Synthesis and Characterization of Stimuli-Responsive Star-Like Polypept(o)ides: Introducing Biodegradable PeptoStars

Article abstract of DOI:10.1002/mabi.201600514

Star-like polymers are one of the smallest systems in the class of core crosslinked polymeric nanoparticles. This article reports on a versatile, straightforward synthesis of three-arm star-like polypept(o)ide (polysarcosine-block-polylysine) polymers, which are designed to be either stable or degradable at elevated levels of glutathione. Polypept(o)ides are a recently introduced class of polymers combining the stealth-like properties of the polypeptoid polysarcosine with the functionality of polypeptides, thus enabling the synthesis of materials completely based on endogenous amino acids. The star-like homo and block copolymers are synthesized by living nucleophilic ring opening polymerization of the corresponding N-carboxyanhydrides (NCAs) yielding polymeric stars with precise control over the degree of polymerization (X_n = 25, 50, 100), Poisson-like molecular weight distributions, and low dispersities (? = 1.06–1.15). Star-like polypept(o)ides display a hydrodynamic radius of 5 nm (μ₂ < 0.05) as determined by dynamic light scattering (DLS). While star-like polysarcosines and polypept(o)ides based on disulfide containing initiators are stable in solution, degradation occurs at 100 × 10^–3m glutathione concentration. The disulfide cleavage yields the respective polymeric arms, which possess Poisson-like molecular weight distributions and low dispersities (? = 1.05–1.12). Initial cellular uptake and toxicity studies reveal that PeptoStars are well tolerated by HeLa, HEK 293, and DC 2.4 cells. (Figure presented.).

Full text of DOI:10.1002/mabi.201600514

Macromolecular
Bioscience
Full Paper
Synthesis and Characterization of
Stimuli-Responsive Star-Like Polypept(o)ides:
Introducing Biodegradable PeptoStars
Regina Holm, Benjamin Weber, Philipp Heller, Kristina Klinker,
Dana Westmeier, Dominic Docter, Roland H. Stauber, Matthias Barz*
Star-like polymers are one of the smallest systems in the class of core crosslinked polymeric
nanoparticles. This article reports on a versatile, straightforward synthesis of three-arm
star-like polypept(o)ide (polysarcosine-block-polylysine) polymers, which are designed to be
either stable or degradable at elevated levels of glutathione. Polypept(o)ides are a recently
introduced class of polymers combining the stealth-like properties of the polypeptoid poly-
sarcosine with the functionality of polypeptides, thus enabling the synthesis of materials
completely based on endogenous amino acids. The star-like homo and block copolymers
are synthesized by living nucleophilic ring opening polymerization of the corresponding
N-carboxyanhydrides (NCAs) yielding polymeric stars with precise control over the degree of
polymerization (Xn = 25, 50, 100), Poisson-like molecular weight distributions, and low dis-
persities (Đ = 1.06–1.15). Star-like polypept(o)ides display a
hydrodynamic radius of 5 nm (μ2 < 0.05) as determined by
dynamic light scattering (DLS). While star-like polysarcosines
and polypept(o)ides based on disulfide containing initiators
are stable in solution, degradation occurs at 100 × 10–3
m
glutathione concentration. The disulfide cleavage yields the
respective polymeric arms, which possess Poisson-like
molecular weight distributions and low dispersities (Đ =
1.05–1.12). Initial cellular uptake and toxicity studies reveal
that PeptoStars are well tolerated by HeLa, HEK 293, and
DC 2.4 cells.
R. Holm, B. Weber, P. Heller, K. Klinker, Dr. M. Barz
Institute of Organic Chemistry
1. Introduction
Johannes Gutenberg-Universität Mainz
Duesbergweg 10-14, 55128 Mainz, Germany
E-mail: barz@uni-mainz.de
Star-like polymers are a particularly interesting class of
polymers, since the size of these nanoparticles can be
precisely controlled from several to several tens of nano-
meters by adjusting the degree of polymerization of the
individual arms. In addition, the microstructure of the
side chains can be controlled to yield core–shell structures
with compartments of different polarity. This precise con-
trol over size and internal structure may provide solutions
to tackle some of the limitations of other drug delivery
P. Heller, K. Klinker
Graduate School Materials Science in Mainz
Staudinger Weg 9, 55128 Mainz, Germany
D. Westmeier, Dr. D. Docter, Prof. R. H. Stauber
Department of Nanobiomedicine/ENT
University Medical Center of Mainz
Langenbeckstr. 1, 55101 Mainz, Germany
Macromol. Biosci. 2017, DOI: 10.1002/mabi.201600514
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(1 of 14) 1600514
DOI: 10.1002/mabi.201600514

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R. Holm et al.
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systems, for example in cancer therapy, where intratu-
moral pressure substantially limits their penetration.[1–3]
In general, a nanodrug delivery system (NanoDDS) for
chemotherapeutics designed with regard to cancer
therapy needs to deliver its cargo to the tumor cells. While
for many solid tumors extravasation into intestinal space
takes place for nanoparticles and retention is ensured by
poor lymphatic drainage,[4,5] deep penetration into the
tumor is hardly realized. The limited accessibility hinders
the drug from reaching the majority of tumor cells and
thus limits the therapeutic outcome. The penetration of
dense tumor tissue highly depends on size and shape of
the carriers.[6,7] Known nano-sized drug delivery systems
like polymeric micelles and polymersomes often fail to
achieve the desired deep tumor penetration due to their
relatively large sizes of 30–100 or even 200 nm. Kataoka
and co-workers nicely demonstrated that the accumula-
tion and the therapeutic outcome of stabilized polymeric
micelles highly depend on the size in poorly permeable
tumors. Only the smallest micelles (Dh < 50 nm) reach the
majority of cancer cells in human pancreatic adenocarci-
noma model and achieve the most pronounced antitumor
effect.[8] For any systemic application and controlled drug
release from a delivery system, however, stable circula-
tion of the respective system turned out to be a require-
ment.[9] Stabilization of micelles can be performed by
crosslinking. To avoid any influence of the crosslinker
itself on the particle corona, it seems beneficial that the
stabilization occurs in the core of the micelle.[9–11] Core-
stabilized micelles have already demonstrated high
stability in blood circulation, enhanced passive tumor
accumulation, and controlled drug release and hence, it is
not much of a surprise that several systems have entered
clinical phases.[12]
The smallest representatives in the class of stable
polymeric core–shell nanoparticles, however, are star-
like block copolymers, in which multiple polymers are
attached to a multivalent core. Such systems can be
synthesized by either grafting onto or grafting from
approaches. Both cases allow to control the size of the
particle as well as the volume of core and corona part by
degree of polymerization of the individual arms.[13] Star-
shaped block copolymers combine small and tunable
sizes between 10 and 40 nm with intrinsic high stability
without additional core crosslinking methods. As men-
tioned above, this size regime may become particularly
interesting for systems taking profit of the enhanced
permeability and retention effect (Dh ≥ 10 nm), but are
still able to achieve deep tumor penetration, which may
enable the drug to be released more uniformly and, thus
enhance therapeutic efficiency. Thereby, star block copoly-
mers seem to be promising candidates for an application
as NanoDDS and consequently, they are currently widely
investigated for the delivery of chemotherapeutics[14]
or genes.[15] The first star-shaped polymer dates back to
1948, while Schaefgen and Flory investigated multichain
polyamides.[16] Since then, more and more research on
star-like polymers and their corresponding features has
been engaged. The branched architectures of these poly-
mers consist of at least three linear arms linked to a cen-
tral core,[17] and thus the resulting 3D globular structure
offers unique chemical and physical properties, namely
smaller hydrodynamic radii, decreased solution viscosi-
ties,[6] high end-group functionalities, and encapsula-
tion capabilities.[14] Star-like polymers that have been
reported in literature are mainly based on polyethers,
polyesters, polyacrylates, polyacrylamides, polyolefines,
polypeptoids,[18] or polypeptides.[19–21] Interestingly, so
far, star-like polypept(o)ides have not been reported in
literature, although this class may possess enormous
potential for biomedical applications.[22–24] Polypept(o)-
ides are block copolymers combining the polypeptoids,
e.g., polysarcosine, with polypeptides and thus lead
to block copolymers completely based on endogenous
amino acids.[25–27] This hybrid system combines the
stealth-like properties of polysarcosine with the mul-
tifunctionality of synthetic polypeptides.[24] Polysarco-
sine has proven to be a highly protein-resistant mate-
rial, which is able to introduce stealth-like properties to
nano- or macroscopic surfaces,[28–30] indicating to be an
alternative to polyethylene glycol (PEG).[31,32] Polypept(o)-
ides are easily accessible by sequential nucleophilic
ring opening polymerization (ROP) of α-amino acid
N-carboxyanhydrides (NCAs) and correspondingly of the
N-substituted glycine N-carboxyanhydride (NNCA).[26]
The quasi-living polymerization provides precise control
of molecular weights, high end-group integrity, and low
polymer dispersity. Furthermore, our group has recently
introduced stimuli-responsive polypept(o)ides, which
react to changes in reduction potential by disulfide
cleavage.[33] The reductive cleavage of the disulfide can
be used to convert an amphiphilic block copolymer into
its individual blocks, which causes destabilization of the
micelle and hence facilitates cargo release.
In this work, we report on the synthesis of polypep-
toid and polypeptide homo arm star polymers as well
as on the synthesis of star-shaped polypept(o)ides (Pep-
toStars) based on three-arm initiators. While one ini-
tiator contains disulfides and is therefore degradable
at elevated glutathione (GSH) levels, the other is stable
under these conditions. The star-like polypept(o)ides
consist of a hydrophobic, protected lysine core with a
hydrophilic polysarcosine shell and are characterized
by 1H and 13C nuclear magnetic resonance (NMR) spec-
1
troscopy, H diffusion ordered spectroscopy (DOSY), size
exclusion chromatography (SEC), multiangle dynamic
light scattering (DLS), and atomic force microscopy
(AFM).
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2.2.1. Boc-Protected Cystamine-Based Initiator: N1,N3,N5-
tris(2-((2-tert-Butyloxycarbonyl-aminoethyl)disulfanyl)-
ethyl)benzene-1,3,5-tricarboxamide
2. Experimental Section
2.1. Materials and Methods
Sarcosine and solvents were purchased from Sigma Aldrich.
Protected amino acids were bought from ORPEGEN and dried
in vacuum before NCA synthesis. Tetrahydrofuran (THF) and
n-hexane were dried over Na/K prior to use. Dimethylforma-
mide (DMF) was purchased from Acros (99.8%, Extra), dried
over CaH2 and molecular sieves (4 Å), and fractionally distilled
in N2 atmosphere. Freshly distilled DMF was stored at −80 °C
under exclusion of light. Before use, DMF was degassed under
vacuum for at least 20 min to remove residual dimethylamine.
Diphosgene was purchased from Alfa Aesar and used without
further purification. 1,3,5-Benzenetricarbonyl trichloride was
purchased from Acros. N-Boc-1,4-Butanediamine was pur-
chased from Sigma Aldrich and N-Boc cystamine was purchased
from Chem-Impex International. Millipore water was prepared
using a MILLI-Q Reference A+ System. Water was used at a
resistivity of 18.2 MΩ cm–1 and total organic carbon of <5 ppm.
1H and 13C NMR spectra were recorded on a Bruker AV 400 at
room temperature. Spectra were calibrated using the solvent
signals (δ = 2.500 ppm for dimethyl sulfoxide-d6 (DMSO-d6),
δ = 7.260 ppm for CDCl3).
N-(tert-Butyloxycarbonyl)cystamine (116 mg, 0.46 mmol, 3.3 eq)
was weighed into a flame-dried two-neck round-bottom flask
and was dissolved in 2 mL abs. THF. Freshly distilled N,N-diiso-
propylethylamine (DIPEA) (78 μL, 0.46 mmol, 3.3 eq) was added
and stirred for 15 min at room temperature. 1,3,5-Benzene-
tricarbonyl trichloride (36 mg, 0.14 mmol, 1 eq) was weighed
into a flame-dried round-bottom flask and was dissolved in 1 mL
abs. THF. The trichloride solution was slowly added to the N-(tert-
butyloxycarbonyl)cystamine mixture via syringe. The progress of
the reaction was monitored by thin-layer chromatography (TLC)
(Rf = 0.53, EtAc/PE 10%). After 4 h, the solvent was evaporated in
vacuo and the residue was dissolved in ethyl acetate. The organic
phase was sequentially washed with milliQ water, 1 m hydro-
chloric acid, and saturated sodium bicarbonate solution. The
organic phase was dried over magnesium sulfate and concen-
trated in vacuo. Purification by column chromatography gave
the desired product as a white foam (92.1 mg, 0.1 mmol, 72%). 1H

NMR (400 MHz, CDCl3) δ [ppm] = 1.38 (s, br, 27H, C(CH3)3), 2.83
3
3

(t, JH,H = 6.40 Hz, 6H, SCH2CH2NHBoc), 2.95 (t, JH,H = 6.00 Hz,



6H, SCH2CH2NHCOC), 3.45 (br, 6H, CH2NHBoc), 3.74–3.78 (m,
Infrared spectroscopy was performed on a Jasco FT/IR-4100
with an attenuated total reflectance (ATR) sampling accessory
(MIRacle, Pike Technologies) using 16 scans per measurement.
IR spectra were analyzed using Spectra Manager 2.0 (Jasco).
NCA polymerization was monitored by Fourier transform
infrared (FT-IR) spectroscopy. Polymerization was judged to
be complete when NCA associated carbonyl peaks at 1853 and
1786 cm−1 had vanished. Size exclusion chromatography (SEC)
in hexafluoroisopropanol (HFIP) was performed with 3 g L–1
potassium trifluoroacetate at 40 °C. The columns were packed
with modified silica (PFG columns; particle size: 7 μm; porosity:
100 and 1000 Å). A refractive index detector (G 1362A RID,
Jasco) and a UV–vis detector (UV-2075 Plus, JASCO) were used
to detect the polymer. Molecular weights were calculated using
calibration performed with poly(methyl methacrylate) (PMMA)
standards (Polymer Standards Services GmbH). As the internal
standard, toluene was used. Melting points were determined on
Mettler Toledo FP62 melting point apparatus at a heating rate


6H, CH2NHCOC), 5.22 (br, 3H, NHBoc), 7.58 (br, 3H, NHCOC),
8.27 (s, 3H, CH); 13C NMR (400 MHz, CDCl3) δ [ppm] = 28.5
C(CH3)3), 38.1 ( SCH2CH2NHCOC), 38.3 ( SCH2CH2NHBoc), 39.4


 
(




(
CH2NHCOC), 39.7 ( CH2NHBoc), 79.8 ( C(CH3)3), 128.7 ( CH),



135.3( CCONH), 156.2 ( NHCOO), 166.6 ( CCONH).
2.2.2. Boc-Protected Butyl-Based Initiator: N1,N3,N5-tris(2-
(2-tert-Butyloxycarbonyl-aminobutyl))benzene-1,3,5-
tricarboxamide
N-(tert-Butyloxycarbonyl)-1,4-butanediamine (121 μL, 0.63 mmol,
3.3 eq) was dissolved in 2 mL abs. dichloromethane. Freshly dis-
tilled DIPEA (107 μL, 0.63 mmol, 3.3 eq) was added and stirred for
15 min at room temperature. 1,3,5-Benzenetricarbonyl trichloride
(51 mg, 0.19 mmol, 1 eq) was weighed into a flame-dried round-
bottom flask and was dissolved in 2 mL abs. dichloromethane.
The trichloride solution was slowly added to the N-(tert-
butyloxycarbonyl)-1,4-butanediamine mixture via syringe. The
progress of the reaction was monitored by TLC (Rf = 0.59, EtAc/
MeOH 10%). After 4 h, the solvent was evaporated in vacuo and
the residue was dissolved in chloroform. The organic phase was
sequentially washed with milliQ water, 1 m hydrochloric acid,
and saturated sodium bicarbonate solution. The organic phase
was dried over magnesium sulfate and concentrated in vacuo.
of 5 °C min–1
.
Dynamic light scattering measurements were first performed
at 25 °C using a Malvern Zetasizer NanoZS with a 633 nm
He/Ne Laser at a fixed scattering angle of 173°. Besides, further
DLS measurements were performed using a Uniphase He/Ne
Laser (l = 632.8 nm, 22 mW), an ALV-SP125 Goniometer, an ALV/
High QE APDAvalanche photodiode with fiber optical detection,
an ALV 5000/E/PCI-correlator, and a Lauda RC-6 thermostat unit
at 20 °C. Angular dependent measurements were carried out in
the range 30° ≤ q ≤ 150° in typically 15° steps.
1
126.5 mg of the pure product was obtained (0.17 mmol, 92%). H

NMR (400 MHz, CDCl3) δ [ppm] = 1.37 (s, 27H, C(CH3)3), 1.59–

1.67 (m, 12H, CH2CH2CH2CH2), 3.13–3.17 (m, 6H, CH2NHBoc),
3.43–3.48 (m, 6H, CH2NHCO), 5.11 (t, 3JH,H = 8.00 Hz, 3H, NHBoc),

7.63 (s, br, 3H, NHCOC), 7.93 (s, 3H, CH); 13C NMR (400 MHz,


2.2. Initiator Synthesis


CDCl3) δ [ppm] = 26.9 ( CH2CH2CH2CH2), 28.6 ( C(CH3)3), 40.0

  
(
CH2NHCOC), 40.5 ( CH2NHBoc), 79.3 ( C(CH3)3), 128.2 ( CH),
The synthesis of the two trimeric amine initiators was adapted
from literature and modified.[34]



135.7 ( CCONH), 156.5 ( NHCOO), 167.1 ( CCONH).
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2.3. Deprotection
2.4.2. N-ε-tert-Butyloxycarbonyl-l-Lysine
N-Carboxyanhydride
2.3.1. Trifluoroacetic acid (TFA) Salt of N1,N3,N5-tris(2-((2-
Aminoethyl)disulfanyl)ethyl)benzene-1,3,5-tricarboxamide
9.8 g (39.8 mmol) of N-ε-Boc-protected lysine was weighed
into a three-neck flask and dried under vacuum for 1 h. The
solid was suspended in 300 mL of absolute THF under a steady
flow of dry nitrogen. Afterwards, 9.9 mL (79.6 mmol) of abso-
lute trimethylsilylchloride and 11.0 mL (79.6 mmol) of abso-
lute triethylamine (TEA) were added slowly via syringe. The
suspension was stirred for 2 h followed by addition of 4.8 mL
(39.8 mmol) of diphosgene, again via syringe. The suspension
was mildly refluxed for another 2 h. Overnight, a steady flow
of dry nitrogen was led through the solution into two gas
washing bottles filled with aqueous NaOH solution removing
excess phosgene and HCl. The suspension was filtered under
dry nitrogen atmosphere to remove the salts of TEA and uncon-
verted amino acid. The filtrate was evaporated under reduced
pressure. Subsequently, absolute THF was added to completely
dissolve the crude reaction product. The solution was precipi-
tated with absolute hexane and stored at 4 °C for 1 h. The solid
was collected by filtration under dry nitrogen atmosphere and
washed with hexane. The crude reaction product was then
recrystallized twice with absolute THF/hexane. 6.52 g of puri-
fied reaction product (23.9 mmol; 68% yield; colorless nee-
dles) was transferred into a Schlenk tube and stored at −80 °C.
Melting point: 138.3 °C (Lit. 134–137 °C).
40 mg (0.04 mmol) of the Boc-protected cystamine-based ini-
tiator was dissolved in dry dichloromethane (1 mL) and 0.5 mL
TFA was added. The reaction was stirred under argon for 60 min
and the completion of the reaction was monitored by TLC. The
solvents were evaporated in vacuo. The TFA salt of the initiator
(38 mg, 0.04 mmol) was obtained in quantitative yields and dried
1
under vacuum. H NMR (400 MHz, D2O) δ [ppm] = 3.00–3.03 (m,
3
12H, CH2CH2SSCH2CH2), 3.38 (t, JH,H = 8.00 Hz, 2H, CH2NHBoc),
3.78 (t, 3JH,H = 8.00 Hz, 2H, CH2NHCO), 8.28 (s, 3H, CH).

2.3.2. TFA Salt of N1,N3,N5-tris(2-(2-Aminobutyl))benzene-
1,3,5-tricarboxamide
20.4 mg (0.03 mmol) of the Boc-protected cystamine-based ini-
tiator was dissolved in dry dichloromethane (1 mL) and 0.5 mL
TFA was added. The reaction was stirred under argon for 60 min
and the completion of the reaction was monitored by TLC. The
solvents were evaporated in vacuo. The TFA salt of the initiator
(22.7 mg, 0.03 mmol) was obtained in quantitative yields and
dried under vacuum. 1H NMR (400 MHz, D2O) δ [ppm] = 1.67–1.79
3
(m, 12H, CH2CH2SSCH2CH2), 3.04 (t, JH,H = 8.00 Hz, 2H, CH2N-
HBoc), 3.46 (t, 3JH,H = 8.00 Hz, 2H, CH2NHCO), 8.24 (s, 3H, CH).

1H NMR (400 MHz, DMSO-d6): δ [ppm] = 1.45–1.12 (13 H, m,









 
CH CH2 CH2 CH2 CH2 NH
and
NH CO O C(CH3)3),







1.79–1.56 (2 H, m, CH CH2 CH2 CH2 CH2 NH ), 2.90 (2 H,
3
3





q, JH,H = 6.03 Hz, CH2 NH CO
6.35 Hz, CO CH CH2 ), 6.95–6.34 (1 H, br, NHCOOC(CH3)3),
O
C(CH3)3), 4.42 (1 H, t, JH,H
=
2.4. Monomer Synthesis





2.4.1. Sarcosine-N-Carboxyanhydride

9.07 (1 H, s, NHCOOCO).
The synthesis of sarcosine NCA was adapted from literature and
modified.[35] Sarcosine (50.96 g, 572 mmol, 1 eq) was weighed
into a predried three neck round-bottomed flask and dried in
vacuo for 1 h. 520 mL abs. THF was added under a steady flow
of nitrogen and diphosgene (55.2 mL, 457.6 mmol, 0.8 eq) was
added slowly via syringe. The colorless suspension was heated
to 70 °C yielding a clear solution after 3 h of stirring. A steady
flow of dry nitrogen was led through the solution into two gas
washing bottles filled with aqueous sodium hydroxide solution
to remove excess HCl and phosgene. The solvent was evapo-
rated under reduced pressure yielding a brownish oil as crude
reaction product. The oil was heated to 50 °C and dried under
reduced pressure (20 mbar, then <10−1 mbar for 2 h) to obtain an
amorphous solid. The crude reaction product was redissolved in
60 mL THF and precipitated with 300 mL dry n-hexane. The solu-
tion was cooled to −18 °C to complete precipitation and was fil-
tered off under dry nitrogen atmosphere. Afterwards, a stream
of dry nitrogen was used to dry the product for 60–90 min to
remove residual traces of THF. The next day the product was
dried in high vacuum for 2 h in the sublimation apparatus.
Then, the crude product was sublimated at 80–85 °C and
<1 × 10−2 mbar. The product was collected from the sublimation
apparatus in a glove box on the same day and aliquoted subse-
quently afterwards when the melting point was above 100 °C.
Colorless crystals were obtained (64%). mp = 103 °C, 1H NMR
(300 MHz, CDCl3) δ [ppm] = 2.86 (s, 3H, NHCH3), 4.22 (s, 2H,
NHCH2CO).
2.4.3. N-ε-Trifluoroacetyl-L-Lysine N-Carboxyanhydride
12.5 g (51.6 mmol) of N-ε-trifluoroacetyl-protected lysine was
weighed into a three-necked flask and dried under vacuum for
1 h. The solid was suspended in 300 mL of absolute THF under
a steady flow of dry nitrogen. The apparatus was connected to
two gas washing bottles filled with aqueous sodium hydroxide
solution. Diphosgene (5.50 mL, 45.6 mmol) was added slowly
via syringe and the nitrogen stream was reduced. The colorless
suspension was mildly refluxed for 2 h, yielding a cloudy solu-
tion. Overnight, a steady flow of dry nitrogen was led through
the solution removing excess phosgene and HCl. The solvent
was evaporated under reduced pressure and the crude reaction
product was redissolved in absolute THF and precipitated with
an excess of dry n-hexane. The solid was collected by filtration
under dry nitrogen atmosphere and washed with n-hexane. The
crude product was recrystallized twice with absolute THF/n-
hexane. 10.43 g of purified product (39.0 mmol; 75% yield;
colorless needles were obtained and stored in a Schlenk-tube at
−80 °C. mp = 101 °C; 1H-NMR (300 MHz, DMSO-d6): δ [ppm] =







1.42–1.24 (2H, m, CH CH2 CH2 CH2 CH2 NH ), 1.53–1.46
3







(2H, quin, JH,H = 7.13 Hz, CH CH2 CH2 CH2 CH2 NH ),








1.79–1.61 (2H, m,
CH CH2 CH2 CH2 CH2 NH ), 3.17
3
3


(2H, q, JH,H = 6.3 Hz, CH2 NH ), 4.43 (1H, t, JH,H = 6.3 Hz,
CO CH CH2 ), 9.09 (1H, s, NH CO








 
O CO ), 9.40 (1H, s,


NH CO CF3).
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

(br, nH, NHCOOC(CH3)3), 7.87–8.38 (br, nH, COCHNH), 8.68 (s,
2.5. Polymer Synthesis

ini, 3H, CH).
All polymerizations with both types of initiator were carried out
in the same way.
SS(PLys(TFA)25)3: 132 mg of Lys(TFA)-NCA (0.49 mmol, 1 eq)
was transferred under nitrogen counter flow into a predried
Schlenk-tube, equipped with a stir bar, and again dried in high
vacuum for 30 min. Then, the NCA was dissolved in 1 mL of dry
DMF. A stock solution of 9.5 mg (0.01 mmol, 1/75 eq) TFA salt of
N1,N3,N5-tris(2-((2-aminoethyl)disulfanyl)ethyl)benzene in 2 mL
DMF was prepared and 5.1 μL (0.03 mmol, 1/25 eq) freshly dis-
tilled DIPEA was added. 1 mL of this stock solution was added
to the monomer solution via syringe. The solution was stirred at
0 °C and kept at a constant pressure of 1.25 bar of dry nitrogen
via the Schlenk-line to prevent impurities from entering the
reaction vessel while allowing CO2 to escape. Completion of
the reaction was confirmed by IR spectroscopy (disappearance
of the NCA peaks (1853 and 1786 cm−1)). Directly after comple-
tion of the reaction, the polymer was precipitated in a mixture
of hexane/diethyl ether (50/50) and centrifuged (4500 rpm
at 4 °C for 15 min). After discarding the liquid fraction, new
hexane/ether was added and the polymer was resuspended
using sonication. The suspension was centrifuged again and
the procedure was repeated. The polymer was then dissolved in
methanol, dialyzed against methanol, and lyophilized to obtain
2.5.1. Polypeptoid Stars
In a typical experiment, 73.4 mg of SarNCA (0.64 mmol) was
transferred under nitrogen counter flow into a predried Schlenk-
tube, equipped with a stir bar and again dried in high vacuum for
30 min. Then, the NCA was dissolved in 0.5 mL of dry DMF. A stock
solution of 4.1 mg (4.3 μmol, 1/300 eq) TFA salt of the N1,N3,N5-
tris(2-((2-aminoethyl)disulfanyl)ethyl)benzene or N1,N3,N5-tris(2-
(2-aminobutyl))benzene (3.3 mg, 4.3 μmol, 1/300 eq) in 2 mL DMF
was prepared and 2.4 μL (14.2 μmol, 1.1/100 eq) freshly distilled
DIPEA was added. 1 mL of this stock solution was added to the
monomer solution via syringe. The solution was stirred at 0 °C
and kept at a constant pressure of 1.25 bar of dry nitrogen via
the Schlenk-line to prevent impurities from entering the reac-
tion vessel while allowing CO2 to escape. Completion of the reac-
tion was confirmed by IR spectroscopy (disappearance of the
NCA peaks (1853 and 1786 cm−1)). Directly after completion of
the reaction, the polymer was precipitated in cold diethyl ether
and centrifuged (4500 rpm at 4 °C for 15 min). After discarding
the liquid fraction, new ether was added and the polymer was
resuspended using sonication. The suspension was centrifuged
again and the procedure was repeated. The polymer was then
dissolved in methanol, dialyzed against methanol, and lyophi-
lized to obtain a fluffy powder (21 mg, 47%). For different chain
lengths, yields ranged from 47% to 50%. 1H NMR: (400 MHz,
1
a fluffy powder (76 mg, 65%). H NMR: (400 MHz, DMSO−d6): δ

[ppm] = 1.20–2.03 (br, 6nH, CHCH2CH2CH2CH2NH), 3.12 (s, 2nH,



CH2NHCOC), 3.75–4.25 (br, nH, COCHNH), 7.90–8.43 (br, nH,


COCHNH), 8.91 (s, ini, 3H, CH), 9.20–9.40 (br, nH, NHCOCF3).
2.5.3. Polypept(o)ide Stars

DMSO−d6): δ [ppm] = 2.74–2.93 (br, 3nH, NCH3), 3.92–4.34 (br,
The syntheses of polypept(o)ide stars were carried out in the
same way as for polypeptoid stars, using polypeptide stars as
macroinitiator.



2nH, COCH2N), 8.42–8.06 (br, 6H, NH), 8.92 (s, ini, 3H, CH).
2.5.2. Polypeptide Stars
(PLys(Boc)25-PSar100)3: In
a
typical experiment, 46.7 mg
as macroinitiator was
(2.61 μmol, 1/300 eq) butyl(PLys(Boc)25 3
)
(PLys(Boc)25)3: In a typical experiment, 176 mg of Lys(Boc)-NCA
(0.65 mmol, 1 eq) was transferred under nitrogen counter flow
into a predried Schlenk-tube, equipped with a stir bar and again
dried in high vacuum for 30 min. Then, the NCA was dissolved
in 1.5 mL of dry DMF. A stock solution of 15.3 mg (0.02 mmol,
1/75 eq) TFA salt of N1,N3,N5-tris(2-(2-aminobutyl))benzene in
2 mL DMF was prepared and 10.2 μL (0.06 mmol, 1/25 eq) freshly
distilled DIPEA was added. 1 mL of this stock solution was added
to the monomer solution via syringe. The solution was stirred at
0 °C and kept at a constant pressure of 1.25 bar of dry nitrogen
via the Schlenk-line to prevent impurities from entering the
reaction vessel while allowing CO2 to escape. Completion of the
reaction was confirmed by IR spectroscopy (disappearance of
the NCA peaks (1853 and 1786 cm−1)). Directly after completion
of the reaction, the polymer was precipitated in a mixture of
hexane/diethyl ether (50/50) and centrifuged (4500 rpm at 4 °C
for 15 min). After discarding the liquid fraction, new hexane/
ether was added and the polymer was resuspended using soni-
cation. The suspension was centrifuged again and the procedure
was repeated. The polymer was then dissolved in methanol,
dialyzed against methanol, and lyophilized to obtain a fluffy
powder (99 mg, 64%). For different initiators, yields ranged from
weighed into a flame-dried Schlenk-flask and dried in high
vacuum for 30 min. The macroinitiator was dissolved in 0.5 mL
of abs. DMF and 1.5 μL (8.61 μmol, 1,1/100 eq) freshly distilled
DIPEA was directly added. The mixture was stirred overnight at
room temperature. The next day, 90 mg (0.78 mmol, 1 eq) SarNCA
was weighed into a flame-dried Schlenk-flask and dried in high
vacuum for 30 min. Then, the NCA was dissolved in 1 mL of dry
DMF. The initiator solution was added to the monomer solution
via syringe. The reaction mixture was stirred at 0 °C and kept at
a constant pressure of 0.25 bar of dry nitrogen via the Schlenk-
line. After completion of the reaction, which was confirmed by
IR spectroscopy, the polymer was precipitated in diethyl ether
and centrifuged (4500 rpm at 4 °C for 15 min). After discarding
the liquid fraction, new ether was added and the polymer was
resuspended. The suspension was centrifuged again and the
procedure was repeated. The polymer was then dissolved in
methanol, dialyzed, and afterwards lyophilized to obtain 76.3 mg
(75%) of a fluffy powder. For different polypeptide-star mac-
1
roinitiators, yields ranged from 72% to 75%. H NMR: (400 MHz,

DMSO−d6): δ [ppm] = 1.10–2.20 (br, 15nH, CHCH2CH2CH2CH2NH


and
and

NHCOOC(CH3)3), 2.72–2.93 (s, 2nH+3mH, CH2NHCOOC
1


COCHNH and
NHCH3), 3.77–4.34 (br, nH+2mH,
61% to 64%. H NMR: (400 MHz, DMSO−d6): δ [ppm] = 1.97–1.14


COCH2NCH3), 6.33–6.66 (br, nH,
NHCOOC(CH3)3), 7.87–8.44


(br, 15nH, CHCH2CH2CH2CH2NH and NHCOOC(CH3)3), 2.87 (s,



(br, nH, COCHNH), 8.67 (s, ini, 3H, CH).
2nH, CH2NHCOOC), 3.77–4.18 (br, nH, COCHNH), 6.33–6.66
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solutions were added respectively to achieve a concentration
of 100 × 10–3 m. After 2 d for polypeptoid stars and 3 d for
polypept(o)ide stars, samples were directly lyophilized and sub-
sequently analyzed by HFIP SEC. Degradation of star polymers
with polypeptoid side chains is conducted in aqueous GSH solu-
tion without the addition of DMF.
(PLys(TFA)25-PSar100)3: 27 mg (1.52 μmol, 1/300 eq) SS(PLys-
(TFA)25)3 as macroinitiator was weighed into a flame-dried Schlenk-
flask and dried in high vacuum for 30 min. The macroinitiator was
dissolved in 0.4 mL of abs. DMF and 0.9 μL (5.02 mmol, 1,1/100 eq)
freshly distilled DIPEA was directly added. The mixture was stirred
overnight at room temperature. The next day, 53 mg (0.46 mmol,
1 eq) SarNCA was weighed into a flame-dried Schlenk-flask and
dried in high vacuum for 30 min. Then, the NCA was dissolved
in 0.5 mL of dry DMF. The initiator solution was added to the
monomer solution via syringe. The reaction mixture was stirred
at 0 °C and kept at a constant pressure of 0.25 bar of dry nitrogen
via the Schlenk-line. After completion of the reaction, which
was confirmed by IR spectroscopy, the polymer was precipitated
in diethyl ether and centrifuged (4500 rpm at 4 °C for 15 min).
After discarding the liquid fraction, new ether was added and the
polymer was resuspended. The suspension was centrifuged again
and the procedure was repeated. The polymer was then dissolved
in methanol, dialyzed, and afterwards lyophilized to obtain
2.8. AFM Measurements
For AFM measurements, a polymer concentration of 0.1 mg mL–1
in methanol was used. AFM was measured on mica in tapping
mode at ambient temperature using a Cypher AFM (Asylum
Research) at a scan rate of 1 Hz. Samples were prepared by
placing a drop of the particle solution onto freshly cleaned mica
via spin-coating. The sample was dried overnight. Images were
evaluated using Gwyddion and diameters of particles were
measured and analyzed using ImageJ.
1
48.5 mg (68%) of a fluffy powder. H NMR: (400 MHz, DMSO−d6):

δ [ppm] = 1.23–2.06 (br, 6nH, CHCH2CH2CH2CH2NH), 2.72–2.93
2.9. DLS Measurements


(s, 3mH, NHCOOC(CH3)3), 3.12 (s, nH, CH2NHCOC), 3.75–4.34
The star polymer solution (3 mg mL–1) was transferred to a dust free
flow box and all samples were filtered (Acrodisc GHP 0.2 μm) into
dust free cylindrical scattering cells (Suprasil, 20 mm diameter,
Hellma, Mühlheim, Germany). For data evaluation, experimental
intensity correlation functions were transformed into amplitude
correlation functions applying the Siegert relation extended to
include negative values after baseline subtraction by calculation
g1(t) = SIGN(G2(t)) · SQRT(ABS((G2(t) − A)/A). All field correlation
functions usually showed monomodal decay and were fitted by


(br, nH+2mH, COCHNH and COCH2NCH3), 7.89–8.43 (br, nH,



COCHNH), 8.91 (s, ini, 3H, CH), 9.20–9.40 (br, nH, NHCOCF3).
2.6. End-Group Modification
2.6.1. N-Acetyl-Polypept(o)ide Stars
All acetylations of polypept(o)ide stars were carried out in the
same way. In a typical experiment, SS(PLys(Boc)25-PSar100 3
)
(38 mg, 0.95 μmol, 1 eq) was weighed into a flame-dried Schlenk-
flask and dried under vacuum for 30 min. The polymer was dis-
solved in 0.5 mL abs. DMF and 2.7 μL (19 μmol, 20 eq) TEA was
added directly with a pipette under nitrogen counter flow. The
mixture was stirred at RT for 20 min to activate the N-termini of
the star polymer. Then, 0.9 μL (9.5 μmol, 10 eq) acetic anhydride
was added to the mixture and left to react at RT for 24 h. For
purification, the polymer was precipitated in diethyl ether and
centrifuged (4500 rpm at 4 °C for 15 min). 34.1 mg (92%) of a
colorless fluffy powder was obtained. For different initiators,
yields ranged from 92% to 95%.
t


a sum of two exponentials
to
g1 (t)= a⋅exp − + c⋅exp(−t/d)




b
take polydispersity into account, and average apparent diffusion
coefficients Dapp were calculated by applying q2 · Dapp = (a · b−1
+
c · d− 1)/(a + c) resulting in an angular-dependent diffusion coeffi-
cient Dapp or reciprocal hydrodynamic radius 〈1/Rh〉app, according
to formal application of the Stokes Einstein law. By extrapolation
−1
of 〈1/Rh〉app to q = 0 z-average hydrodynamic radii Rh = 〈1/Rh〉z
were obtained (uncorrected for c-dependency). For DLS at a
single-angle, star polymer solutions (1 mg mL–1 in methanol or
HFIP) were measured in triplicates.
2.6.2. End-Group Labeling of Polypeptoid Stars and
Polypept(o)ide Stars with Alexa Fluor 647
2.10. Cellular Uptake and Toxicity Assay of Polypeptoid
and Polypept(o)ide Stars
In
a typical experiment, SS(PLys(Boc)25-PSar100)3 (12 mg,
0.30 μmol, 1 eq) was dissolved in 0.5 mL of abs. DMF under dry
nitrogen atmosphere. 3 eq of Alexa Fluor 647 N-hydroxysuccin-
imide (NHS) ester dissolved in abs. DMF were added via syringe.
The solution was protected from light and stirred for 3 d at RT.
After that, the labeled polymer was directly dialyzed against
methanol and purified by Amicon Ultra Centrifugal Filters. Lyo-
philization yielded 12 mg (yield: 93%) of labeled polymer as
fluffy, blue solid.
HeLa (ATCC CCL-2.1), HEK 293, and DC 2.4 cells were maintained
in Dulbecco’s modified Eagle’s medium (DMEM, Sigma Aldrich)
supplemented with 10% fetal calf serum (FCS), 1% l-glutamine
and 1% Pen/Strep (100 U/100 μg mL–1) as described.[36] All
cells were used up to passage eight for the study. For cellular
uptake and cytotoxicity studies, cells (2 × 104 cell per well)
were seeded from a confluent culture flask in μ-slide eight-well
plates (ibidi) or in 96-well plates (Greiner) and were cultivated
for 24 h. Subsequently, cells were exposed to the respective
polymer concentrations for 24 h under standard cell culture
conditions. Cell viability was measured using the CellTiter-
Glo Luminescent Cell Viability Assay (Promega) according to
manufacture instruction. For cellular uptake studies, cells
were washed twice with phosphate-buffered saline (PBS), fixed
2.7. Reduction of Disulfide-Containing Star Polymers
For reduction of star polymers with polypept(o)ide side chains
linked to the core by disulfides, the polymers (about 2 mg)
were first dissolved in 200 μL DMF. Freshly prepared GSH
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with 4% paraformaldehyde, and stained with Hoechst 33342.
Images of fixed cells were captured using the AxioVert 200M
microscope (Carl Zeiss). Image analysis and presentation were
performed as described in detail using the Axiovision software
(Carl Zeiss).[36]
and led to trifunctional cores with equally reactive initia-
tion sites enabling simultaneous initiation. Thereby, the
direct synthesis of three-arm star polymers by the core-
first approach is feasible. In addition, the cystamine con-
taining initiator features disulfides, which can be cleaved
under reductive conditions. Consequently, the three-arm
stars would disassemble into the multifunctional core and
the respective polymeric arms. As an advantage, the linear
arms can be characterized to identify their molecular
weight distribution and ensure a preceded successful ini-
tiation and well-controlled polymerization.
With respect to the evaluation of the initiating capa-
bilities of the formed multifunctional cores, polysarcosine
stars have been synthesized. The three primary amines
of the cores were used to initiate simultaneously the ring
opening polymerization of a sarcosine (Sar) NCA. Three
different chain lengths for the individual arms have
been chosen to investigate the influence of the degree
of polymerization (Xn) on the polymerization itself and
on polymer properties. The Xns were set to be 25, 50,
and 100. After full monomer conversion was verified by
IR spectroscopy, the polypeptoid stars were precipitated
and characterized by 1H NMR as well as HFIP SEC (see
Figure 1). Analytical data are summarized in Table 1. The
molecular weight and hence the chain lengths of the indi-
vidual arms from 25 to 100 could be controlled by the
monomer-to-initiator ratio and are perfectly in line with
the analytical data. The degree of polymerization was
3. Results and Discussion
In order to establish a synthetic pathway to star-like
polypept(o)ides (PeptoStars), multivalent initiators with
three primary amine functionalities are required (see
Scheme 1). For this purpose, the N1,N3,N5-tris(2-((2-ami-
noethyl) disulfanyl)ethyl)benzene and the N1,N3,N5-tris(2-
(2-aminobutyl))benzene initiators have been synthesized
according to the literature.[34] These compounds were
obtained by a simple aminolysis of 1,3,5-benzenetricar-
bonyl trichloride with mono-Boc-protected diamines,
N-Boc-cystamine, or N-Boc-butanediamine, in the presence
of DIPEA. Column chromatography resulted in the purifica-
tion of the Boc-protected triamines, as confirmed by 1H and
13C NMR (see the Supporting Information), and avoids the
presence of undesired side products like mono- or difunc-
tional types of cores. To obtain the free amine functionali-
ties, removal of the Boc-protecting groups was conducted
under acidic conditions in a mixture of dichloromethane
with trifluoroacetic acid and proceeded in a quantitative
manner. The successful deprotection was verified by TLC
Scheme 1. Synthesis of stable and disulfide containing star-like polymers: PSar stars, polypeptide stars and polypept(o)ide stars.
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Figure 1. Characterization of star-like polypeptoids and polypeptides: A) 1H NMR analysis of star-like polysarcosines of different chain
lengths; B) size exclusion chromatography (SEC) of star-like polysarcosines in HFIP; and C) size exclusion chromatography of star-like
polylysines with TFA or Boc-protective groups in HFIP.
1
calculated by H NMR by comparing the aromatic signals
of the core with those of the repeating units. Only minor
deviation of around 5% between adjusted and achieved
degree of polymerization was found (see Table 1), under-
lining the quasi-living nature of the polymerization. The
star-like Sar polymers display monomodal Poisson-like
molecular weight distributions with low dispersities
(Đ ≤ 1.08) as determined by HFIP SEC analysis. These results
indicate that the living nature of the polymerization of
NNCAs is preserved for multivalent (three-arm) initiators.
The next step toward star-like polypept(o)ides was the
preparation of polypeptide stars. In our case, we have
chosen N-ε-t-butyloxycarbonyl-l-lysine NCA (Lys(Boc)
NCA) and N-ε-trifluoroacetyl-l-lysine NCA (Lys(TFA)
NCA) monomers to achieve three-arm Boc- and TFA-pro-
tected polylysine stars. While for the N1,N3,N5-tris(2-(2-
aminobutyl))benzene initiator both protective groups can
be cleaved using standard conditions, the deprotection of
the P(Lys(TFA)) block in N1,N3,N5-tris(2-((2-aminoethyl)-
disulfanyl)ethyl)benzene-based stars is critical, since this
can lead to disulfide cleavage under basic conditions.[37]
The deprotection of these polymers is, however, possible
under acidic conditions and the disulfides remain stable.
The required Lys(Boc)NCA was synthesized under acid-
free conditions according to a recently published strategy,
applying trimethylsilyl chloride and trimethylamine as
HCl scavenger.[38] Thus, the Boc-protecting group will not
be cleaved by HCl, emerging during NCA formation.
also provides a rational for the slight low molecular
weight tailing visible in all SEC plots (see Figure 1C).
Furthermore, the hydrodynamic volume of PLys(TFA) is
smaller than the one of PLys(Boc), although the degree
of polymerization determined by 1H NMR is even higher
(see Table 1). Interestingly, the initiator itself does not
have an influence on the molecular weight distribution
determined by SEC. As demonstrated in Figure 1C, the
PLys(Boc) stars result in almost identical SEC plots, which
leads to the conclusion that initiator effects are absent
and that the polymerization is very well controlled and
molecular weights can be precisely adjusted.
In order to form PeptoStars, the purified polylysine
stars were used as macroinitiators to generate star-
shaped block copolymers; star-like polypept(o)ides. The
accessible N-termini of the polylysine arms initiate the
polymerization of the sarcosine NCA. With a view toward
proper shielding of the PSar corona, a block length of
100 per arm was chosen. SEC analyses display a clear shift
from polypeptide stars to PeptoStars (see Figure 2A and
Supporting Information) and 1H DOSY experiments indi-
cate the presence of a single diffusing species, which can be
attributed to the PeptoStar since it contains the expected
signals for both PSar and PLys(Boc) blocks (see Figure 2B).
Molecular weights of 54 154 g mol–1 for butyl(PLys(Boc)25
PSar100 3
and 53 179 g mol–1 for SS(PLys(Boc)25-PSar100 3
-
)
)
(relative to PMMA standards) were determined, indi-
cating only a slight but detectable increase in hydro-
dynamic volume compared to the corresponding PSar
stars. The PeptoStars display low dispersity indices of
1.14 (see Table 1) for both systems. Interestingly, the SEC
plots are identical for both PeptoStars (see Figure 2C).
These data clearly indicate the successful formation of
star block polypept(o)ides, reported for the first time.
Furthermore, we would like to report that the direct syn-
thesis of PeptoStars by sequential in situ polymerization
is also feasible by addition of the second monomer after
complete consumption of the first one and hence, the
extra purification step of the polypeptide macroinitiator
Polymerization of the Lys(Boc)NCA was initiated by
the disulfide-containing and the noncleavable core. In
general, the desired block length of 25 per arm can be
1
obtained as indicated by H NMR with slight deviation of
7% (see Table 1). Dispersity indices determined by HFIP
SEC were Đ = 1.16–1.17 and hence, higher than the one for
the polysarcosine stars. This can be attributed to the coil
to helix transition of polylysine as reported by Huesmann
et al.[39] The coexistence of secondary structures with dif-
ferent hydrodynamic volumes (coil vs rod) broadens the
molecular weight distribution obtained by SEC, which
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Table 1. Characteristics of stable and disulfide-containing star-like polymers: PSar stars, polypeptide stars, and polypept(o)ide stars.
a)
b)
c)
d)
Polymers
Xn
Xn
Mn
Ðc)
Dh
(calc.)
(found)
[g mol−1
21 240
41 856
51 241
53 474
24 161
25 013
21 716
54 154
53 179
55 409
]
[nm]
SS(PSar25 3
SS(PSar50 3
SS(PSar100 3
Butyl(PSar100 3
Butyl(PLys(Boc)25 3
SS(PLys(Boc)25 3
SS(PLys(TFA)25 3
Butyl(PLys(Boc)25-PSar100 3
SS(PLys(Boc)25-PSar100 3
SS(PLys(TFA)25-PSar100 3
)
75
79
1.08
1.06
1.08
1.08
1.15
1.14
1.15
1.12
1.13
1.11
4
)
150
143
7
)
300
285
10
10
6
)
300
323
)
75
84
)
75
87
7
)
75
84
8
)
75–300
75–300
75–300
84–323
87–320
84–331
10
10
10
)
)
a)Calculated degree of polymerization using Xn = [M]/[I]; b)Determined by 1H NMR in DMSO-d6; c)Determined by SEC in HFIP using PMMA
standards; d)Determined by dynamic light scattering (NanoZS) at 173° scattering angle in methanol.
is not required, if the characterization of polymerization
intermediates is not required.
For further characterization of PeptoStars, the hydro-
dynamic diameter Dh was analyzed in methanol by
single-angle and multiangle DLS. Angular dependent DLS
measurements were carried out in the range from 30°
to 150° in 15° steps. As visible in Figure 3A, no angular
dependency was detected for PeptoStars with a hydrody-
namic radii (Rh) of 5 nm. In addition, μ2 values were below
0.05, indicating uniform populations of PeptoStars. These
findings were also supported by AFM measurements (see
Figure 3B). The average diameter determined by AFM
was around 20 nm (see the Supporting Information). This
increase by a factor of 2 can be attributed to flattening and
aggregation effects during drying of the sample. Due to the
small diameter and the absence of angular dependency
ensured by multiangle DLS, the use of single-angle DLS
(NanoZS, 173° detection angle) led to identical results for
the particle radii. The obtained hydrodynamic diameters
Dh were 10 nm (Rh = 5 nm). Therefore, we have chosen to
apply the Zetasizer NanoZS setup for degradation studies,
In the next step, the end groups of the polypept(o)ide
arms were capped by using an excess of acetic anhy-
dride in the presence of TEA. Acetylation did not influ-
ence the SEC elugrams of both PeptoStars but a detailed
quantification via 1H NMR was not possible since the
signal intensity was low compared to the backbone
signals. End-group modification can also be realized
straight after complete monomer conversion. Accu-
rate purification of star polymers by precipitation and
dialysis is necessary particularly after end-group cap-
ping but result in reduction of yields under these condi-
tions. Instead of acetylation, labeling polypeptoid stars
and polypept(o)ide stars with dyes was conducted with
Alexa Fluor 647 NHS ester. Further modifications at
the N-termini with functional end groups or targeting
structures are easily accessible as reported by Klinker
et al.[40]
Figure 2. Characterization of star-like polypept(o)ides: A) SEC in HFIP indicating successful chain extension from star-like PLys(Boc) to star-
like PLys(Boc)-block-PSar polymers; B) 1H DOSY NMR spectra of PLys(Boc)-block-PSar polymers displaying a single diffusing polymer species;
and C) SEC of non-degradable (butyl) or degradable star-like polypept(o)ides (PLys(Boc)-block-PSar side chains) in HFIP.
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Figure 3. Characterization of star-like polypept(o)ides (PLys(Boc)-block-PSar side chains) by A) multiangle dynamic light scattering, B) atomic
force microscopy (AFM) height image of star-like polypept(o)ides on mica, and C) single-angle dynamic light scattering.
because it allows to reduce the sample volume and thus,
the amount of sample required for the measurements (10
to 30 times less sample required).
hindered by solubility effects of the individual arms. After
2 d, the solutions were lyophilized and analyzed by HFIP
SEC. The elugrams of (PSar25)3 stars clearly demonstrate
Degradation studies were performed in the presence
of GSH in aqueous solution (see Scheme 2). We have con-
ducted these studies for two reasons. First, it has to be
ensured that the individual arms have grown equally
and hence, showing comparable length and displaying
Poisson-like distributions.
complete degradation of polymeric stars into the indi-
vidual arms (see Figure 4A). In addition, it is clearly vis-
ible that the individual arms are narrowly distributed
(Đ = 1.05–1.1; see Table 2) following a Poisson-like mole-
cular weight distribution as indicated by the highly sym-
metrical peak at an elution volume of 18–20 mL (see
Figure 4A). In parallel, the hydrodynamic volume of poly-
peptoid stars before and after degradation was monitored
by DLS. For comparability, SEC and DLS studies were per-
formed in HFIP (3 g L–1 potassium trifluoroacetate). In line
with the SEC results, a clear reduction in size from 4 nm
Second, intracellular degradation, e.g., in antigen pre-
senting cells, has to be ensured for further use of star-
like polymers as biodegradable delivery system of drugs
or probes.[41] The GSH levels inside cells can reach up to
100 × 10–3 m depending on cell type and cellular compart-
ment.[42] Therefore, GSH concentrations of 100 × 10–3
m
for (PSar25)3 stars to 2 nm for the individual arms is vis-
were chosen and a complete degradation in the individual
arms is feasible for further characterization. As internal
control, non-degradable N1,N3,N5-tris(2-(2-aminobutyl))-
benzene-based PeptoStars and polypeptoid stars were
used. As expected, these star polymers remained stable
in solution in the presence or the absence of GSH (see
the Supporting Information). In the absence of GSH, the
N1,N3,N5-tris(2-((2-aminoethyl)disulfanyl)ethyl)benzene-
based stars also remained stable.
The polysarcosine stars with chain lengths of Xn = 75,
150, and 300 were exposed to aqueous 100 × 10–3 m GSH
solutions. PSar is very well soluble in aqueous medium
and hence, disintegration of polypeptoid stars is not
ible, which indicates degradation of PSar stars into the
individual arms (see Figure 4B). The same behavior was
observed for (PSar50)3 and (PSar100)3 stars (see Figure 4C, D
and Supporting Information). For (PSar50)3 stars, the par-
ticle size was reduced from 7 to 4 nm and from 10 to 6 nm
for (PSar100)3 stars (see Table 2).
In the case of PeptoStars, SS(PLys(Boc)25-PSar100)3 and
SS(PLys(TFA)25-PSar100)3 were dissolved in DMF and exposed
to the aqueous 100 × 10–3 m GSH solution for 3 d instead
of 2. Due to the amphiphilic nature of polypept(o)ide
arms, the dissociation observed is slower than for PSar-
based stars (see the Supporting Information). The hydro-
phobic part of the individual arms tends to aggregate in
Scheme 2. Schematic depiction of GSH-induced degradation of disulfide-containing star-like polymers.
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Figure 4. GSH-induced degradation of disulfide-containing star-like polypeptoids and polypept(o)ides (PLys(Boc)-block-PSar side chains) in
aqueous solution monitored by SEC and single-angle dynamic light scattering (DLS) before and after exposure in HFIP.
aqueous solution impairing further degradation of the
PeptoStars. All other steps were carried out as described
for PSar stars. We observed a distinct shift in elution time
of the PeptoStar peak from 15–17.5 mL before to 16–19 mL
after GSH exposure (see Figure 4E). Furthermore, the peak
at 16–19 mL elution volume is still narrowly distributed
(Đ = 1.15; see Table 2) and symmetrical, indicating that also
polypept(o)ide side chains have grown uniformly during
block copolymer synthesis by sequential ROP. DLS analysis
revealed comparable behavior. Before GSH exposure, Pepto-
Stars displayed an average size of 10 nm, while afterwards,
particles with a size of 5 nm are visible (see Figure 4F and
Table 2). This clearly indicates cleavage of the disulfide
bonds and degradation of the polypept(o)ide stars.
degradable in the presence of GSH and the incorporation
of disulfides does not influence the high level of control in
star polymer synthesis.
After detailed studies on synthesis and degradation,
polypeptoid and polypept(o)ide stars were subjected
to biological evaluation. Therefore, studies on cellular
uptake and cytotoxicity were carried out in HeLa cells (see
Figures 5 and 6).
The uptake of polypeptoid stars by HeLa cells was inves-
tigated by fluorescence microscopy using Alexa Fluor 647
labeled star polymer derivatives. Cells were incubated
with a polypeptoid star concentration of 0.5 mg mL−1 for
24 h. By microscopic analysis, a considerable intracellular
fluorescence was observed all over the cell (see
Figure 5A, B) and consequently proved cellular uptake of
polypeptoid stars by HeLa cells. The same uptake behavior
Therefore, both PSar stars and PeptoStars can be
synthesized with uniform arms to be either stable or
Table 2. Characteristics of disulfide-containing star-like polysarcosine and polypept(o)ide polymers before and after reduction with
glutathione.
Before reduction
After reduction
a)
b)
a)
b)
Polymer
Ða)
Mn
Dh
Ða)
Mn
Dh
[g mol–1
21 240
41 856
51 241
53 179
]
[nm]
[g mol–1
5 764
]
[nm]
SS(PSar25 3
SS(PSar50 3
SS(PSar100 3
SS(PLys(Boc)25-PSar100 3
)
1.08
1.06
1.08
1.13
4
1.10
1.05
1.05
1.12
2
3
6
6
)
7
13 925
22 315
22 654
)
10
10
)
a)Determined by SEC in HFIP using PMMA standards; b)Determined by dynamic light scattering (NanoZS) at 173° scattering angle in
HFIP.
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Figure 5. Microscopy images confirming cellular uptake of A) polypeptoid stars SS(PSar50)3 and B) SS(PSar100)3 in HeLa cells after incubation
for 24 h.
was observed for a dendritic cell line (DC 2.4) as well as in
human embryonic kidney (HEK 293) cells.
polypeptides with Xn 25 or 75 nor PLys(Boc)25-PSar100 do
display any detectable cellular toxicity even at higher
concentrations.[40] Although the exact reasons remain
unclear, these findings indicate that the trimeric acid core
seems to influence cytotoxicity more than the individual
polymer side chains.
Nevertheless, these initial experiments encourage fur-
ther research on polypept(o)idic star polymers for appli-
cation as nano-sized drug delivery or imaging systems,
since a total concentration of more than 1 g L–1 corre-
sponds to 200 μg per 20 000 cells.
Having ensured cellular uptake, cellular viability assays
were performed using different polymer concentrations
from 0.1 up to 1 g L−1. In general, none of the star-like
polymers reached the IC50 value for cell viability at the
applied concentration range in all cell lines (see Figure 6).
Especially in the dendritic cell line DC 2.4, all polymers at
all concentrations did not decrease cell viability or only
led to a minor decrease. The most pronounced decrease in
viability of about 28% compared to the PBS treated con-
trol was detected for (PSar25)3. Interestingly, the effects
on cell viability were slightly more pronounced for HeLa
and HEK cells, which may relate to differences in cell
metabolism and robustness (see Figure 6). In these cases,
again (PSar25)3 turned out to induce the most pronounced
effects by reducing the cell viability down to 56% of the
4. Conclusions
In this work, we have demonstrated an easy synthetic
pathway to generate polypeptoid, polypeptide, and
polypept(o)ide stars (PeptoStars). We have successfully
synthesized well-defined, biodegradable stars via the
core-first approach. Sequential ROP of NCAs allowed
adjustable block length and polymers with high end-group
PBS treated control. But also the SS(PLys(Boc)25-PSar100 3
)
and (PLys(Boc)25-PSar100)3 PeptoStars displayed a decrease
in viability of 41% and 39% relative to the control. These
findings are particularly interesting since neither linear
(PLys
(Boc)25-
SS(PLys
(Boc)25-
(PLys
(PSar25)3 (PSar100 3
)
Conc.
PBS
(TFA)25- DMSO
PSar100 3
PSar100 3
)
PSar100 3
)
)
Hela
0.1 g/L
0.5 g/L
1.0 g/L
HEK
0.1 g/L
0.5 g/L
1.0 g/L
DC 2.4
0.1 g/L
0.5 g/L
1.0 g/L
Figure 6. Concentration-dependent cell viability of HeLa, HEK, and DC.2.4 cells after incubation with star-like polypeptoids and polypept(o)-
ides for 24 h. Colour code for cell viability corresponds to 100%–80% (■, dark green), 80%–65% (■, light green), 65%–50% (■, yellow),
50%–30% (■, orange), 30%–0% (■, red).
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[6] L. Y. Qiu, Y. H. Bae, Pharm. Res. 2006, 23, 1.
[7] S. D. Perrault, C. Walkey, T. Jennings, H. C. Fischer,
W. C. W. Chan, Nano Lett. 2009, 9, 1909.
integrity, Poisson-like molecular weight distribution, and
low dispersity indices between 1.05 and 1.15. The star-like
polypept(o)ides (PeptoStars) were characterized by multi-
angle DLS and AFM, proving the existence of uniform
core–shell nanoparticles with sizes of 5 nm (μ2 < 0.05).
Moreover, a disulfide-containing initiator core provides
intrinsic stimuli-responsiveness at glutathione levels
of 100 × 10–3 m. Degradation of PSar- and polypept(o)-
ide-based star-like polymers revealed that the individual
arms have grown uniformly during the sequential nucle-
ophilic ring opening polymerization. Most importantly,
the reported synthetic pathway enables a simple scal-
able access to PeptoStars consisting of a hydrophobic
N-ε-tert-butyloxycarbonyl-l-lysine (PLys(Boc)) or N-ε-tert-
trifluoroacetyl-l-lysine (PLys(TFA)) core and a hydrophilic
polysarcosine shell. In addition, in vitro cytotoxicity—if
detectable—was low for all systems in HeLa, HEK, as well as
DC 2.4 cells. Therefore, the introduced star-like polypept(o)-
ides (PeptoStars) seem to be a promising platform for nano-
drug delivery systems by incorporation or conjugation of
hydrophobic drugs. In addition, the deprotected PLys core of
PeptoStars may be used to entrap small negatively charged
cargo such as siRNA.
[8] H. Cabral, Y. Matsumoto, K. Mizuno, Q. Chen, M. Murakami,
M. Kimura, Y. Terada, M. R. Kano, K. Miyazono, M. Uesaka,
N. Nishiyama, K. Kataoka, Nat. Nanotechnol. 2011, 6, 815.
[9] M. Talelli, M. Barz, C. J. F. Rijcken, F. Kiessling, W. E. Hennink,
T. Lammers, Nano Today 2015, 10, 93.
[10] Y. Shi, R. Van Der Meel, B. Theek, E. Oude Blenke,
E. H. E. Pieters, M. H. A. M. Fens, J. Ehling, R. M. Schiffelers,
G. Storm, C. F. Van Nostrum, T. Lammers, W. E. Hennink, ACS
Nano 2015, 9, 3740.
[11] C. Deng, Y. Jiang, R. Cheng, F. Meng, Z. Zhong, Nano Today
2012, 7, 467.
[12] Clinical studies: https://clinicaltrials.gov/ (November 2016).
[13] N. Hadjichristidis, H. Iatrou, M. Pitsikalis, J. Mays, Prog.
Polym. Sci. 2006, 31, 1068.
[14] H. Gao, Macromol. Rapid Commun. 2012, 33, 722.
[15] T. K. Georgiou, Polym. Int. 2014, 63, 1130.
[16] J. R. Schaefgen, P. J. Flory, J. Am. Chem. Soc. 1948, 70, 2709.
[17] Definition of star macromolecule: http://goldbook.iupac.
org/S05935.html (November 2016).
[18] K. Aoi, T. Hatanaka, K. Tsutsumiuchi, M. Okada, T. Imae,
Macromol. Rapid Commun. 1999, 382, 378.
[19] A. Duro-castano, R. M. England, D. Razola, E. Romero,
M. Oteo-vives, M. A. Morcillo, J. Vicent, Mol. Pharm. 2015, 12,
3639.
[20] M. Byrne, P. D. Thornton, S.-A. Cryan, A. Heise, Polym. Chem.
2012, 3, 2825.
[21] A. Sulistio, P. A. Gurr, A. Blencowe, G. G. Qiao, Aust. J. Chem.
2012, 65, 978.
[22] C. Secker, S. M. Brosnan, R. Luxenhofer, H. Schlaad,
Macromol. Biosci. 2015, 15, 881.
[23] R. Luxenhofer, C. Fetsch, A. Grossmann, J. Polym. Sci. Part A
Polym. Chem. 2013, 51, 2731.
Supporting Information
Supporting Information is available from the Wiley Online
Library or from the author.
Acknowledgements: The authors thank Prof. Angelika Kühnle for
providing AFM facilities and Prof. Manfred Schmidt for support
during multiangle dynamic light scattering measurements. M.B.
acknowledges the financial support from the German Research
Council (DFG, SFB1066-1). D.D acknowledges the financial
support from the German Research Council (DFG, GZ DO
1910/2-1), Peter und Traudl Engelhorn Foundation, and the Fonds
der chemischen Industrie. D.W. would like to thank the Joachim
Herz Foundation for financial support. R.S. acknowledges the
financial support from BMBF-NanoBEL and BMBF-DENANA.
K.K would like to thank the HaVo Foundation and the graduate
school MAINZ (DFG/GSC 266) for financial support.
[24] K. Klinker, M. Barz, Macromol. Rapid Commun. 2015, 36,
1943.
[25] S. Kimura, T. Kidchob, Y. Imanishi, Polym. Adv. Technol. 2001,
12, 85.
[26] A. Birke, D. Huesmann, A. Kelsch, M. Weilbächer, J. Xie,
M. Bros, T. Bopp, C. Becker, K. Landfester, M. Barz,
Biomacromolecules 2014, 15, 548.
[27] P. Heller, A. Birke, D. Huesmann, B. Weber, K. Fischer,
A. Reske-Kunz, M. Bros, M. Barz, Macromol. Biosci. 2014, 14,
1380.
[28] R. G. Chapman, E. Ostuni, S. Takayama, R. E. Holmlin, L. Yan,
G. M. Whitesides, J. Am. Chem. Soc. 2000, 122, 8303.
[29] K. H. A. Lau, C. Ren, T. S. Sileika, S. H. Park, I. Szleifer,
P. B. Messersmith, Langmuir 2012, 28, 16099.
[30] M. Schneider, Z. Tang, M. Richter, C. Marschelke, P. Förster,
E. Wegener, I. Amin, H. Zimmermann, D. Scharnweber,
H. G. Braun, R. Luxenhofer, R. Jordan, Macromol. Biosci. 2016,
16, 75.
Received: December 10, 2016; Revised: January 13, 2017;
Published online: ; DOI: 10.1002/mabi.201600514
Keywords: block
copolypept(o)ides;
NCA
polymerization;
polypept(o)ide stars; redox-sensitive polypept(o)ide stars;
star polymers
[31] M. Barz, R. Luxenhofer, R. Zentel, M. J. Vicent, Polym. Chem.
2011, 2, 1900.
[32] D. Huesmann, A. Sevenich, B. Weber, M. Barz, Polymer 2015,
67, 240.
[1] A. Duro-Castano, J. Movellan, M. J. Vicent, Biomater. Sci.
2015, 3, 1321.
[33] R. Holm, K. Klinker, B. Weber, M. Barz, Macromol. Rapid
Commun. 2015, 36, 2083.
[2] M. Byrne, R. Murphy, A. Kapetanakis, J. Ramsey, S. Cryan,
A. Heise, Macromol. Rapid Commun. 2015, 36, 1862.
[3] W. Wu, W. Wang, J. Li, Prog. Polym. Sci. 2015, 46, 55.
[4] Y. Matsumura, H. Maeda, Cancer Res. 1986, 46, 6387.
[5] H. Maeda, H. Nakamura, J. Fang, Adv. Drug Delivery Rev.
2013, 65, 71.
[34] N. Trouche, S. Wieckowski, W. Sun, O. Chaloin, J. Hoebeke,
S. Fournel, G. Guichard, J. Am. Chem. Soc. 2007, 129,
13480.
[35] C. Fetsch, R. Luxenhofer, Polymers 2013, 5, 112.
[36] D. Wünsch, V. Fetz, D. Heider, S. Tenzer, C. Bier, L. Kunst,
S. Knauer, R. Stauber, Blood Cancer J. 2012, 2, e77.
Macromol. Biosci. 2017, DOI: 10.1002/mabi.201600514
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Macromolecular
Bioscience
R. Holm et al.
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[37] M. V. Trivedi, J. S. Laurence, T. J. Siahaan, Curr. Protein Pept.
Sci. 2009, 10, 614.
[40] K. Klinker, R. Holm, P. Heller, M. Barz, Polym. Chem. 2015, 6,
4612.
[38] P. Heller, B. Weber, A. Birke, M. Barz, Macromol. Rapid
Commun. 2015, 36, 38.
[41] A. Pompella, A. Visvikis, A. Paolicchi, V. De Tata, A. F. Casini,
Biochem. Pharmacol. 2003, 66, 1499.
[39] D. Huesmann, A. Birke, K. Klinker, S. Türk, H. J. Räder,
M. Barz, Macromolecules 2014, 47, 928.
[42] D.
Montero,
C.
Tachibana,
J.
Rahr
Winther,
C. Appenzeller-Herzog, Redox Biol. 2013, 1, 508.
Macromol. Biosci. 2017, DOI: 10.1002/mabi.201600514
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