Supporting Information
Stabilization of Factor VIII by poly(2-oxazoline) hydrogelsa
Matthias Hartlieb, Stephanie Schubert, Kristian Kempe, Norbert Windhab, Ulrich S.
Schubert*
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M. Hartlieb, K. Kempe, U. S. Schubert
Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University
Jena, Humboldtstrasse 10, 07743, Jena, Germany
E-mail: ulrich.schubert@uni-jena.de
M. Hartlieb, S. Schubert, K. Kempe, Prof. U. S. Schubert
Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Humboldtstrasse 10,
07743, Jena, Germany
S. Schubert
Institute of Pharmacy, Department of Pharmaceutical Technology, Friedrich Schiller
University Jena, Otto-Schott-Straße 41, 07745 Jena, Germany
N. Windhab
Evonik Röhm GmbH, Kirschenallee 45, 64293 Darmstadt, Germany
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1.
Materials and instrumentation
All chemicals and solvents were purchased by Sigma-Aldrich, Merck, Fluka, and Acros. 2Ethyl-2-oxazoline (EtOx) and methyltosylate (MeTos) were distilled to dryness prior use.
Dichloromethane (CH2Cl2) for polymerization was obtained from Roth (99.9%) and dried
using a solvent purification system (Four Solvent Pure Solv 400 liter solvent purifier from
DeMaCo). Phenyl-1,4-bis-oxazoline (BisOx) (1) was synthesized according to literature,[29]
recrystallized from iso-propanol and dried in high vacuum (1∙10-2 mbar) at 50 °C for 3 days.
Human FVIII was purchased from Biotest Pharma GmbH. The assay for FVIII activity
determination (BIOPHEN FVIII:C) was purchased from HYPHEN BioMed. Filter well plates
were purchased from PALL (AcroPrep Advance, 96 well, 1.2 mL, Supor (poly(ethersulfone))
filled poly(propylene)). General well plates were obtained from Greiner Bio-One
(CELLSTAR, 96 well, F-bottom). Centrifuge filter tubes were purchased from Costar (SpinX, Cellulose Acetate membrane).
a
Supporting Information is available online from the Wiley Online Library or from the author.
-1-
The Initiator Sixty single-mode microwave synthesizer from Biotage, equipped with a
noninvasive IR sensor (accuracy: 2%), was used for polymerizations under microwave
irradiation. Microwave vials were heated overnight to 110 °C and allowed to cool to room
temperature under argon atmosphere before usage. All polymerizations were carried out with
temperature control. Size-exclusion chromatography (SEC) was performed on a Shimadzu
system equipped with a SCL-10A system controller, a LC-10AD pump, a RID-10A refractive
index detector and a PSS SDV column with chloroform triethylamine 2-propanol (94:4:2) as
eluent. The column oven was set to 50 °C. Poly(styrene) samples were used as calibration
standards. Proton and carbon NMR spectroscopy (1H- and
13
C-) measurements were
performed on a Bruker AC 300 MHz spectrometer at room temperature, with CDCl3 as
solvent. The chemical shifts are given in ppm relative to the signal from residual nondeuterated solvent. Solid state NMR measurements were performed on a Bruker Avance
400 MHz spectrometer equipped with widebore magnet and double resonance probe head. For
dry samples, 4 mm zircon-oxid rotors were used at 12.5 kHz. Swollen samples were measured
using Kel-F rotors from Bruker-Biospin at 6 kHz. Tetrakis(trimethylsilyl)silan (1H) and
adamantan (13C) were used as standards. Measurement times for all experiments were 6 s.
FTIR spectroscopy was performed on an Affinity-1 FT- IR from Shimadzu, using the
reflection technique. Gas chromatography (GC) measurements were performed on a GC-2010
from Shimadzu. Anisole was used as internal standard to determine the monomer conversion.
Absorption in 96 wellplate formats was recorded on a FLUOstar UV/Vis plate reader from
OPTIMA at 405 nm (BIOPHEN assay) and 595 nm (Bradford assay). For centrifugation of
wellplates, Centrifuge 5804 equipped with an A2 DWP rotor from Eppendorf was used.
Centrifugation of filter tubes was performed using a Multifuge 1S-R from Thermo Scientific.
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2.
Synthesis of linear poly(2-ethyl-2-oxazoline) (PEtOx) (2, 3)
In a microwave vial, MeTos (21 mg, 0.1 mmol) was mixed with EtOx (387 mg, 40 mmol)
under argon atmosphere. After addition of CH2Cl2 (0.481 mL) (and in the case of polymer 3
anisole (0.1 mL, reaction molarity 4 mol/L)), the vial was sealed and heated in a microwave
synthesizer under stirring at 140 °C for 5 min. After cooling to room temperature, the reaction
mixture was diluted with a saturated aqueous solution of Na2CO3 and stirred for 12 h at 80 °C.
The polymer was precipitated in ice cold diethyl ether (100 mL), filtered off, and dried under
high vacuum. 1H-NMR (CDCl3, 300 MHz): δ = 3.45 (s, 78 H), 2.99 (m, 2 H), 2.36 (s, 39 H),
1.11 (s, 59 H) ppm. SEC (eluent: CHCl3/i-propanol/Et3N): Polymer 2) Mn = 3900 g/mol, Mw
= 4,600 g/mol, PDI = 1.19; polymer 3) Mn = 3900 g/mol, Mw = 4,600 g/mol, PDI = 1.19
3.
Hydrogel synthesis: General procedure (4-7)
In a microwave vial, MeTos (15 mg, 0.08 mmol) was mixed with BisOx (173 mg, 0.8 mmol)
and EtOx (1.5 g, 15.2 mmol) under argon atmosphere. After addition of CH2Cl2 (2.4 mL,
reaction molarity 4 mol/L), the vial was sealed and heated in a microwave synthesizer under
stirring at 140 °C for 30 min. The yellow gel was extracted with CH2Cl2 in a soxhlet
apparatus for 24 h, dried in vacuum, swollen in water, and freeze dried. 1H-NMR (250 MHz,
CDCl3): δ = 8.11 (s), 7.55 (s), 4.56 (s), 4.18 (s), 3.58 (s), 2.53 (s), 2.43 (s), 1.24 ppm.
13
C-
NMR (250 MHz, CDCl3): δ = 174, 138, 126, 47, 26, 10 ppm. FTIR: ν (cm-1) = 2978, 2936,
1628, 1474, 1420, 1373, 1246, 1192, 1060.
4.
Kinetic studies of the gelation
Kinetic samples were prepared from a stock solution of EtOx, MeTos, CH2Cl2 and anisol
(reaction molarity 4 mol/L). BisOx was added to every vial separately due to solubility
reasons. After microwave-assisted polymerization (140 °C, varying polymerization times),
the sample (1 mL of stock solution) was diluted with CH2Cl2 to 30 mL to dissolve remaining
-3-
cross-linker. Monomer conversion of EtOx and BisOx was determined using GC-MS with
anisole as internal standard. To prove that anisole does not affect the polymerization, two
linear PEtOxs were synthesized in presence as well as absence of anisole. The comparison of
the NMR spectra and the PDI values determined by SEC (1.17 for polymer 2 and 1.19 for
polymer 3) shows that anisole is suitable as an internal standard for the kinetic measurements.
The polymerization time was varied between 0 and 18 min with an increment of 3 min. To
calculate the rate constant of each monomer, Equations 1 and 2 were applied. The reaction
velocity constant kP of the monomers was determined using Equations 1 and 2 assuming that
the slope of the plot of ln([M]0/[M]t) = f(t) complies with keff.
ln 𝑀0 − ln 𝑀t = 𝑘𝑒𝑓𝑓 ∙ 𝑡
(1)
𝑘𝑒𝑓𝑓 = 𝑘𝑃 ∙ [𝐼]
(2)
Figure 1. First-order kinetic plots of the copolymerization of EtOx and BisOx under
microwave irradiation in dichloromethane at 140 °C using methyltosylate as initiator.
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Figure 2. SEC traces of the purified PEtOx samples obtained from the kinetic experiment
(eluent: CHCl3/i-propanol/NEt3).
Figure 3. Pictures of the different states during hydrogels synthesis: A) Polymerization
solution at room temperature with undissolved BisOx (white precipitate) in microwave vial;
B) Reaction solution during polymerization in the microwave at 140°C with moving stirrer;
C) Increased viscosity at the gel point stops the stirrer; D) Finished gel sample swollen in
CH2Cl2 direct after polymerization.
5.
Solid state NMR of PEtOx hydrogels
Sample 7 was analyzed in dry state using magic angle (54.6°) spinning. The
13
C-NMR
spectrum is depicted in SI Figure 4. At 174 ppm, the signal of the carbonyl carbon is visible.
Also aromatic peaks, resulting from the cross-linker, are present (138 and 127 ppm) as well as
the signals of the polymer backbone (47 ppm) and, of the ethyl side chain (26 and 10 ppm).
For 1H-NMR measurements, the gel was swollen inside a Kel-F rotor in CDCl3. The increased
movement of the polymer chains in the swollen state combined with magic angle spinning
leads to a higher resolution of the 1H-NMR spectrum (SI Figure 5). The signals of the ethyl
-5-
group and the backbone are clearly visible. In the zoomed area, also signals for the aromatic
region are observable at 7.54 and 8.10 ppm. At 4.56 and 4.18 ppm, two small peaks are
present. These signals are associated to the CH2 groups of the oxazoline rings and prove the
incomplete conversion of BisOx in the hydrogel. 1H-NMR spectra of the other samples
showed no significant difference of the peak arrangement. All gel NMR spectra posses signals
of cross-linker beside the peaks of the polymer backbone. The intensity of these signals is
enhanced with increasing content of BisOx relative to the signals of the backbone.
Unfortunately, the resolution of the spectra is insufficient for an integration of the signals, so
that the amount of non-reacted cross-linker cannot be quantified.
Figure 4. Solid state 13C-NMR spectrum of a PEtOx hydrogel (7) (no solvent, 100 MHz).
Figure 5. Solid state 1H-NMR spectrum of PEtOx hydrogel (6) swollen in CDCl3 at 400
MHz.
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6.
Swelling studies
The water uptake of the freeze dried hydrogel samples was measured gravimetrically using
centrifuge filter tubes. The filter tube was saturated with water and the excess solvent was
removed by centrifugation (3,000 rpm, 10 min). The tube was weighted to yield m0. After
addition of the hydrogel sample (10 to 20 mg), the tube was weighted again (m0,gel) and the
sample weight (mgel) was determined using Equation 3.
mgel = m0,gel − m0
(3)
After swelling of the sample in water for 24 h the filter tube was centrifuged (3,000 rpm,
10 min) and weighted (mwet) to determine the mass of the swollen gel (msw) with Equation 4.
msw = mwet − mgel
(4)
The swelling degree (Qeq) was calculated according to Equation 5.
𝑄𝑒𝑞 =
msw −mgel
msw
∙ 100%
(5)
The gelated polymer fraction (FP) was determined using Equation 6.
m
FP = m d ∙ 100%
(6)
th
mth: Theoretical mass of the polymer in the gel (based on the mass of initiator and
monomers),
md: Mass of the dried sample.
7.
Protein stabilization measurements
The amount of hydrogel used for stabilization measurements was determined using the
swelling degree and Equation 7.
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V
mHG = Q ∙ (100% − Qeq )
(7)
eq
The hydrogel was weighted in and placed into a filter well plate. The gel was swollen in trisbuffer (1 M aqueous solution of 2-amino-2-hydroxymethyl-propane-1,3-diol, pH = 7.3,
200 μL) for 1 h and washed by centrifugation (3,000 rpm, 10 min, gradient). To the wet gel
sample, 200 μL of a freshly prepared FVIII solution (1 mg/mL) were added. For the reference
sample without gel, the solution was added to an empty well, which was washed before under
the same conditions. The remaining solution was stored at room temperature, to serve as
reference value. The solution was prepared from a stock solution (2 mg/mL), which was
stored to serve as calibration for the Bradford assay. After a defined waiting time, the protein
solution was gathered by centrifugation (3,000 rpm, 10 min, gradient). The total amount of
protein was determined by the Bradford assay (Bio-Rad Protein Assay), using the micro assay
procedure. The ratio between sample and test solution was 1:1 (120 to 120 μL). The activity
of FVIII was measured by the BIOPHEN assay. The calibration was done with BIOPHEN
PLASMA CALIBRATOR. The activity of FVIII, related to the detected amount of protein,
was calculated using Equation 8.
A
AR = 100% ∙ 𝑐
(8)
𝑃
AR: Relative activity of FVIII,
A: Activity of FVIII determined by BIOPHEN assay,
cP: Detected amount of protein related to reference sample in %.
For all tests, double determinations were performed. The reference of untreated FVIII at t = 0
was set to 100%. The activity is correlated to the total protein amount, which was detected by
the Bradford assay. In detail, a value of 70% implies that 70% of the FVIII, collected after
centrifugation, is still active.
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8.
Concentration effect of protein stabilization
In order to determine the loading capacity of the PEtOx hydrogels and to verify the
concentration dependence of the inactivation of FVIII in aqueous solution, the stabilization
experiments were repeated with different concentrations of FVIII (1, 4, 8, and 16 mg/mL)
using the hydrogel with the best stabilizing ability (6). The results of the stabilization with the
hydrogel were compared with the activity of the protein, which was stored in the filter well
plate without a gel, and with the reference value. The deactivation was observed over 7 d at
three measuring points. The results for the FVIII concentration of 1 mg/mL confirm the trend,
which was already found before.
The incorporation into the polymer scaffold decelerated the inactivation of the protein. No
significant difference is visible between the reference sample and the sample, which was
stored in the filter well plate without a hydrogel. This behavior changes with higher protein
concentrations. At 4 mg/mL, nearly no difference is visible between stabilized and nonstabilized sample.
Figure 6. Concentration dependent stabilization measurement of FVIII in presence of a
hydrogel in a filter matrix (gel), a filter matrix (no gel), and no matrix (reference). For all
measurements with gel, PEtOx hydrogel 6 was used.
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