polymers

Article
Enzymatic Degradation of Poly(butylene succinate)
Copolyesters Synthesized with the Use of
Candida antarctica Lipase B
Aleksandra Wcisłek 1 , Agueda Sonseca Olalla 1,2 , Andrew McClain 3 , Agnieszka Piegat 1 ,
Peter Sobolewski 1 ID , Judit Puskas 3 and Miroslawa El Fray 1, * ID
1 Division of Functional Materials and Biomaterials, Faculty of Chemical Technology and Engineering,
West Pomeranian University of Technology, Szczecin, Al. Piastow 45, 71-311 Szczecin, Poland;
[email protected] (A.W.); [email protected] (A.S.O.); [email protected] (A.P.);
[email protected] (P.S.)
2 Instituto de Ciencia y Tecnología de Polímeros, ICTP-CSIC, Calle Juan de la Cierva 3, 28006 Madrid, Spain
3 Department of Chemical and Biomolecular Engineering, The University of Akron, Akron, OH 44325, USA;
[email protected] (A.M.); [email protected] (J.P.)
* Correspondence: [email protected]; Tel.: +48-91-4494828




Received: 15 May 2018; Accepted: 16 June 2018; Published: 20 June 2018


Abstract: Biodegradable polymers are an active area of investigation, particularly ones that can be
produced from sustainable, biobased monomers, such as copolymers of poly(butylene succinate)
(PBS). In this study, we examine the enzymatic degradation of poly(butylene succinate-dilinoleic
succinate) (PBS-DLS) copolymers obtained by “green” enzymatic synthesis using lipase B from
Candida antarctica (CALB). The copolymers differed in their hard to soft segments ratio, from 70:30
to 50:50 wt %. Enzymatic degradation was carried out on electrospun membranes (scaffolds) and
compression-moulded films using lipase from Pseudomomas cepacia. Poly(ε-caprolactone) (PCL) was
used as a reference aliphatic polyester. The degradation process was monitored gravimetrically via
water uptake and mass loss. After 24 days, approx. 40% mass loss was observed for fibrous materials
prepared from the PBS-DLS 70:30 copolymer, as compared to approx. 10% mass loss for PBS-DLS 50:50.
Infrared spectroscopy (FTIR) and size exclusion chromatography (SEC) analysis were used to examine
changes in chemical structure. Differential scanning calorimetry (DSC) and scanning light microscopy
(LSM) revealed changes in degree of crystallinity, and changes in surface morphology, consistent
with a surface erosion mechanism. We conclude that the obtained copolymers are suitable for tissue
engineering applications thanks to tuneable degradation and lack of acidification during breakdown.

Keywords: polyester copolymers; enzymatic degradation; nanofibers; electrospinning; fatty acid;

poly(butylene succinate) (PBS)

1. Introduction
Biodegradable polymers are being constantly investigated for medical applications [1], especially
for tissue engineering (TE). Such polymers need to meet many important requirements to
be successfully applied in TE. These include having suitable chemical properties (non-toxicity,
biodegradability, and ease of sterilization), physical properties (nanofibrous, elastic, and complaint),
and biological properties (biocompatibility and biofunctionality) [2]. Currently, attention is primarily
focused on a large group of aliphatic polyesters, including poly(lactic acid) (PLA), poly(glycolic
acid) (PGA), poly(ε-caprolactone) (PCL) [3], and polyhydroxyalkanoates (PHA) [4], such as
poly(hydroxybutyrate) (PHB). Overall, these biobased polymers see wide use in tissue engineering due
to their biodegradability, good biocompatibility, bioresorbability, and suitable mechanical properties.

Polymers 2018, 10, 688; doi:10.3390/polym10060688 www.mdpi.com/journal/polymers

Polymers 2018, 10, 688 2 of 18

However, since the 1990’s, poly(butylene succinate) (PBS) and its copolymers have
gained (renewed) prominence within this aliphatic polyester family, particularly for biomedical
applications [5–7]. PBS offers good mechanical properties and biocompatibility, as well as better
processability, easier obtainability, and higher elongation at break than other aliphatic polyesters. In
particular, its high ductility suggests that PBS might be a good candidate for soft tissue repair. In terms
of degradation, much like the other aliphatic polyesters, PBS is susceptible to hydrolysis and, in the
environment, degradation of PBS by enzymes and microorganisms yields harmless products (carbon
dioxide and water). However, (but again similarly to PLA, PGA, PCL and PHB) the crystallinity of PBS
plays a major role in the degradation process [8] and may limit its suitability for soft-tissue repair and
tissue engineering, due to its relatively slow degradation and resorption rate. In these applications, the
polymer should combine an appropriate mechanical performance and degradation profile; maintaining
strength until the formation of new tissue can occur to replace the degraded material.
It is well known that polyester properties are influenced by many variables, some of which
are molar mass, chemical structure, stereochemistry, hydrophilic/hydrophobic balance or chain
mobility [9,10]. Consequently, copolymerization [11–14], as well as nanoadditives [15], are
well-established strategies for tailoring the properties of PBS to suit various biomedical applications.
Previously, we demonstrated that copolymerization of PBS with an increasing amount of dilinoleic acid
(a dimerized fatty acid derivative) yielded lower crystallinity, higher susceptibility to degradation, and
increased elasticity [16]. These fatty acid derivatives are obtained from vegetable oils and, thus, this
new family of PBS copolymers could be entirely biobased, because PBS monomers (diol, diacid/diester)
are readily available from refined biomass and biobased PBS (as well as various copolymers) is already
commercially accessible [17].
Sustainability and “green chemistry” [18] can be further embraced in polyester synthesis by
employing enzymatic catalysts [19], which offer much greater selectivity. This approach offers several
additional advantages compared to traditional melt polycondensation [20], such as mild conditions
reactions (low temperature and pressure), and no toxic organometallic initiators—particularly relevant
for biomedical applications. Lipases, in particular, have been demonstrated to be powerful biocatalysts
for the synthesis of polyesters when the proper conditions are used [21–23]. In previous work
performed in our laboratory using immobilized lipase B from Candida antarctica (CALB) as a catalyst,
we obtained PBS copolymers containing dimer linoleic diol (poly(butylene succinate-co-dilinoleic
succinate)), (PBS-DLS) with well-defined structures and high reproducibility of the initial monomer
feed ratio [24].
Our primary goal has been to obtain unique PBS-DLS copolymers, able to form flexible and soft
materials for application in scaffolds for soft tissue engineering. The introduction to fatty acid moieties
(dilinoleic diol), as well as using enzymatic catalysis, can be expected to significantly influence the
degradation profile of these copolymers. In this work, we examine the enzymatic degradation of
PBS-DLS copolymers differing in the amount of dilinoleic diol building up the soft segments (30 and
50 wt %, respectively). In the body, a range of hydrolase enzymes, present in fluids and tissues or
as part of the inflammatory response, can be involved in increasing the in vivo degradation rate, as
compared to hydrolysis alone [25]. It is impossible to adequately mimic these complex (and dynamic)
conditions in vitro, thus it is common and useful to use model enzyme systems to gain insight in to the
degradation behavior of new materials and plan future in vivo studies. Lipase, a hydrolase present in
human serum and macrophages, is a common model enzyme system, as it is well-known to hydrolyze
ester bonds of aliphatic polyesters in aqueous media [25] (in addition to the previously mentioned
catalytic activity towards ester formation). In the present study, we utilized lipase from Pseudomonas
cepacia, one of the most commonly used lipases for in vitro enzymatic degradation, thanks to sufficient
stability and activity to yield reproducible studies [11]. The degradation process was monitored by
weight loss, water uptake, and changes in medium pH as a function of degradation time, as well
as Fourier Transform Infrared Spectroscopy (FTIR) and Laser Scanning Microscopy. Additionally,
Polymers 2018, 10, x FOR PEER REVIEW 3 of 18
Polymers 2018, 10, 688 3 of 18

Laser Scanning Microscopy. Additionally, Differential Scanning Calorimetry (DSC) and Size
Exclusion Chromatography
Differential analysis
Scanning Calorimetry wereSize
(DSC) and performed to compare samples
Exclusion Chromatography before
analysis and after
were performed
degradation.
to compare samples before and after degradation.

2. Materials and Methods

2.1. Materials
Two aliphatic
aliphatic PBS
PBS copolyesters
copolyesters containing
containing dimer linoleic diol moieties (PBS-DLS), differing in
hard to soft segments ratios (70:30 and 50:50 50:50 wtwt %),
%), were
were synthesized
synthesized usingusing immobilized
immobilized CALB
(Fermase CALB™10000, purchased from Fermenta Biotech Ltd. Mumbai Mumbai and kindly provided by
Enzyme
Enzyme Catalyzed
CatalyzedPolymers
PolymersLLC, LLC,Akron,
Akron, OH,OH,USA)USA)as aascatalyst. The The
a catalyst. synthesis was carried
synthesis out using
was carried out
ausing
two stage method and worked-up accordingly to the procedure described
a two stage method and worked-up accordingly to the procedure described previously [24] to previously [24] to obtain
samples with a white
obtain samples with apowder appearance
white powder after 96 after
appearance h. Briefly,
96 h. 1,4-butanediol (BD, ≥99%,
Briefly, 1,4-butanediol (BD,Sigma-Aldrich,
≥99%, Sigma-
TM 2033,TM
Pozna
Aldrich,ń, Poland), dimer linoleic
Poznań, Poland), dimerdiol (DLA-OH,
linoleic diol (DLA-OH, dimer≥alcohol
dimer alcohol 96.5%, Pripol
≥96.5%, Pripol Croda,
2033, Gouda,
Croda,
The Netherlands), and diethyl succinate (DS, ≥ 99%, Sigma-Aldrich, Pozna
Gouda, The Netherlands), and diethyl succinate (DS, ≥99%, Sigma-Aldrich, Poznań, Poland) were ń, Poland) were dissolved
in diphenylinether
dissolved (DE, ≥ether
diphenyl 99%, (DE,
Sigma-Aldrich, Poznań, Poland)
≥99%, Sigma-Aldrich, (in appropriate
Poznań, Poland) (inratios to obtainratios
appropriate desired
to
hard
obtaintodesired
soft segment
hard to ratio
softofsegment
the copolymer)
ratio of with the additionwith
the copolymer) of CALB as the catalyst.
the addition of CALB The
as mixture was
the catalyst.
reacted at atmospheric
The mixture was reacted pressure at 80 ◦ C under
at atmospheric N2 . The
pressure temperature
at 80 °C under was slowly
N2. The increased to
temperature 95 ◦slowly
was C and
after 2 h (half
increased to 95of°C
theand
theoretical
after 2 hamount
(half of oftheethanol was collected)
theoretical amount ofthe pressure
ethanol waswas reduced
collected) thetopressure
600 Torr.
After 18 h theto
was reduced pressure
600 Torr. was gradually
After 18 h the reduced
pressure to was
2 Torr and maintained
gradually reducedfor to 272Torr
h (96 h of
and total reaction
maintained for
time).
72 h (96 After
h ofthis
totaltime, the cooled
reaction time). products
After thisweretime,dissolved
the cooledinproducts
chloroformwere and filtered to
dissolved in remove
chloroformthe
CALB. Products
and filtered to were
remove concentrated
the CALB.using a rotary
Products evaporator
were and precipitated
concentrated using a rotaryby drop-wise
evaporator addition
and
into cold methanol (MeOH: ≥ 99.8%, POCh, Gliwice, Poland), under
precipitated by drop-wise addition into cold methanol (MeOH: ≥99.8%, POCh, Gliwice, Poland),continuous stirring. Finally, the
polymers were filtered, washed well ◦
under continuous stirring. Finally, thewith cold methanol,
polymers and dried
were filtered, washed in well
a vacuum
with coldovenmethanol,
at 40 C. The and
mass
dried feed of all monomers
in a vacuum oven at 40 was °C.varied in order
The mass feedtoofobtain two materials
all monomers with butylene-succinate
was varied in order to obtain (BS) two
units as hard
materials withsegments and succinate-dimer
butylene-succinate (BS) unitslinoleic
as harddiol units (DLS)
segments as soft segmentslinoleic
and succinate-dimer in approximately
diol units
50:50
(DLS)and 70:30
as soft wt % ratios.
segments The obtained50:50
in approximately copolymers
and 70:30 withwthard to soft
% ratios. The segments
obtainedratios of 70:30with
copolymers and
50:50 wt % will be abbreviated in this work as PBS-DLS 70:30 and PBS-DLS
hard to soft segments ratios of 70:30 and 50:50 wt % will be abbreviated in this work as PBS-DLS 70:30 50:50, respectively. The
structural
and PBS-DLS formula
50:50,ofrespectively.
the copolymers Thecomprising hard and
structural formula of soft segments respectively
the copolymers comprisingishard illustrated in
and soft
Figure
segments 1. respectively is illustrated in Figure 1.

O H3C O

HO O O CH 3
O O
n m
O CH3 O

Poly(butylene succinate-co-dilinoleic succinate)

Figure 1. Chemical structure of synthesized

synthesized PBS-DLS copolymers showing the composition of hard
segments (red) and soft segments
segments (blue).
(blue).

2.2. Characterization of PBS-DLS Enzymatically Obtained Copolymers

chemicalstructures
The chemical structuresof of purified
purified PBS-DLS
PBS-DLS 70:3070:30 and 50:50
and 50:50 copolymers
copolymers were verified
were verified by
by nuclear
nuclear magnetic
magnetic resonance (1 H NMR,
resonance (1H 13
NMR,
C NMR)13C NMR) and Fourier transform infrared spectroscopy (FTIR).
and Fourier transform infrared spectroscopy (FTIR). The
1The 1H NMR (128 scans, 1 s relaxation delay)13
H NMR (128 scans, 1 s relaxation delay) and and 13C NMR (5120 scans, 1 s relaxation delay) spectra
C NMR (5120 scans, 1 s relaxation delay) spectra were
were recorded
recorded in chloroform-d
in chloroform-d (CDCl3 :(CDCl
99.8%3:atom
99.8%
D) atom
on a TMD) Bruker
on a TM DPXBruker DPX
400 (400 MHz) 400spectrometer
(400 MHz)
spectrometer
(Bruker, Germany) (Bruker, Germany) with(TMS)
with tetramethylsilane tetramethylsilane (TMS) FTIR
as internal reference. as internal reference.
transmission FTIR
spectra (4000
−
transmission 1 −
spectra1 (4000 to 400 cm −1, 2 cm−1 resolution, 32 scans), were obtained on a Bruker ALPHA
to 400 cm , 2 cm resolution, 32 scans), were obtained on a Bruker ALPHA spectrometer with an
spectrometer
Attenuated with
Total an Attenuated
Reflectance (ATR)Total Reflectance
cell. Intrinsic (ATR)[η]cell.
viscosity Intrinsic viscosity
measurements [η] measurements
were performed using an
were performed
Ubbelohde using
capillary an Ubbelohde
viscometer (Labit, capillary viscometer
Koczargi Nowe, (Labit,
Poland) (K =Koczargi
0.00342) Nowe,
immersed Poland) (K =
in a water
0.00342) immersed in a water bath at 25 °C. Samples were dissolved in chloroform (0.125 g/25 cm3)
and results were calculated using the Solomon–Ciuta Equations (1)–(3) [26].
Polymers 2018, 10, 688 4 of 18

bath at 25 ◦ C. Samples were dissolved in chloroform (0.125 g/25 cm3 ) and results were calculated
using the Solomon–Ciuta Equations (1)–(3) [26].
p
2(ηw − ln ηr )
η= (1)
c
η w = ηr − 1 (2)
t1
ηr = (3)
to
where:
to —average flow time for solvent [s]
t1 —average flow time for polymer solution [s]
c—concentration of polymer solution [g/100 mL]
Qualitative size exclusion chromatography (SEC) measurements were performed in order to
better understand and confirm the degradation results, in a set-up consisting of an Agilent 1260 Infinity
Isocratic Pump (Agilent Technologies, Santa Clara, CA, USA), a Wyatt OPTILAB T-rEX interferometric
refractometer (Wyatt Technology, Santa Barbara, CA, USA), a Wyatt DAWN HELOS-II multi-angle
static light scattering detector (MALS) (Wyatt Technology, Santa Barbara, CA, USA) with a built-in
dynamic light scattering (DLS) module, an Agilent 1260 Infinity Standard Autosampler (Agilent
Technologies, Santa Clara, CA, USA) and 6 StyragelVR columns (HR6, HR5, HR4, HR3, HR1, and
H0.5). The columns were thermostatted at 35 ◦ C and tetrahydrofuran (THF), continuously distilled
from CaH2 , was used as the mobile phase at a flow rate of 1 mLmin−1 . Previous to the analysis, samples
were dissolved in THF (the same employed as mobile phase in the instrument) at 2–3 mg·mL−1 and
filtered. The dRI signal was analyzed qualitatively.

2.3. Sample Preparation for Degradation Studies

Commercial PCL was used as a reference and positive control (CAPA 6430, Perstorp,
Mn = 43,000 g/mol), as it has well-established biomedical applications [3], while PBS homopolymer
is very resistant to enzymatic degradation [10,27] due to combination of high crystallinity and a
high melting temperature. The PCL and enzymatically synthesized PBS-DLS 70:30 and 50:50 were
processed by both hot-press melting and electrospinning, in order to assess the degradation profiles of
the materials in the shape of films and fibrous membranes (scaffolds). Thin polymer films (100 µm)
were prepared in a Remi-Plast hot-pressing machine (Remi-Plast, Czerwonak, Poland) at temperature
of 120 ◦ C. Electrospun mats of similar thickness (~100–150 µm) were prepared by means of a standard
horizontal electrospinning setup equipped with a ASCOR MED AP14 (Ascor Med, Warsaw, Poland)
pump, a Gamma High Voltage Research (Gamma High Voltage, Ormond Beach, FL, USA) voltage
source connected to a 21 gauge needle, and a grounded flat collector covered with aluminum foil.
PCL polymeric solution was prepared by dissolving 20 wt % of the material in a DMF:DCM 5:5 v/v
solvent mixture. PBS-DLS 70:30 and PBS-DLS 50:50 solutions were prepared by dissolving 25 and
35 wt % respectively in a CHCl3 :MeOH 7:3 v/v solvent mixture. All solutions were stirred at room
temperature overnight and polymers were pre-dried in a vacuum oven before their preparation. The
electrospinning process parameters are summarized in Table 1. The concentrations and parameters
used are optimal for obtaining fibers free of defects or drops. The obtained mats were dried under
vacuum for 48 h in order to eliminate any residual solvents. Samples in the shape of discs (6 mm
diameter) were punched-out from the polymeric film and mats to perform the degradation studies.
Polymers 2018, 10, 688 5 of 18

Table 1. Electrospinning conditions for PCL, PBS-DLS 70:30 and PBS-DLS 50:50 copolymers.

Distance Voltage Solvents/Concentration Humidity

Material Rate [mL/h] Temp [◦ C]
[cm] [kV] (v/v, wt %) [%]
PBS-DLS 70:30 1 15 15 CHCl3 :DCM(7:3)/25 23 34
PBS-DLS 50:50 1 15 15 CHCl3 :DCM(7:3)/35 23 28
PCL 3 18 18 DMF:DCM(1:1)/20 25 30

2.4. Enzymatic Degradation

Enzymatic degradation was carried out inside a laminar flow cabinet [28]. The degradation
media consisted of a solution of lipase enzyme (EC3.1.1.3, from Pseudomonas cepacia (Sigma Aldrich,
Poznań, Poland) (25 units/mL) prepared in Dulbecco’s PBS buffer (Sigma Aldrich, Poznań, Poland)
(pH range 7.1–7.2), with sodium azide added to avoid bacterial contamination. Each sample, in the
form of a 6 mm diameter disc, was placed in the well of a 48-well plate and covered with 1.5 mL of
degradation media. Plates were then incubated at 37 ◦ C on a rocker (60 rpm) for 24 days and samples
were collected for analysis at 24 h, 48 h and every 96 h thereafter. In order to maintain enzymatic
activity, the degradation medium was changed every 48 h. At the specific time points, samples were
removed, washed with distilled water, and weighed (n = 3).

2.5. Characterization of Processed Materials before and after Degradation

Water uptake and weight loss were determined gravimetrically using an analytical balance with
an accuracy of 0.01 mg. At the specific time points, samples were removed from the media, washed
thoroughly with distilled water, and weighed wet. Then discs dried at 40 ◦ C under a pressure of
~30 mmHg for 10 days and weighed dry. The initial mass, before degradation, was indicated as m0 ,
while the wet mass, immediately after degradation was indicated as mw (wet), and the mass after
drying as md .
The water uptake (P) was then calculated from equation:

mw − md
P= · 100% (4)
md

The weight loss (D) was calculated from equation:

m0 − m d
D= · 100% (5)
m0

Changes in the chemical structure of films and fiber membranes (scaffolds) before and
after degradation were evaluated by ATR-FTIR spectroscopy (measured under previously
mentioned conditions).
TA Instruments Q1000 (TA Instruments, New Castle, DE, USA). Approximately 3 mg of sample
(sealed in aluminum pans) was subjected to a triple heating-cooling-heating cycle −90 to 190 ◦ C at
a rate of 10 ◦ C/min under N2 atmosphere. Glass transition temperature (Tg ), melting temperature
(Tm ) and crystallinity were evaluated from the heating run and before/after degradation. In all the
cases, glass transition temperature (Tg ) was calculated as the midpoint of the transition. The crystalline
phase content in the hard segments (Xc,h %) for PBS-DLS 70:30 and 50:50 was calculated following the
Equations (6) and (7), while the total content of crystalline phase in PCL (Xc,tot %), was calculated from
Equation (8):
Xc,h % = (0.7 × ∆Hm /∆H0 m ) × 100 (6)

Xc,h % = (0.5 × ∆Hm /∆H0 m ) × 100 (7)

Xc,tot % = ∆Hm /∆H0 m × 100 (8)

Polymers 2018, 10, 688 6 of 18

where ∆H0 m is ∆Hm =110.3 J/g−1 for 100% crystalline PBS [29] and ∆Hm =135.3 J/g−1 for 100%
crystalline PCL [30].
Changes in the surface morphology (roughness, cracks, pores) were examined by Laser Scanning
Microscopy (LSM) using a Keyence VK9700 LSM equipped with a 408 nm violet laser (Keyence, Itasca,
IL, USA). Images were collected at maximum resolution (2048 × 1536) with a 10% and 30% intensity
Polymers of
setting 2018,
the10,laser
x FORfor
PEER 20× (0.3 NA) and 50× (0.95 NA) objectives respectively.
theREVIEW 6 of 18

3. Results

3.1. Chemical Structure

3.1. Chemical Structure Analysis
Analysis
11 H NMR and 13 C
13 NMR results confirmed the successful synthesis of the CALB catalyzed PBS:DLS
H NMR and C NMR results confirmed the successful synthesis of the CALB catalyzed
copolymers. Figure 2 presents 1 H NMR spectrum
PBS:DLS copolymers. Figure 2the
presents of PBS-DLS
the 1H NMR spectrum 70:30 copolymer.
of PBS-DLS 70:30 copolymer.

O H3 C O
b a
e
HO O O CH3
O g O
l c n d f m k
h
O CH3 O
j n+m
i

b f+h
a c

g+e i+j
k d l

Figure 2. 11H
H NMR
NMR of
of poly(butylene
poly(butylene succinate-co-dilinoleic
succinate-co-dilinoleic succinate) 70:30 copolyester.

In 11H
In NMR, signals
H NMR, signalsappearing
appearingat: at:δ1δH
1H = 4.12 ppm are due to four protons from BD adjacent to
= 4.12 ppm are due to four protons from BD adjacent to the
the oxygen
oxygen (a), δ(a),
1H δ=H
1 = 2.63
2.63 ppmppm are signals
are signals from from four protons
four protons from DSfrom(c),DS (c),
δ1 H δ1H ppm
= 1.72 = 1.72are
ppmfromareBD
from
(b).
BD (b). Following signal are characteristic for soft segments DLS1 units: δ1 H
Following signal are characteristic for soft segments DLS units: δ H = 1.62 ppm (20H, from DLA-OH, = 1.62 ppm (20H, from
DLA-OH,
g + e), δ1 Hg=+1.25
e), δ(52H,
1H = 1.25 (52H, from DLA-OH, f + h), 0.85 (10H, from DLA-OH, i + j). Low intensity
from DLA-OH, f + h), 0.85 (10H, from DLA-OH, i + j). Low intensity resonance
resonance ascribed to
ascribed to the BD end-groupsthe BD end-groups
appears at appears
3.68 ppm at 3.68
(2H,ppm (2H, l, normalized
l, normalized to valueto2.00)
value(132.00)
C NMR(13C
NMR confirmed
confirmed the absence
the absence of DLA-OHof DLA-OH end groups.
end groups. Figures S2 Figures S2 and
and S3). S3).succinate
Diethyl Diethyl succinate
end-groups end-at
groups at 4.16 ppm and at 1.25 are partially/total overlapped with
4.16 ppm and at 1.25 are partially/total overlapped with BD and DLA-OH signals respectively. BD and DLA-OH signals
respectively.
As detailed in the Supplementary Materials, analysis of the 1H NMR spectra showed that the
final achieved copolymer compositions were in a good agreement with the initial monomer feed
(BD:DS:DLA-OH molar feed/compositions; PBS-DLS 70:30 initial 45:50:5, final 45:50:5; PBS-DLS 50:50
initial 50:39:11, final 50:41:9) (Supplementary Materials Figures S1and S2; Supplementary Materials
Tables S1 and S2) [24]. In addition, the number-average molecular weight (Mn) calculated from 1H
Polymers 2018, 10, 688 7 of 18

As detailed in the Supplementary Materials, analysis of the 1 H NMR spectra showed that the
final achieved copolymer compositions were in a good agreement with the initial monomer feed
(BD:DS:DLA-OH molar feed/compositions; PBS-DLS 70:30 initial 45:50:5, final 45:50:5; PBS-DLS
50:50 initial 50:39:11, final 50:41:9) (Supplementary Materials Figures S1 and S2; Supplementary
Materials Tables S1 and S2) [24]. In addition, the number-average molecular weight (Mn ) calculated
from 1 H NMR (Supplementary Materials) resulted in an Mn = 11,590 g/mol for PBS-DLS 70:30 and
Mn = 27,440 g/mol for PBS-DLS 50:50. The higher value obtained for PBS-DLS 50:50 is on account of
the higher amount of DLA-OH present in the polymer backbone. An analogous relationship was also
observed for the intrinsic viscosity measurements, where for PBS-DLS 70:30 η = 0.40 dL/g and for
50:50 copolymer η = 0.56 dL/g.
Taking into account the related literature and our previous experience with enzymatically
synthesized PBS and PBS copolymers, lowering of the vacuum in a stepwise manner and the usage of
high boiling point and high log P (octanol/water partition coefficient of diphenyl ether 4.21) solvent,
helps the formation of a high molecular weight materials avoiding premature deactivation of the
catalyst and precipitation of the system for medium conversions [24,31–33]. Thus, the synthesis
conditions chosen helped to retain the initial desired ratio of hard to soft segments and favor the
formation of high molecular weight PBS-DLS materials that ensured their further processing into fibers
and films.

3.2. Enzymatic Degradation of Film and Fibrous Materials

The progress of enzymatic degradation was monitored for film and fibrous materials differing
in segmental composition. The surface area available for degradation plays an important role, as in
contrast to hydrolytic degradation, which for PBS copolymers is typically a bulk process, enzymatic
degradation can be expected to occur via surface erosion [5]. Here, the mass loss and water uptake
values for bulk materials (films, ~100 µm) were low and almost constant over the observation period
of 24 days: Mass loss was at the same level of 3.2% for both PBS-DLS 70:30 and PBS-DLS 50:50
copolymers, while water uptake was higher for PBS-DLS 70:30 (8.7%) than for PBS-DLS 50:50 (5.4%).
The difference in water uptake, while small, can be explained by the higher content of hydrophobic
fatty acid sequences in PBS-DLS 50:50 and higher Mn as compared to PBS-DLS 70:30. In the case of
electrospun mats, the fibrous morphology had a very pronounced effect on water uptake and mass
loss—dramatically increasing both, as compared to bulk materials—as can be seen from Figure 3a. For
the PBS-DLS 70:30 copolymer, containing a higher amount of short butylene succinate hard segments,
it can be clearly seen, that the much greater surface area of fibrous materials along with the segmental
composition has resulted in a much higher water uptake, ranging from 400% to 600%, as compared to
approx. 100% for PBS-DLS 50:50, which contains more long chain hydrophobic fatty acid sequences.
As can be seen from Figure 3b, fibrous mats show increasing mass loss over time: after 24 days, mass
loss exceeds 40% for PBS-DLS 70:30 and is approx. 10% for PBS-DLS 50:50. The much more rapid
degradation of fibrous mats, as compared to films, is likely due to the much greater effective surface
area, in agreement with a surface erosion mechanism.
Importantly, no acidification of the degradation media was observed during the degradation
process of films (Figure 4) and electrospun mats (Figure 5), for either copolymer composition. The
reference material and positive control PCL exhibits very fast degradation, along with a sharp drop in
pH (as low as pH = 6). Again, the degradation proceeds much faster for electrospun PCL mats; these
materials completely disintegrate after 24 h.
 much more rapid degradation of fibrous mats, as compared to films, is likely due to the much greater
effective surface area, in agreement with a surface erosion mechanism.
Importantly, no acidification of the degradation media was observed during the degradation
process of films (Figure 4) and electrospun mats (Figure 5), for either copolymer composition. The
reference material and positive control PCL exhibits very fast degradation, along with a sharp drop
in pH (as low as pH = 6). Again, the degradation proceeds much faster for electrospun PCL mats;
Polymers 2018, 10, 688 8 of 18
these materials completely disintegrate after 24 h.

Polymers 2018, 10, x FOR PEER REVIEW (a) 8 of 18

Polymers 2018, 10, x FOR PEER REVIEW 8 of 18

(b)
Figure 3. Water uptake (a) and mass loss (b) for(b)
electrospun fiber mats of PBS-DLS 70:30 and PBS-
Figure 3. Water uptake
DLS 50:50 (mean(a) and
± SD, n =mass
3). loss (b) for electrospun fiber mats of PBS-DLS 70:30 and PBS-DLS
Figure
50:50 (mean 3. Water
± SD, uptake (a) and mass loss (b) for electrospun fiber mats of PBS-DLS 70:30 and PBS-
n = 3).
DLS 50:50 (mean ± SD, n = 3).

Figure 4. Changes in the pH of degradation media during degradation for PBS-DLS 70:30, PBS DLS
50:50, and PCL films.
Figure 4. Changes in the pH of degradation media during degradation for PBS-DLS 70:30, PBS DLS
Figure 4. Changes in the
50:50, and PCL pH of degradation media during degradation for PBS-DLS 70:30,
films. PBS DLS
50:50, and PCL films.

In order to reveal changes in Mn of degraded samples, SEC was carried out and the dRI signal
was analyzed qualitatively. Figure 6 shows the traces before and after degradation for films materials.
The trace of PBS-DLS 70:30 before degradation is multimodal, clearly showing the populations with
various molecular weights, typical of polycondensation reactions. After degradation (24 days), no
differences are observed for PBS-DLS 50:50 (Figure 6b), while PBS-DLS 70:30 (Figure 6a) showed
some molecular weight breakdown, especially from the higher molecular weight populations. These
results are consistent with a surface erosion mechanism, as one would expect small molecular weight
breakdown products to be dissolved in the media.
Figure 5. The changes in the pH of degradation media during degradation for PBS-DLS 70:30, PBS-
DLS 50:50, and PCL electrospun fiber mats.
Figure 5. The changes in the pH of degradation media during degradation for PBS-DLS 70:30, PBS-
DLS 50:50, and PCL electrospun fiber mats.
Polymers 2018, 10, 688 9 of 18
Figure 4. Changes in the pH of degradation media during degradation for PBS-DLS 70:30, PBS DLS
50:50, and PCL films.

Polymers 2018, 10, x FOR PEER REVIEW 9 of 18

In order to reveal changes in Mn of degraded samples, SEC was carried out and the dRI signal
was analyzed qualitatively. Figure 6 shows the traces before and after degradation for films materials.
The trace of PBS-DLS 70:30 before degradation is multimodal, clearly showing the populations with
various molecular weights, typical of polycondensation reactions. After degradation (24 days), no
differences are observed for PBS-DLS 50:50 (Figure 6b), while PBS-DLS 70:30 (Figure 6a) showed
some molecular weight breakdown, especially from the higher molecular weight populations. These
Figure 5. The changes in the pH of degradation media during degradation for PBS-DLS 70:30, PBS-
Figureresults
5. Theare consistent
DLS changes
50:50, and in
PCL
with
the pHa surface erosion mechanism, as one would expect small molecular weight
of degradation
electrospun fiber mats. media during degradation for PBS-DLS 70:30, PBS-DLS
breakdown
50:50, and products to fiber
PCL electrospun be dissolved
mats. in the media.

(a)

(b)

Figure 6. High
Figureresolution SEC chromatograms
6. High resolution SEC chromatogramsof of films (a)PBS-DLS
films (a) PBS-DLS 70-3070-30 and
and (b) (b) PBS-DLS
PBS-DLS 50:50, 50:50,
before (solid line,(solid
before red)line,
andred)
after
and(dashed line,
after (dashed blue)
line, blue)degradation
degradation (24(24 days).
days).
 (here we show only PBS-DLS 70:30, Figure 7). Three characteristic regions of both PBS-DLS 70:30 and
PBS-DLS 50:50 copolyesters are highlighted: (1) the two peaks at 2920 and 2853 cm−1 corresponding
to absorption by methylene –CH2– stretching groups of the aliphatic soft segments, (2) the peak at
1718 cm−1 corresponding to absorption by carbonyl C=O bond stretching, and (3) the peak at 1153
cm−1 corresponding to the ester C–O–C bond stretching. As can be seen from Figure 7 (PBS-DLS 70:30,
Polymers 2018, 10, 688
bulk material 10 of 18
(film) in region 1, at 2920 and 2853 cm−1, there is only a slight increase in absorbance
after 24 days of degradation, which is consistent with the SEC observation where we do not see
changes in Mn and confirms that the polymer is degrading via surface erosion.
The chemical structure of materials was monitored with infrared spectroscopy (ATR–FTIR) (here
Figure 8 shows changes in IR spectra for films prepared from PCL homopolymer. As can be seen
we show only PBS-DLS 70:30, Figure 7). Three characteristic regions of both PBS-DLS 70:30 and
from this figure in region 1, two peaks, 2942, 2862cm−1, indicate the absorbance −by stretching of
PBS-DLS 50:50 copolyesters are highlighted: (1) the two peaks at 2920 and 2853 cm 1−1 corresponding
methylene –CH2– groups of aliphatic soft segments. The peak in region 2, at 1731 cm , and the peak
to absorption−1by methylene –CH2 – stretching groups of the aliphatic soft segments, (2) the peak at
at 1159 cm , corresponds to C=O and C–O–C bonds, respectively, confirming the presence of
1718 cm−1 corresponding to absorption by carbonyl C=O bond stretching, and (3) the peak at 1153 cm−1
carbonyl bonds of ester groups. Due to rapid degradation of the material, it was only possible to
corresponding to the ester C–O–C bond stretching. As can be seen from Figure 7 (PBS-DLS 70:30, bulk
obtain spectra after 24 and 48 h. For PCL films, the absorbance in region 1, at 2942 and 2862 cm−1,
material (film) in region 1, at 2920 and 2853 cm−1 , there is only a slight increase in absorbance after
increases following degradation. In region 2, with the peak at 1731 cm−1, there is a shift of the
24spectrum
days of degradation,
to the lower which
values is consistent
and with cm
band at 1159 the−1SEC observation
, showing where
an increase inwe do not see
absorbance aschanges
observedin
Mafter
n and24confirms that the
h of degradation. polymer is degrading via surface erosion.

Figure 7. FTIR-ATR spectra of PBS DLS 70:30 films, before and after the enzymatic degradation. Inset
Figure 7. FTIR-ATR spectra of PBS DLS 70:30 films, before and after the enzymatic degradation. Inset
(1): 2920, 2853 cm−1−1correspond to aliphatic soft segments backbone. Inset (2): peaks corresponding to
(1): 2920, 2853 cm correspond to aliphatic−soft segments backbone. Inset (2): peaks corresponding to
ester carbonyl backbone: 1718 and 1731 cm −1.1.
ester carbonyl backbone: 1718 and 1731 cm

Figure 8 shows changes in IR spectra for films prepared from PCL homopolymer. As can be
seen from this figure in region 1, two peaks, 2942, 2862cm−1 , indicate the absorbance by stretching
of methylene –CH2 – groups of aliphatic soft segments. The peak in region 2, at 1731 cm−1 , and the
peak at 1159 cm−1 , corresponds to C=O and C–O–C bonds, respectively, confirming the presence of
carbonyl bonds of ester groups. Due to rapid degradation of the material, it was only possible to obtain
spectra after 24 and 48 h. For PCL films, the absorbance in region 1, at 2942 and 2862 cm−1 , increases
following degradation. In region 2, with the peak at 1731 cm−1 , there is a shift of the spectrum to
the lower values and band at 1159 cm−1 , showing an increase in absorbance as observed after 24 h
of degradation.
Polymers 2018, 10, 688 11 of 18
Polymers 2018, 10, x FOR PEER REVIEW 11 of 18

Figure 8. FTIR-ATR spectra of PCL films, before and after the enzymatic degradation, Inset (1) show
Figure 8. FTIR-ATR spectra of PCL films, before and after the enzymatic degradation, Inset (1) show
changes in peaks at 2942 and 2862 cm−−11 ascribed to aliphatic chains. Insets 2: Peak corresponding to
changes in peaks at 2942 and 2862 cm ascribed to aliphatic chains. Insets 2: Peak corresponding to
C=O bonds of ester groups.
C=O bonds of ester groups.
3.3. Thermal Properties and Surface Morphology of Films and Fiber Materials
3.3. Thermal Properties and Surface Morphology of Films and Fiber Materials
Table 2 summarizes the thermal properties of the synthesized copolymers and Figure 9 shows
Table
their DSC2 traces.
summarizesAs canthe be thermal
seen, both properties
copolymers of thearesynthesized
semicrystalline copolymers and Figure
as the thermal 9 shows
behavior is
their DSC traces.
characterized by Asthe can be seen,
presence of aboth
glasscopolymers
transition atare semicrystalline
around −40 °C andasan the thermal behavior
endothermic peak atis
characterized by the presence
higher temperatures related with of athe glass
meltingtransition at around
of crystalline part.− 40 ◦ C and
However, an endothermic
compared peakofat
to the values
higher temperatures (T
PBS homopolymer related
m = 113 with
°C)the melting
[34], of crystalline
the increase in DLA–OH part. However,
content leads compared to the point
to a melting values
ofdecreasing
PBS homopolymer
in temperature(Tm =value113 ◦ C)
and[34],
lower theinincrease
enthalpy in(∆H
DLA–OH
m). Further,content leads 50:50
PBS-DLS to a melting point
has a broad
decreasing in temperature value and lower in
melting peak and reaches the lowest value of melting enthalpy, enthalpy (∆H m ). Further, PBS-DLS 50:50 has
probably due to a decrease in the a broad
melting peak
perfection of and reaches the
the crystalline lowest
phase. value of the
In addition, melting enthalpy, step
glass transition probably
heightdue to aevident
is more decrease forin the
this
system due
perfection to the
of the higher amount
crystalline phase. In of addition,
amorphous thepart
glass(soft segments).
transition stepAlthough PBS homopolymer,
height is more evident for this
as welldue
system as both
to thecopolymers,
higher amount have the capability topart
of amorphous crystallize before melting
(soft segments). (cold-crystallization),
Although PBS homopolymer, the
ascopolymerization with DLA–OH
well as both copolymers, have theaffects the areatounder
capability the exothermic
crystallize before meltingpeak, (cold-crystallization),
(∆Hcc) lowering the
ability
the of the butylene
copolymerization withsuccinate
DLA–OH sequences
affects the to crystallize.
area underIn theaddition,
exothermic the peak,
DSC curve
(∆Hcc )for PBS-DLS
lowering the
50:50 shows a reduction in the cold crystallization temperature at 3
ability of the butylene succinate sequences to crystallize. In addition, the DSC curve for PBS-DLS°C, suggesting that the highest
amount
50:50 showsof DLA-OH
a reduction increased the chain
in the cold mobility oftemperature
crystallization the system [35]. ◦ C, suggesting
at 3Finally, from thethatcooling
the run,
highestit
can be seen
amount that the Tincreased
of DLA-OH c behaves the similarly
chain to the Tm, decreasing
mobility of the system as a[35].
function of the
Finally, DLA–OH
from content.
the cooling run,
Regarding the T g values, the DSC results showed just one Tg value for the PBS-DLS 70:30 and PBS-
it can be seen that the Tc behaves similarly to the Tm , decreasing as a function of the DLA–OH
DLS 50:50 copolymersthe T(Table 2), with both having an intermediate value between Tg of PBS
content. Regarding g values, the DSC results showed just one T g value for the PBS-DLS 70:30
homopolymer (~−43 °C)
and PBS-DLS 50:50 copolymers (Table[27] and neat DLA2), with (~55both
°C) [36].
havingDueantointermediate
this, and taking intobetween
value accountTtheof
g
synthesis
PBS procedure,
homopolymer (~ the
−43most◦ C) likely
[27] and segmental
neat DLA distribution for the
(~55 ◦ C) [36]. Dueobtained
to this,copolymers
and takingisinto a random
account
distribution.
the synthesis procedure, the most likely segmental distribution for the obtained copolymers is a
random distribution.
 Polymers 2018, 10, x FOR PEER REVIEW 12 of 18
Polymers 2018, 10, 688 12 of 18

Table 2. Thermal behavior of PBS-DLS 70:30 and 50:50 copolymers as synthesized (powder).
Table 2. Thermal behavior ofTPBS-DLS
c ∆H70:30
c IIand
Tg 50:50 copolymers
II T cc II ∆Hccas synthesized
II Tm (powder).
II ∆H m a Χc,h
Sample
(J/g) (J/g) (°C) (°C) (J/g) (°C) (J/g) (%)
II T (◦ C)
II T cc II ∆Hcc IITm II ∆Hm aXc,h
PBS-DLS 70:30 powder ∆H c (J/g)62.6
T c (J/g) 51.8 g −43.3 78.8 (J/g)4.4 101.3 57.3 33.6
Sample
(◦ C) ◦
( C) (J/g) (%)
PBS-DLS
PBS-DLS 50:50
70:30 powder
powder 51.8 13.962.6 43.5−43.3−46.4 78.82.9 4.45.8 84.2
101.3 45.1
57.3 17.8
33.6
Crystallization
PBS-DLS temperature
50:50 powder 13.9 (Tc); glass
43.5 transition
−46.4 temperature
2.9 (Tg);5.8cold-crystallization
84.2 temperature
45.1 17.8
(Tcc); meltingtemperature
Crystallization temperature (T(T m); crystallization, cold-crystallization and melting enthalpies (∆Hc, ∆Hcc
c ); glass transition temperature (T g ); cold-crystallization temperature (T cc ); melting
and ∆Hm); (T
temperature crystallinity from hard
m ); crystallization, segments (Χc,h); and
cold-crystallization crystallinity total content
melting enthalpies c , ∆H
(∆H(Χ c,tot); and ∆Hm );from
cccalculated the
crystallinity
from hard segments II calculated from
DSC cooling run;(XIIc,hcalculated
); crystallinity
fromtotal
thecontent
second (XDSC
c,tot ); calculated
heating from
run; the DSC cooling
a calculated from run;
the second DSC
the second DSC heating run; a calculated from the second DSC heating run using Equations (6) and (7).
heating run using Equations (6) and (7).

Figure 9. DSC thermograms. Left: first (solid line) and second heating (dashed line) scans of polymer
Figure 9. DSC thermograms. Left: first (solid line) and second heating (dashed line) scans of polymer
powders (after synthesis) of PBS-DLS 50:50 (blue) and PBS-DLS 70:30 (red) copolymers. Right: cooling
powders (after synthesis) of PBS-DLS 50:50 (blue) and PBS-DLS 70:30 (red) copolymers. Right: cooling
scan of polymer powders (after synthesis) PBS-DLS 50:50 (blue) and PBS-DLS 70:30 (red) copolymers.
scan of polymer powders (after synthesis) PBS-DLS 50:50 (blue) and PBS-DLS 70:30 (red) copolymers.
With respect to the processed materials, films and fibers, before and after degradation, the
With behavior
thermal respect towas
the analyzed
processedfrommaterials,
the firstfilms and The
heating. fibers, before and transition
characteristic after degradation, the thermal
temperatures and
behavior was analyzed
DSC traces fromin
are presented theTable
first heating.
2 and FigureThe characteristic
10 (films) and transition
Figure 11temperatures
(fibers). Theand DSC traces
processing
areparameters
presented(hot melt2pressing
in Table and Figure versus electrospinning
10 (films) and Figurefrom solution)
11 (fibers). did
The not strongly
processing affect the(hot
parameters
melting
melt pressingtemperature, Tm. However,
versus electrospinning fromthe processing
solution) did notof strongly
materials didthe
affect have an effect
melting on theTm .
temperature,
crystallization
However, temperature
the processing Tc, which was
of materials foundan
did have toeffect
be higher for crystallization
on the PBS-DLS 70:30 temperature
film, as compared to
Tc , which
the fibrous mat fabricated from this copolymer (Table 3). On the other hand, for
was found to be higher for PBS-DLS 70:30 film, as compared to the fibrous mat fabricated from this PBS DLS 50:50, with
a lower content
copolymer of On
(Table 3). crystallizable hard for
the other hand, segments,
PBS DLS a 50:50,
higherwithTc was observed
a lower contentforoffibrous and film
crystallizable hard
materials, as compared to the as-synthesized copolymer (powder). Regarding
segments, a higher Tc was observed for fibrous and film materials, as compared to the as-synthesized the crystallinity, it
appears that during the electrospinning process the molecules of PBS-DLS had less time to crystallize,
copolymer (powder). Regarding the crystallinity, it appears that during the electrospinning process
compared to the film forming process, which allows for the crystallization of the PBS, as can be seen
the molecules of PBS-DLS had less time to crystallize, compared to the film forming process, which
from the calculated Χc,h values and the DSC cooling process of the powder materials (Figure 9) that
allows for the crystallization of the PBS, as can be seen from the calculated Xc,h values and the DSC
can simulate the cooling during the hot-press film forming [35].
cooling process of the powder materials (Figure 9) that can simulate the cooling during the hot-press
film forming
Table[35].
3. Thermal behavior of PBS-DLS 70:30 and 50:50 copolymers before and after degradation.
Before degradation After degradation
Sample ITg I Tm I ∆Hm a Χc,h b Χc,tot Tc IITg I Tg I Tm I ∆Hm Tc II Tg

(°C) (°C) (J/g) (%) (%) (°C) (°C) (°C) (°C) (J/g) (°C) (°C)
PCL film −61.2 65.0 77.8 - 57.5 24.9 −71.2 n.o. 61.4 58.7 30.1 n.o.
PBS-DLS
−42.8 97.0 83.3 53.0 - 51.7 −43.3 - - - - -
70:30 powder
PBS-DLS −35.1
−43.0 99.0 50.1 33.0 - 44.9 −43.8 100.8 56.8 50.2 -
70:30 fiber −57.9
PBS-DLS
−40.1 102.0 52.6 33.4 - 51.0 −42.4 −43.7 100.5 60.8 49.5 −43.5
70:30 film
PBS-DLS
−47.0 84.2 61.1 27.7 - 14.0 −46.4 - - - - -
50:50 powder
 on the Tg of the fibers, compared to the as-synthesized powder. In particular, for the PBS-DLS 70:30
fibers, there is a clear split into 2 different Tg, one at −35 °C, closer to that of PBS homopolymer, and
the other at −58 °C, closer to that of neat DLA. This is direct evidence of the hydrolysis of the linkage
between copolymer hard and soft segments contributing to the highest degradation rate observed for
this material.
Polymers 2018, 10, 688This phenomenon appears to start to occur in the PBS-DLS 50:50 fibers 13 after
of 18
degradation, but is not as evident as in the PBS-DLS 70:30.

Figure 10. Left: First heating scan of films before (solid line) and after (dashed line) degradation with
Figure 10. Left: First heating scan of films before (solid line) and after (dashed line) degradation with a
a Tg region detail of degraded samples. Right: Cooling scan of films before (solid line) and after
Tg region detail of degraded samples. Right: Cooling scan of films before (solid line) and after (dashed
(dashed line) degradation. PBS-DLS 50:50 (blue) and PBS-DLS 70:30 (red) copolymers.
line) degradation. PBS-DLS 50:50 (blue) and PBS-DLS 70:30 (red) copolymers.
Polymers 2018, 10, x FOR PEER REVIEW 14 of 18

Figure 11. Left: First heating scan of fibers before (solid line) and after (dashed line) degradation with
Figure 11. Left: First heating scan of fibers before (solid line) and after (dashed line) degradation with a
a Tg region detail of degraded samples. Right: Cooling scan of fibers before (solid line) and after
Tg region detail of degraded samples. Right: Cooling scan of fibers before (solid line) and after (dashed
(dashed line) degradation. PBS-DLS 50:50 (blue) and PBS-DLS 70:30 (red) copolymers.
line) degradation. PBS-DLS 50:50 (blue) and PBS-DLS 70:30 (red) copolymers.

Changes in the surface morphology of samples before and after degradation are shown in
Figures 12–15. Polymeric films were prepared by melting in hot press, followed by cooling. The LSM
micrographs of both bulk materials (Figures 12A and 13A) before degradation show surfaces with
cracks, reflecting the Teflon foil surface used during hot pressing. After degradation (Figures 12B and
13B), the PBS-DLS 70:30 copolyester exhibits banded morphology, attributed to the spherulites
formed during the crystallization. However, the typical periodic distance along the radial direction
is not observed. It is possible that the amorphous region of the spherulites degraded, while the
Polymers 2018, 10, 688 14 of 18

Table 3. Thermal behavior of PBS-DLS 70:30 and 50:50 copolymers before and after degradation.

Before degradation After degradation

Sample
I Tg ITm I ∆Hm a Xc,h b Xc,tot Tc II Tg I Tg ITm I ∆Hm Tc II Tg
(◦ C) (◦ C) (J/g) (%) (%) (◦ C) (◦ C) (◦ C) (◦ C) (J/g) (◦ C) (◦ C)
PCL film −61.2 65.0 77.8 - 57.5 24.9 −71.2 n.o. 61.4 58.7 30.1 n.o.
PBS-DLS 70:30 powder −42.8 97.0 83.3 53.0 - 51.7 −43.3 - - - - -
−35.1
PBS-DLS 70:30 fiber −43.0 99.0 50.1 33.0 - 44.9 −43.8 100.8 56.8 50.2 -
−57.9
PBS-DLS 70:30 film −40.1 102.0 52.6 33.4 - 51.0 −42.4 −43.7 100.5 60.8 49.5 −43.5
PBS-DLS 50:50 powder −47.0 84.2 61.1 27.7 - 14.0 −46.4 - - - - -
PBS-DLS 50:50 fiber −44.1 89.3 41.6 18.8 - 21.0 −44.6 −41.6 84.7 47.7 36.0 −45.7
PBS-DLS 50:50 film −40.6 84.8 59.6 27.0 - 24.6 −44.7 −42.2 84.0 60.4 29.2 −44.1
Glass transition temperature (Tg ); crystallization temperature (Tc ); melting temperature (Tm ); melting enthalpie
(∆Hm ); crystallinity from hard segments (Xc,h ); crystallinity total content (Xc,tot ); I calculated from the first DSC
heating run; calculated from the DSC cooling run; II calculated from the second DSC heating run; a calculated from
the first DSC heating run using Equations (6) and (7); b calculated from the first DSC heating run using Equation (8);
n.o.-transition not observed on the thermograms.

Meanwhile, as can also be seen from DSC thermograms (Figures 10 and 11), the degradation
process has an effect on melting enthalpy, ∆Hm . In general, for processed PBS-DLS 70:30 fibers and
films after degradation, there is an increase in the melting enthalpy, as a direct consequence of the
degradation affecting mainly the amorphous part. The same trend occurs for the PBS-DLS 50:50 fibers;
however, after degradation the films retain the same ∆Hm as before, which is in agreement with the
overall results (mass loss, LSM and SEC) indicating slower degradation for this material. Interestingly,
as can be seen from the inset in Figure 11, the degradation process has a greater effect on the Tg of
the fibers, compared to the as-synthesized powder. In particular, for the PBS-DLS 70:30 fibers, there is
a clear split into 2 different Tg , one at −35 ◦ C, closer to that of PBS homopolymer, and the other at
−58 ◦ C, closer to that of neat DLA. This is direct evidence of the hydrolysis of the linkage between
copolymer hard and soft segments contributing to the highest degradation rate observed for this
material. This phenomenon appears to start to occur in the PBS-DLS 50:50 fibers after degradation, but
is not as evident as in the PBS-DLS 70:30.
Changes in the surface morphology of samples before and after degradation are shown in
Figures 12–15. Polymeric films were prepared by melting in hot press, followed by cooling. The LSM
micrographs of both bulk materials (Figures 12A and 13A) before degradation show surfaces with
cracks, reflecting the Teflon foil surface used during hot pressing. After degradation (Figures 12B
and 13B), the PBS-DLS 70:30 copolyester exhibits banded morphology, attributed to the spherulites
formed during the crystallization. However, the typical periodic distance along the radial direction
is not observed. It is possible that the amorphous region of the spherulites degraded, while the
crystalline component still remains after the degradation. The surface morphology of the PBS-DLS
50:50 copolyester does not show as pronounced spherulitic forms (Figure 13B). Further, this copolyester
shows holes due to the degradation and many un-banded structures. This surface morphology can be
related to higher content of hydrophobic fatty acid soft segments, which may be resistant to enzymatic
lysis. The LSM micrographs are consistent with the DSC analysis, which indicated lower crystallinity
of PBS-DLS 50:50 copolyester.
crystalline component still remains after the degradation. The surface morphology of the PBS-DLS
50:50 copolyester does not show as pronounced spherulitic forms (Figure 13B). Further, this
copolyester shows holes due to the degradation and many un-banded structures. This surface
morphology can be related to higher content of hydrophobic fatty acid soft segments, which may be
resistant
Polymers to 10,
2018, enzymatic
688 lysis. The LSM micrographs are consistent with the DSC analysis, which
15 of 18
indicated lower crystallinity of PBS-DLS 50:50 copolyester.

A B

Polymers 2018, 10, x FOR PEER REVIEW 15 of 18

A B

Figure 12. LSM micrographs of PBS-DLS 70:30 films (A) before and (B) after 24 days of degradation.
Figure
Polymers 10, xLSM
2018, 12. FOR micrographs
PEER REVIEWof PBS-DLS 70:30 films (A) before and (B) after 24 days of degradation.
15 of 18

A B
Figure 13. LSM micrographs of PBS-DLS 50:50 films (A) before and (B) after 24 days of degradation.

The morphology of electrospun mats is presented in Figures 14 and 15. Following degradation,
micrographs of PBS-DLS 70:30 copolyester (Figure 14) reveal many regions with disrupted fibers,
thus reflecting the observations from mass loss and water uptake, which indicated more pronounced
degradation of these samples after 24 days. In the case of PBS-DLS 50:50 (Figure 15), more fibers are
visible and fewer differences from the pre-degradation micrograph can be observed. This is
consistent with the previous results and explained by the greater proportion of hydrophobic fatty
Figure
Figure 13.
13. LSM micrographs
LSMthat
micrographs of
of PBS-DLS
PBS-DLS 50:50
50:50 films
films (A)
(A) before
before and (B) after
and (B) after 24
24 days
days of
of degradation.
degradation.
acid soft segments may segregate to the surface.
The morphology of electrospun mats is presented in Figures 14 and 15. Following degradation,
micrographs of PBS-DLS 70:30 copolyester (Figure 14) reveal many regions with disrupted fibers,

A B
thus reflecting the observations from mass loss and water uptake, which indicated more pronounced
degradation of these samples after 24 days. In the case of PBS-DLS 50:50 (Figure 15), more fibers are
visible and fewer differences from the pre-degradation micrograph can be observed. This is
consistent with the previous results and explained by the greater proportion of hydrophobic fatty
acid soft segments that may segregate to the surface.

A B
Figure 14. LSM micrographs of PBS DLS 70:30 electrospun fibers (A) before and (B) after 24 days of
Figure 14. LSM micrographs of PBS DLS 70:30 electrospun fibers (A) before and (B) after 24 days
degradation.
of degradation.

A B

Figure 14. LSM micrographs of PBS DLS 70:30 electrospun fibers (A) before and (B) after 24 days of
degradation.

A B
Figure 15. LSM micrographs of PBS-DLS 50:50 electrospun fibers (A) before and (B) after 24 days of
degradation.
 Figure
Polymers 2018, 14. LSM micrographs of PBS DLS 70:30 electrospun fibers (A) before and (B) after 24 days of
10, 688 16 of 18
degradation.

A B

Figure
Figure 15.
15. LSM
LSMmicrographs
micrographsofofPBS-DLS
PBS-DLS50:50
50:50electrospun
electrospunfibers (A)
fibers before
(A) and
before (B)(B)
and after 24 24
after days of
days
degradation.
of degradation.

The morphology of electrospun mats is presented in Figures 14 and 15. Following degradation,
micrographs of PBS-DLS 70:30 copolyester (Figure 14) reveal many regions with disrupted fibers,
thus reflecting the observations from mass loss and water uptake, which indicated more pronounced
degradation of these samples after 24 days. In the case of PBS-DLS 50:50 (Figure 15), more fibers are
visible and fewer differences from the pre-degradation micrograph can be observed. This is consistent
with the previous results and explained by the greater proportion of hydrophobic fatty acid soft
segments that may segregate to the surface.

4. Conclusions
Our work describes the enzymatic degradation of poly(butylene succinate-co-dilinoleic succinate)
(PBS-DLS) copolymers, which can be synthesized from sustainable, biobased monomers using
enzymatic catalysis. The incorporation of dimer fatty acid soft segments yields thermoplastic
elastomers with good processability, well-suited to biomedical applications. The enzymatic
degradation studies presented here indicate that an increase in fatty acid soft segments is associated
with lower water uptake and mass loss, indicating slower degradation. Additionally, preparing
samples in the form of electrospun fiber mats had a marked influence on degradation, indicating
that the effective surface area plays a pronounced role, consistent with an erosive degradation
mechanism. Importantly, we do not observe any trend towards acidification of the media during the
degradation process. Overall, we conclude that these copolymers are well suited to applications in
biomedicine, such as tissue engineering, thanks to their attractive processability (melt compression
moulding and electrospinning) and tuneable degradation behaviour, which does not generate acidic
breakdown products.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4360/10/6/688/

s1.
Author Contributions: M.E.F., P.S., A.W. and J.P. conceived and designed the experiments; A.W., A.M.C., and
A.S.O. performed the experiments; A.W., A.S.O., A.M.C., J.P., A.P., P.S. and M.E.F. analyzed the data; P.S., A.W.,
A.S.O. and M.E.F. wrote the paper.
Acknowledgments: This work was financially supported by the National Science Centre, Poland, under
the HARMONIA program under agreement No.UMO-2014/14/M/ST00610. ASO acknowledge the
“Juan de la Cierva”·(FJCI-2015-24405) contract from the Spanish Ministry of Economy, Industry and
Competitiveness (MINECO).
Conflicts of Interest: The authors declare no conflicts of interest
Polymers 2018, 10, 688 17 of 18

References
1. Manavitehrani, I.; Fathi, A.; Badr, H.; Daly, S.; Negahi Shirazi, A.; Dehghani, F. Biomedical Applications of
Biodegradable Polyesters. Polymers (Basel) 2016, 8, 20. [CrossRef]
2. Chen, Q.-Z.; Harding, S.E.; Ali, N.N.; Lyon, A.R.; Boccaccini, A.R. Biomaterials in cardiac tissue engineering:
Ten years of research survey. Mater. Sci. Eng. R Rep. 2008, 59, 1–37. [CrossRef]
3. Woodruff, M.A.; Hutmacher, D.W. The return of a forgotten polymer—Polycaprolactone in the 21st century.
Prog. Polym. Sci. 2010, 35, 1217–1256. [CrossRef]
4. Chen, G.-Q.; Wu, Q. The application of polyhydroxyalkanoates as tissue engineering materials. Biomaterials
2005, 26, 6565–6578. [CrossRef] [PubMed]
5. Gigli, M.; Fabbri, M.; Lotti, N.; Gamberini, R.; Rimini, B.; Munari, A. Poly(butylene succinate)-based
polyesters for biomedical applications: A review. Eur. Polym. J. 2016, 75, 431–460. [CrossRef]
6. Li, H.; Chang, J.; Cao, A.; Wang, J. In vitro Evaluation of Biodegradable Poly(butylene succinate) as a Novel
Biomaterial. Macromol. Biosci. 2005, 5, 433–440. [CrossRef] [PubMed]
7. Wang, H.; Ji, J.; Zhang, W.; Zhang, Y.; Jiang, J.; Wu, Z.; Pu, S.; Chu, P.K. Biocompatibility and bioactivity
of plasma-treated biodegradable poly(butylene succinate). Acta Biomater. 2009, 5, 279–287. [CrossRef]
[PubMed]
8. Mochizuki, M.; Hirami, M. Structural Effects on the Biodegradation of Aliphatic Polyesters.
Polym. Adv. Technol. 1997, 8, 203–209. [CrossRef]
9. Rizzarelli, P.; Puglisi, C.; Montaudo, G. Soil burial and enzymatic degradation in solution of aliphatic
co-polyesters. Polym. Degrad. Stab. 2004, 85, 855–863. [CrossRef]
10. Gigli, M.; Negroni, A.; Soccio, M.; Zanaroli, G.; Lotti, N.; Fava, F.; Munari, A. Influence of chemical and
architectural modifications on the enzymatic hydrolysis of poly(butylene succinate). Green Chem. 2012,
14, 2885. [CrossRef]
11. Marten, E.; Müller, R.-J.; Deckwer, W.-D. Studies on the enzymatic hydrolysis of polyesters I. Low molecular
mass model esters and aliphatic polyesters. Polym. Degrad. Stab. 2003, 80, 485–501. [CrossRef]
12. Gualandi, C.; Soccio, M.; Govoni, M.; Valente, S.; Lotti, N.; Munari, A.; Giordano, E.; Pasquinelli, G.;
Focarete, M.L. Poly(butylene/diethylene glycol succinate) multiblock copolyester as a candidate biomaterial
for soft tissue engineering: Solid-state properties, degradability, and biocompatibility. J. Bioact. Compat. Polym.
2012, 27, 244–264. [CrossRef]
13. Gigli, M.; Lotti, N.; Gazzano, M.; Finelli, L.; Munari, A. Synthesis and characterization of novel poly(butylene
succinate)-based copolyesters designed as potential candidates for soft tissue engineering. Polym. Eng. Sci.
2013, 53, 491–501. [CrossRef]
14. Gualandi, C.; Soccio, M.; Saino, E.; Focarete, M.L.; Lotti, N.; Munari, A.; Moroni, L.; Visai, L. Easily
synthesized novel biodegradable copolyesters with adjustable properties for biomedical applications.
Soft Matter 2012, 8, 5466. [CrossRef]
15. Nerantzaki, M.; Koliakou, I.; Kaloyianni, M.G.; Koumentakou, I.; Siska, E.; Diamanti, E.; Karakassides, M.A.;
Boccaccini, A.R.; Bikiaris, D.N. A biomimetic approach for enhancing adhesion and osteogenic differentiation
of adipose-derived stem cells on poly(butylene succinate) composites with bioactive ceramics and glasses.
Eur. Polym. J. 2017, 87, 159–173. [CrossRef]
16. Kozłowska, A.; Gromadzki, D.; El Fray, M.; Štêpánek, P. Morphology evaluation of biodegradable
copolyesters based on dimerized fatty acid studied by DSC, SAXS and WAXS. Fibres Text. East. Eur.
2008, 16, 85.
17. Nakajima, H.; Dijkstra, P.; Loos, K. The Recent Developments in Biobased Polymers toward General
and Engineering Applications: Polymers that are Upgraded from Biodegradable Polymers, Analogous to
Petroleum-Derived Polymers, and Newly Developed. Polymers (Basel) 2017, 9, 523. [CrossRef]
18. Anastas, P.; Eghbali, N. Green chemistry: Principles and practice. Chem. Soc. Rev. 2010, 39, 301–312.
[CrossRef] [PubMed]
19. Varma, I.K.; Albertsson, A.-C.; Rajkhowa, R.; Srivastava, R.K. Enzyme catalyzed synthesis of polyesters.
Prog. Polym. Sci. 2005, 30, 949–981. [CrossRef]
20. Gross, R.A.; Ganesh, M.; Lu, W. Enzyme-catalysis breathes new life into polyester condensation
polymerizations. Trends Biotechnol. 2010, 28, 435–443. [CrossRef] [PubMed]
Polymers 2018, 10, 688 18 of 18

21. Puskas, J.E.; Sen, M.Y.; Kasper, J.R. Green polymer chemistry: Telechelic poly(ethylene glycol)s via enzymatic
catalysis. J. Polym. Sci. Part A Polym. Chem. 2008, 46, 3024–3028. [CrossRef]
22. Puskas, J.E.; Sen, M.Y.; Seo, K.S.; Khang, G.; Bang Lee, H.; Suk Kim, M. Green Polymer Chemistry Using
Nature’s Catalysts, Enzymes. J. Polym. Sci. Part A Polym. Chem. 2009, 47, 2959–2976. [CrossRef]
23. Castano, M.; Seo, K.S.; Guo, K.; Becker, M.L.; Wesdemiotis, C.; Puskas, J.E. Green polymer chemistry:
Synthesis of symmetric and asymmetric telechelic ethylene glycol oligomers. Polym. Chem. 2015, 6, 1137–1142.
[CrossRef]
24. Sonseca, A.; El Fray, M. Enzymatic synthesis of an electrospinnable poly(butylene succinate-co-dilinoleic
succinate) thermoplastic elastomer. RSC Adv. 2017, 7, 21258–21267. [CrossRef]
25. Azevedo, H.; Reis, R. Understanding the Enzymatic Degradation of Biodegradable Polymers and Strategies
to Control Their Degradation Rate. In Biodegradable Systems in Tissue Engineering and Regenerative Medicine;
CRC Press: Boca Raton, FL, USA, 2004.
26. Solomon, O.F.; Ciutǎ, I.Z. Détermination de la viscosité intrinsèque de solutions de polymères par une simple
détermination de la viscosité. J. Appl. Polym. Sci. 1962, 6, 683–686. [CrossRef]
27. Papageorgiou, G.Z.; Bikiaris, D.N. Synthesis, cocrystallization, and enzymatic degradation of novel
poly(butylene-co-propylene succinate) copolymers. Biomacromolecules 2007, 8, 2437–2449. [CrossRef]
[PubMed]
28. Skrobot, J.; Ignaczak, W.; El Fray, M. Hydrolytic and enzymatic degradation of flexible polymer networks
comprising fatty acid derivatives. Polym. Degrad. Stab. 2015, 120, 368–376. [CrossRef]
29. Correlo, V.M.; Boesel, L.F.; Bhattacharya, M.; Mano, J.F.; Neves, N.M.; Reis, R.L. Properties of melt processed
chitosan and aliphatic polyester blends. Mater. Sci. Eng. A 2005, 403, 57–68. [CrossRef]
30. Jiang, S.; Ji, X.; An, L.; Jiang, B. Crystallization behavior of PCL in hybrid confined environment.
Polymer (Guildf) 2001, 42, 3901–3907. [CrossRef]
31. Poojari, Y.; Clarson, S.J. Thermal stability of Candida antarctica lipase B immobilized on macroporous acrylic
resin particles in organic media. Biocatal. Agric. Biotechnol. 2013, 2, 7–11. [CrossRef]
32. Azim, H.; Dekhterman, A.; Jiang, Z.; Gross, R.A. Candida a ntarctica Lipase B-Catalyzed Synthesis of
Poly(butylene succinate): Shorter Chain Building Blocks Also Work. Biomacromolecules 2006, 7, 3093–3097.
[CrossRef] [PubMed]
33. Debuissy, T.; Pollet, E.; Avérous, L. Enzymatic Synthesis of a Bio-Based Copolyester from Poly(butylene
succinate) and Poly((R)-3-hydroxybutyrate): Study of Reaction Parameters on the Transesterification Rate.
Biomacromolecules 2016, 17, 4054–4063. [CrossRef] [PubMed]
34. Mazzocchetti, L.; Scandola, M.; Jiang, Z. Enzymatic synthesis and structural and thermal properties of
poly(ω-pentadecalactone-co-butylene-co-succinate). Macromolecules 2009, 42, 7811–7819. [CrossRef]
35. Arrieta, M.P.; López, J.; López, D.; Kenny, J.M.; Peponi, L. Biodegradable electrospun bionanocomposite
fibers based on plasticized PLA–PHB blends reinforced with cellulose nanocrystals. Ind. Crops Prod. 2016, 93,
290–301. [CrossRef]
36. Piegat, A.; El Fray, M. Poly(ethylene terephthalate) modification with the monomer from renewable resources.
Polimery 2007, 52, 885–888.

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