Self-assembled spin-labeled nanoparticles based on poly(amino acids)

Article abstract of DOI:10.1016/j.reactfunctpolym.2016.01.018

The development of detectable nanoparticles for controlled drug delivery systems has tremendous practical importance regarding the monitoring of drug pathway in organism. Self-assembly amphiphilic block-copolymer poly(l-glutamic acid)-b-poly(l-phenylalanine) (pGlu-b-pPhe) was chosen for the preparation of discussed nanoparticles. The synthesis of blocks was carried out using ring-opening polymerization (ROP) of N-carboxyanhydrides of mentioned amino acids. To introduce the spin label at C-terminal position of hydrophilic block, (4-amino-2,2,6,6-tetramethylpiperidin-1-yl)oxyl (4-amino-TEMPO) was applied as ROP initiator and the polymerization of hydrophobic block was carried out with previously synthesized macroinitiator. The results obtained by transmission electron microscopy clearly showed that TEMPO-pGlu-b-pPhe polymer was really capable to self-assembling in aqueous solutions followed by polymersome formation. The mean size of nanoparticles was increased in a range of TEMPO-pGlu₄₃-b-pPhe₁₂ < TEMPO-pGlu₄₃-b-pPhe₂₉ < TEMPO-pGlu₄₃-b-pPhe₄₉ as 60 < 200 < 280 nm, respectively. EPR spectroscopy of the solutions of spin-labeled homopolymer TEMPO-p-γ-Glu(Bzl), block copolymers TEMPO-p-γ-Glu(Bzl)-b-pPhe and suspension of polymersomes formed from TEMPO-p-Glu-b-pPhe was performed and the results were compared. It was proved that in the case of nanoparticles EPR detectable spin labels are located on polymersome surface. The experiments in cell culture demonstrated the absence of cytotoxicity of labeled nanoparticles. Additionally, it was shown that TEMPO-label can be detected inside the cell by EPR method.

Full text of DOI:10.1016/j.reactfunctpolym.2016.01.018

Self-assembled spin-labeled nanoparticles based on poly(amino acids)
A.V. Hubina, A.A. Pogodaev, V.V. Sharoyko, E.G. Vlakh, T.B. Ten-
nikova
PII:
S1381-5148(16)30017-7
DOI:
Reference:
doi: 10.1016/j.reactfunctpolym.2016.01.018
REACT 3626
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Received date:
Revised date:
Accepted date:
20 July 2015
22 January 2016
28 January 2016
Please cite this article as: A.V. Hubina, A.A. Pogodaev, V.V. Sharoyko, E.G. Vlakh,
T.B. Tennikova, Self-assembled spin-labeled nanoparticles based on poly(amino acids),
(2016), doi: 10.1016/j.reactfunctpolym.2016.01.018
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ACCEPTED MANUSCRIPT
Self-assembled spin-labeled nanoparticles based on poly(amino acids)
a,b
a
a
a
a,*
A.V. Hubina , A.A. Pogodaev , V.V. Sharoyko , E.G. Vlakh and T.B. Tennikova
a
Institute of Chemistry, Saint-Petersburg State University,
Universitesky pr. 26, 198504 St. Petersburg, Russia
b
Institute of Macromolecular Chemistry, National Academy of Science of Ukraine,
Kharkivske shose 48, 02160, Kyiv, Ukraine
*
Corresponding author: Prof. Dr. Chem. Sci. Tatiana B. Tennikova
E-mail: tennikova@mail.ru
Tel. (++ 7 812) 323 1050
Fax: (++7 812) 328 6869
Abstract
The development of detectable nanoparticles for controlled drug delivery systems has tremend-
ous practical importance regarding the monitoring of drug pathway in organism. Self-assembly
amphiphilic block-copolymer poly(L-glutamic acid)-b-poly(L-phenylalanine) (pGlu-b-pPhe) was
chosen for the preparation of discussed nanoparticles. The synthesis of blocks was carried out
using ring-opening polymerization (ROP) of N-carboxyanhydrides of mentioned amino acids. To
introduce the spin label at C-terminal position of hydrophilic block, (4-amino-2,2,6,6-
tetramethylpiperidin-1-yl)oxyl (4-amino-TEMPO) was applied as ROP initiator and the polyme-
rization of hydrophobic block was carried out with previously synthesized macroinitiator. The
results obtained by transmission electron microscopy clearly showed that TEMPO-pGlu-b-pPhe
polymer was really capable to self-assembling in aqueous solutions followed by polymersomes
formation. The mean size of nanoparticles was increased in a range TEMPO-pGlu -b-pPhe <
4
3
12
TEMPO-pGlu -b-pPhe
TEMPO-pGlu -b-pPhe as 60 < 200 < 280 nm, respectively. EPR
4
3
29 <
43 49
spectroscopy of the solutions of spin-labeled homopolymer TEMPO-p-γ-Glu(Bzl), block copo-
lymers TEMPO-p-γ-Glu(Bzl)-b-pPhe and suspension of polymersomes formed from TEMPO-p-
Glu-b-pPhe was performed and the results were compared. It was proved that in the case of na-
noparticles EPR detectable spin labels are located on polymersome surface. The experiments in
cell culture demonstrated the absence of cytotoxity of labeled nanoparticles. Additionally, it was
shown that TEMPO-label can be detected inside the cell by EPR method.
Keywords: poly(amino acids); spin label; polymersomes; ring-opening polymerization of N-
carboxyanhydrides.
1

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1
.
Introduction
In recent decades the preparation of nanoparticles for controlled drug delivery is on fron-
tier of biomedical chemistry challenges. A wide variety of nanoparticles composed of lipids,
natural and synthetic polymers, as well as inorganic materials have been developed [1]. To date,
the known nanoparticulate drug delivery systems, depending on their nature and method of prep-
aration, can have different design. For example, these particles can represent solid beads, nanos-
pheres, nanocapsules, as well as self-assembled nanostructures of different morphology [1, 2].
Self-assembling has revolutionized soft materials research by enabling the efficient and
high-throughput fabrication of ordered nanostructures for drug delivery [3]. For decades, self-
assembled vesicles comprised low-molecular mass compounds such as phospholipids (lipo-
somes) [4] or surfactants [5]. More recently, amphiphilic polymers have been shown to form
very elaborated architectures and to serve as useful nanocontainers in aqueous solution [6, 7].
The self-assembly of amphiphilic block copolymers in appropriate media can form a variety of
supramolecular assemblies including spherical, worm and multicompartment micelles, as well as
vesicles (or polymersomes). Polymersomes represent the liposome-like polymeric spheres with
aqueous core surrounded by double layer polymer membrane. The aqueous core can be utilized
for the encapsulation of hydrophilic therapeutics such as low-molecular mass substances, pep-
tides, proteins (including enzymes), oligonucleotides and nucleic acids [8, 9]. In its turn, the hy-
drophobic part of membrane can integrate hydrophobic drugs [10].
Comparing to liposomes, polymersomes have thicker membrane resulting in less permea-
bility for outside medium that, in turn, leads to their higher stability, storage capabilities [11, 12],
as well as prolonged circulation time [13]. Moreover, block-copolymer chemistries can be tuned
through polymer synthesis to yield polymersomes with diverse functionality [14, 15]. The com-
position and molecular weight of these polymers can be varied that leads to the formation of po-
lymersomes not only with different chemical nature, size and responsiveness to stimuli but also
different membrane thickness and permeability [16].
2

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A lot of synthetic polymers are discussed in the literature regarding to their ability to
serve as building blocks for polymersomes [17, 18]. Nevertheless, poly(amino acids) and poly-
peptides can be related to one of the most promising types of macromolecules. These bio-
inspired polymers possess biodegradability and biocompatibility that, in particular, are the main
features for construction of modern drug delivery systems. Besides, further modification of po-
lymersomes is possible due to functional groups on the surface [19-21].
Drug delivery and release is a complex process, which demands powerful modern tools
for tracking in time drug delivery carrier’s diffusion and stability. Electron Paramagnetic Reson-
ance (EPR) spectroscopy represents direct, sensitive and non-invasive method, which can be ap-
plied both in vitro and in vivo. Furthermore, electron paramagnetic resonance imaging allows
visualizing traces and spatial distribution of drug carriers [22, 23]. The EPR spectroscopy con-
cerns investigation of the compounds that contain paramagnetic centers. In the case if the poly-
mer does not have such centers, the spin-labeling approach can be used. Stable nitroxide radicals
of various structure and functionality are widely used as spin probes or spin labels for biochemi-
cal applications, including investigation of biopolymers (proteins) [22-26]. They demonstrate
stability at physiological temperature and pH, and the versatility of functional groups, which they
can contain. It opens a lot of pathways to attach the paramagnetic label to the polymer.
In current literature (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) and its derivatives
are mostly mentioned as spin probe/spin label or capping agents and mediators in free radical
polymerization and oxidative processes [27, 28, 29]. In [30] the method of fabrication of EPR
detectable amphyphilic block copolymers was proposed. The spin label, namely, 4-amino-
TEMPO was attached to the hydrophilic block of synthesized poly(N-isopropyl methacrylamide-
co-N-isopropyl maleamic acid-co-10-undecenoic acid). The pH- and thermo-sensitive micelles
were assembled in aqueous solution, and the EPR was applied to control the process of micelle
formation and destruction under various pH and temperature conditions. Polymer-coated TEM-
PO-labeled inorganic nanoparticles were also synthesized and investigated [31]. The possibility
3

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of EPR-imaging of spatial distribution of nanoparticles obtained was shown using EPR in L-
band region. However, there is no reference in the literature to application of spin label as initia-
tor of ring-opening polymerization (ROP) of N-carboxyanhydrides of amino acids.
In this paper the formation of polymer nanoparticles from amphiphilic copolymers based
on poly(L-glutamic acid)-b-poly(L-phenylalanine) (pGlu-b-pPhe) with covalently attached 4-
amino-TEMPO spin label is discussed. The mentioned label was used as initiator for ROP of -
N-carboxyanhydride of γ-benzyl-L-glutamate. This approach allowed the introduction of spin
label into C-terminal position of synthesized poly(amino acid). The labeled macroinitiator was
further used for copolymerization of hydrophobic pPhe block. Finally, after amphiphilic copo-
lymer self-assembly the label appeared to be located on the outer (hydrophilic) nanoparticle sur-
face that is very important for sensitive ESR-imaging.
2
.
Experimental section
2
.1. Materials
γ-benzyl-L-glutamate, L-phenylalanine, triphosgene, -pinene, 4-amino-TEMPO, (3-
aminopropyl)triethoxysilane (APTES), trifluoromethanesulfonic acid (TFMSA), trifluoroacetic
acid (TFA) and other reagents were purchased from Sigma–Aldrich (Germany) and used as re-
ceived. 1,4-Dioxan, n-hexan, N,N-dimethylformamide (DMF), ethyl acetate and other solvents
were purchased from Acros (Russia) and distilled before application.
2
.2. Methods
2
.2.1. Synthesis of N-carboxyanhydrides (NCAs)
NCAs of γ-Glu-(Bzl) and Phe were synthesized using routine procedure reported, in par-
ticular, in [32]. Dioxane was used as a solvent, and both acquired NCA were purified by recrys-
tallization from ethyl acetate/n-hexane. Yields: NCA of γ -Glu-(Bzl) – 75%, NCA of Phe –
1
7
0%. Structure and purity level of synthesized NCAs were confirmed by H NMR. Spectra were
4

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recorded at room temperature (298 K) using Bruker 400 MHz Avance instrument (Germany) and
1
the samples dissolved in СDCl . H-NMR: NCA of γ -Glu-(Bzl) – 2.05 – 2.39 (m, 2 H), 2.63 (t,
3
2
2
H), 4.39 (t, 1 H), 5.17 (s, 2 H), 6.40 (br. s., 1 H), 7.39 (m, 5 H), NCA of Phe – 2.94–3.35 (m,
H), 4.55 (m, 1H), 5.60 (s, 1H), 7.19–7.41 (m, 5H).
2
.2.2. Polymerization
The homopolymer of γ-Glu(Bzl) was synthesized via NCA polymerization in dry dioxane
using 4-amino-TEMPO as initiator. The monomer/initiator molar ratio was equal to 25. After
reaction for 24 hours at 30C, the product was precipitated in excess of diethyl ether and dried.
Precipitation procedure from dioxane/diethyl ether was performed twice. The yield of the homo-
polymer was 67%. The APTES initiated polymerization of γ-Glu(Bzl) NCA was carried out in
the same way with the yield of the homopolymer product equal to 75%.
Both prepared TEMPO-p-γ-Glu(Bzl) and APTES-p-γ-Glu(Bzl) were used as macroinitia-
tor for Phe-NCA polymerization. For that, three polymerization mixtures differed with molar
monomer/initiator ratios (100, 150 and 200), featuring TEMPO-p-γ-Glu(Bzl) as macroinitiator
were prepared in anhydrous and amine free DMF and incubated at 30C for 24 hours. Then reac-
tion product was isolated by precipitation in diethyl ether, purified twice using DMF/diethyl eth-
er mixture. Finally, p-γ-Glu(Bzl)-b-pPhe was collected after centrifugation and dried. The same
procedure was carried out for APTES-p-γ-Glu(Bzl)/Phe-NCA taken as 1:100.
2
.2.3. Polymer characterization
The gel permeation chromatography (GPC) measurements were performed on a Shimad-
zu LC-20 Prominence system with refractometric RID 10-A detector (Japan) using 7.8×300 mm
Styragel Column, HMW 6E, 15-20 µm bead size (Waters, USA). The analysis was carried out at
6
0°C using DMF with 0.1 M LiBr as eluent. The mobile phase flow rate was 0.3 mL/min. Mole-
cular weights and molecular weight distributions for γ-Glu(Bzl) homopolymer were calculated
5

ACCEPTED MANUSCRIPT
using poly(methyl methacrylate) standards in M range from 17,000 to 250,000 with polydisper-
w
sity lower than 1.14. The calculations were carried out with GPC LC Solutions software (Shi-
madzu, Japan).
The contribution of hydrophobic block was evaluated using chromatographic amino acid
analysis after total hydrolysis of polymer samples. The hydrolysis of 1 mg of a sample was car-
ried out in 2 mL of 6 M HCl with 0.0001% phenol in vacuum-sealed ampoule during 4 days. The
solvent was evaporated several times with water to eliminate HCl and finally to reach neutral
pH. The products of hydrolysis were analyzed by reversed phase (RP) HPLC using LCMS-8030
Shimadzu system with triple quadruple mass-spectrometry detection (LC-MS) (all from Shimad-
zu, Japan), equipped with 2x150 mm Luna C column, packed with 5 μm particles. The isocratic
1
8
elution mode was applied and 0.1% acetonitrile/НСООН in ratio 5/95 wt% was used as eluent.
The mobile phase flow rate was equal to 0.3 mL/min.
2
.2.4. Preparation of polymersomes
The Bzl-protection group of p-γ-Glu(Bzl)-b-pPhe was removed by TFMSA/TFA mixture
in ratio 1:10 at 22C. Then the product was dispersed in DMSO, put into dialysis membrane bag
MWCO 1000 and dialyzed against 0.1 M Na-borate buffer solution, pH 8.6, for 1 day. After 2
days of freeze drying, pGlu-b-pPhe was collected.
Polymer nanoparticles were prepared by dispersing the copolymer in 0.1 M Na-borate
buffer solution, pH 8.6, at a concentration of 0.5 and 0.25 mg/mL. The dispersion was sonicated
o
at 30 C for 2 hours.
2
.2.5. Dynamic Light Scattering (DLS)
DLS measurements were performed on Laser Particle Analazer SZ100 (Horiba Jobin
Yvon, Japan) at scattering angle 90 at 25°C. The range of concentrations of nanoparticles in Na-
borate buffer solution, pH 8.7, was 1.0, 0.5 and 0.25 mg/mL.
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2
.2.6. Transmission electron microscopy (TEM)
The nanoparticles obtained were analyzed using a transmission electron microscope (Jeol
JEM-2100, Japan) operated at an acceleration voltage of 160 kV, after negatively staining the
solution with 2% (w/v) uranyl acetate.
2
.2.7. EPR spectroscopy
The homopolymer, block-copolymer and deprotected block-copolymer were investigated
by EPR spectroscopy. The spin probe TEMPO was non-covalently incorporated into APTES-p-
γ-Glu(Bzl), APTES-p-γ-Glu(Bzl)-b-pPhe and APTES-pGlu-b-pPhe in vapors at 50°C as it was
described in [33]. Nanoparticle suspensions were prepared in concentration 0.5 mg/mL. EPR
spectra were recorded at 25°C using Brucker Elexsys 580 spectrometer (X-band) (Germany)
with high sensitivity resonator. General instrument settings were as follows:
Solid samples with spin probe: microwave power 150 mkW, microwave frequency 9.87 GHz,
modulation frequency 100 kHz, modulation amplitude 1.0 G, sweep width 100 G, central field
value 3370 G, sweep time 200 s. Measurements were performed using 50 mL capillary tubes.
Nanoparticle suspension: microwave power 751.8 mkW, microwave frequency 9.77 GHz, mod-
ulation frequency 100 kHz, modulation amplitude 1.5 G, sweep width 75 G, central field value
3
482.5 G; sweep time 20.40 s. Measurements were performed using 500 μL tube for liquid sam-
ples.
The rotation correlation time was calculated using the equation:
I
10

 6.65 H (
1) 10
(1)
I
where ΔH is line width of high-field component, I - line height of high-field component I -
+
+
-
line height of low-field component.
For the calculation of the anisotropy parameter ε, the following equation was used:
7

ACCEPTED MANUSCRIPT
I0


1
1
I
I


(2)
I0
where I , I and I are line heights of central, high-field and low-field component, respectively.
o
+
-
To evaluate the size of nanoparticles, Stokes-Einstein equation was applied:
3
4R


(3)
3kT
where where η is solvent viscosity, R is effective hydrodynamic radius of the molecule, k is
Boltzmann constant and T is temperature.
The EPR experiment with nanoparticle\cell suspension was carried out under the follow-
ing EPR spectrometer tunings: microwave power 751.8 mkW, microwave frequency 9.77 GHz,
modulation frequency 100 kHz, modulation amplitude 1.5 G, sweep width 100 G, central field
value 3482 G; sweep time 25 s. The measurements were performed using 500 μL tube for liquid
samples.
6
Incubation mixtures consisted of U937 cells resuspended to 1×10 cells/ml in RPMI 1640
medium in new disposable tubes. The suspension of TEMPO-pGlu -b-pPhe (final concentra-
4
3
29
tion is 0.5 mg/mL) was added to cell suspension. The contents of the tube were gently mixed by
o
vortexing, then transferred to an EPR flat cell and incubated at 37 C during 1 hour. EPR spectra
were recorded each 40 minutes within 24 hours.
2
.2.8. Cytotoxicity assay
Cells were routinely cultured at 37°C in humidified atmosphere containing air and 5%
CO . Human embryonic kidney cells (HEK 293), human epithelial colorectal adenocarcinoma
2
cells (Caco-2) and human lung lymphoblasts (U937) were obtained from BioloT (Saint Peters-
burg, Russian Federation) and were grown in Dulbeccos Modified Eagle’s Medium (BioloT)
containing 10% (v/v) heat-inactivated fetal bovine serum (FBS, HyClone Laboratories, UT,
8

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USA), 1% L-glutamine, 1% sodium pyruvate, 50 U/mL penicillin, and 50 μg/mL streptomycin
(BioloT).
Toxicity of polymer nanoparticles was assessed using a standard colorimetric assay with
tetrazolium dye – 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The
cells were incubated for 48 h with fresh medium containing different concentrations of polymer
nanoparticles. Following treatment, Dulbeccos Modified Eagle’s Medium (100 μL/ well) and 20
μL of a 2.5 mg/mL MTT solution were added and cells were incubated for 1 h at 37 °C. The
3
used cell density was 10 × 10 cells/200 μL/well in 96-well microtiter plates. After careful re-
moval of the supernatants, the MTT-formazan crystals formed by metabolically viable cells were
dissolved in DMSO (100 μL/well) and absorbance was measured at 540 nm and 690 nm in a
multiwell plate reader. Values measured at 540 nm were subtracted for background correction at
6
90 nm, and the data were plotted as a percent of control samples using Microsoft Excel.
2
.2.9. Dye encapsulation and cell imaging
To investigate the cellular uptake the TEMPO-pGlu -b-pPhe sample was stained with
4
3
29
©
the photosensitizer Fotoditazin . For this, 0.2 mL of dye was dissolved in 0.8 mL of distilled wa-
ter. 2.0 mg of TEMPO-pGlu -b-pPhe suspended in 1.0 mL of 0.1 M PBS solution, pH 7.4,
4
3
29
was mixed with 1.0 mL dye solution and sonicated for 2 h. Excess of dye was removed via di-
alysis through membrane (12 kDa MWCO).
6
Then, the stained sample was added to the U937 cells suspension (1×10 cells/ml) in
RPMI 1640 medium to obtain the final concentration of polymersomes 0.5 mg/mL. The sample
obtained was incubated during 1 hour and then the images were taken using multiphotonic con-
focal scanning microscope Leica TCS SP5 MP at 662 nm.
3
.
Results and discussion
3
.1. Synthesis and investigation of spin labeled poly(amino acids)
9

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The localization of the spin label in block copolymers is of the great importance because
it brings information about the way the self-assembly was performed [26]. The terminal resi-
dence of spin label attached to the end of the hydrophylic block of the self-assembling block co-
polymer opens the possibility to obtain detectable nanoparticles with the label located directly on
the surface of the polymeric vesicle. Following this idea, 4-amino-TEMPO was applied as initia-
tor for the NCA polymerization of γ-Glu(Bzl) (Scheme 1). The TEMPO-p-γ-Glu(Bzl) with mo-
lecular weight 10225 (43 amino acid residues) and narrow molecular weight distribution (M /M
w
n
=
1.1) was synthesized. At second step the TEMPO-p-γ-Glu(Bzl) was used as macroinitiator for
NCA polymerization of Phe. The synthesized samples were insoluble in organic solvents and the
characterization of hydrophobic block length of co-polymers by GPC or in solution NMR was
impossible. Thus, to evaluate the hydrophobic block length, the resulting copolymers were total-
ly hydrolyzed as it was described in 2.2.3. Then the hydrolyzed samples were investigated by RP
HPLC (see Supplementary, Figure S1). Afterwards, using L-glutamic acid and L-phenylalanine
concentration obtained in each sample (see Supplementary, Table S1) along with the GPC data
of TEMPO-p-γ-Glu(Bzl), the contribution of hydrophobic block was evaluated. Particularly,
three samples of block copolymer with different length of hydrophobic block, namely, TEMPO-
p-γGlu(OBzl) -b-pPhe , TEMPO-p-γGlu(OBzl) -b-pPhe and TEMPO-p-γGlu(OBzl) -b-
4
3
49
43
29
43
pPhe were obtained. The formation of amphiphilic block copolymers was achieved after depro-
1
2
tection of Bzl-protective group (Scheme 1).
To compare the results on magnetic properties of copolymers containing terminal spin
label (4-amino-TEMPO) strictly located in hydrophilic part of the block copolymer to those
bearing non-covalently incorporated paramagnetic molecule (spin probe TEMPO), the samples
of diamagnetic p-γGlu(OBzl)-b-pPhe for spin probe incorporation was synthesized using APTES
as initiator. The APTES- p-γGlu(OBzl) molecular mass characteristics were the following:
M =13800 (62 amino acid residues) and molecular weight distribution M /M = 1.2. The final
n
w
n
block copolymer was of the following structure APTES-p-γGlu -b-pPhe .
6
2
29
1
0

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The EPR spectra were recorded for all the intermediate and resulting polymer com-
pounds: TEMPO-p-γ-Glu(BzL), TEMPO-p-γ-Glu(BzL)-b-pPhe and deprotected TEMPO-pGlu-
b-pPhe in solid, as well as for the same series of model APTES-initiated analogue block copo-
lymers (APTES-p-γ-Glu(Bzl), APTES-p-γ-Glu(Bzl)-b-pPhe and APTES-pGlu-b-pPhe) with
spin-probe incorporated into polymers in vapors.
The line shape, symmetry, and width of the EPR spectra for spin probe non-covalently
introduced into APTES-initiated polymer matrices differ sufficiently from the spectra of the
spin-labeled polymer samples obtained at polymerization step (Figure 1). The parameters of
2
A and g were calculated and given in Table 1.
║
First, the data for spin probe samples were analyzed. The spectra of TEMPO in APTES-
p-γ-Glu(BzL), APTES-p-γ-Glu(Bzl)-b-pPhe and APTES-pGlu-b-pPhe are the triplets with
asymmetric widened components (Figure 1a). The line shape of the all spectra revealed the ani-
sotropy of the constant of hyperfine coupling. All the spectra have split high-field and low-field
components that is the evidence of the existing at least two different populations of paramagnetic
particles [34]. It may be caused by the localization of the TEMPO in the domains with different
molecular dynamics. For APTES-p-γ-Glu(BzL) and APTES-pGlu(BzL)-b-pPhe the contribution
of one type (A ) prevails against another (A ) while for APTES-pGlu-b-pPhe no noticeable
║1
║2
dominance of any of the types was observed. Thus, the character of the spin probe behavior is
similar both in hydrophobic protected polymer matrix and in block copolymer containing two
hydrophobic blocks. After deprotection the remarkable difference between two populations of
nitroxide radical is, mainly, explained by the presence of hydrophilic and hydrophobic blocks in
APTES-pGlu-b-pPhe. Still the g-factor is also changed from APTES-p-γ-Glu(BzL) to APTES-
pGlu-b-pPhe.
The spectrum of poly(γ-benzyl ester of glutamic acid) synthesized via ROP with 4-
amino-TEMPO has broad line shape. In contrary to the free radical spectrum has poorly realized
high-field component (Figure 1b). A number of publications [35; 36] confirms that such kind of
1
1

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spectrum line shape is common for a spin-label covalently attached to macromolecular chain,
which results into highly hindered rotation of the spin label due to the fixed position of the label.
Considering that 4-amino-TEMPO initiates ROP of γ-Glu(Bzl) NCA as primary amine,
the amine mechanism of ROP is realized and the initiator is located at the C-terminal position of
the poly(amino acid) chain [33]. The line shape and spectra parameters such as g-factor and A║
did not changed after co-polymerization and further deprotection (Table 1). Besides, according
to [35 - 37] the line shape of the EPR spectrum of the spin-labeled protein is in dependence on
position of the spin label in the molecule. Considering on the constant spectra parameters for all
spin-labeled samples, it is obvious that EPR detectable fragment preserves the location in the end
of the hydrophilic chain of the final amphiphilic block copolymer, and no radicals are distributed
in other domains of the polymer matrix.
3
.2. Morphology and size distribution of self-assembled nanoparticles
The morphology of the prepared nanoparticles was investigated by TEM. As it can be
seen on the Figure 2, the formed particles can be identified as polymer vesicles with double-
layered membrane. Considering that the hydrophobic block is presented by poly(phenylalanine),
the observed structure can be formed due to π-π interactions between aromatic fragments of hy-
drophobic chains. Though the particle size differs with the growth of hydrophobic part of the
macromolecule, the morphology for all samples remains the same. According to the published
data, the liposome-like nanoparticles (polymersomes) can be prepared with the hydrophilic part
contribution under 45% [38], but for the nanoparticles obtained in present research the polymer-
somes were assembled even at the dominance of hydrophilic block part.
As it can be seen from DLS data (Figure 3), the hydrodynamic diameter of prepared na-
noparticles inversely depended on the length of the hydrophobic block. The mean nanoparticle
size increased in the row pGlu -b-pPhe < pGlu -b-pPhe < pGlu -b-pPhe as 60 < 200 <
4
3
12
43
29
43
49
2
80 nm, respectively. According to ζ-potential values that were about -20 mV at pH 8.6 at poly-
1
2

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mer concentration 0.5 mg/mL, all samples formed stable nanoparticles with negative surface
charge.
All samples exhibited evident dependence of particle size distribution on polymer con-
centration. Thus, with the concentration growth from 0.25 mg/mL to 1.00 mg/mL the mean size
of nanoparticles increased.
3
.3. EPR investigation of self-assembled nanoparticles
After the self-assembly was performed in sodium borate buffer, pH 8.6, at polymer con-
centration of 0.5 mg/mL, the EPR spectra of suspensions obtained were recorded. Comparing to
the solid samples, the EPR spectra of all samples changed sufficiently comparing to the solid
sample spectra (Figure 4). The spectra of suspensions are isotropic and symmetric with three
well-defined components and are similar to the line shape of the spectrum of free 4-amino-
TEMPO radical diluted in the same buffer solution (Figure 4). The values of τ were high (Table
2
). Such line shape and line width prove the absence of spin-spin interactions between neighbor-
ing spin-labels both in the membrane of one nanoparticle and between 4-amino-TEMPO residues
from different nanoparticles. All spectra are characterized with similar value of hyperfine coupl-
ing constant a ≈ 17.9 G. It is known from the literature that the value of hyperfine coupling con-
N
stant is the characteristic of medium polarity [26]. The values calculated for all samples confirm
that the spin labeled end of polymer chain is located on the surface of the nanoparticle in polar
medium (Figure 4). Considering that only spin-labels located on the surface are EPR-responsive,
the amount of the responsive centers can be valued from EPR data via calculating the integral
intensity of the each sample spectrum and comparing it to the standard solutions of the 4-amino-
TEMPO. The concentration of spin labels in nanoparticle suspension was calculated using the
linear approximation of integral intensity dependence on the concentration of 4-amino-TEMPO
solutions.
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The integral intensity of the EPR spectrum of nanoparticle suspensions increased in the
raw pGlu -b-pPhe to pGlu -b-pPhe . This result corresponds to the change of nanoparticle
4
3
29
43
49
hydrodynamic diameter (DLS data), revealing that the growth of nanoparticle diameter leads to
increasing of the concentration spin labels on the surface.
The possibility of nanoparticle hydrodynamic diameter determination via EPR data was
discussed in the work [26]. Particularly, the correlation rotation time value calculated from EPR
spectrum can be applied in Stokes-Einstein equation to evaluate the nanoparticle size (see Ex-
perimental part). This method is efficient in the case if only the motion of the whole nanoparticle
is considered, while the self-rotation of the spin probe against the nanoparticle is neglected. Us-
ing this assumption, the hydrodynamic radiuses of the samples were calculated (Table 2). The
results were opposite to the TEM and DLS data obtained. It means that τ values obtained from
EPR data cannot characterize the motion and the size parameters of the nanoparticles.
Then the approach of determination of the segmental mobility discussed in [40] was ap-
plied. According to the above-mentioned work, the covalently attached spin label simultaneously
takes part in two types of motion: oscillation near the segment and in slow isotropic motion with
the segment of the macromolecule. Considering on the dependence of rotation correlation time
on viscosity of the solvent, the Stokes-Einstein equation (3) can be applied to determine the ef-
fective radius of the segment. Thus, the insufficient changes of τ values can be explained by dif-
ferent size of spin labeled macrochain fragment and its distance from the hydrophobic part of the
membrane due to the dependence of the membrane thickness on the hydrophilic/hydrophobic
block balance.
3
.4. Cell experiments
It was a principal part of the research to investigate if the TEMPO-bearing nanoparticles
can be detected inside the cells via EPR method.
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First of all, the TEMPO-bearing nanoparticles obtained were tested regarding to their cy-
totoxicity using standard MTT assay and three cell lines. Suspensions of TEMPO-pGlu -b-
4
3
pPhe nanoparticles at four different concentrations ranging from 0.125 to 2.00 mg/mL were in-
x
cubated with cells within 48 h. As can be seen from Figure 5 illustrating the data for pGlu -b-
4
3
pPhe the cell viability was higher than 85% for all tested concentrations that means the absence
4
9
of cytotoxicity during the studied time period.
To detect if the nanoparticles penetrate into the cells, the TEMPO-pGlu -b-pPhe poly-
4
3
29
mersomes containing encapsulated fluorescent dye Fotoditazin were incubated with the U937
cells and then investigated by confocal microscopy. The coloration of inner space of cells by en-
capsulated dye proved the cellular uptake of nanoparticles developed (Figure 6).
Figure 7 presents the EPR spectrum of TEMPO-pGlu -b-pPhe after its penetration into
4
3
29
U937 cells. The spectrum has the line shape similar to the spectrum of the respective initial na-
noparticle suspension (Figure 4). The hyperfine coupling constant value a is also about 17.5 G,
N
which is similar to the nanoparticle suspensions. The calculated value of rotation correlation time
-10
(
τ) is 5.02×10 s that does not differ from the value of τ of the nanoparticle suspension. Thus,
the approach suggested can be applied as an effective imaging tool in biomedicine, particularly,
for monitoring of new drug system distribution in tissues at bioapprobation step.
4
. Conclusions
A new approach of synthesis of spin labeled poly(amino acid)-based nanoparticles was
developed. The series of amphiphilic block copolymers with different hydrophobic part length
was synthesized using the spin label 4-amino TEMPO as initiator of NCA ROP at the first stage
of synthesis. The spin label was strictly located in the С-terminal position of hydrophilic
poly(amino acid) chain of the resulting block copolymer.
The synthesized pGlu-b-pPhe samples were able to self-assembling in aqueous solutions
forming polymersomes. The diameter of prepared nanoparticles depended on the length of hy-
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drophobic block and polymer concentration. The increasing of the hydrophobic block contribu-
tion into polymer molecular weight led to the growth of nanoparticle size.
In suspension spin labels are located on polymersome surface and make them EPR de-
tectable. The nanoparticle size defines the amount of the spin labels and depends on the hydro-
phobic/hydrophilic blocks balance. The rotation correlation time calculated from EPR data for all
samples were the values of one order of magnitude and insignificantly grew in the row pGlu -b-
4
3
pPhe ˂pGlu -b-pPhe ˂pGlu -b-pPhe showing the dependence of TEMPO containing frag-
4
9
43
29
43
12
ment size on the contribution of increasing hydrophobic block.
The spin-labeled nanoparticles were found to be biocompatible and successfully detected
by EPR after penetrating the cell membrane. Thus, it makes possible to use the developed poly-
mersomes for bioimaging purposes.
Acknowledgements
This work was supported by grant of Russian Scientific Foundation (# 14-50-00069). The
participation in this work of Dr. A.V. Hubina (monthly salary) was supported by postdoc pro-
gram from St. Petersburg State University (# 12.50.1193.2014).
GPC and LC-MS were carried out in Chemical Analysis and Materials Research Centre.
NMR and EPR experiments were performed in St. Petersburg State University Resource Center
for Magnetic Resonance. The authors are very grateful to Dr. S.M. Sukharzhevskii for EPR mea-
surements and result discussion. Cell experiments were carried out using the equipment of the
Research Center on Development of Molecular and Cell Technologies of St. Petersburg State
University.
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Scheme 1. Synthesis of TEMPO-pGlu-b-pPhe block copolymers.
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0

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Figure 1. EPR spectra of non-covalently introduced spin-probe (A) 1 - APTES-p-γ-Glu(Bzl),
2
- APTES-p-γ-Glu(Bzl)-b-pPhe, 3 - APTES-pGlu-b-pPhe; and covalently attached spin label
(B) 1- TEMPO-p-γ-Glu(Bzl), 2 - TEMPO-p-γ-Glu(Bzl)-b-pPhe, 3 - TEMPO-pGlu-b-pPhe.
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Figure 2. TEM image of pGlu -b-pPhe (A) and pGlu -b-pPhe (B) polymersomes.
4
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2
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Figure 3. Dependence of (A) particle size distribution on hydrophobic part contribution at
nanoparticle concentration 0.5 mg/mL 1 – TEMPO-pGlu -b-pPhe , 2 - TEMPO-pGlu -b-
4
3
12
43
pPhe , 3 - TEMPO-pGlu -b-pPhe ; and (B) particle size on the concentration of TEMPO-
2
9
43
49
pGlu -b-Phe 1 – 0.25 mg/mL, 2 – 0.5 mg/mL, 3 – 1.00 mg/mL.
4
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3

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Figure 4. EPR spectra of 0.5 mg/mL suspensions of 1 – TEMPO-pGlu -b-pPhe , 2 - TEM-
43
12
PO-pGlu -b-pPhe , 3 - TEMPO-pGlu -b-pPhe .
4
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2
4

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Figure 5. Viability of different cells incubated with TEMPO-pGlu -b-pPhe polymersomes.
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5

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Figure 6. Confocal fluorescent (A) and bright-field images of U937 cell containing stained
TEMPO-pGlu -b-pPhe particles.
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3
420
3440
3460
3480
3500
3520
3540
Field, G
Figure 7. EPR spectrum of TEMPO-pGlu -b-pPhe polymersomes after their penetration
43
29
into U937 cells.
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Table 1. Parameters of EPR spectra of the samples.
Sample
2A║1
G
A║2
G
g1
g2
ε
APTES-p-γ-
Glu(Bzl)
44.2
65.4
2.0061
2.0070
2.0029
2.0068
0.338
0.269
APTES-p-γ-
Glu(Bzl)-b-pPhe
35.1
64.4
APTES-pGlu-b-
pPhe
TEMPO-p-γ-
35.6
68.1
-
2.0059
2.0060
2.0024
-
0.097
-
65.20
Glu(Bzl)
TEMPO-p-
γGlu(OBzl) -b-
65.09
67.09
-
-
2.0061
2.0060
-
-
-
-
4
3
pPhe49
TEMPO-p-
γGlu -b-pPhe
49
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8

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Table 2. Parameters calculated from the nanoparticle suspensions EPR spectra
Sample
aN
Τ
s
Rh, (Stokes-Einstein)
C
m
mole/L
-10
-10
-10
-10
-6
TEMPO-pGlu -b-pPhe
16.94
17.07
17.08
7.7x10
9.34 x10
2.4x10
1.4x10
2.0x10
4
3
12
29
49
-10
-5
-5
TEMPO-pGlu -b-pPhe
4.5x10
4.1x10
4.88x10
4
3
-10
TEMPO-pGlu -b-pPhe
2.68x10
4
3
2
9