3 h to let native chemical ligation proceed in the micellar core.
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Figure 4: A) Size of the micelles as a function of temperature, before and after lyophilization. B) Effect of
repeated temperature cycles from 10 to 37 ºC on micelle size and PDI.
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crosslinking of the micellar core by native chemical ligation52. To confirm that crossꢀlinking occurred via
native chemical ligation (NCL), the formed micelles were passed through a HiTrapTM desalting column to
separate micelles from ethyl thioglycolate, which is the byproduct of the NCL reaction. The Ellman’s
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assay was used to quantify the concentration of released ethyl thioglycolate solution revealing that at least
23% of HPMAꢀETSA monomer contributed to crosslinking. Due to the volatile nature of ethyl
thioglycolate (boiling point = 54 °C) and therefore partial loss of this compound during the workup the
actual percentage of reacted thioester groups is likely much higher. Moreover, the effectiveness of the
crosslinking method was examined by lowering the temperature below the LCST of the PNC and PNE
polymers to remove the effect of heatꢀinduced micelle formation53. By lowering the temperature below the
LCST (10 °C), the size of the micelles increased to a diameter of 90 nm due to rehydration of
thermosensitive chains resulting in swelling of the micelles. However, the extent of micelle swelling is
limited because of the presence of permanent crosslinks. To investigate the reversibility of this behavior,
the temperature was changed between 10 and 37 °C repeatedly (below and above LCST of PNE and PNC,
respectively). As expected, the size of the micelles changed reversibly as a function of temperature, but
notably, the PDI was barely affected (Fig. 4B). This “sponge” behavior can be explained by the structure
of the polymer as determined from the polymerization kinetics study. According to kinetics study, most
HPMAꢀCys/ETSA monomers are located next to the PEG chain in both polymers, which results in a
crosslinking layer between shell (PEG) and core (PNIPAM segment) of micelles. The PNIPAM segments
in the core are relatively flexible and can adjust their conformation depending on temperature resulting in
a relatively large difference in size below and above the LCST. The sponge behavior of the micelles is an
interesting feature and may be used for loading desirable cargo.
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For storage reasons and to remove the released byꢀproduct of NCL, ethyl thioglycolate, the micelles
were dialyzed against water and lyophilized. Interestingly, freeze drying, even without a cryoꢀprotectant,
hardly affected the micellar size upon resuspension in buffer (Fig. 4A).
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The CryoTEM images confirmed the formation of micelles with uniform spherical shape (Fig. 5A). The
size of micelles reported by this method varies from about 50 to 70 nm, which coincides with the data
obtained by DLS. The tomographic view reveals that the polymeric micelles have dark patches mostly
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located in the corona and some inside the micelle together with lighter areas filling the micellar space
(Fig. 5B), similar as reported before by Berlepsch et al. for double hydrophobic triblock copolymers54.
According to an earlier review, the dark patches can be attributed to less hydrated areas in the triblock
micelle structure55. A closeꢀup of a representative micelle (Fig. 5C) shows the distribution of dark patches
in the micelle which is unlike the completely dark micellar core that has been observed in, for example,
PEOꢀPB micelles56. The surface representation of an extricated micelle (Fig. 5C) shows that these patches
are distributed throughout the crosslinked space of the micelle. This can be explained by the fact that
during polymer synthesis, most HPMAꢀCys/ETSA (crosslinkable monomers) were polymerized close to
the PEG block. After micellization and crosslinking, these monomers will be located closer to the corona
of micelles and form dense and hydrophobic areas which can be seen as dark patches in tomographic
view. The low contrast part in the core of micelles could be assigned as the PNIPAM polymeric chains
since they remain hydrated at the temperature of sample preparation (room temperature).
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spherical micelle form instead of a solid single core54.
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Figure 5: A: CryoTEM images of uniformly sized spherical micelles at a concentration of 3 mg/mL in
water on graphene oxide grid. B: 5 nm thick tomographic reconstruction of the spherical micelles shown
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in A. C: A cutꢀoff of the tomographic reconstruction of the selected particle from B in 3D, XꢀY, YꢀZ and
XꢀZ views. The bounding box in C is of 55 × 55 × 30 nm3.
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The micelles were studied by HꢀNMR in D2O at 25 and 37 °C, which showed that the signals
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corresponding to PNIPAM at 1.04 and 3.8 ppm were suppressed by increasing the temperature above the
LCST, while the signal corresponding to the protons of PEG at 3.6 ppm remain visible at both
temperatures. The disappearance of these characteristic signals confirmed the proposed structure of the
micelles with dehydrated PNIPAM hydrophobic cores and hydrated PEG hydrophilic shells (Fig. 6)57.
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The micelles’ average molecular weight (Mw) and aggregation number (Nagg) was measured at different
temperatures of 10, 25, 37, and 45 °C by static light scattering (SLS) (table 2). The obtained data by SLS
revealed that the micelles’ Nagg and Mw did not change significantly with increasing temperature.
Considering the number of PNE polymers in each micelle according to Nagg and crosslink density
calculated based on the Ellman’s assay, the minimum number of crosslinking points in each micelle is
approximately 1000. This number of crosslinks corroborates with the high stability of the micelles even at
low temperatures.
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The ratio of the radius of gyration to the hydrodynamic radius (Rg/Rh) is an important parameter to
understand the conformation of the nanoparticles in solution. Rg/Rh values of 0.773, ∼ 1.8, and ∼ 2 have
been reported for an uniform sphere, a polydisperse linear coil, and a rodꢀlike linear chain,
respectively58,59. In this study, the found Rg/Rh ratios for the micelles (0.83–0.95) were also similar at the
various temperatures and close to the theoretical limit for spherical structures. Furthermore, the increase in
the density (
ρ) of micelles with increasing temperature, clearly shows shrinking of micelles at
temperatures above the LCST of the polymers.
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Table 2: Characteristics of core crosslinked polymeric micelles consisting of PNE and PNC (1:1)
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measured by DLS and SLS in PBS.
a
b
c
Rg
Rh
Mw (mic.)
ρ (mic.)
d
Temperature
Rg/Rh
Nagg
(nm)
(nm)
(106 Da)
(g.cmꢀ3)
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29,7
29,9
48,2
42,8
35,8
35,0
0,95
0,84
0,83
0,85
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16,20
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14,24
0,06
0,14
0,21
0,20
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379
10°C
25°C
37°C
45°C
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a Radius of gyration extrapolated to zero concentration. b Hydrodynamic radius extrapolated to zero
concentration and zero scattering angle. c Density of the micelles. d Aggregation number of the micelles.
Figure 6: 1HꢀNMR spectra of the core crosslinked micelles in D2O at 25 and 37 °C. The peak shifts are
due to the change in temperature.
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To investigate possible biomedical applications of the developed micelles, cellular uptake was studied
on HeLa and A549 cells. To this end, two kinds of dyes which also represent model cargos, having
maleimide and amine functionalities, were used for labeling the micelles. An NHS modified Alexa fluor
647 was used for conjugation to cysteine moieties while a maleimideꢀmodified Alexa fluor C5 568 was
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used for conjugation to free thiol groups remaining present after native chemical ligation or of nonꢀreacted
cysteine moieties. To conjugate NHS modified dyes, micelles with a molar ratio of 3: 2 for PNC/PNE
were formed. This ratio ensures that sufficient cysteine groups are present in the core of the micelles,
which were used for covalent attachment of dye. For the conjugation of maleimideꢀfunctionalized dye,
micelles with 1:1 molar ratio of PNC and PNE were used. To perform the conjugation, the crosslinked
micelles were swollen in DMSO, therefore cysteine functionalities or thiol moieties became available for
conjugation to the dyes. The swelling of micelles in organic solvent did not result in dissociation due to
the presence of covalent crosslinks and no aggregation was observed after extensive dialysis against water
to remove excess dye and solvent. The successful covalent conjugation of these two kinds of dyes
demonstrated the accessibility of functional groups in the core of micelles for conjugation to desired cargo
molecules having different functional groups.
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Cells were incubated with labeled micelles for 2, 4, and 24 h, and eventually washed with PBS before
imaging. The confocal images in figure 7 showed punctate fluorescence close to the nuclei confirming the
internalization of micelles by cells after 24 h at a concentration of 400 µg/mL. At lower concentrations
and shorter incubation times, the presence of micelles inside cells was hardly observed, which can be
expected for micelles with PEG corona without targeting ligand60.
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In addition, cytotoxicity of the micelles was studied on HeLa cells. An MTS assay was performed to
assess the metabolic activity of cells in the presence of the micelles. In living cells, mitochondrial
enzymes reduce the MTS tetrazolium compound and generate a colored formazan that can be quantified
by a colorimetric method61. Figure 8 shows that no change in mitochondrial activity was observed upon
incubation of HeLa cells with micelles at concentrations ranging from 11 µg/mL to 1.5 mg/mL after 24 h.
To study possible cell membrane damage upon exposure to the micelles, an LDH assay was performed. In
damaged cells, lactate dehydrogenase (LDH) is released, which catalyzes a series of reactions that
eventually cause reduction of a tetrazolium salt to a highlyꢀcolored formazan, which absorbs strongly at
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490ꢀ520 nm62. The results demonstrated no damage to the cell membrane upon incubation with micelles
over a wide range of concentrations (SIꢀFig. 5). These results confirm the cytocompatibility of these
micelles.
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Figure 7: Core crosslinked micelle internalization study. Laser confocal scanning microscopy images of
HeLa cells and A549 incubated for 24 h with (A and C) cell culture medium (B and D) fluorescentlyꢀ
labeled micelles at a concentration of 400 µg/mL. Cell nuclei are stained with Hoechst (blue) while the
micelles are visualized by NHSꢀAlexa fluor 647 in B (red) and maleimideꢀAlexa flour C5 568 in D
(orange).
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Figure 8: In vitro cytotoxicity (MTS assay) on HeLa cells after 24 h incubation with micelles across a
135ꢀfold concentration range (0.011 – 1.5 mg/mL). Data are presented as mean values ± SD of three
independent experiments.
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Conclusion
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In this study, native chemical ligation was introduced as a mild, chemoselective method for the
crosslinking of micelles in an aqueous medium. Mixing two complementary PNIPAM based ABA
polymers, containing either HPMAꢀCys or HPMAꢀETSA, and subsequently, increasing the temperature
above the LCST resulted in flowerꢀlike micelles with a size of 65 nm at 37 °C. Reducing the temperature
to 10 °C resulted in a change in the size of the micelles to 90 nm, but hardly showed any change in
polydispersity and aggregation number, thus confirming permanent crosslinking of the micelles. The
uniform spherical shape of micelles was confirmed by CryoꢀTEM. Interestingly, in tomographic view of
the micelles, dark patches were seen close to the corona of micelles, which according to the kinetics study
corresponds to the area with the highest content of crosslinkable monomers. Therefore, the dark patches
can be interpreted as the crosslinked area of the micelles while the inner low contrast part of micelles is
composed of the more hydrated PNIPAM chains. In addition, by adjusting the molar ratio between PNC
and PNE polymers during micelle formation, nucleophilic (cysteine) or electrophilic (thioester) sites can
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be introduced within the micellar core. Here, we have shown that the presence of cysteine and thiol
moieties remaining after crosslinking could be used for conjugation of an NHS or maleimide
functionalized dyes respectively. These functional sites may also be used for further modification of the
micelle to introduce (pro) drugs or charge in the core. Notably, this crosslinking method resulting in the
presence of thiol moieties in the micellar core also provides the possibility for conjugation of cargo via a
disulfide bond, which may be interesting for intracellular drug delivery. Moreover, the high cell viability
and observed cellular uptake of these micelles by HeLa cells show a good cytocompatibility profile and
potential of this nanocarrier for drug delivery applications.
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Supporting information
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NMR spectrums (1HNMR and CNMR) of HPMAꢀETSA (SIꢀFig. 1A, B); H NMR spectrum of PNE
(SIꢀFig. 2); 1H NMR spectrum of PNC (SIꢀFig. 3); GPC chromatograms of PNE and PNC (SIꢀFig. 4 A, B)
and LDH assay on HeLa cells (SIꢀFig. 5).
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Acknowledgments
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The Netherlands Organization for Scientific Research (NWO/VIDI 13457 and NWO/Aspasia
015.009.038) is acknowledged for funding.
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