γ-Butyrolactone Copolymerization with the Well-Documented Polymer Drug Carrier Poly(ethylene oxide)-block-poly(ε-caprolactone) to Fine-Tune Its Biorelevant Properties

Article abstract of DOI:10.1002/mabi.201900408

Polymeric drug carriers exhibit excellent properties that advance drug delivery systems. In particular, carriers based on poly(ethylene oxide)-block-poly(ε-caprolactone) are very useful in pharmacokinetics. In addition to their proven biocompatibility, there are several requirements for the efficacy of the polymeric drug carriers after internalization, e.g., nanoparticle behavior, cellular uptake, the rate of degradation, and cellular localization. The introduction of γ-butyrolactone units into the hydrophobic block enables the tuning of the abovementioned properties over a wide range. In this study, a relatively high content of γ-butyrolactone units with a reasonable yield of ≈60% is achieved by anionic ring-opening copolymerization using 1,5,7-triazabicyclo[4.4.0]dec-5-ene as a very efficient catalyst in the nonpolar environment of toluene with an incorporated γ-butyrolactone content of ≈30%. The content of γ-butyrolactone units can be easily modulated according to the feed ratio of the monomers. This method enables control over the rate of degradation so that when the content of γ-butyrolactone increases, the rate of degradation increases. These findings broaden the application possibilities of polyester-polyether-based nanoparticles for biomedical applications, such as drug delivery systems.

Full text of DOI:10.1002/mabi.201900408

Full PaPer
www.mbs-journal.de
γ-Butyrolactone Copolymerization with the
Well-Documented Polymer Drug Carrier Poly(ethylene
oxide)-block-poly(ε-caprolactone) to Fine-Tune Its
Biorelevant Properties
Tomáš Urbánek, Jiˇrí Trousil, Dmytro Rak, Kristýna Gunár, Rafał Konefał, Miroslav Šlouf,
Marián Sedlák, Olga Šebestová Janoušková, and Martin Hrubý*
1. Introduction
Polymeric drug carriers exhibit excellent properties that advance drug
delivery systems. In particular, carriers based on poly(ethylene oxide)-
block-poly(ε-caprolactone) are very useful in pharmacokinetics. In
addition to their proven biocompatibility, there are several requirements
Amphiphilic polymeric nanoparticles
(NPs) are nanomaterials generated by the
self-association of polymeric amphiphiles
above a critical aggregation concentra-
for the efficacy of the polymeric drug carriers after internalization, e.g.,
nanoparticle behavior, cellular uptake, the rate of degradation, and cellular
localization. The introduction of γ-butyrolactone units into the hydrophobic
block enables the tuning of the abovementioned properties over a wide
range. In this study, a relatively high content of γ-butyrolactone units
with a reasonable yield of ≈60% is achieved by anionic ring-opening
copolymerization using 1,5,7-triazabicyclo[4.4.0]dec-5-ene as a very efficient
catalyst in the nonpolar environment of toluene with an incorporated
γ-butyrolactone content of ≈30%. The content of γ-butyrolactone units can
be easily modulated according to the feed ratio of the monomers. This
method enables control over the rate of degradation so that when the
content of γ-butyrolactone increases, the rate of degradation increases.
These findings broaden the application possibilities of polyester-
polyether-based nanoparticles for biomedical applications, such as drug
delivery systems.
tion, and their use of intervention is
based on their physicochemical properties
and architecture; the array of hydrophilic
and hydrophobic domains can vary.[1–3]
A
class of important biodegradable and/or
biocompatible polymers, aliphatic poly-
esters, have been widely studied due to
their large-scale accessibility by the ring-
opening polymerization (ROP) of cyclic
esters or lactones.[4] In contrast to the
commonly used lactones or esters for
the synthesis of aliphatic copolyesters,
γ-butyrolactone (γBL) is a very promising
monomer, as introduction of γBL into
other aliphatic polyesters might modify
the degradation rate to meet the desired
application demand.[5–8] Notably, the bio-
compatibility of poly(γ-butyrolactone)
(P4HB) has been shown to be better
than that of poly(glycolic acid) and
poly(lactide-co-glycolide), which are copolymers that have been
widely studied in terms of drug delivery in vivo.[7,9] Given this,
γBL has been suggested to be a suitable comonomer for drug
delivery system tuning.
T. Urbánek, J. Trousil, Dr. K. Gunár, Dr. R. Konefał, M. Šlouf,
Dr. O. Šebestová Janoušková, Dr. M. Hrubý
Institute of Macromolecular Chemistry
Czech Academy of Sciences
Heyrovského námeˇstí 2, 162 00 Prague 6, Czechia
E-mail: mhruby@centrum.cz
Although γBL seems to be the perfect choice as a monomer
for polymers in medical applications, its thermodynamic
parameters do not allow for it to undergo ROP to a sufficient
extent.[10,11] In the 1930s, γBL was proclaimed as a nonpolymeriz-
able substance[12] and the corresponding research slowed. Polym-
erization conditions have been considered the key factors that
determine the polymerizability of a substance, and in the 1960s,
γBL was successfully converted into low-molecular-weight poly-
ester (1200–3500 g mol−1) under extreme conditions (2 GPa,
165 °C);[13] the high-pressure homopolymerization of γBL has
been reported several times, and the molecular weight was
increased by acid catalysis.[14] Attempts were made to prepare
high-molecular-weight P4HB under less extreme conditions, and
in 2016, these attempts resulted in success when a lanthanum
J. Trousil
Department of Analytical Chemistry
Charles University
Faculty of Science
Hlavova 8, 128 43 Prague 2, Czechia
Dr. D. Rak, Dr. M. Sedlák
Institute of Experimental Physics
Slovak Academy of Sciences
Watsonova 47, 040 01 Košice, Slovakia
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/mabi.201900408.
DOI: 10.1002/mabi.201900408
©
1900408 (1 of 15)
Macromol. Biosci. 2020, 1900408
2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.mbs-journal.de
complex was employed as a catalyst.[15] However, currently,
as biologic and electronic applications are at the forefront of
interest, the purity of polymers is taken into account, and thus,
nonmetallic catalysts are preferred.[16–21] A viable strategy for the
incorporation of γBL into a polymeric chain is its copolymeriza-
tion with thermodynamically favorable monomers.[8,22,23]
Amphiphilic diblock copolymers composed of hydrophobic
biodegradable polyester blocks and hydrophilic polymer blocks
are very suitable for the construction of micellar/nanoparticle
drug delivery systems with core-corona structures because
hydrophobic drugs can be encapsulated into micelle cores,
while the hydrophobic portion can be degraded into low-
molecular-weight fragments after drug release, allowing for
elimination from the system of the organism while the hydro-
philic corona-forming block protects the core from unwanted
interactions in the organism. Among such copolymers,
methoxy poly(ethylene oxide)-block-poly(ε-caprolactone) is very
popular for the construction of drug delivery systems. However,
the tuning of its properties is limited by the chemical proper-
ties of the polymer block from which it is composed.
In this work, we report the synthesis and characteriza-
tion of an amphiphilic block copolymer composed of mPEO
and poly(ε-caprolactone-co-γ-butyrolactone) (PCL-co-P4HB)
possessing a favorable amount of P4HB. This copolymer
undergoes self-association in an aqueous medium and forms
spherical amphiphilic assemblies, as determined by cryo-
transmission electron microscopy (cryo-TEM). This interven-
tion was found to be enzymatically degradable by two different
approaches (dynamic light scattering, DLS, and flow cytometry)
and noncytotoxic. In addition, the low-molecular-weight hydro-
lytic products of the polyester block (6-hydroxyhexanoic acid
and 4-hydroxybutyric acid) are fully metabolizable into carbon
dioxide and water. The novel copolymerization of P4HB into
the hydrophobic polyester block of mPEO-PCL block copoly-
mers of this article was found to be an excellent tool for the
adjustment of the properties critical for its use in drug delivery
systems (NP behavior, cell uptake, biodegradation rate, and
intracellular localization) over a very wide range, strongly
increasing the variety of possible uses.
ether, 2 m), lipase from Pseudomonas sp. (type XIII) (Sigma-
Aldrich Ltd.), paraformaldehyde, 1,5,7-triazabicyclo[4.4.0]dec-
5-ene (TBD, 98%), 7-(diethylamino)coumarin-3-carbonyl azide
(DACCA), and γ-hydroxybutyric acid sodium salt were purchased
from Sigma-Aldrich Ltd. and used as received.
7-Aminoactinomycin
D
(7AAD),
amphotericin
B
(250 µg mL−1), CellMask Deep Red, Dulbecco’s modified Eagle
medium (DMEM, high glucose, GlutaMAX), fetal bovine serum
(FBS, heat-inactivated), LysoTracker Deep Red, penicillin-strep-
tomycin solution (10 000 U mL−1), and Triton X-100 solution
(1%) were purchased from Life Technologies Ltd. (Prague,
Czechia).
2.2. Synthesis of N-(3,5-bistrifluoromethyl)phenyl-N′-
cyclohexylthiourea (TU)
Cyclohexylamine (210 µL, 1.84 mmol) was added to 10 mL of
dry dichloromethane (DCM) under an argon atmosphere. This
solution was cooled in an ice bath and 3,5-bis(trifluoromethyl)
phenyl isothiocyanate (337 µL, 1.85 mmol) was added.[24] The
reaction was maintained overnight (16 h) at room tempera-
ture. The product precipitated from the solution, the solvent
was evaporated under reduced pressure and the product was
purified by recrystallization from petroleum ether with a yield
of 91%. The product was further used as a cocatalyst of DBU
for the ROP of γBL with εCL.
2.3. Synthesis of Poly(ε-caprolactone-co-γ-butyrolactone)
(PCL-co-P4HB)
Polymerization reactions (Figure 1A) were carried out in dry
vessels under an argon atmosphere for 24 h. The desired
amounts of initiator (BA), catalyst (HCl/Et2O, DPP, TBD or
DBU/TU), and solvent were added into the vessels equipped
with magnetic stirring bars. The solutions were stirred while
the monomers (εCL and γBL) were added. The feed ratios are
listed in the Tables S1 and S2 in the Supporting Information.
The products were washed with ice-cold Et2O.
2. Experimental Section
2.4. Synthesis of Poly[ethylene oxide-block-(ε-caprolactone-co-γ-
butyrolactone)] [mPEO-b-(PCL-co-P4HB)]
2.1. Materials
γBL (≥99%) and ε-caprolactone (εCL, 97%) were purchased
from Sigma-Aldrich Ltd. (Prague, Czechia), dried over calcium
hydride for 24 h, subsequently distilled under reduced pres-
sure, and stored under an inert atmosphere prior to use.
Benzyl alcohol (BA, ≥99%, Sigma-Aldrich Ltd.) was dried
over calcium oxide for 24 h and subsequently distilled under
reduced pressure twice and stored under an inert atmos-
phere prior to use. Poly(ethylene oxide) monomethyl ether, 2
and 5 kDa (mPEO45 and mPEO114, Sigma-Aldrich Ltd.), were
azeotropically dried with toluene prior to use.
Polymerization reactions (Figure 1B) were carried out in the
same manner as the polymerization of PCL-co-P4HB. In this
case, the poly(ethylene oxide) monomethyl ethers were used as
the macroinitiators and only TBD was used as the catalyst. Feed
amounts are listed in Table S3 in the Supporting Information.
2.5. Fluorescent Labeling of mPEO-b-(PCL-co-P4HB)
Chosen samples of the prepared copolymers were labeled
with DACCA for biological analysis, synthesis scheme shown
in Figure S12 in the Supporting Information. Each sample of
80 mg of mPEO-b-(PCL-co-P4HB) was dissolved in 10 mL of
toluene, and 2 molar equivalents of DACCA were added to this
3,5-Bis(trifluoromethyl)phenyl isothiocyanate (98%), 1-(4,5-
dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT, BioReagent),
cyclohexylamine (≥99.9%), 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU), diphenyl phosphate (DPP), hydrochloric acid (in diethyl
©
1900408 (2 of 15)
Macromol. Biosci. 2020, 1900408
2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.mbs-journal.de
Figure 1. Synthesis scheme of A) copolymer PCL-co-P4HB and B) block copolymer mPEO-b-(PCL-co-P4HB).
solution. The reactions were maintained at 80 °C under an
argon atmosphere for 24 h.[25] The products were purified by
dialysis and freeze-dried. The UV–vis spectra were measured
with a UV–vis spectrophotometer (Evolution 220 Spectrometer,
Thermo Scientific, USA). Fluorescence spectra of the samples
were recorded (90° angle geometry, 1 × 1 cm quartz cell) with
an Aminco Bowman Series 2 spectrofluorometer (Aminco,
Lake Forest, CA, USA) at an excitation wavelength of 420 nm
and are shown in Figure S13 in the Supporting Information.
using an ATR-Specac MKII Golden Gate Single Reflection
system with a diamond crystal and an angle of incidence of 45°.
The samples were characterized in powder form.
Physical characterization of the synthetized copolymers
was determined by SEC. Namely, the mass-average molar
mass Mn, the number-average molar mass Mw, and their
ratio Mw/Mn provided information about the chain length
distribution–dispersity, Ð. The separation was carried out using
a DeltaChrom pump (Watrex Ltd., Prague, Czechia), a Midas
Spark autosampler (DataApex Ltd., Prague, Czechia), and two
PLgel MIXED-B-LS columns. The samples were dissolved in
tetrahydrofuran (THF), which served as the mobile phase in
the chromatographic system with a flow rate of 1 mL min−1
at 25 °C. A PL ELS-1000 evaporative light scattering detector
(Polymer Laboratories, Varian Inc., Amherst, USA) was used
for the detection. The evaluation of the sample chromatograms
was performed using Clarity Software Version 6.0.0.295 (Data-
Apex Ltd.) with polystyrene standard calibration.[27]
2.6. Preparation of Nanoformulations
Nanoparticles were prepared by the nanoprecipitation method.
Samples of mPEO-b-(PCL-co-P4HB) (10 mg) were diluted in
acetone (1 mL), and subsequently, an aqueous phase [distilled
water or phosphate-buffered saline (PBS)] (10 mL) was rapidly
added while stirring at 200 rpm. The excess acetone was evapo-
rated under reduced pressure. The final concentrations of the
samples were 1 mg mL−1. Nanoformulations were always pre-
pared fresh for each characterization.
2.8. Characterization of the Nanoparticles Prepared by
mPEO-b-(PCL-co-P4HB)
2.7. Characterization of PCL-co-P4HB and
mPEO-b-(PCL-co-P4HB)
Different techniques were used for the characterization of
the nanoparticles. DLS measurements were carried out on a
ZEN3600 Nano-ZS instrument (Malvern Instruments Ltd., UK)
using an angle of 173°. A He–Ne laser (4.0 mW, operating at
633 nm) was used. The hydrodynamic diameter of the particles
was calculated from the diffusion coefficient using the Stokes–
Einstein equation.
Proton nuclear magnetic resonance (1H NMR), size-exclusion
chromatography (SEC), and Fourier transform infrared spec-
troscopy (FTIR) were used for fundamental characterization of
the synthetized copolymer structures.
A Bruker AVANCE DPX 300 (and/or AVANCE III 600 MHz)
spectrometer (Bruker Daltonik GmbH, Bremen, Germany)
operating at 300.1 or 600.2 MHz was utilized for chemical struc-
ture determination. All samples were dissolved in deuterated
chloroform at 25 °C. The chemical shifts were relative to tetra-
methylsilane (TMS) using hexamethyldisiloxane (HMDSO,
δ = 0.05 ppm from TMS) as the internal standard.[26] 13C
NMR spectrum was recorded using Bruker AVANCE III
600 MHz operating at 150.9 MHz in deuterated chloroform at
25 °C. The chemical shift was relative to TMS using HMDSO
(δ = 2.00 ppm from TMS) as the internal standard.
Static light scattering (SLS) measurements were performed
on an ALV-6010 instrument (ALV GmbH, Germany) equipped
with a 22 mW He–Ne laser in the angular range of 30°–150°.
The data were analyzed by using a Zimm plot. The dn/dc values
were measured as described below.
The additional method for characterizing the prepared
nanoparticles was asymmetric-flow field-flow fractionation
(AF4). The instrument setup for AF4 consisted of an Eclipse
3+ separation system (Wyatt Technology Europe, Dern-
bach, Germany) coupled to a 1260 Infinity isocratic pump
and degasser (Agilent Technologies, Santa Clara, CA, USA),
a DAWN HELEOS II MALS detector with a 120 mW gal-
lium-arsenide laser at a wavelength of 661 nm (Wyatt Tech-
nology, Santa Barbara, CA, USA), and an RI-101 RI detector
(Shodex, Munich, Germany). Prepared nanoparticles in filtered
A Perkin Elmer PARAGON 1000 PC FTIR spectrometer was
utilized for chemical structure confirmation. The spectrometer
had a resolution of 4 cm−1, 32 accumulated scams, and strong
apodization. The reflective measurements were performed
©
1900408 (3 of 15)
Macromol. Biosci. 2020, 1900408
2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.mbs-journal.de
deionized water underwent measurements using a 275 mm
long trapezoidal channel consisting of a 490 µm spacer and a
regenerated cellulose membrane with a 5 kDa cutoff. Collected
data were processed using ASTRA 6 software.
The measurements followed the established procedure in
ref. [28]. As a result, the mass-average molar mass Mw and the
diameter of gyration Dg were obtained.
The refractive index increment dn/dc of the solutions
was measured with a BI-DNDCW differential refractometer
(Brookhaven Instruments Corporation, USA). The dn/dc values
were obtained using a potassium chloride calibration solution.
reaching 10–6 mg mL−1 were obtained by serial dilution in PBS.
A solution of Nile red in ethanol was added to each sample
to obtain a final concentration of 10–6 mol L−1. A Synergy H1
Hybrid Reader instrument (BioTek, Winooski, USA) was uti-
lized to record the fluorescence of the samples. The excitation
wavelength was 550 nm and the emission intensity was meas-
ured at 650 nm. The CACs were determined as the intersection
points of the lines from two different parts of the graph of fluo-
rescence intensity versus the negative logarithm of the concen-
trations, as seen in Figure S8 in the Supporting Information.[28]
2.12. Hemolysis assay
2.9. Morphology of the NPs
The determination of blood compatibility with the prepared NPs
was carried out following the procedure in ref. [28]. Fresh human
blood was collected for the analysis into heparin-coated vacu-
tainers (Becton Dickinson Czechia Ltd., Prague, Czechia). Red
blood cells (RBCs) were obtained by centrifugation of the blood at
3000 rpm for 10 min. The plasma was removed and RBCs were
washed with PBS. The final concentration of RBCs in PBS for the
hemolysis measurements was at a full blood dilution of 1:49.
To 0.3 mL of diluted RBCs, 1.2 mL of the appropriately
diluted nanoformulation (in PBS) was added. With this pro-
cedure, several dilutions of NPs were prepared (100, 200, 400,
and 800 µg mL−1). Triton X-100 (1% in PBS) and PBS served as
positive and negative controls, respectively.
After incubation at 37 °C for 8 h, the samples were centri-
fuged (3000 rpm, 10 min). Subsequently, the supernatants were
collected and subjected to absorbance measurement at 541 nm
on a Synergy H1 Hybrid Reader instrument (BioTek, Winooski,
Vermont, USA). The percent hemolysis of RBCs in each sample
was calculated by the following Equation (1)
The nanoparticle morphology was observed by transmission elec-
tron microscopy. To characterize the morphology of the nanopar-
ticles, 3 µL of sample solution was applied to a formvar-carbon
electron microscopy grid. Samples were observed without staining
at 100 kV using an FEI Morgagni TEM equipped with a MegaView
III side-entry charge-coupled device camera. For cryo-TEM, where
the hydrated samples were imaged without the perturbation gen-
erated by drying, the sample solution was applied to a carbon-
covered polymer-grid supporting film (lacey-carbon grids, Electron
Microscopy Sciences, Hatfield, USA) and glow discharged for 40 s
with a 5 mA current. Most of the sample was removed by blot-
ting and the grid was immediately plunged into liquid ethane at
−183 °C. The sample was then transferred without rewarming
into a Tecnai Sphera G20 electron microscope (FEI) using a Gatan
626 cryo-specimen holder (Gatan Inc., Pleasanton, CA, USA).
Images were recorded at a 120 kV accelerating voltage using a
Gatan UltraScan 1000 slow-scan charge-coupled device camera
(achieving a final pixel size from 2 to 0.7 nm) in low-dose mode.
sample absorbance − negative control
positive control − negative control
Hemolysis =
× 100
(1)
2.10. Enzymatic Degradation of NPs
1
The H NMR and SEC measurements were used to determine
The results of the hemolysis assays were expressed as a
percentage of the positive control (1% Triton X-100 in PBS),
which was considered 100%. Hemoglobin release up to 2%
was classified as nonhemolytic, according to the ASTM F756-08
standard.[29] The results of this assay are expressed as mean
values of triplicate analysis.
the enzymatic degradation of the prepared nanoparticles and the
procedure is described in ref. [28]. The degradation was studied
in the presence of lipase from Pseudomonas sp. The appropriate
amount of lipase (aqueous solution in PBS, 4 U mL−1) was added
to the nanoformulation (1 mg mL−1 in PBS) to initiate the degra-
dation process, giving final concentrations of the copolymer and
lipase of 0.98 mg mL−1 and 0.06 U mL−1, respectively. The degra-
dation was performed at 37 °C inside an NMR cuvette. In case of
SEC measurements, the purification of the organic content was
needed to be done after the degradation period. The PBS solu-
tion of the sample incubated with the lipase was poured into
excess of cold diethyl ether, so the organic content precipitated.
The precipitate was separated by centrifugation at 7000 rpm and
0 °C and dried. This material was dissolved in mobile phase of
SEC and the analysis was performed.
2.13. Cell Culture
The murine monocyte-macrophage (Mφ) cell line J774A.1[30] was
purchased from Sigma-Aldrich Ltd. The cells were maintained in
DMEM supplemented with FBS (10%), penicillin (100 U mL−1),
streptomycin (100 µg mL−1), and amphotericin B (2.5 µg mL−1)
in a humidified atmosphere containing 5% CO2 at 37 °C.
2.14. Cytotoxicity Study
2.11. Critical Aggregation Concentration (CAC) Determination
Cytotoxicity was evaluated using the MTT assay. For this purpose,
J774A.1 Mφs were seeded in 96-well plates at a density of 5000 cells
per well. The cells were then incubated overnight at 37 °C and 5%
The NP formulations of the synthesized copolymers were pre-
pared by the protocol described above. Series of concentrations
©
1900408 (4 of 15)
Macromol. Biosci. 2020, 1900408
2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.mbs-journal.de
CO2 with subsequent replacement of the medium with 100 µL of
fresh culture medium containing the tested formulations. After
24 h of incubation, the medium was replaced with 100 µL of MTT
solution (0.5 mg mL−1 in PBS), and the plates were incubated for
2–4 h prior to aspiration of the medium and dimethyl sulfoxide
addition (100 µL). After 15 min of blue formazan solubilization
(37 °C), a Synergy H1 Hybrid Reader instrument (Biotek, Win-
ooski, USA) was used to assess cell viability by spectrophotometry
at 570 nm. The results of the MTT assay were expressed as a per-
cent of the control (cells in control medium), which was consid-
ered 100 %. A reduction in cell viability by more than 30% was
considered a cytotoxic effect according to the ISO 10993-5.[31] The
tests were performed in at least three separate experiments.
DACCA-labeled NPs was measured via a BD FACSVerse flow
cytometer (BD Biosciences, Franklin Lakes, NJ, USA). For this
purpose, J774A.1 cells were incubated at 37 °C and 5% CO2 in
full DMEM. The overnight culture was seeded at a density of
200 000 cells per sample. NPs were added at a final concentra-
tion of 2 nmol mL−1 to the wells and incubated for 1, 5, 20,
40, 60, or 80 min. After washing with warm PBS, the cells
were suspended in 0.5% bovine serum albumin (BSA, in PBS
solution). Dead cells were stained with 7AAD according to the
manufacturer’s protocol. The measurement was repeated inde-
pendently three times and in duplicate. Cell-associated fluo-
rescence was analyzed using FlowJo 10.5.0 software (Tree Star
Inc., Ashland, OR, USA).
Intracellular degradation estimation was based on a previ-
ously described method.[28] The overnight J774A.1 culture was
seeded at a density of 200 000 cells per sample. NPs at a con-
centration of 2 nmol mL−1 were added to the appropriate wells
and incubated for 60 min in full DMEM (37 °C, 5% CO2). The
incubation length was chosen according to the above-described
internalization-related experiment, where the macrophages
showed the maximum internalized NPs between 60 and 80 min
of incubation. After washing with warm PBS, the cells were fur-
ther incubated in particle-free full DMEM for 0, 10, 20, 30, 40,
or 80 min. Cells were washed with PBS and suspended in 0.5%
BSA (PBS solution); dead cells were stained with 7AAD and
measured by BD FACSVerse. The measurement was repeated
independently three times and in duplicate. Cell-associated flu-
orescence was analyzed using FlowJo 10.5.0 software.
2.15. Microscopic Investigation of Cellular Uptake
Confocal laser scanning microscopy (CLSM) was used to study
cellular uptake and lysosome-nanoparticle colocalization. For the
uptake study, J774A.1 cells were seeded at the bottom of ibidi
µ-dishes (ibidi GmbH, Planegg/Martinsried, Germany) and left
to adhere overnight. Subsequently, the medium was replaced
with fresh medium (full DMEM) containing DACCA-labeled
NPs (2.8 nmol mL−1). After 40 min of incubation, the cells were
washed with prewarmed PBS (37 °C) three times, stained with
CellMask Deep Red according to the manufacturer’s protocol
for 10 min, washed with warm PBS, fixed using a paraformalde-
hyde solution (4% in PBS), and kept under PBS until subsequent
analysis. Cells were visualized on an Olympus FV 10 confocal
laser scanning microscope (Olympus Czech Group Ltd., Prague,
Czech Republic) using a 60 × oil objective. Signal detection was
observed in channels 1 (DACCA, exc. 405 nm, em. 425–475 nm)
and 2 (CellMask Deep Red, exc. 650 nm, em. 655–750 nm).
Similarly, the lysosome-nanoparticle colocalization study was
carried out using overnight-adhered macrophages (Mφs). The
medium was replaced with fresh DMEM containing DACCA-
labeled NPs (2.8 nmol mL−1) and LysoTracker (0.0125 × 10−6 m)
and incubated for 40 min. Subsequently, the cells were washed
and fixed as described above. Visualization was carried out using
the same instrument under the same conditions. Before image
analysis, the images were converted to eight-bit grayscale pictures,
and the levels of colocalization were determined using ImageJ
software. An image of the product of the differences from the

3. Results and Discussion
3.1. Synthesis and Content of 4HB in PCL-co-P4HB
Previous studies that have focused on the polymerization of five-
membered lactones have shown their unwillingness to undergo
ring-opening polymerizations due to the low ceiling tempera-
ture.[10] However, this does not mean that the ring of five-mem-
bered lactones cannot be opened and that only the cyclic form
is thermodynamically preferred. These rings do open, but there
is no impetus to form high-molecular-weight polymers under
normal conditions.[32,33] This is the reason why these com-
pounds are difficult to homopolymerize but, on the other hand,
are easy to incorporate into a polymer chain when copolymeri-
zation occurs with another appropriate heterocycle with a high
ceiling temperature. Moreover, the extent of incorporation can
also be tuned by the polymerization conditions.[10,34]
Hence, for the systematic investigation of incorporating γBL
into polymer chains during copolymerization with εCL, a series
of copolymerizations initiated by benzyl alcohol were per-
formed (Figure 1A) with different copolymerization conditions.
The polymerization parameters and resulting characterizations
are given in Tables S1 and S2 in the Supporting Information.
The data in Table S1 in the Supporting Information show the
parameters depending on the feed ratio of monomers, whereas
the data in Table 2 show the parameters depending on the
nature of the catalyst and the solvent and the initial monomer
concentrations. In total, four different catalytic systems were
evaluated (structures shown in Figure 2A).
mean (PDM), i.e., for each pixel: (blue intensity
mean blue
intensity) × (red intensity − mean red intensity), was used to ana-
lyze the colocalization. In addition, Pearson correlation coefficient
(PCC) values were calculated using the same software. For this
purpose, at least six fields were used for the PCC calculation. PCC
is a positive correlation between different channels that generates
values ranging from −1 to 1. A value of 1 suggests a perfect posi-
tive linear correlation. Hence, in this case, a value of 1 would indi-
cate total colocalization of the DACCA-labeled nanobead-based
intervention with LysoTracker-positive compartments.
2.16. Flow Cytometry Investigations
Cellular internalization and intracellular degradation were
studied by flow cytometry. Cell uptake of the fabricated
©
1900408 (5 of 15)
Macromol. Biosci. 2020, 1900408
2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.mbs-journal.de
Figure 2. Copolymerization optimization. A) Different catalysts were used in subsequent studies of the content of 4HB in copolymers. B) Different
initial monomer concentrations with DCM as the solvent as well as C) different solvents possessing various dipole moments were studied with TBD
used the catalyst. D) The relationship between monomer consumption and the content of 4HB in the polymer chain and molar mass was studied. The
εCL/γBL molar feed ratio was in all cases 1.15. Abbreviations: TBD, 1,5,7-triazabicyclo[4.4.0]dec-5-ene; DPP, diphenyl phosphate; Et2O, diethyl ether;
DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; TU, N-(3,5-bistrifluoromethyl)phenyl-N′-cyclohexylthiourea; DCM, dichloromethane; THF, tetrahydrofuran.
The data in Table S1 in the Supporting Information show the
relationship between the monomer feed ratio and the amount
of incorporated γBL. Not surprisingly, the higher the ratio of
γBL in the feed, the more γBL was incorporated. The disadvan-
tage of copolymerization with high amounts of γBL is its rela-
tively low yield. The difference in the incorporated amount of
γBL is remarkable when different catalysts were used (P1–P4
catalyzed by HCl/Et2O and P5–P8 by TBD).
The data schematically plotted in Figure 2B show a sig-
nificant difference in the incorporated amount of γBL when
different catalysts were used. A higher content of 4HB units
in the prepared copolymers was observed when anionic
catalysts (TBD and DBU/TU) were used compared to that
observed in the copolymers prepared with cationic catalysts
(HCl/Et2O and DPP). This fact implies that different polym-
erization mechanisms significantly affect the amount of
incorporated γBL.
The bulk copolymerization catalyzed by DBU/TU resulted in
a yield of 39% and a copolymer containing 27% 4HB units but
there was no high-molecular-weight copolymer formed when
solution copolymerization was carried out at any concentration.
TBD was identified as the most efficient and versatile catalytic
system in this study. TBD is able to incorporate a relatively high
amount of γBL at any initial monomer concentration. The trend
of increasing incorporated γBL amount with increasing initial
monomer concentration is evident in Figure 2B.
Solvent polarity is a considerable parameter when a certain
amount of γBL incorporation is needed in the polymer chain.
As shown in Figure 2C, there was a marked difference in 4HB
content in the copolymer composition of PCL-co-P4HB when
the nonpolar solvent toluene was used compared to the 4HB
content when polar acetonitrile was used. It should be noted
that TBD is a bifunctional catalyst, in which the base site works
as a H-bonding acceptor to activate the alcohol initiator, while
©
1900408 (6 of 15)
Macromol. Biosci. 2020, 1900408
2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.mbs-journal.de
the H-bonding donor component can activate the monomer via
the coordination of the ester functionality. Thus, the noncoordi-
nating solvent toluene enables higher incorporation of γBL in
the evaluated copolymerization reaction.
used for copolymerization, the assemblies do not show any tox-
icity at reasonable concentrations and are efficient when loaded
with hydrophobic drugs,[27] suggesting an attractive polymeric
matrix for drug delivery system preparation.
The prepared copolymers were characterized by 1H NMR
and FTIR spectroscopy, and their structures were confirmed.
Thus, this work aimed to study the impact of 4HB distur-
bance on hydrophobic block regularity and on the biorelevant
properties of mPEO-based assemblies. Given this, the most
efficient conditions for incorporating γBL via the copolymeri-
zation initiated by BA were implemented (see above) and the
copolymerization was conducted via ROP in the same manner
and in the presence of the macroinitiator mPEO (Figure 1B).
The macromolecular parameters of the prepared block copoly-
mers are listed in Table 1. Samples B2–B5 differ in hydrophobic
ratio and macromolecular molar mass (i.e., mPEO) and in the
molar mass of the samples themselves. The 1H NMR spectrum
of mPEO-b-(PCL-co-P4HB) with signal assignment is shown
in Figure S6 in the Supporting Information. Moreover, the 13C
NMR spectrum in Figure S14 in the Supporting Information
gave as detailed information about the copolymer microstruc-
ture. It proved that the distribution of CL and 4HB units along
polyester block is very likely random.[38]
Sample mPEO44-b-PCL81 B1 was synthesized in the absence
of γBL for comparison of the properties with those of samples
B2 and B3. The critical aggregation concentrations were deter-
mined and the values are listed in Table 1. The CAC-related
curves are shown in Figure S8 in the Supporting Information.
The one-step synthesis of labeled mPEO-b-(PCL-co-P4HB)
with fluorescent dye was performed. DACCA was used for this
purpose and the reaction scheme is shown in Figure S12 in the
Supporting Information. The efficiencies of the reactions were
determined by the standard addition method (free 7-(diethyl-
amino)coumarin-3-carboxylic acid) and were found to be ≈45%
by UV–vis spectroscopy. The absolute amounts of DACCA occur-
ring in the polymeric matrices are listed in Table 1. The excitation
and emission spectra at the excitation wavelength of 420 nm of
free 7-(diethylamino)coumarin-3-carboxylic acid and samples of
B1–B5 are shown in Figure S13 in the Supporting Information.
1
The H NMR spectra and FTIR spectra are shown in Figures
S2 and S3 in the Supporting Information, respectively. The
table of polymerization parameters and copolymer character-
istics obtained by 1H NMR spectroscopy and size exclusion
chromatography can be found in Table S2 in the Supporting
Information.
1
In addition, a H NMR spectroscopic kinetic study was per-
formed to understand the copolymerization behavior. TBD was
used as the catalyst for this purpose and deuterated DCM was
used as the solvent. The polymerization was performed in an
1
NMR cuvette in situ and H NMR spectra were taken periodi-
cally over time. The relative intensities of peaks corresponding to
the γBL unit (δ 1.91 ppm) and the εCL unit (δ 1.33 ppm) were
integrated and the following parameters were calculated. The
resulting graph in Figure 2D shows the dependence of the content
of 4HB and molar mass on the conversion. A moderate decrease
in the content of 4HB units in the growing polymer chain with
increasing conversion was observed, and the linear dependence
of molar mass on conversion indicated the controlled manner of
ring opening copolymerization. Additionally, the rate constants of
the incorporation of γBL and εCL were determined. As the graphs
in Figure S7 in the Supporting Information of the supplemental
information show, εCL has been incorporated according to first-
order kinetics with a rate constant of kεCL = 4.3 × 10−5 s−1, and
the incorporation of γBL follows second-order kinetics with a rate
constant of kγBL = 5.8 × 10–6 dm3 mol−1 s−1.
3.2. Synthesis of mPEO-b-(PCL-co-P4HB)
Many studies have shown that assemblies based on
poly(ethylene oxide)-block-polycaprolactone can be success-
fully used as drug carriers for many medical applications.[35,36]
Copolymerization with mPEO and other hydrophilic poly-
mers ensures or enhances the solubility of these assemblies
based on the copolymers and improves the biocompatibility/
nonimmunogenicity of the whole system.[37] When mPEO is
3.3. Characterization of the Nanoparticles
The fabricated copolymers (B1–B5) were used for NP prepara-
tion. For this purpose, the nanoprecipitation method described
Table 1. Composition and characteristics of polymers initiated by poly(ethylene oxide) monomethyl ether (mPEO) using TBD as the catalyst.
No.
B1
B2
B3
B4
B5
Copolymer
HH-ratioa)
4.6
Yield [%]
ÐSECd)
1.21
1.45
1.58
1.25
1.61
fγBLb) [%]
CACe) [µg mL−1
]
DACCAf) [µg mg−1
]
MnNMRc)
11 200
7500
MnSECd)
8300
mPEO45-b-PCL81
92
62
60
63
59
0
9
20
9
mPEO45-b-(PCL39-co-P4HB13
)
)
2.8
20
21
18
21
6700
15
11
14
10
mPEO45-b-(PCL55-co-P4HB19
4.0
9900
10 500
7600
10
7
mPEO114-b-(PCL41-co-P4HB12
)
1.1
10 800
23 000
mPEO114-b-(PCL124-co-P4HB45
)
3.6
12 300
4
a)The hydrophobic ratio was defined as (Mn, hydrophobic block)/(Mn, hydrophilic block), where the Mn of the hydrophilic block was held at either 2000 or 5000 depending
on which macroinitiator was used; b)The polymer composition ratios of hydrophobic blocks were calculated using 1H NMR data by dividing the integrated peaks of the
repeating unit of γBL (δ 1.95 ppm) by the sum of the integrated peaks of γBL and εCL (δ 1.37 ppm); c)The molecular weights were calculated using 1H NMR by inte-
grating the methoxy peak of mPEO at 3.37 ppm and the peak for the repeating units of εCL and γBL; d)Determined by SEC using tetrahydrofuran as the eluent; e)Critical
aggregation concentrations were determined at room temperature in PBS; f)When DACCA-labeled copolymers were used, the content of DACCA was determined by UV–vis
spectroscopy.
©
1900408 (7 of 15)
Macromol. Biosci. 2020, 1900408
2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.mbs-journal.de
Figure 3. A) Cryo-TEM images and B) curves obtained from DLS and AF4.
in detail in the Experimental Section was used. The physico-
chemical characterization of the NPs was performed via cryo-
TEM, DLS, SLS, and AF4 experiments.
depending on the block lengths. To verify these findings, we per-
formed additional AF4 experiments that provided the actual sizes
and molecular weights. These values are in mutual agreement
with those obtained by DLS or SLS. It should be noted, however,
that light scattering techniques usually overestimate the size of
particles.[36,40] In addition, the samples for the AF4 measure-
ments were filtered through large-pore polyvinylidene fluoride
filters (0.8 µm), as AF4 is not as sensitive to dust as conventional
light scattering techniques. Thus, any influence of filtration on
the size distribution of the nanoparticles can be excluded.
The DLS and AF4 results together with the cryo-TEM inves-
tigations allowed for us to obtain insight into the structure of
the NPs. In the literature,[39,41,42] mPEO-b-PCL-based assem-
blies are often stated to be micelles. The AF4-estimated mole-
cular weight values (Table 2), however, indicate that ≈400–9000
polymer chains comprise one nanoparticle. Micelles, in con-
trast, contain ≈10–100 polymer chains.[43]
It was hypothesized that the supramolecular structure of
the mPEO-b-(PCL-co-P4HB)-based assemblies correlated to the
structure of mPEO-b-PCL in some respects, as the only differ-
ence was the aliphatic chain length between the ester bonds in
the repeating units. In the context of mPEO-b-PCL assemblies,
several architectures were described in the literature, depending
on the hydrophobic/hydrophilic ratio.[35,39] Note that if the sol-
vent switch is fast during NP preparation, block copolymers tend
to form spherical structures instead of cylindrical or planar ones
because this is the fastest way to reduce the interfacial area.[28]
This is in line with the cryo-TEM investigations that revealed that
all of the samples inspected were found to largely be spherical
polymeric nanoparticles (Figure 3A) with diameters in mutual
agreement with those found by DLS, SLS, and AF4 (Table 2).
The DLS distribution functions (Figure 3B) are portrayed as rel-
atively narrow peaks, with diameters ranging from ≈40–110 nm
The apparent structural density (ρ) of the particles was calcu-
lated as an equivalent average density from the average of the
Table 2. Characterization of nanoparticles.
SLSc) [g mL−1
0.029
]
ρ
AF4c) [g mL−1
0.324
]
DHDLSa) [nm]
DGSLS b) [nm]
MWSLS
DGAF4
MWAF4
N
agg
AF4 d)
b) [kDa]
14 900
b) [kDa]
101 400
b) [nm]
77
Sample
PDIa) DLS
ρ
B1
B2
B3
B4
B5
109
41
0.113
0.188
0.166
0.109
0.111
91
40
9050
710
5800
13 800
1000
0.134
26
8160
24 700
4180
0.690
71
120
48
0.012
85
0.060
1110
420
36
71
0.013
47
0.060
170
38 100
0.011
103
66 700
0.091
2510
a)The Z-average of the hydrodynamic diameter DH and polydispersity index PDI; b)The molecular weight MW and the Z-average of the diameter of gyration DG; c)The
apparent structural density was calculated from the equation ρ = 6Mw/πNAD3 considering a spherical shape (D = 1.29·DG); d)The aggregation number is the quotient of the
MW of the particles (AF4) and the MW of the polymer (SEC).
©
1900408 (8 of 15)
Macromol. Biosci. 2020, 1900408
2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.mbs-journal.de
molecular weight Mw using the model of a sphere with a diam-
eter of D = 1.29 DG; therefore, ρ = 6Mw/πNAD3. On average, the
density of the mPEO-b-(PCL-co-P4HB) nanoparticles was three
times lower than the density of the mPEO-b-PCL nanoparticles.
Given this finding, we hypothesized that the 4HB-possessing
NPs were less dense due to their irregular structure (two
repeating units) within the hydrophobic core. This also means
that the core of the mPEO-b-(PCL-co-P4HB) nanoparticles was
less hydrophobic than that of the mPEO-b-PCL nanoparticles,
which may influence cargo encapsulation. However, this issue
was not addressed further.
One of the advantages of the AF4 method over conventional
light scattering methods or cryo-TEM is the capability of AF4 to
detect eventual nonassembled polymers in solution. The AF4
investigations revealed that no free nonassembled polymer was
found in any of the samples investigated in this work. This is
probably due to the favorably low estimated CAC values (i.e.,
9–15 µg mL−1) and supports the above-discussed fact that our
assemblies are not micelles, which are known to be in thermo-
dynamic equilibrium with unimers, but instead are probably
nanogel-like polymeric NPs.
broader signals obtained from the PCL-co-P4HB hydrophobic
block and the strong signals obtained from the hydrophilic
mPEO demonstrate the presence of core–shell formation in
the deuterated PBS system. These results demonstrate that the
PCL-co-P4HB protons were restricted in mobility in the mod-
erately hydrated solid-like core of the NPs, while the mPEO
blocks created a liquid-like shell. After the addition of lipase,
the intensity of the side methyl signals from the PCL-co-P4HB
monomer unit (“i + l” and “j, e” protons, see Figure S9, Sup-
porting Information) decreased, while the singlets related to
the “f + h” and “k” protons split and the mPEO signals (“a”,
“b”) remained unchanged. The second effect observed in this
spectrum was the appearance of new signals related to the
degradation products, which were identified as 6-hydroxyhexa-
noic acid and 4-hydroxybutanoic acid (Figure S9, Supporting
Information).
Additionally, due to the appearance of the signals at 4.1,
2.2, and 2.0 ppm in Figure S9 in the Supporting Information,
there was confirmation of an intermediate product during the
degradation appeared before final the products (6-hydroxyhexa-
noic acid and 4-hydroxybutyric acid). From the chemical shifts
of signals of this intermediate product, especially the peak at
4.1 ppm, which was shifted downfield to its respective signal
from the polymer (i + l at 4.0 ppm), there was a high proba-
bility that this compound was a small cycle made from a small
amount of CL and BL units. To support these considerations,
one sample was chosen for 1H 2D diffusion-ordered NMR spec-
troscopy experiments, which were measured before lipase addi-
tion (Figure S11, Supporting Information red spectrum) and
24 h after lipase addition (Figure S11, Supporting Information
blue spectrum). From those spectra it followed that the inter-
mediate product was still not fully degraded after this time and
had a slightly lower self-diffusion coefficient (higher molecular
weight) than that of the final products (6-hydroxyhexanoic acid).
The degradation process of the copolymer was confirmed by
SEC analysis. The products of degradation were collected in dif-
ferent times. As evident in Figure S15 in the Supporting Infor-
mation, the degradation of the copolymer was already signifi-
cant in the first hour of the action of the lipase.
The described complex characterization of NPs allowed for
us to fine-tune the nanoparticle parameters so that the particles
could be designed more precisely in the future according to the
particular application requirements.
3.4. 1H NMR-Assessed Degradation
The enzyme-catalyzed degradation of the NPs was investigated
1
using H NMR. The degradation was observed as a decrease
in the intensity of the proton signal of ε-caprolactone units
in sample B1 and ε-caprolactone and γ-butyrolactone units in
sample B3 during the experiment. Samples B1 and B3 were
chosen for this more detailed study due to the comparable sizes
of the blocks in these copolymers. For the quantitative charac-
terization of the enzymatic degradation of the PCL and PCL-co-
P4HB blocks, the time dependence of PCL and PCL-co-P4HB
consumption was calculated for the measured samples. The
integral intensity of the signal marked as “g” (Figure S9, Sup-
porting Information) was defined as 2 (there are two equivalent
protons) and used as an internal standard for monitoring deg-
radation in the NMR spectra. Subsequently, the integral inten-
sity of the signal “i + l” (Figure S9, Supporting Information)
was used to calculate the percentage of PCL and PCL-co-P4HB
blocks according to the relationship [(Iti+l/I0i+l) × 100], where the
subscripts 0 and t represent the time points during degradation
with t = 0 (i.e., before lipase addition) and t = t (i.e., after lipase
addition), respectively.
3.5. Cytotoxicity Evaluation
To assess the biorelevant properties of the manufactured
NPs, their cytotoxicity and hemolytic activity were analyzed
in terms of their interactions with red blood cells (RBCs)
and their behavior in monocyte-macrophage cultures, as
these cells are one of the first contact partners after systemic
administration. In addition, the goal of this evaluation was
to answer the questions of whether our synthetized NPs are
safe and whether the biocompatibility of P4HB-based assem-
blies is affected when P4HB is introduced within the Food
and Drug Administration-approved mPEO-b-PCL copol-
ymer.[44] The mPEO-b-PCL copolymer was found to possess
no or only minor toxic effects on the cell viability of different
cells, such as the murine monocyte-macrophage cell line Raw
264.7,[27,28] zebrafish embryonic fibroblast cell line ZF4,[28]
human hepatocellular carcinoma cell line HepG2,[42,45] and
prostate cancer cell lines LNCaP and PC-3.[46]
The results are shown in Figure S10 in the Supporting
Information. It is evident that sample B1 degraded faster than
sample B3. Figure S9 in the Supporting Information shows
1
the H NMR spectra of B3-based NPs that were measured in
deuterated PBS at 37 °C before (A) and after (B, C) lipase addi-
tion. The chemical structures and signal assignments are pre-
sented in the same figure. The comparison between the spectra
was recorded in a solvent suitable for all of the blocks (deuter-
ated chloroform, cf. Figure S6, Supporting Information) and
PBS (Figure S9, Supporting Information), showing that the
©
1900408 (9 of 15)
Macromol. Biosci. 2020, 1900408
2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.mbs-journal.de
successfully internalized in Mφs (Figure 5), suggesting that
such systems are usable as a logical strategy for effectively
killing intracellular microbes.[48] Note that slight differences
between the intracellular distribution patterns were evident.
In other words, some of the samples revealed bright cytosolic
localization (e.g., B4) compared to the vesicle-localized samples
(e.g., B1). These findings seem to be in line with our previous
mPEO-b-PCL-focused study,[28] which confirmed that control
of the NP physicochemical properties also allows for control of
their biorelevant behavior.
Given this, for a better view of the interactions between the
NPs and J774A.1 cells, a marker for low pH compartments
(i.e., presumably lysosomes) was applied to visualize the
colocalization of the NPs. The cells were incubated for 40 min
with DACCA-labeled NPs and LysoTracker Deep Red. These
experiments indicated that all of the NP samples tested (B1–B5)
were able to target acidic organelles (Figure 6), which was also
proven by the analysis in terms of the PDM images and PCC
value calculations. However, the DACCA-labeled nanoparticle
colocalization with lysosomes was not found to be exclusive,
as suggested by the PCC values found (0.43–0.59, Figure 6).
As mentioned above, Pearson correlation analysis generates
values ranging from −1 to 1, where a value of 1 suggests full
colocalization. In other words, these results suggest that the
nanocarriers are within low-pH compartments. It should be
noted that this finding seems to be in line with our previous
study.[27,28] Ultrastructural analysis by TEM revealed that
mPEO-b-PCL nanoparticles can be found both within vesic-
ular structures and freely in the cytoplasm within Raw 264.7
macrophage-like cells.
To study the cellular internalization kinetics of the NPs
within J774A.1 Mφs, DACCA-labeled NPs (2 nmol mL−1)
were added to Mφ monolayers and incubated for different
times. Subsequently, the cellular uptake was analyzed using
flow cytometry (Figure 7A). All of the obtained cell-associ-
ated fluorescence data were normalized to the maximum
fluorescence values to ensure the comparability of the results
determined from samples with variable total cell fluorescence
intensities.[28]
Figure 4. Viability of J774A.1 cells as detected by an MTT assay. Mφs were
incubated with different concentrations of the fabricated NPs for 24 h
and their viability was analyzed via measurement of the concentration of
metabolically produced formazan. The horizontal line in the panel indi-
cates the level at which, in terms of cell viability, the distinction between
cytotoxic (above) and noncytotoxic (below) was made.
First, we studied the effects of the prepared formulations
B1–B5 in terms of possible cytotoxicity by using the MTT
method after a 24 h incubation of J774A.1 Mφs with each
prepared nanoformulation. As evident in Figure 4, the cell
viability was influenced very little. The copolymer cytotoxicity
was nearly negligible at clinically relevant concentrations. In
the hemolytic study, none of the analyzed formulations (B1–
B5) induced hemolysis within the tested concentration range
(100–800 µg mL−1) after an 8 h incubation at 37 °C.
Not surprisingly, the use of nanomedicines bears the risk
of unwanted side effects.[44] Hence, an important step in the
development of nanobead-based products is the assessment of
any safety risks. Considering this, our results demonstrate that
P4HB introduction within the copolymeric matrix did not affect
its acute biocompatibility with both J774A.1 cells and RBCs.
As shown in Figure 7A, the fabricated NPs were successfully
taken up by J774A.1 cells, and the cell-associated fluorescence
intensity increased as the incubation time increased for all of
the samples tested. A maximum cell fluorescence intensity
was reached after 60–100 min of incubation (Figure 7A); the
data were normalized to the plateau-corresponding values of
fluorescence, individually. Subsequently, internalization half-
time values (Figure 7A) were calculated based on the inter-
nalization time course by curve-fitting of the data using the
following equation[49]
3.6. Uptake and Intracellular Degradation Study
Our research group strives to rationally design nanobead-based
interventions to benefit from the combination of the specific
properties of NPs and those of biocompatible and biodegrad-
able polymers in one self-assembled formulation allowing for
drug delivery. Given this background, one of the crucial studied
aspects was the question of whether the 4HB unit-possessing
NPs could be taken up by J774A.1 Mφs as a model cell type,
which is an important subject of drug delivery research. Mφs
are the main hosts of intracellular pathogens and, thus, are
pursued as a therapeutic target for the intracellular delivery of
antibiotics. In addition, Mφs have gained increasing interest
as a therapeutic target for cancer immunotherapy due to its
complex roles in the tumor microenvironment.[47]
F
(
t
)
= F0 +(Fplateau − F ) 1− e−tk
(2)
(
)
0
where F(t) is the cell fluorescence signal at time t, F0 and
Fplateau are the initial fluorescence signal and the maximum
signal, respectively, and k is the internalization rate constant.
The half-time of internalization (τ1/2) was calculated as the ratio
of ln 2 and k.
The DACCA-labeled copolymers were visualized inside the
J774A.1 cells using CLSM after an incubation time of 40 min.
The microscopy study showed that all of the formulations
tested, i.e., mPEO-b-PCL and mPEO-b-(PCL-co-P4HB), were
The cellular uptake of nanobeads, in general, depends
on several factors, such as the size, charge, shape, and
©
1900408 (10 of 15)
Macromol. Biosci. 2020, 1900408
2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.mbs-journal.de
Figure 5. Study of DACCA-labeled NP uptake by J774A.1 cells. Fluorescence and differential interference contrast (DIC) images of J774A.1 cells are
shown 40 min after the addition of DACCA-labeled NPs (2.8 nmol mL−1, blue fluorescence). The plasma membrane-related CellMask Deep Red signal
is pseudocolored in yellow. Scale bars: 20 µm.
©
1900408 (11 of 15)
Macromol. Biosci. 2020, 1900408
2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.mbs-journal.de
Figure 6. Study of DACCA-labeled NP localization within J774A.1 cells. CLSM images of J774A.1 Mφs 40 min after the addition of DACCA-labeled NPs
(2.8 nmol mL−1) and LysoTracker. The analysis of colocalization was allowed by both the product of the differences from the mean (PDM) images
and Pearson correlation coefficient (PCC) values. The PDM images are pseudocolored; each pixel is equal to the PDM value at that location and a
PDM scale bar was inserted. The orange color indicates colocalized pixels and the blue color suggests segregation. Scale bars: 20 µm. DIC denotes
differential interference contrast.
hydrophilic/hydrophobic ratio of the NPs.[42,50] Not surprisingly,
the resulting uptake kinetics are then based on a combination
of such parameters. Thus, a group of nanocarriers possessing
several different variable properties makes interpretation of
the kinetic results harder.[28] Despite this fact, the kinetics
study carried out revealed a phenomenon worth mentioning;
it was found that the P4HB-free mPEO45-b-PCL81-based sample
B1 exhibited a considerably high internalization τ1/2 value
(≈54 min) compared to that of the rest of the samples analyzed.
Notably, the mPEO45-b-(PCL55-co-P4HB19) copolymer (B3)
exhibited an internalization τ1/2 of ≈9.5 min, even when
having the same hydrophilic/hydrophobic ratio as that of B1
as well as the same molecular weight. This may be due to
the change in crystallinity and/or NP size, one of the main
physicochemical aspects that influence cellular uptake. This
finding suggests that the introduction of P4HB can strongly
affect the biorelevant properties of such nanobead-based inter-
ventions. Such a hypothesis, however, must be investigated in
©
1900408 (12 of 15)
Macromol. Biosci. 2020, 1900408
2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.mbs-journal.de
Figure 7. Study of DACCA-labeled NP fate. A) Time course of cell-associated fluorescence after the addition of 2 nmol mL−1 DACCA-labeled formula-
tions. The data were normalized to the maximum fluorescence values (i.e., the plateau values reached). B) Time course of NP degradation observed
after the monolayers were incubated with 2 nmol mL−1 DACCA-labeled formulations for 60 min. Both half-time and R squared values are shown. Note
that B1 exhibited different internalization as well as degradation kinetics compared to those of formulation B3, even when having the same hydrophilic/
hydrophobic ratio as well as the same molecular weight.
more detail to come to any specific conclusions in terms of
this phenomenon.
most distinct, as it revealed the slowest degradation rate (τ1/2 of
≈16 min), which also suggests that the introduction of P4HB
seems to be able to tune the biorelevant properties of the NPs.
Similar to our recent study,[28] the flow cytometry results were
not in line with the above-described 1H NMR degradation
study. This is probably because the 1H NMR study was car-
ried out in PBS only (i.e., without the presence of FBS or cell
proteins), as protein-NP interactions play a crucial role in the
biorelevant behavior. For the same reason, the flow cytometry
results may be deemed to be more relevant, because they reflect
interactions in biorelevant environments (i.e., in the presence
of serum and intracellular proteins).
It is worth mentioning that enzymatic degradability suggests
favorable behavior in vivo, as it can be assumed that the mPEO-
b-(PCL-co-P4HB) copolymers undergo degradation to mPEO,
6-hydroxyhexanoic acid (the degradation product of PCL) and
4-hydroxybutyric acid. Both mPEO44 (2000 Da) and mPEO113
(5000 Da) do not exceed the threshold for the renal filtration
of polymers 30–50 kDa.[52] The biocompatible 6-hydroxyhexa-
noic acid is converted to adipic acid by ω-oxidation in the
endoplasmic reticulum of liver and kidney cells in vivo. Adipic
acid is then metabolized by β-oxidation and the Krebs cycle
to carbon dioxide and water.[53] Similarly, 4-hydroxybutyric
acid is a ubiquitous molecule in vivo, derived both endog-
enously and exogenously and is catabolized by processes
including β-oxidation to acetyl-CoA and glycolate, α-oxidation
to 3-hydroxypropionate-CoA and formate, and cleavage of C-4
to yield 3-hydroxypropionate and carbon dioxide.[54]
As mentioned above, a plateau in the cell-associated fluo-
rescence was reached after 60–100 min of incubation with the
DACCA-labeled formulations. Subsequently, a decrease in the cell-
associated fluorescence values was observed (data not shown), as
the enzymatic degradation of the DACCA-labeled matrix leads to
quenching of its fluorescence.[27] Hence, to study the intracellular
degradation kinetics, DACCA-labeled NPs (2 nmol mL−1) were
added to Mφ monolayers and incubated for 60 min for each of
the formulations tested. Subsequently, the cells were washed with
PBS (i.e., time 0) and analyzed using flow cytometry at different
time points (Figure 7B). The degradation rate constant values
were determined by fitting the data to a single exponential decay
model. The half-time of degradation was calculated as the ratio of
ln 2 and the degradation rate constant.
In our recent study,[28] we suggested that there is a relation-
ship between the lysosomal localization of a nanocarrier and
its degradation kinetics in vitro. In other words, mPEO-b-PCL-
based NPs with cytosolic localization within Raw 264.7 Mφs
exhibited slow intracellular degradation, and, vice versa, fast
intracellular degradation was observed in the case of NPs
colocalized with lysosomes, i.e., lipase-rich compartments.[51]
It is evident from Figure 7B that all of the nanoformulations
analyzed (i.e., B1, B2, B3, B4, and B5) revealed similar degra-
dation patterns, probably because all of the samples exhibited
similar colocalization within low-pH compartments. Similar
to the uptake kinetics study, the P4HB-free sample B1 was the
©
1900408 (13 of 15)
Macromol. Biosci. 2020, 1900408
2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.mbs-journal.de
the paper. J.T. carried out the cytotoxicity and confocal microscopy
experiments and wrote the paper. D.R. performed the AF4 experiments.
K.G. performed the flow cytometry investigations. R.K. performed and
evaluated the 1H NMR analysis. M.Š. supervised the TEM investigations.
M.S. supervised the AF4 experiments and evaluated the results. O.Š.J.
supervised the flow cytometry investigations and cell-associated
fluorescence data fitting. M.H. supervised the project and contributed to
the final version of the paper.
4. Conclusions
The aim of this work was to describe, fundamentally charac-
terize, and perform biorelevant analyses of biocompatible and
biodegradable nanocarriers based on mPEO-b-(PCL-co-P4HB),
where the γBL introduction would hopefully serve as a biorele-
vant behavior-controlling mechanism.
First, it has been shown that the copolymer composition of
PCL-co-P4HB can be tuned not only by the feed ratio of the
monomers but also by choosing a feasible catalyst. The dif-
ference in γBL incorporation was demonstrated when anionic
or cationic catalysts were employed. A notable increase in
incorporation was observed when anionic catalysis in a non-
polar environment were employed. The content of incorpo-
rated γBL was ≈30% when TBD was used as a catalyst of the
copolymerization in toluene.
Keywords
biodegradation,
γ-butyrolactone
macrophages,
nanomedicine,
nanoparticles,
Received: November 28, 2019
Revised: January 22, 2020
Published online:
Additionally, we studied the biorelevant properties of P4HB-
based nanocarriers. Their biocompatibility, cellular uptake,
and degradation were evaluated. By combining flow cytom-
etry and CLSM, we demonstrated that these nanocarriers can
be internalized by macrophage-like cells in which the NPs
underwent intracellular degradation. The influence of the pres-
ence of the γBL units on the biological behavior was observed.
This method enabled control over the rate of degradation so
that when the content of γ-butyrolactone increased, the rate of
degradation increased.
[1] V. P. Torchilin, Pharm. Res. 2006, 24, 1.
[2] R. Palao-Suay, L. G. Gómez-Mascaraque, M. R. Aguilar,
B. Vázquez-Lasa, J. S. Román, Prog. Polym. Sci. 2016, 53, 207.
[3] I. Schlachet, J. Trousil, D. Rak, K. D. Knudsen, E. Pavlova,
B. Nyström, A. Sosnik. Carbohydr. Polym. 2019, 212, 412.
[4] A. C. Albertsson, I . K. Varma, Biomacromolecules 2003, 4, 1466.
[5] P. Markland, V. C. Yang, Encyclopedia of Pharmaceutical Technology,
Vol. 1, 3rd ed. (Ed: J. Swarbrick), Informa Helthcare USA, New York
2007.
[6] L. S. Nair, C. T. Laurencin, Prog. Polym. Sci. 2007, 32, 762.
[7] T. Moore, R. Adhikari, P. Gunatillake, Biomaterials 2005, 26, 3771.
[8] A. Nakayama, N. Kawasaki, S. Aiba, Y. Maeda, I. Arvanitoyannis,
N. Yamamoto, Polymer 1998, 39, 1213.
Given our findings, this study provides a generalizable
strategy for the future improvement of polyester nanobead-
based interventions in application fields where fine-tuned
properties are needed.
[9] D. P. Martin, S. F. Williams, Biochem. Eng. J. 2003, 16, 97.
[10] P. Olsén, K. Odelius, A. C. Albertsson, Biomacromolecules 2016, 17,
699.
[11] D. Myers, A. Cyriac, C. K. Williams, Nat. Chem. 2015, 8, 3.
[12] W. H. Carothers, Chem. Rev. 1931, 8, 353.
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
[13] F. Korte, W. Glet, J. Polym. Sci, Part B: Polym. Lett. 1966, 4, 685.
[14] K. Yamashita, K. Yamamoto, J.-i. Kadokawa, Chem. Lett. 2014, 43,
213.
[15] M. Hong, E. Y.-X. Chen, Nat. Chem. 2015, 8, 42.
[16] M. Hong, E. Y.-X. Chen, Angew. Chem., Int. Ed. 2016, 55, 4188.
[17] N. Zhao, C. Ren, H. Li, Y. Li, S. Liu, Z. Li, Angew. Chem., Int. Ed.
2017, 56, 12987.
[18] L. Lin, D. Han, J. Qin, S. Wang, M. Xiao, L. Sun, M. Yuezhong, Mac-
romolecules 2018, 51, 9317.
[19] C.-J. Zhang, L.-F. Hu, H.-L. Wu, X.-H. Cao, X.-H. Zhang, Macromol-
ecules 2018, 51, 8705.
[20] P. Walther, W. Frey, S. Naumann, Polym. Chem. 2018, 9, 3674.
[21] Y. Shen, Z. Zhao, Y. Li, S. Liu, F. Liu, Z. Li, Polym. Chem. 2019, 10, 1231.
[22] M. Hong, X. Tang, B. S. Newell, E. Y. X. Chen, Macromolecules 2017,
50, 8469.
[23] P. Walther, S. Naumann, Macromolecules 2017, 50, 8406.
[24] N. Dolan, D. P. Gavin, A. Eshwika, K. Kavanagh, J. McGinley,
J. C. Stephens, Bioorg. Med. Chem. Lett. 2016, 26, 630.
[25] L. Luo, J. Tam, D. Maysinger, A. Eisenberg, Bioconjugate Chem.
2002, 13, 1259.
Acknowledgements
Financial supports from the Czech Science Foundation (Grants Nos.
17-07164S and 17-09998S) the Ministry of Education, Youth and Sport
of the Czech Republic (National sustainability program I, grant #
POLYMAT LO1507) are gratefully acknowledged. T.U. and R.K. thank
1
Dr. Sabina Nováková for her kind assistance with H NMR analysis. J.T.
acknowledges support from Charles University (Project No. SVV260440)
and would like to thank Dr. Tomáš Mazel for his expert flow cytometry
data fitting advice and Dr. Jakub Hranícˇek (Department of Analytical
Chemistry, Faculty of Science, Charles University) for his kindness
in assisting with the instrumentation necessary for fluorescence
spectroscopy. D.R. and M.S. acknowledge support from the Scientific
Grant Agency VEGA and the Slovak Research and Development Agency
(Project Nos. 2/0177/17 and 16-0550). The authors would like to thank
Ewa Pavlova, who performed the TEM measurements.
Conflict of Interest
The authors declare no conflict of interest.
[26] S. Petrova, D. Klepac, R. Konefał, S. Kereïche, L. Kovácˇik,
S. K. Filippov, Macromolecules 2016, 49, 5407.
[27] J. Trousil, S. K. Filippov, M. Hrubý, T. Mazel, Z. Syrová, D. Cmarko,
S. Svidensk, J. Mateˇjková, L. Kovácˇik, B. Porsch, R. Konefał,
R. Lund, B. Nyström, I. Raškab, P. Šteˇpánek, Nanomedicine 2017,
13, 307.
Author Contributions
T.U. synthesized the polymers, carried out the physicochemical
characterizations of both the polymers and nanoparticles, and wrote
©
1900408 (14 of 15)
Macromol. Biosci. 2020, 1900408
2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.mbs-journal.de
[28] J. Trousil, Z. Syrová, N.-J. K. Dal, D. Rak, R. Konefał, E. Pavlova,
J. Mateˇjková, D. Cmarko, P. Kubícˇková, O. Pavliš, T. Urbánek,
M. Sedlák, F. Fenaroli, I. Raška, P. Šteˇpánek, M. Hrubý, Biomacro-
molecules 2019, 20, 1798.
[29] M. Bauer, C. Lautenschlaeger, K. Kempe, L. Tauhardt,
U. S. Schubert, D. Fischer, Macromol. Biosci. 2012, 12, 986.
[30] P. Ralph, M. A. Moore, K. Nilsson, J. Exp. Med. 1976, 143, 1528.
[31] Biological Evaluation of Medical Devices – Part 5: Tests for In Vitro
Cytotoxicity, International Organization for Standardization, Geneva,
Switzerland 2009.
[41] M. Ukawala, T. Rajyaguru, K. Chaudhari, A. S. Manjappa, S. Pimple,
A. K. Babbar, R. Mathur, A. K. Mishra, R. S. Murthy, Drug Delivery
2012, 19, 155.
[42] Z. Zhang, Q. Qu, J. Li, S. Zhou, Macromol. Biosci. 2013, 13,
789.
[43] K. Kolouchova, O. Sedlacek, D. Jirak, D. Babuka, J. Blahut, J. Kotek,
M. Vit, J. Trousil, R. Konefał, O. Janouskova, B. Podhorska,
M. Slouf, M. Hruby, Biomacromolecules 2018, 19, 3515.
[44] P. Grossen, D. Witzigmann, S. Sieber, J. Huwyler, J. Controlled
Release 2017, 260, 46.
[32] A. Duda, S. Penczek, P. Dubois, D. Mecerreyes, R. Jérôme, Mac-
romol. Chem. Phys. 1996, 197, 1273.
[33] M. Danko, J. Mosnácˇek, Polimery 2017, 62, 272.
[45] P. Grossen, G. Québatte, D. Witzigmann, C. Prescianotto-
Baschong, L.-H. Dieu, J. Huwyler, J. Nanomater. 2016, 2016,
13.
[34] A. Bhaw-Luximon, D. Jhurry, S. Motala-Timol, Y. Lochee, Macromol.
Symp. 2005, 231, 60.
[46] J. Jin, B. Sui, J. Gou, J. Liu, X. Tang, H. Xu, Y. Zhang, X. Jin, PLoS
One 2014, 9, e112200.
[35] M. Dionzou, A. Morère, C. Roux, B. Lonetti, J. D. Marty,
C. Mingotaud, P. Joseph, D. Goudounèche, B. Payré, M. Léonettie,
A.-F. Mingotaud, Soft Matter. 2016, 12, 2166.
[36] R. F. Domingos, M. A. Baalousha, Y. Ju-Nam, M. M. Reid,
N. Tufenkji, J. R. Lead, G. G. Leppard, K. J. Wilkinson, Environ. Sci.
Technol. 2009, 43, 7277.
[37] O. Sedlacek, B. D. Monnery, S. K. Filippov, R. Hoogenboom,
M. Hruby, Macromol. Rapid Commun. 2012, 33, 1648.
[38] S. Agarwal, X. Xie, Macromolecules 2003, 36, 3545.
[39] A. Ianiro, J. Patterson, Á. González García, M. M. J. van Rijt, M. M.
R. M. Hendrix, N. A. J. M. Sommerdijk, I. K. Voets, A. C. C. Esteves,
R. Tuinier, J. Polym. Sci., Part B: Polym. Phys. 2018, 56, 330.
[40] S. Zolls, R. Tantipolphan, M. Wiggenhorn, G. Winter, W. Jiskoot,
W. Friess, A. Hawe, J. Pharm. Sci. 2012, 101, 914.
[47] Y. Pei, Y. Yeo, J. Controlled Release 2016, 240, 202.
[48] S. M. Abaza, Parasitol. United J. 2016, 9, 1.
[49] I. Vainshtein, L. K. Roskos, J. Cheng, M. A. Sleeman, B. Wang,
M. Liang, Pharm. Res. 2015, 32, 286.
[50] S. Behzadi, V. Serpooshan, W. Tao, M. A. Hamaly, M. Y. Alkawareek,
E. C. Dreaden, D. Brown, A. M. Alkilany, O. C. Farokhzad,
M. Mahmoudi, Chem. Soc. Rev. 2017, 46, 4218.
[51] M. J. Czaja, A. M. Cuervo, Autophagy 2009, 5, 866.
[52] M. E. Fox, F. C. Szoka, J. M. J. Fréchet, Acc. Chem. Res. 2009, 42,
1141.
[53] A. Orchel, K. Jelonek, J. Kasperczyk, Z. Dzierzewicz, Acta Pol.
Pharm. 2010, 67, 710.
[54] S. Sadhukhan, G.-F. Zhang, G. P. Tochtrop, ACS Chem. Biol. 2014,
9, 1706.
©
1900408 (15 of 15)
Macromol. Biosci. 2020, 1900408
2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim