Cinnamyl modified polymer micelles as efficient carriers of caffeic acid phenethyl ester

Article abstract of DOI:10.1016/j.reactfunctpolym.2020.104763

A synthetic strategy towards modular polymer platform capable of further tunable degree of modification is presented. Initially, a well-defined amphiphilic block copolymer with alkyne side groups distributed along the biodegradable hydrophobic block was successfully prepared applying controlled ring-opening copolymerization of D,L-lactide and an acetylene-functional cyclic carbonate initiated by polyoxyethylene macroinitiator. In the second synthetic step a desired number of cinnamyl pendant groups was introduced into the hydrophobic block via “click” reaction. The functional block copolymers self-associated in aqueous media into stable micelles with narrow size distribution and average diameters of around 50 nm. The micelles' functional hydrophobic cores were loaded with caffeic acid phenethyl ester (CAPE) as bioactive compound with great potential for therapeutic application. It was demonstrated that the initial drug-release profiles, hence the stability of the loaded nanocarriers during circulation, can be modulated via the number of cinnamyl groups into the micelles core. Initial in-vitro evaluations were preformed on empty and drug-loaded functional polymer micelles indicating their potential for application in nanomedicine as safe and biocompatible drug-delivery nanovehicles with enhanced stability.

Full text of DOI:10.1016/j.reactfunctpolym.2020.104763

Reactive and Functional Polymers 157 (2020) 104763
Contents lists available at ScienceDirect
Reactive and Functional Polymers
journal homepage: www.elsevier.com/locate/react
Cinnamyl modified polymer micelles as efficient carriers of caffeic acid
phenethyl ester
Radostina Kalinovaa, Yordan Yordanovb, Borislav Tzankovb, Virginia Tzankovab,
,
*
Krassimira Yonchevab, Ivaylo Dimitrova
a Institute of Polymers, Bulgarian Academy of Sciences, Akad. G. Bonchev St., Bl 103A, 1113 Sofia, Bulgaria
b Faculty of Pharmacy, Medical University of Sofia, 2 Dunav St., 1000 Sofia, Bulgaria
A R T I C L E I N F O
A B S T A R C T
Keywords:
A synthetic strategy towards modular polymer platform capable of further tunable degree of modification is
presented. Initially, a well-defined amphiphilic block copolymer with alkyne side groups distributed along the
biodegradable hydrophobic block was successfully prepared applying controlled ring-opening copolymerization
of D,L-lactide and an acetylene-functional cyclic carbonate initiated by polyoxyethylene macroinitiator. In the
second synthetic step a desired number of cinnamyl pendant groups was introduced into the hydrophobic block
via “click” reaction. The functional block copolymers self-associated in aqueous media into stable micelles with
narrow size distribution and average diameters of around 50 nm. The micelles’ functional hydrophobic cores
were loaded with caffeic acid phenethyl ester (CAPE) as bioactive compound with great potential for therapeutic
application. It was demonstrated that the initial drug-release profiles, hence the stability of the loaded nano-
carriers during circulation, can be modulated via the number of cinnamyl groups into the micelles core. Initial in-
vitro evaluations were preformed on empty and drug-loaded functional polymer micelles indicating their po-
tential for application in nanomedicine as safe and biocompatible drug-delivery nanovehicles with enhanced
stability.
Polymer micelles
Functional
Stability
Drug delivery
Caffeic acid phenethyl ester
1. Introduction
delivery characteristics of polymeric micelles include biodegradability,
low toxicity, high drug loading capacity, improved drug pharmacoki-
A widely applied technique to improve bioavailability of poorly
soluble active substances while preserving their activity is the use of
appropriate drug delivery systems [1]. The nanometer size range of
these systems would be perfect for circulating in the blood stream,
passing through tissues, and finally entering the cells [2,3]. Thus, being
nanosized colloidal systems with unique physicochemical nature, poly-
meric micelles are extensively used in drug delivery systems [4,5]. They
are formed through self-association of amphiphilic block or graft co-
polymers in aqueous media. The size of the polymeric micelles, which is
in the 10–100 nm range can be controlled by their formation method,
aggregation number, the copolymers molar mass, and hydrophilic-
hydrophobic balance [6]. The micelles size is an important factor for
targeting to the tumor site via the so-called enhanced permeability and
retention (EPR) effect [7–9]. The smaller size of the polymeric micelles
avoids the reticuloendothelial system (RES) uptake resulting in pro-
longed blood circulation [10]. Other essential for successful drug
netics and pharmacodynamic profiles [11]. The drug loaded micelles’
stability is also an important factor for efficient delivery. Upon entering
the bloodstream polymeric micelles are exposed to various environ-
mental changes such as dilution, pH and salt changes, contact with
proteins and cells [12]. In order to be suitable for drug delivery appli-
cations polymeric micelles should be capable of preserving their cargo
during the circulation by preventing disintegration whereas releasing
the drug at the target site. The stability of polymeric micelles corre-
sponds directly to the length of copolymer’s hydrophobic block and
their dissociation tendency is related to the composition, physical state
and cohesion of the hydrophobic core [13,14]. The micelles’ core usu-
ally consists of a biodegradable hydrophobic polymer such as polylactic
acid (PLA), polyglycolic acid (PGA), poly(lactic-co-glycolic acid)
(PLGA), polycaprolactone (PCL) or poly(amino acid) [15]. The hydro-
philic part of the copolymer, most commonly polyethylene glycol (PEO),
forms the shell and ensures the so-called stealth behavior of the micelles
* Corresponding author.
E-mail address: dimitrov@polymer.bas.bg (I. Dimitrov).
https://doi.org/10.1016/j.reactfunctpolym.2020.104763
Received 10 June 2020; Received in revised form 21 September 2020; Accepted 15 October 2020
Available online 20 October 2020
1381-5148/© 2020 Elsevier B.V. All rights reserved.

R. Kalinova et al.
Reactive and Functional Polymers 157 (2020) 104763
preventing their opsonization and uptake by the RES, thus prolonging
the circulation time [16]. Other suitable hydrophilic biocompatible
polymers include poly(N-vinyl-2-pyrrolidone), poly(2-hydroxyethyl
methacrylate), poly(2-oxazoline)s, poly(acrylic acid) [17–20].
recrystallized from toluene/ethyl acetate mixture (95:5 v/v). Triethyl-
amine (TEA, 99%) was distilled from potassium hydroxide. Methox-
ypolyoxyethylene (MPEO-5 K, Mn = 5000 g molꢀ 1) was freeze-dried
from toluene. 2,2-Bis(hydroxymethyl)propionic acid (98%), propargyl
bromide (80 wt% in toluene), ethyl chloroformate (97%), 4-dimethyla-
minopyridine (DMAP, >99%), CuI (≥99.5%), N,N-diisopropylethyl-
amine (DIPEA, ≥99%), potassium hydroxide, Na2SO4 (≥99.0%), diethyl
ether (for analysis), dichloromethane (DCM, ≥99.5%), hexane (>99%),
acetone (≥99.5%) and acetonitrile, (99.8%) were used as received.
Cinnamyl azide was obtained reacting cinnamyl chloride and NaN3 in
water/acetone mixture [35]. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) was purchased from Sigma. Ea.hy926
human endothelial hybrid cell line was purchased from ECACC (Salis-
bury, Wiltshire, England). The cells were maintained in DMEM medium,
supplemented with fetal calf serum, 2 mmol Lꢀ 1 glutamine and HAT at
37 ◦C, humidified air and 5% CO2. The cells were subcultured when they
reach 70–80% confluence by trypsinization.
Caffeic acid phenethyl ester (CAPE) is a natural, bioactive hydro-
phobic, polyphenolic compound found in various plants and is a key
constituent of propolis [21,22]. It can also be prepared by various
chemical and enzyme-catalyzed methods [22,23]. CAPE has attracted an
increasing interest due to its antioxidant, anti-inflammatory, antimi-
crobial, antiviral and anticancer activities, as well as for its neuro-
protective, cardioprotective, and hepatoprotective properties [24–26].
A number of in vitro studies demonstrated CAPE’s ability to suppress the
proliferation of several human cancer cell lines, whereas in vivo evalu-
ations revealed that CAPE prevents cancer initiation, tumor growth, and
cancer metastasis of the colon, liver, and breast cancers in animal
models [27]. The cytotoxic effects of CAPE are usually selective against
cancer cells [28]. However, in order to achieve the therapeutic effect,
CAPE should be able to reach the target tissues at a specific concentra-
tion, which has to be maintained for a prolonged period of time. A
contemporary approach to overcome the drug’s poor water solubility is
to load it into the hydrophobic core of amphiphilic copolymer micelles,
thus achieving increased solubility and stability. Various nanocarriers
formed from biodegradable and biocompatible polymers were used to
encapsulate CAPE. Thus, CAPE loaded PLGA micelles showed sustain-
able and prolonged drug release and enhanced antibacterial and anti-
parasitic activity [29,30]. CAPE-loaded PCL-based amphiphilic diblock
and triblock copolymers with PEO hydrophilic blocks showed higher
anticancer and antioxidant activity, enhanced stability and compati-
bility between CAPE and the micellar core [31–33]. Polymeric micelles
from the amphiphilic diblock copolymers consisting of hydrophilic
polyglycidol and hydrophobic poly(allylglycidyl ether) were also used
as CAPE carriers [34]. Overall, CAPE-incorporated polymer nano-
carriers were found to be more efficient as compared to the drug itself
due to the improved properties (e.g. solubility) and effects.
2.2. Synthetic procedures
2.2.1. Synthesis of 5-methyl-5-propargyloxycarbonyl-1,3-dioxane-2-one
(PC) [36]
2,2-Bis(hydroxymethyl)propionic acid (6 g, 44.7 mmol) and potas-
sium hydroxide (2.74 g, 48.8 mmol) were dissolved in 33 mL of DMF.
The mixture was stirred for 1.5 h at 100 ◦C. Propargyl bromide (8.2 mL,
80 wt% solution in toluene) was then added dropwise and the mixture
was stirred at 70 ◦C for another 40 h. After the mixture was cooled to
room temperature, the solids were filtered off and the filtrate was
concentrated under vacuum. The residue was dissolved in 10 mL of
distilled water and extracted with DCM (3 × 30 mL). The organic phase
was dried over Na2SO4 and DCM was removed in vacuum yielding
propargyl-2,2-bis(hydroxymethylpropionate) as a clear viscous liquid
(6.3 g, 82%).
Propargyl-2,2-bis(hydroxymethylpropionate) (6.3 g, 37 mmol) and
ethyl chloroformate (8.03 g, 74 mmol) were dissolved in 100 mL of THF
at 0 ◦C and the mixture was stirred for 30 min followed by a dropwise
addition of TEA (8.99 g, 88 mmol) over a period of 30 min. After the
mixture was stirred at room temperature for 24 h, the NEt3.HCl salts
were filtered off and the filtrate was concentrated in vacuum to obtain a
viscous liquid. The product was recrystallized from diethyl ether to give
light brown crystals. Yield: 40%. 1H NMR (600 MHz, CDCl3, δ, ppm):
1.37 (s, 3H, -CH3), 2.54 (t, 1H, -CH2-C ≡ CH), 4.23–4.24 (d, 2H, -CH2-C
(CH3)-CH2-), 4.72–4.74 (d, 2H, -CH2-C(CH3)-CH2-), 4.79 (d, 2H, -CH2-C
≡ CH).
The properties of the reported so far polymer nanocarriers of CAPE
were adjusted just through variation of the hydrophilic-hydrophobic
balance as a result of series of polymerizations. Herein, we present a
novel synthetic strategy towards well-defined multifunctional amphi-
philic PLA-PEO-based copolymers with finely tunable number of cin-
namyl side groups distributed along the hydrophobic block. Thus, with
no need of synthesizing a series of amphiphilic copolymers with
different hydrophilic-hydrophobic ratio the delicate balance between
enhanced stability of drug loaded polymer micelles and the successful
cargo release into the target site could be achieved just by attaching the
desired number of cinnamyl side groups onto the same reactive func-
tional block copolymer precursor. A controlled ring-opening copoly-
merization of cyclic monomers was initiated by PEO-macroinitiator
followed by the attachment of the desired amount of side groups to the
copolymers’ hydrophobic segment via highly efficient “click” reactions.
The copolymers self-associated in aqueous media to form micelles
intended for CAPE-loading. Modification of PLA-core with cinnamyl
groups was designed aiming to enhance micelles’ and CAPE stability
2.2.2. Synthesis of MPEO-b-(PLA-co-PPCA) (ВА)
Initially, MPEO-5 K (1 g, 0.2 mmol), D,L-lactide (0.8 g, 5.6 mmol), PC
(0.2 g, 1 mmol) and DMAP (48.9 mg, 0.4 mmol) were dried under high
vacuum for 2 h followed by flushing the flask with an argon. The
polymerization was carried out in bulk at 125 ◦C for 24 h. The crude
product was extracted with 2-propanol, filtered and dried under vacuum
at 30 ◦C. The product was purified by precipitation from DCM/diethyl
ether. Yield: 1.5 g (75%). 1H NMR (600 MHz, CDCl3, δ, ppm): 1.29 (t,
3H, -CH3PPC), 1.56 (t, 3H, -CH3PLA), 2.52 (s, 1H, -CH2-C ≡ CH), 3.38 (s,
3H, CH3-O), 3.65 (s, 4H, -O-CH2-CH2-O-), 4.30–4.35 (d, 2H, -O-CH2-
CH2-O(C=O)- + 4H, (-O-CH2-C(CH3)-CH2-O), 4.73 (s, 2H, -CH2-C ≡ CH),
5.17 (m, 1H, CH-(CH3)-O-).
through π-π stacking interactions between the aromatic groups. The
safety of the developed polymer micelles was evaluated by measuring
their hemolytic potential in freshly isolated erythrocytes and by scratch
test in vitro. The cytotoxic effects of the empty and CAPE-loaded micelles
on endothelial cell line were investigated aiming to explore their po-
tential as drug delivery system.
2.2.3. “Click” reaction of cinnamyl azide with MPEO-b-(PLA-co-PPCA)
block copolymer (BC)
2. Materials and methods
Typically, MPEO-b-(PLA-co-PPCA) (0.4 g, 0.035 mmol) and CuI
(0.027 g, 0.14 mmol) were dried under high vacuum for 30 min followed
by flushing the flask with argon. Then, a solution of cinnamyl azide
(0.086 g, 0.54 mmol) in 2 mL THF was added to the reaction vessel with
a glass syringe and the reaction mixture was degassed by bubbling argon
for 30 min. Finally, DIPEA (0.1 mL, 0.074 g, 0.57 mmol) was added and
2.1. Materials and reagents
All chemicals were purchased from Sigma-Aldrich. Tetrahydrofuran
(THF, >99%) and N,N-dimehylformamide (DMF, ≥99.5%) were
distilled from calcium hydride prior to use. D,L-lactide (LA) was
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R. Kalinova et al.
Reactive and Functional Polymers 157 (2020) 104763
reaction was conducted at 35 ◦C for 24 h. The reaction mixture was
diluted with THF and the catalyst complex was removed by passing the
polymer solution through a short column packed with an aluminum
oxide. The solvent was evaporated, resulting material was precipitated
from DCM/hexane, filtered and was dried in a vacuum oven at 30 ◦C.
Yield: 0.31 g, (76%). 1H NMR (600 MHz, CDCl3, δ, ppm): 1.26 (t, 3H,
-CH3PPC), 1.54 (t, 3H, -CH3PLA), 3.38 (s, 3H, CH3-O), δ 3.65 (s, 4H, -O-
CH2-CH2-O-), 4.32 (s, 2H, -O-CH2-CH2-O(C=O)- + 4H, (O-CH2-C(CH3)-
CH2-O- + 2H, ꢀ NCH2-), 5.14 (m, 1H, CH-(CH3)-O-)), 6.37 (s, 1H,
CH=CH-Ar), 6.65 (s, 1H, CH=CH-Ar), 7.3–7.4 (m, 5H, ArH), 7.53 (s, 1H,
triazole ring).
2.5. Critical micelle concentration (CMC) determination
The amphiphilic block copolymers’ concentration at and above
which micelles are formed was evaluated applying the dye solubilization
method described by Alexandridis et al. [37]. Samples of block co-
polymers in aqueous media (1 mL) with concentrations ranging from
0.001 to 2.0 mg mLꢀ 1 were prepared followed by the addition of hy-
drophobic dye 1,6-diphenyl-1,3,5-hexatriene (DPH, 10 μL from 0.4 mM
solution in methanol). The dye is water-insoluble but it solubilizes into
the hydrophobic micellar core once block copolymer self-association
takes place, giving a characteristic UV-spectrum with absorption
maximum at 356 nm. The intensity of λmax for the series of samples was
plotted against the copolymers’ concentration. The CMC was estimated
from the intersection point of the base line and the straight line obtained
from the UV absorption of the increasing micellar concentrations.
2.2.4. Synthesis of MPEO-b-PLA
MPEO-5 K (1 g, 0.2 mmol), D,L-lactide (1.3 g, 9.0 mmol), and DMAP
(49 mg, 0.4 mmol) were dried under high vacuum for 1 h followed by
flushing the flask with an argon. The polymerization proceeded in bulk
at 125 ◦C (oil bath) for 2 h. The crude product was extracted with 2-
propanol, filtered and dried to yield MPEO-b-PLA diblock copolymer
(1.5 g, 67% yield). 1H NMR (600 MHz, CDCl3, δ, ppm): 5.17 (CH-(CH3)-
O), 4.33 (CH-(CH3)-OH), 3.67 (O-CH2-CH2-O), 3.37 (CH3-O), 1.60 (CH-
(CH3)-O).
2.6. Drug loading and in vitro release
The preparation procedure of CAPE loaded micelles was similar to
that of the unloaded ones. Typically, CAPE was dissolved in acetone (1
mg mLꢀ 1) and 1 mL from solution was added to dissolve 10 mg of the
block copolymer. Then, 0.5 mL from the copolymer/CAPE solution was
added to 3 mL of water under stirring and after the acetone removal on a
rotary evaporator the concentration was adjusted to 1 mg mLꢀ 1 (mi-
celles to CAPE ratio – 10:1 w/w). The micellar dispersion was filtered
2.3. Characterization
1H NMR spectra were recorded in CDCl3 on a Bruker Avance II+ 600
MHz instrument. The average molecular weight and dispersity of the
polymers were determined by gel permeation chromatography using
Shimadzu Nexera XR HPLC chromatograph, equipped with quaternary
pump, degasser, automatic injector, column heater, UV/Vis (SPD-20A)
(0.45 μm), lyophilized and then resuspended in acetonitrile for UV–vis
analysis at a wavelength of 319 nm. The extinction coefficient value, ε =
16770 Mꢀ 1 cmꢀ 1 (λmax = 319 nm) of CAPE obtained by calibration curve
in acetonitrile was used to determine the drug content into the micelles.
The drug loading efficiency (DLE) and drug loading capacity (DLC) were
calculated according to the following equations:
detector, differential refractive index (RID-20A) detector, 10
μm PL gel
mixed-B, 5 m PL gel 500 Å and 50 Å columns. The mobile phase was
μ
tetrahydrofuran (THF) with a flow-rate of 1.0 mL minꢀ 1. The system was
calibrated versus polyoxyethylene narrow molar mass standards. UV–vis
spectra were taken on a DU 800 Beckman Coulter spectrometer. Atomic
force microscope (AFM) images were taken on a Bruker NanoScope V9
instrument with a 1.00 Hz scan rate under ambient conditions. Obser-
vations were performed in ScanAsyst (Peak Force Tapping) mode.
Transmission electron microscope (TEM) images were obtained using
HRTEM JEOL JEM-2100 (200 kV) instrument equipped with CCD
camera GATAN Orius 832 SC1000 and GATAN Microscopy Suit Soft-
ware. The size distribution of the prepared micelles was determined by
dynamic light scattering (DLS) using a NanoBrook Plus PALS instrument
(Brookhaven Instruments), equipped with a 35 mW solid-state laser
operating at λ = 660 nm at a scattering angle of 90o. The particles’
hydrodynamic diameters (dH) were determined according to the Stokes-
Einstein equation:
DLE(wt%)=(am ountofCAPEinm icelles/totalinitialam ountofCAPE)×100
(2)
DLC (wt%) = (am ount of CAPE in m icelles/am ount of the m icelles) × 100
(3)
The in vitro release of CAPE from the polymer micelles was studied in
a phosphate buffer (pH = 7.4) by a dialysis. The micellar dispersions
were placed into a dialysis membrane (MWCO = 6000–8000 Da) and
immersed into 100 mL of the buffer supplemented with ethanol (10% v/
v). The studies were conducted at 37 ◦C and stirring of the release me-
dium (50 rpm). At certain time intervals samples were withdrawn from
the buffered media and the concentration of the released CAPE was
determined by UV–vis spectroscopy (λ = 330 nm).
2.7. Hemolysis assay
dH = kT/(3πηD)
(1)
By a series of centrifugation steps, erythrocytes were isolated and
resuspended in phosphate buffers with pH values of 7.4, 6.2 and 5.6 that
mimic the conditions in extracellular, early endosomal, and late endo-
lysosomal compartments [38]. The isolated red blood cells were pipet-
where k is the Boltzmann’s constant, T is the absolute temperature,
η
is
the viscosity, D is the diffusion coefficient.
The size measurements were carried out in an automated mode in
triplicate and recorded as an average of 3 runs. The polymer dispersions
ted in 96-well plates and co-incubated with empty (6.25–50
μ
g mLꢀ 1
)
were passed through Millipore® 0.45 μm pore-sized syringe filters prior
micelles in buffers at defined pH-values. Negative control wells with
diluent and positive control wells with a final concentration of 2%
TritonX-100 were included. After an hour of incubation, each plate was
centrifuged and supernatant aliquots were transferred to an empty plate.
The amount of hemoglobin released in supernatant was spectrophoto-
metrically measured at 405 nm. Hemolysis percentage was calculated as
the fraction of lyzed erythrocytes, according to the following equation:
to measurements.
2.4. Preparation of micelles
The block copolymer micelles were prepared by the nano-
precipitation technique. The block copolymer was first dissolved in
acetone (10 mg mLꢀ 1). Then, 0.5 mL of the polymer solution was added
dropwise to approx. 3 mL of ultrapure water (18.2 MΩ cm) under
vigorous stirring. Finally, acetone was removed on a rotary evaporator
and the concentration of the micellar dispersions was adjusted to 1 mg
mLꢀ 1 by the addition of an ultrapure water in a volumetric flask.
Hem olysis(%) = (Abs(sam ple)ꢀ Abs(negative control))/(Abs(TritonX100)
ꢀ Abs(negative control))×100
(4)
3

R. Kalinova et al.
Reactive and Functional Polymers 157 (2020) 104763
Scheme 1. Synthetic route to functional amphiphilic copolymers with pendant cinnamyl groups.
2.8. Scratch assay
reference. After 72 h of treatment, the contents of all wells were aspi-
rated and replaced with medium, containing 0.5 mg mLꢀ 1 MTT. After 3
h of incubation at 37 ◦C, humidified air and 5% CO2, the intracellular
L929 mouse fibroblast cells were plated at a density of 3 × 105 cells/
well in a 24 well plate and allowed to attach overnight. A wound was
created by scratching the monolayer with a pipette tip [39]. Wells were
washed twice with PBS and then treatment dispersions of BA-4 and BC-2
formazan crystals were solubilized by adding 100 μL DMSO per well.
The absorbance was determined at 570 and 690 nm for background
absorbance on a multiplate reader Synergy 2 (BioTek Instruments).
(25
μ
g mLꢀ 1) in culture medium were added. Photomicrographs were
taken after 0, 3, 6, 8 and 10 h. Scratch areas were estimated by means of
ImageJ software and its MRI Wound Healing Tool. The area of the
scratch, taken by migrating cells was estimated for each time. Linear
regression with curve fitting was carried out for all the samples and the
slope for each sample was calculated as an estimate for the rate of wound
closure. Then, groups were compared by means of Kruskal-Wallis one-
way analysis of variance.
2.10. Statistical analysis
All statistical analyses of experimental data have been done on
GraphPad Prism 6 Software. Comparisons between groups, for which
normal distribution was expected, have been done by applying one-way
Anova with Dunnet’s multiple comparisons post-test and for all other
comparisons, Kruskal-Wallis test with Dun’s post-test was used.
2.9. Cell viability assay
3. Results and discussion
The effects of the empty and CAPE-loaded micelles on cell viability
were evaluated by MTT assay [40]. Ea.hy926 cells were plated at a
density of 1 × 104 cells/well in 96-well plates and allowed to attach
overnight. The medium was discarded and the cells were incubated for
3.1. Synthesis of cinnamyl-functionalized amphiphilic block copolymers
The synthetic route to functional amphiphilic diblock copolymers
bearing pendant cinnamyl functions intended to enhance both the
compatibility between CAPE and hydrophobic core and the micelles’
stability is presented in Scheme 1. Initially, the cyclic functional
72 h with the empty (20–160
μ
g mLꢀ 1) or CAPE-loaded micelles (2–16
μ
g CAPE mLꢀ 1). CAPE dissolved in DMSO (0.1%) was applied as a
4

R. Kalinova et al.
Reactive and Functional Polymers 157 (2020) 104763
Fig. 1. 1H NMR (600 MHz) spectra in CDCl3 of (a) functional amphiphilic copolymer with acetylene pendant groups (BA-4), and (b) cinnamyl-functionalized
copolymer BC-2.
Fig. 2. GPC elugrams of MPEO-5 K-macroinitiator (Mn = 4800 g molꢀ 1, Ð = 1.08) and the corresponding acetylene-functional copolymer BA-4 (Mn = 8800 g molꢀ 1
Ð = 1.52) in THF (vs. polyoxyethylene standards).
,
5

R. Kalinova et al.
Reactive and Functional Polymers 157 (2020) 104763
Fig. 3. Size-distribution curves from DLS measurements of 1 mg mLꢀ 1 aqueous micellar dispersions of: (a) functional amphiphilic copolymer with pendant acetylene
groups BA-4 (d = 50 nm, PdI: 0.098); and (b) cinnamyl-modified functional amphiphilic copolymer BC-2 (d = 46 nm, PdI: 0.096).
monomer 5-methyl-5-propargyloxycarbonyl-1,3-dioxane-2-one (PC)
carrying an acetylene group was synthesized in a two-step procedure as
previously described with slight modifications (Scheme 1a) [36].
Briefly, 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) was used to
obtain propargyl-2,2- bis(hydroxymethylpropionate) (P-bis-MPA) by
reacting the formed potassium salt of bis-MPA with propargyl bromide.
The P-bis-MPA was further reacted with ethyl chloroformate using
triethylamine as a base to obtain the cyclic carbonate monomer (PC).
The product was purified by recrystallization from diethyl ether and the
chemical structure of cyclic carbonate was confirmed by 1H NMR. In the
next synthetic step the functional cyclic monomer PC was copoly-
merized with D,L-lactide using MPEO-5 K as a macroinitiator applying
the metal-free organocatalyzed controlled ring-opening polymerization
proposed by Nederberg et al. [41]. The polymerization was carried out
in bulk at 125 ◦C with DMAP as a catalyst (Scheme 1b). The crude
product was extracted with 2-propanol, filtered, dried under vacuum
and purified by precipitation from DCM/diethyl ether. The obtained
amphiphilic copolymer with randomly distributed in the hydrophobic
block acetylene side groups was characterized by 1H NMR spectroscopy.
The average degree of LA and PC polymerization (DP = 30 and 10,
respectively) was estimated from the relative intensities of methyne
protons from the polyester repeating units at 5.15 ppm (for PLA), the
methylene protons located next to the terminal alkyne group at 4.75
ppm (for PPCA) and those corresponding to oxyethylene protons of
MPEO-5 K at 3.65 ppm (Fig. 1a). The successful polymerization was also
confirmed by FTIR spectroscopy (Fig. S1b). The strong bands at 1188
and 1748 cm, corresponding to C–O–C (symmetric and asymmetric) and
copolymer was confirmed by 1H NMR spectroscopy. For example, in the
spectrum of the fully functionalized copolymer BC-2 the resonances at
2.52 ppm and 4.73 ppm corresponding to alkyne protons and methylene
protons next to the alkyne end-functionality, respectively disappeared
while new resonances at 6.4 and 6.6 ppm corresponding to protons of
cinnamyl double bonds, at 7.3–7.4 ppm corresponding to the aromatic
protons of cinnamyl units and at 7.53 ppm for triazole ring proton
appeared (Fig. 1b). Moreover, the band at 3277 cmꢀ 1 corresponding to
the stretching vibrations of C ≡ C–H completely disappeared from the
FTIR-spectrum of the modified product (Fig. S1c).
3.2. Synthesis of non-functionalized amphiphilic diblock copolymer
To perform comparative studies, an amphiphilic methoxypolyoxy-
ethylene-b-poly(D,L-lactide) non-functionalized diblock copolymer
(MPEO-b-PLA) was also synthesized. A commercially available MPEO-5
K was used to initiate the metal-free organocatalyzed controlled ring-
opening polymerization of D,L-lactide in bulk to obtain well-defined
amphiphilic MPEO-b-PLA diblock copolymer. The LA degree of poly-
merization was 30 as calculated from the product’s 1H NMR spectrum,
while the formation of diblock architecture and the monomodal molar-
mass distribution with dispersity Ð = 1.12 were estimated from the
block copolymer’s GPC elugram (Fig. S2).
3.3. Self-association of the amphiphilic block copolymers
Due to the block copolymers insolubility in aqueous media the mi-
celles were prepared applying the nanoprecipitation technique. A pre-
determined amount of the block copolymers was dissolved in acetone
which is miscible with water and a good solvent for all copolymer seg-
ments. The polymer solution was added dropwise to vigorously stirred
water followed by evaporation of the organic solvent and adjusting the
dispersion to the desired concentration.
–
–
C
O stretching vibrations were assigned to polyester and poly-
carbonate blocks, while those at 3277 cmꢀ 1 were attributed to stretching
vibrations of C ≡ C–H from the alkyne side groups.
The GPC analysis demonstrates the formation of block copolymer
architecture showing a clear shift towards lower elution time (higher
molar mass) for the amphiphilic copolymer BA-4 in comparison with the
corresponding macroinitiator (Fig. 2). Although, not as monodisperse as
MPEO-5 K, the copolymer still exhibits monomodal molar-mass
distribution.
Critical micelle concentration (CMC) is a fundamental parameter
used to characterize the stability of micelles. The CMC values of the
synthesized functional and non-functional amphiphilic copolymers were
assessed following the increasing UV-absorption intensity of the hy-
drophobic dye DPH solubilized in micelles core as a function of co-
polymers concentration in water (Fig. S3). The estimated CMC values for
In the final synthetic step, MPEO-b-(PLA-co-PPCA) was fully or
partially functionalized with pendant cinnamyl groups via the highly
efficient copper catalyzed 1,3-dipolar cycloaddition of azides and al-
kynes [42,43]. The “click” reaction between the alkyne side groups of
BA-4 and cinnamyl azide was performed at 35 ◦C using the CuI/DIPEA
catalytic complex (Scheme 1b). The complete attachment of the desired
number of cinnamyl functions to the hydrophobic segment of the
all copolymers were in the 0.009–0.040 mg mLꢀ 1 (0.7–4.5
μM) range.
The results are consistent with the copolymers’ hydrophilic-
hydrophobic balance. Thus, the lowest CMC value (hence the highest
micelles stability) of the densely grafted with cinnamyl groups BC-2
6

R. Kalinova et al.
Reactive and Functional Polymers 157 (2020) 104763
Fig. 4. AFM and TEM images of: a) and c) functional amphiphilic copolymer with pendant acetylene groups BA-4; b) and d) cinnamyl-modified functional
amphiphilic copolymer BC-2.
copolymer (0.009 mg mLꢀ 1) might be attributed to its higher hydro-
The DLS measurements performed on the block copolymer aqueous
dispersions revealed the presence of nanosized particles with average
diameters between 30 and 52 nm and narrow size distribution. The
parent functional amphiphilic copolymer with acetylene side groups BA-
4 forms micelles in aqueous media with an average diameter of 50 nm
phobicity combined with the π-π stacking interactions between the ar-
omatic side groups into the core. For the further analyses the block
copolymer micelles in aqueous media were prepared at a concentration
of 1 mg mLꢀ 1 which is much higher than the estimated CMC values.
Fig. 5. In vitro release profiles of CAPE from non-functionalized (MPEO-b-PLA) and cinnamyl-functionalized (BC-2 and BC-4) copolymer micelles in a phos-
phate buffer.
7

R. Kalinova et al.
Reactive and Functional Polymers 157 (2020) 104763
(Fig. 3a). The average micelles’ size decreases to 46 nm for the highly
cinnamyl-functionalized copolymer BC-2 indicating the formation of
more compact hydrophobic core as a result of π-π stacking interactions
between the closely situated pendant aromatic rings (Fig. 3b). On the
other hand, the less cinnamyl-functionalized copolymer BC-4 forms
micelles with an average dimeter of 52 nm which is close but slightly
bigger than that of the parent copolymer (Fig. S4a). Obviously, the
presence of just two bulky side groups into the BC-4 hydrophobic seg-
ments is not enough to shrink the micelles core via stacking interactions,
but rather leads to its slight expansion. The non-functionalized MPEO-b-
PLA block copolymer forms compact micelles with an average diameter
of 30 nm (Fig. S4b). The measured micelles’ average diameters for the
functional copolymers might be beneficial for potential anticancer
nanomedicine applications since it has been clearly demonstrated that
nanoparticles with sizes of around 50 nm can take full advantage of EPR
effect for optimal cell-uptake efficiency compared to their smaller and
bigger sized analogues [44].
Fig. 6. Release of hemoglobin from lyzed erythrocytes, treated with BA-4 and
μ
g mLꢀ 1) for 1 h. One-way Anova, Dunnet’s post-
The copolymer micelles’ morphology was visualized by AFM and
TEM. The images obtained from both analyses show spherical particles
formed from all studied copolymers with sizes that are bigger or close to
those obtained from DLS measurements (Fig. 4 and Fig. S5).
BC-2 micelles (6.25–50
hoc test.
cinnamyl groups into the micelles core, an optimal balance between
drug loading efficiency, nanocarrier stability during blood circulation
and drug release into the target site could be achieved.
3.4. CAPE loading into copolymer micelles and in vitro release study
The copolymer micelles (functionalized and non-functionalized)
were loaded with the phenolic compound CAPE applying the same
nanoprecipitation procedure as that for the empty micelles’ formation.
The corresponding block copolymer was dissolved together with drug in
the common solvent acetone. The solution was added dropwise to
vigorously stirred water, followed by acetone evaporation and adjusting
the loaded micelles’ concentration to 1 mg mLꢀ 1. In order to calculate
DLE the filtered and lyophilized micelles were resuspended in acetoni-
trile instead of acetone due to the overlapping of the absorption bands of
the latter and CAPE. Thus, to quantify the amount of loaded CAPE into
the micelles by UV–vis measurements, a calibration curve of different
CAPE concentrations in acetonitrile was constructed (Fig. S6). The
estimated DLE was between 77 and 100% for the different loaded
polymer micelles. The lowest DLE was estimated for the micelles con-
taining the highest amount of cinnamyl groups in the core. This might be
attributed to the steric hindrance in the core resulted from the densely
grafted cinnamyl side groups. The calculated values for DLC were 9.0,
7.0, 9.7 and 9.0% for BA-4, BC-2, BC-4 and MPEO-b-PLA, respectively.
All types of drug loaded polymer micelles were evaluated by DLS mea-
surements. No significant changes in the micelles’ average diameters
and size distributions were detected. The characteristics of amphiphilic
block copolymers and the corresponding micelles used for CAPE loading
are presented in Table S1.
3.5. Toxicological evaluation of the polymer micellar nanocarriers
The synthesis of new copolymers requires evaluation of their safety
profile because every new modification in the copolymer’s structure
could result in changes in biological performance. Therefore, initial
toxicological evaluation of the newly synthesized copolymer micellar
carriers was performed by two in vitro approaches – hemolysis and
scratch assay. The parent alkyne functionalized block copolymer (BA-4)
and its fully modified with cinnamyl side groups analogue (BC-2) were
chosen for the study. Evaluating the hemolytic potential is important for
substances for venous administration because the erythrocytes, among
other blood components are the first targets of interaction. The percent
hemolysis values after treating human erythrocytes with micellar dis-
persions of BA-4 and BC-2 were lower than 0.5% (Fig. 6), which was far
from the conditional hemolysis limit of 5% [45]. Thus, these results
indicate that both copolymers could be appropriate for parenteral
application as drug carriers.
The scratch assay was performed on L929 fibroblast cells, which is
related to the fact that the fibroblasts are ubiquitously found in all or-
gans and their migration is paramount for regenerative processes.
Although L929 cell line originates from cells, which have undergone
malignant transformation, they retain to some extent the morphological
and biochemical characteristics of normal fibroblasts [46]. The results in
the present study showed that BA-4-treated cells have a decreased
migration rate, compared to BC-2 treated ones (*p < 0.05). However,
there was no statistically significant difference between the effect of
both polymeric micelles and control (Figs. 7 and 8). Thus, none of the
polymers negatively affects fibroblast structure and function, related to
cell migration and reorganization capability.
The in vitro dissolution tests in a phosphate buffer revealed an
achievement of sustained release of CAPE (Fig. 5). The profiles were
biphasic with a burst release in the initial stage and sustained release
after the 8th h of the studies. However, the burst effect was lower in the
case of CAPE loaded into the BC-2 micelles compared to MPEO-b-PLA
and BC-4 micelles. In particular, 19% of CAPE were released for 8 h
from BC-2 micelles, whereas for the same time the released CAPE from
MPEO-b-PLA and BC-4 micelles was 48 and 39%, respectively. The
higher burst release from MPEO-b-PLA and BC-4 micelles might be
partially due to the higher CAPE loading compared to BC-2 micelles
(Table S1). It might be also speculated that a high percent of CAPE was
located closely to the border between the core and shell of the micelles
formed from MPEO-b-PLA copolymer giving rise to fast initial release.
The presence of increasing number of cinnamyl groups into the micelles
core for BC-4 and BC-2 copolymers led to an enhanced interaction with
CAPE. As a result of cohesive forces between the functional groups and
the active compound the latter was located entirely into the micelle’s
core and a significant decrease of the initial release rate for the densely
cinnamyl grafted copolymer occurred. Thus, by tuning the number of
3.6. In-vitro cytotoxicity assessment of micellar and free CAPE
The next task in the present study was to evaluate the cytotoxic ef-
fects of CAPE loaded into the different types of polymer micelles. The
cytotoxic effects of CAPE have been reported in studies on cancer cell
lines of different origin [47–49]. Its antineoplastic as well as its anti-
inflammatory effects are related to its inhibitory action on the activa-
tion of nuclear factor kappa-light-chain-enhancer of activated B cells
(NF-κB) [50]. As an inhibitor of NF-κB, CAPE is also expected to block
the endothelial activation and proliferation as part of the inflammatory
8

R. Kalinova et al.
Reactive and Functional Polymers 157 (2020) 104763
Fig. 7. Photomicrographs of scratch on L929 cells’ monolayers, treated with 25
μ
g mLꢀ 1 BA-4 and BC-2 micelles, taken at different time points. Bar = 100
μm.
the block copolymer micelles were related only to CAPE itself and not to
the carriers since all the empty micelles were not cytotoxic in wide
concentration interval (Fig. 9b).
4. Conclusions
Functional amphiphilic block copolymers comprising biocompatible
polyoxyethylene hydrophilic block and biodegradable poly(D,L-lactide)-
based hydrophobic block modified with desirable number of randomly
distributed cinnamyl pendant groups were successfully synthesized. A
PEO-macroinitiator was used for the ring-opening copolymerization of
D,L-lactide and an acetylene-functional cyclic carbonate yielding well-
defined amphiphilic copolymer bearing alkyne side groups randomly
distributed into the hydrophobic block. The obtained functional
modular platform was further modified with desired number of cin-
namyl pendant groups via “click” chemistry. The newly synthesized
copolymers self-associated into stable micelles with average diameters
of around 50 nm. They were used to encapsulate caffeic acid phenethyl
ester into their functional cores. It was demonstrated that the nano-
carriers’ drug-loading efficiency and release profile can be modulated
through the number of compatibilizing groups introduced into the mi-
celles’ hydrophobic core. Moreover, the initial in-vitro evaluations
qualify the synthesized functional block copolymers as safe, biocom-
patible and suitable for further studies as efficient drug delivery
vehicles.
Fig. 8. Cell migration rates of L929 cells, treated with BA-4 and BC-2 micelles.
Kruskal-Wallis analysis of variance with Dunn’s post-hoc test, *p < 0.05.
process [51]. In our study on endothelial hybrid cell line (Ea.hy926),
both the pure and micellar CAPE exerted cytotoxic effect on the prolif-
erating cells (Fig. 9a). This observation suggested that the effect of CAPE
was retained after micellization although some differences between the
CAPE loaded micelles were observed. The effects after the treatment
with CAPE loaded in cinnamyl modified micelles were less pronounced
comparing to the other two types of micelles obtained from the non-
functional and the alkyne functionalized precursor (Fig. 9a). This
might be due to the slower release of CAPE from these micelles (Fig. 5).
The observed effects indicate that in the concentration interval studied
CAPE is safely preserved into the micelles core of the densely modified
with cinnamyl groups copolymer BC-2 as a result of cohesive in-
teractions. The effects of aqueous micellar dispersions of CAPE with
MPEO-b-PLA and BA-4 demonstrated similar cytotoxicity as free CAPE
dissolved in DMSO. The observed cytotoxic effects of CAPE loaded into
Data availability statement
The raw/processed data required to reproduce these findings cannot
be shared at this time as the data also forms part of an ongoing study.
Declaration of Competing Interest
The authors declare no conflict of interest.
9

R. Kalinova et al.
Reactive and Functional Polymers 157 (2020) 104763
Fig. 9. Cytotoxic effects of free and micellar CAPE (a) and empty micelles (b) on Ea.hy926 cells after 72 h of treatment. Values are presented as percentage of
untreated controls and expressed as mean ± SEM (n = 6).
10

R. Kalinova et al.
Reactive and Functional Polymers 157 (2020) 104763
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Acknowledgements
This research was financially supported by the National Science Fund
of Bulgaria through project DN09-1/2016. The authors are grateful to
Prof. Vassya Bankova form The Institute of Organic Chemistry with
Centre of Phytochemistry, Bulgarian Academy of Sciences for providing
the CAPE.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.reactfunctpolym.2020.104763.
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