Injectable Thermoreversible HydrogelsBased on Amphiphilic Polyurethanes:Structure-Property Correlations
C. Ciobanu1, L. M. Gradinaru1�∗, M. Drobota1, F. Quaini3, A. Falco2, C. Frati3, G. Graiani3,D. Madeddu3, C. Lagrasta2, S. Vlad1, M. Bercea1, and L. Sacarescu11“Petru Poni” Institute of Macromolecular Chemistry, 700487 Iasi, Romania2Department of Biomedical, Biotechnological and Translational Sciences, University of Parma, 43126 Parma, Italy3Department of Clinical and Experimental Medicine, University of Parma, 43126 Parma, Italy
A new series of amphiphilic polyurethanes (PUs) based on different polyols and aliphatic diisocyanates was synthesizedand characterized. The chemical structure and molecular characteristics were investigated by ATR-FTIR and GPC anal-ysis. Wetting properties were evaluated by static contact angle determinations. Aqueous solution of these PUs presentsthermoreversible behavior at a temperature close to the human body. Water-soluble PUs present very low critical micelleconcentration as determined by surface tension measurements. The temperature-induced micellization is a progressiveprocess followed by gelation which was due to the ordered packing of micelles under a cubic or hexagonal phase. Theassociation of micelles in ordered crystalline structures was confirmed by ATR-FTIR and SAXS experiments. The aver-age size and distribution of micelles of PUs macromolecules in water as a function of temperatures were measuredusing DLS technique. The self-aggregation and temperature induced gelation was investigated through 1H-RMN anddynamic oscillatory measurements. In vitro studies show that thermoreversible polyurethane hydrogels are biocompatibleand biodegradable. These results suggest that injectable thermoreversible PUs can be used as scaffolds in restoring theheart muscle.
INTRODUCTIONInjectable thermoreversible hydrogels with low criticalsolution temperature (LCST), about 37 �C, are highlydesirable clinically as they can be introduced into a bodyusing endoscopic or percutaneous procedures.1 They alsopresent a great interest in drug delivery, tissue engineer-ing owing to their low interfacial tension, high oxygenpermeability and mechanical properties like those of nat-ural tissues.2–4 In situ gelling hydrogels have been widelystudied in many pharmaceutical and biomedical applica-tions such as delivery systems of drugs, cells, proteins andgenes, scaffolds for tissue engineering.1�5–8 The character-istic property of these polymers is their ability to formlow viscosity aqueous solutions at ambient temperature
∗Author to whom correspondence should be addressed.Email: [email protected]: 10 December 2013Accepted: 24 June 2014
and gelled at a temperature close to that of the humanbody. Thermoreversible hydrogels can be easily loadedwith bioactive molecules or cells, playing a key role inminimally invasive surgery, thereby surgical proceduresare obviated.9–14 Such an instance of thermoreversiblepolymers are triblock copolymers of poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide) (PEO–PPO–PEO), commercially known as Pluronic or Poloxamer,which have been widely investigated for controlled drugdelivery,15�16 tissue engineering.17 However, their lowmechanical strength and poor stability make their hydro-gels inadequate for some applications.Lately, polyurethanes have been considered one of the
most versatile materials used for a broad range of biomed-ical applications.18–23 Generally, polyurethane is obtainedby polyaddition reaction between a macrodiol, a diiso-cyanate and a chain extender. By changing the type ofmonomer and their ratio, a wide range of materials withdifferent characteristics can be achieved. Due to their
12 J. Hydrogels 2015, Vol. 1, No. 1 2377-6293/2015/1/012/014 doi:10.1166/jh.2015.1004
Ciobanu et al. Injectable Thermoreversible Hydrogels Based on Amphiphilic Polyurethanes: Structure-Property Correlations
biocompatibility, low cytotoxicity and good mechanicalproperties, polyurethanes are very attractive for biomedi-cal applications.24�25 By combining the triblock copolymerthermoreversibility and polyurethane properties biomateri-als with improved properties can be provided to be suc-cessfully used in various applications, especially in drugdelivery systems26–31 and tissue engineering.32
The aim of this work was the synthesis of some newthermoreversible injectable polyurethane hydrogels and thestudies of gelation, micellization, crystallization and bio-compatibility. Thus, three polyurethanes were synthesizedstarting from isopropyl ricinoleat diol, bis[2-(2-hydroxy-ethyldimethylamino)ethyl](polyethylenoxy)diphosphate(bifunctional derivative of phosphatidylcholine (BPHC)),poly(ethylene oxide)–poly(propylene oxide)–poly(ethyleneoxide) triblock copolymer-ricinoleat diol, poly(ethyleneoxide)–poly(propylene oxide)–poly(ethylene oxide) tri-block copolymer diol and two diisoyanates. For thein vitro study, polyurethane hydrogels were loaded withRGD (Arg-Gly-Asp), soluble elastin from bovine neckligament, Hepatocyte Growth Factor human (HGF) andHuman Bone Morphogenetic Protein 4 (BMP4). Thebehavior of polyurethanes and their aqueous solutionswere investigated by gel permeation chromatography(GPC), ATR-FTIR spectroscopy, rheometry, 1H-NMRspectroscopy, tensiometry, dynamic light scattering (DLS),small-angle X-ray scattering (SAXS) and fluorescencemicroscopy.
EXPERIMENTAL DETAILSMaterialsIsopropyl ricinoleat diol with hydroxyl number of 189 mgKOH/g (Mn 358 Da) (IzPR), poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide)-ricinoleatdiol with Mn 6140 Da (P-RD) and bis[2-(2-hydroxy-ethyldimethylamino)ethyl] (polyethylenoxy)diphosphate(bifunctional derivative of phosphatidylcholine (BPHC)with Mn 592 Da were synthesized in our laboratory.Poly(ethylene oxide)–poly(propylene oxide)–poly(ethyleneoxide) (PEO–PPO–PEO) or Pluronic P 123 with Mn
5800 Da (P) was purchased from Sigma-Aldrich and usedas received. Ethyl ester L-lysine diisocyanate (LDI) wassupplied by Infine Chemicals, China. 1,6-Hexamethylenediisocyanate (HDI) was purchased from Aldrich. Thediisocyanates were freshly distilled before synthesis. Allthe other chemicals and reagents were of the highestpurity available and used without further purification.
Synthesis of PolyurethanesThe detailed experimental procedure of the polyurethanesynthesis has been previously described.26�27 Briefly, poly-ols in 1:1 molar ratio were dehydrated at 80 �C and0.1 mmHg for 3 hours. Then the reactor was broughtto atmospheric pressure with purified N2. The requiredamount of diisocyanate was added dropwise to the mixture
so that the molar ratio NCO:OH was 0.9:1. The reac-tion between diisocyanate and macrodiol took place for8 h under stirring and dry nitrogen atmosphere at 70 �C.Figure 1 shows the structure of these polyurethanes.
Gel AchievementWater solutions (%wt) were prepared by mixing the poly-mer with distilled water and periodically stirring in an icewater mixture at ∼2 �C, until complete dissolution. Thesolutions were then refrigerated at 4 �C for 1–2 days toensure that all polymers were fully dissolved. Gels werethen generated by raising temperature.
Characterization of Polyurethanes andTheir SolutionsThe characterization of functional groups was carried outby FTIR analysis using a Bruker Vertex 70 type spectrom-eter (U.S.), equipped with a diamond ATR device (GoldenGate, Bruker) and provided with software for spectral pro-cessing. The sample surface was scanned in the range400–4000 cm−1, at a 45� angle. The FTIR spectra wererecorded at a constant temperature of 25 �C. For the kinet-ics study, the experiment was performed at a temperaturebetween 25 and 40 �C using a Specac’s High Tempera-ture Golden Gate™ ATR Accessory which is a high per-formance single reflection (45� angle) monolithic diamondATR product offer for spectroscopic sample analysis.
1H-NMR spectra were acquired using a Bruker AvanceDRX 400 MHz Spectrometer equipped with a 5 mm QNPdirect detection probe and z-gradients. The spectra wererecorded in D2O at different temperatures. For each tem-perature, the solution was equilibrated for 10 min beforeacquiring the data. The chemical shifts are reported as� values (ppm) relative to the peak of TSP (0 ppm).The average molecular weight was determined by
gel permeation chromatography (GPC) at 25 �C witha GPC PL-EMD 950 evaporative mass detector. Sam-ples were eluted with DMF at 0.7 mL/min. Calibrationwas performed with narrow polydispersity polystyrenestandards (Polymers Laboratories Ltd.). Computer anal-ysis of the elution data was based on normalization ofchromatograms.
Surface Property CharacterizationThe PUs surface was evaluated by static contact anglemeasurements using sessile-drop method and a CAM-101(KSV Instruments, Helsinki, Finland) contact angle mea-surement system equipped with a liquid dispenser, videocamera and drop-shape analysis software. For each drop10 photos were recorded at an interval of 0.016 s. Allthe measurements were performed at room temperature.The solvents used for these studies were double distilledwater, ethylene glycol and di-iodomethane. For each liq-uid, three different surface regions were selected to obtaina statistical result.
J. Hydrogels 1, 12–25, 2015 13
Injectable Thermoreversible Hydrogels Based on Amphiphilic Polyurethanes: Structure-Property Correlations Ciobanu et al.
P 1 : A I1 E I1 A I1 E
P 3 : D I1 E I1 D I1 E
P 2 : A I2 E I2 B I2 E
where:
A = IzRD: CH3 CH2 CH CH2
OH
CH CH CH2 C
O
O CH
CH3
CH2 OH5 7
O C N CH2 CH2 CH2 CH2 CH N
COO
C O
CH2 CH3
I1 = LDI:
B = BPHC: HO CH2 CH2 N
CH3
CH3
CH2 CH2 O P
O
O
O
CH2 CH2 O P
O
O
O
CH2 CH2 N
CH3
CH3
CH2 CH2 OHn
n = 4.7
D = P-RD:57
HO CH2 CH2 O CH
CH3
CH2 O CH2 CH2 On m n
C
O
CH2 CH CH CH2 CH
OH
CH2 CH3
E = P: HO CH2 CH2 O CH
CH3
CH2 O CH2 CH2 On m n
H
I2 = HDI: O C N CH2 CH2 CH2 CH2 CH2 CH2 N C O
Figure 1. Structures of polyurethanes.
Critical Micelle Concentration DeterminationThe critical micelle concentrations (CMC) of the syn-thesized polyurethanes were investigated by surface ten-sion measurements of initial 0.1%wt aqueous solutions ofpolyurethanes at different temperatures, using Wilhelmymethod and a Sigma 700 tensiometer (KSV Instruments,Finland). The automatic tensiometer was equipped witha platinum plate which was flamed, cleaned and washedwith double distilled water and ethanol several times. Dis-tilled water was used to calibrate the instrument. In thisstudy, the average value was 73± 0.2 mN/m at roomtemperature.
Dynamic Light Scattering MeasurementsThe average size and distribution of micelles of PUsmacromolecules in water as a function of temperatureswere measured using dynamic light scattering (DLS) tech-nique (Zetasizer model Nano ZS (Malvern Instruments,UK)) with red laser 633 nm (He/Ne). The system uses non-invasive back scatter (NIBS) technology wherein opticsare not in contact with the sample, back scattered lightbeing detected. The use of NIBS technology reduces mul-tiple scattering effects and, consequently, size distribu-tions in higher concentrations of sample can be measured.This is the system for which the Mie method is appliedover the whole measuring range from 0.6 nm to 6 �m.Using this technique, the random movement of particlesis monitored according to fluctuations in the intensity ofthe scattered light. Analysis of these fluctuations allows
for the determination of diffusion coefficients, which, inturn, yield the particle size through the Stokes-Einsteinequation:
DH = kBT
3��D(1)
where DH is the hydrodynamic diameter, kB the Boltzmannconstant, T the temperature, � the viscosity and D thediffusion coefficient.A concentration (0.01%wt) around CMC was chosen to
study micellar aggregation as a function of temperature.The instrument was operated at four different tempera-tures: 25, 37, 38 and 40 �C.The Zeta potential was estimated with the same equip-
ment (Zetasizer model Nano ZS; Malvern Instruments,UK). The instrument works with the technique of laserDoppler electrophoresis (4 mW He–Ne, 633 nm). Resultswere automatically calculated from electrophoretic mobil-ity using the Smoluchowski equation:
� = ��/� and k� 1 (2)
where � is the zeta potential, � the viscosity, � the elec-trophoretic mobility, � the dielectric constant of themedium and k, the Debye–Huckel parameter and theparticle radius, respectively.
Rheological MeasurementsSteady shear and dynamic oscillatory investigations wereperformed with a Bohlin CVO rheometer using a parallel-plate geometry (the upper plate radius was 30 mm and
14 J. Hydrogels 1, 12–25, 2015
Ciobanu et al. Injectable Thermoreversible Hydrogels Based on Amphiphilic Polyurethanes: Structure-Property Correlations
the gap between the plates 500 �m). The rheometer wasequipped with a Peltier temperature controller. To preventwater evaporation, an anti-evaporation device was used.Prior to oscillatory measurements, the amplitude sweeptests were made at a constant frequency () of 1 rad/sto establish the linear viscoelastic range when the stor-age (G′) and loss (G′′) moduli as well as the loss tangent(tan�) were independent of the strain amplitude. The lin-ear viscoelastic regime is reached for shear stress lowerthan 1 Pa. For the temperature sweep tests, the temperaturerange was between 4–70 �C. To determine the behaviorof the polyurethane samples in conditions close to thatof the human body (37 �C), the evolution of viscoelas-tic parameters in time was followed at constant frequencyof oscillation ( = 1 rad/s) and shear stress (1 Pa). Forrheological measurements, hydrogels aqueous solutions of15%wt concentration were prepared and then stored at4 �C overnight before the experiments were performed.
SAXS MeasurementsSAXS experiments were performed using a NanostarU-Bruker system equipped with a Vantec 3000 detector(diameter of 200 mm) and an X-ray I�S microsource withcopper anode. The wavelength of Cu K was �= 1:54 Åand the X-ray beam was collimated by three pinholes.The sample-to-detector distance was 68 cm allowing mea-surements with qmin (scattering vector) of 0.01 Å−1. Theangular scale was calibrated by the scattering peaks ofsilver beheaded. The data analysis was carried out usingATSAS 2.5.1 software packages PRIMUS and PEAK.33
For the study of crystalline structures,34 the measurementswere performed at a concentration of 30%wt. The PU solu-tion were sealed in a quartz capillary and then brought totemperature with a heating rate of 5 �C/min. The sampleswere equilibrated for 5 min and exposed for 3000 s.
In Vitro StudiesFor biological characterization, aqueous solutions of P1and P2 (152 mg/mL) were loaded with soluble elastin(46 �g/mL), RGD (6 �g/mL), HGF human (0.1 �g/mL)and BMP-4 human (0.2 �g/mL).
Cardiac Progenitor Cells (rCPCs) were isolated from3 months old male Wistar rat hearts according to a method-ology often employed by our laboratory35 and originallydescribed by Beltrami et al.36 To allow cell tracking,rCPCs were also isolated from the heart of rats carry-ing the transgene encoding for the Green Fluorescent Pro-tein (EGFP), kindly provided by Okabe.37 Briefly, therat heart was quickly excised from anaesthetized animalsand hanged by an aortic cannula to the perfusion sys-tem. The heart was dissociated with Collagenase type II(Worthington Biochemical Corporation, USA) at 37 �C for20 min and minced. After centrifugation at 300 rpm toseparate cardiomyocytes, the cell supernatant was placedon Percoll (Sigma, Italy) gradient and the cell layer visual-ized at the interface of the desired gradient was centrifuged
at 1000 rpm. Cells were resuspended in 10 mL IMDMculture medium supplemented with 1% P/S, 1% I/T/S,10% FBS and cultured in Petri dishes (Corning, USA)at 37 �C–5% CO2 for their amplification. Daily, micro-scopic observation of cultures showed the growth of twodifferent adherent cell populations, one with fibroblast-likeand one with monomorphic blast-like characteristics. Thislatter population constitutes the so-called Cardiac Pro-genitor Cells (CPCs) provided by clonogenic growth andmultipotency. CPCs at P1 to P2 were employed for thisstudy. These cells were also amplified for several passagesand cryo-preserved in aliquots in a medium composed ofFBS supplemented with 1% Dimethylsulphoxide (DMSO,Sigma, Italy). CellTracker CM-DiI (Invitrogen, C-7001) isa DiI derivative that is more water-soluble than DiI andcontains a thiol-reactive chloromethyl moiety (CM) thatallows the dye to covalently bind to cellular thiols. Thus,unlike other membrane stains, this label is well retainedin the cells throughout several mitotic divisions and cellto cell contact does not allow dye diffusion. rCPCs wereincubated in 1–2 �M working solution for 15 min at 37 �C,and then for an additional 15 min at 4 �C. Incubation atthis lower temperature appears to allow the dye to labelthe plasma membrane but slows down endocytosis, thusreducing dye localization into cytoplasmic vesicles. Afterlabeling, cells were washed with phosphate-buffered saline(PBS), resuspended in fresh medium and used for experi-mental plan.In order to study the cell culture on both scaffolds, ster-
ile thermoreversible hydrogels (250 �L/well) were put in a24-well culture plate (Corning, USA) and left overnight inthe incubator to solidify. DiI labelled rCPCs were seededat 25× 103 cells/cm2 concentration onto P1 or P2 scaf-folds. Cells cultured in standard conditions were consid-ered as control. Cell loaded hydrogels were evaluated for48 hours, 7 days and 14 days after cell plating. At eachtime, point hydrogels were dissolved by adding a largevolume of PBS and cells were collected. Cell suspensionwas centrifuged at 1100 rpm for 5 min at 4 �C to main-tain hydrogels in a liquid state. Afterwards, rCPCs werere-suspended in 1 ml of fresh medium, 10 �L of cellsuspension were collected and diluted twice with TrypanBlue (Sigma Aldrich, Milan, Italy) to evaluate cell via-bility. Cells were counted using a hemocytometer and thefraction of viable cells was calculated.
RESULTS AND DISCUSSIONCharacterization of Polyurethane HydrogelsThe average numbers of molecular weights (Mn) weredetermined using gel permeation chromatography (GPC).Table I shows the molecular weights of the synthe-sized polyurethanes. The average numbers of molecularweight (Mn) of these polyurethanes ranges from 17.50 to19.06 KDa. P3 has the lowest average number of molecu-lar weight, and, in contrast, P2 has the highest Mn.
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Injectable Thermoreversible Hydrogels Based on Amphiphilic Polyurethanes: Structure-Property Correlations Ciobanu et al.
Table I. Molecular characteristic and surface parameters of PUs samples.
Notes: bEach value is expressed as mean±standard error; eDetermined by GPC measurements.
Surface Morphological StudiesThe biomaterial surface is the first component of theimplant that comes into contact with biological cells orfluids. Thus, biocompatibility will be influenced mainlyby surface characteristics of the biomaterial, wettabilityand surface energy characteristics, in particular.38 Sur-face thermodynamics can be evaluated by contact anglemeasurements at the liquid–solid interface. The surfacehydrophilicity of dry PUs, as characterized by static watercontact angle, is reported in Table I.Contact angles are usually calculated using the Young
equation:�LV cos = �SV−�SL (3)
where �SV is the surface energy, �SL is the solid—dropinterfacial tension, �LV is the liquid–vapor surface tension,and cos is the drop—surface contact angle.39–41
Table I shows that P1 and P3 have water contact anglevalues of 82.74 and 83.18 degree, respectively, while P2has a 92.96 degree, although P2 has a ionic structureand is more soluble in water as compared to the othertwo polymers. The surfaces of these polymers becomemore hydrophobic in contact with air and also, eventhe hydrophobicity of P2 increased, is more hydrophilic.Based on these observations, and considering the papers ofHolly42 and Yasuda,42a this phenomenon can be explainedby the fact that the surface structure (namely, the ratio ofpolar and hydrophobic groups on the surface of PU) isdictated both by macromolecular chain mobility and themedium it comes in contact with.When the static contact angle was measured on the
PUs gel surfaces, its values decreased (P1= 40, P2 = 45,P3= 35 degree), therefore the surfaces are hydrophilic.This is due to the high mobility of macromolecules fromthe polymer surfaces.Surface free energy was determined from the con-
tact angle measurements using Owens-Wendt-Rabel andKaelbe methods.39 The surface tensions can be dividedinto two components: the polar component (�p) includ-ing two types of coulomb interactions, i.e., dipole–dipoleand dipole-induced dipole, and dispersive components (�d)represent the van der Waals interactions.
Wa = 2�√�dSV�
dLV+
√�pSV�
pLV� (4)
�SV = �pSV+�d
SV (5)
where Wa is the work of adhesion.
The calculations of these two components of surfacefree energy have given more detailed information on PUsurface properties. It is clear from the results (Table I) thatthe dispersive component is higher than the polar one atthe samples for which only the diisocyanate differs (P1and P3). The P2 sample has a higher value of the polarcomponent than the dispersive one, but the surface freeenergy is lesser as compared with other samples. This phe-nomenon is due to the fact that any increase in contactangle values leads to a decrease in surface energy, P2 hasa higher water contact angle value. The work of adhe-sion (Wa) of liquid to polymer surface can be calculatedfrom the contact angle data. As can be seen, Wa of the PUsamples decreases with increasing contact angle values.The interfacial solid–liquid tension (�SL) is given by the
equations:
�SV = �SV+�LV−2�√�pSV�
pLV+
√�dSV�
dLV� (6)
From Eqs. (1) and (4) results:
�LV�1+ cos �= 2√�pSV�
pLV+2
√�dSV�
dLV (7)
The interfacial solid–liquid tension (�SL) has higher val-ues at the samples P1, P2 (20–24 mN/m) and a valueof 17 mN/m at the sample P3. The values of interfacialtension could be high or low, depending on the attrac-tive forces between molecules of the liquid and solid.The lower the attraction between the liquid and the solid,the higher the interfacial tension and the less spreadingof the liquid over the solid surface.38
Critical surface tension (�c) is a graphically extrapolatedcharacteristic for any solid material, at cos = 1, whenthe liquid wets the surface perfectly.21–23�43 This parameterdefines the wettability of a solid by noting the lowest sur-face tension at which a liquid can exhibit a contact anglegreater than zero on that solid.44 P1 and P3 have a criticalsurface tension between 31–37 mN/m, and for the P2, thevalue is around 20 mN/m (19.97 mN/m), as a consequenceof its ionic structure.These data and the literature,38 explain why these
materials are generally recommended in biomedicalapplications.
Study of the Gelation ProcessATR-FTIR StudyThe contribution of water to form hydrogels as a func-tion of temperature increase, in 28–40 �C region, were
16 J. Hydrogels 1, 12–25, 2015
Ciobanu et al. Injectable Thermoreversible Hydrogels Based on Amphiphilic Polyurethanes: Structure-Property Correlations
investigated by means of ATR-FTIR analyses to threesolutions of polyurethanes 15%wt in water with differentpolyurethanes named P1, P2, P3 (Figs. 2–4). These figureslist polyurethane and their gels, and each polyurethane iscontrol spectrum.
The characteristic bands of P1, P2, and P3 control sam-ples are illustrated in Table II.
The bands at 3505 (P1), 3514 (P2) and 3500 (P3) cm−1
of controls, corresponding to the O–H symmetrical stretch-ing vibrations of water,45�46 shifted to lower wavenumbersin hydrogels with 144 (P1) and 108 cm−1, respectivelywhile the peak of P3 increases with 32 �C in the end(Figs. 2–4). The surfaces of these bands increased up to38 �C, then gels began to degrade over this temperature forP1 and P2 and the surface increased up to 33–34 �C, thendecreased up to 36 �C and it remained constant between37–40 �C. It can be seen that the wavenumber decreasedalong with an increase in temperature for P1 and P3 gels,while for P2 it decreased slightly to 31 �C, then increasedup to 36 �C and decreased slightly to 39 �C (Figs. 5 and 6).This behavior of gels is given by phosphate ion of polymerlength.
The bands at 2781, 2690 (P1) and 2780, 2697 (P3) cm−1
show a phases separation as a temperature function, yetthis phenomenon has not been observed for the P2 sam-ple; this is, perhaps, due to the strongest intermolecularforces produced by the phosphate ions of phosphatidyl-choline. It can also be observed that �O–H bendingvibration of water at 1640, 1642, 1648, cm−1 shifted to1628, 1637 and 1628 cm−1, respectively and the amide IIat 1550 (P1), 1530 (P2) and 1548 (P3) cm−1 of con-trol samples shifted to higher wavenumbers with 31, 18and 32 cm−1 and the asymmetric and symmetric stretch-ing vibrations (CH2–O–CH2 and CH2–CH(CH3�–O–CH2�bands shifted slightly to lower wavenumbers with 15 (P1),24 (P2) and 20 (P3) cm−1, respectively.
3500 3000 1500 1000
0.0
0.2
0.4
0.6
0.8
3505
Ab
sorb
ance
, a.u
.
Wavenumber, cm–1
28º29º30º31º32º33º34º35º36º37º38º39º40ºSMP- P1
33663265
29692938
2903
2874
27802696
1735 1640 1550
16281581
1457
1509
1374
13451296
1254
1115
1012933
842
1100
Figure 2. ATR-FTIR spectra of P1 sample (at room tempera-ture) and its hydrogels at 28–40 �C.
3500 3000 1500 1000
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Ab
sorb
ance
, a.u.
Wavenumber, cm–1
28º29º30º31º32º33º34º35º36º37º38º39º40ºSMP- P 2
3514
3486
2972
2931
2894
2870
17221637
1642 1530
1458
1374
1344
12961243
1108
1094
1017932
844
Figure 3. ATR-FTIR spectra of P2 sample (at room tempera-ture) and its hydrogels at 28–40 �C.
These results suggest that in the polyurethane structureoccur higher molecular geometry changes and an orderedgel is formed with the water molecules which depends ofthe chemical polyurethane structure and temperature.
1H-NMR StudyNuclear magnetic resonance (NMR) spectroscopy is apowerful method for investigations of stimuli-responsivepolymer systems in aqueous solutions and gels.51–53 Themethod uses the weak interaction of nuclear spins withtheir molecular environment to give information aboutmolecular structure and dynamics.54–57 1H NMR is a suit-able technique used to analyze the variation of chemi-cal shifts,58�59 spin relaxation times,60 or intensity61–63 ofthe NMR signals along with temperature, concentration,
3500 3000 1500 1000
0.0
0.2
0.4
0.6
0.8
1.0
Ab
sorb
ance
a.u
.
Wavenumber, cm–1
28º29º30º31º32º33º34º35º36º37º38º39º40ºSMP-P3
3500
33923264
2977
2935
2894
2864
2780
2696
1722
1648
1628
1539
1580
1115
1095
1458
1373
13491295
1244 1010938
840
Figure 4. ATR-FTIR spectra of P3 sample (at room tempera-ture) and its hydrogels 28–40 �C.
J. Hydrogels 1, 12–25, 2015 17
Injectable Thermoreversible Hydrogels Based on Amphiphilic Polyurethanes: Structure-Property Correlations Ciobanu et al.
Table II. The characteristic bands of PUs control samples.
Wavenumber, cm−1
P1 P2 P3 Assignments References
3505 3514 3500 �O–Hsym of water; these polyurethanes can absorb moisture from the air [45, 46]3375sh 3350sh 3350 �N–H hydrogen-bonded [47–50]2970 2970 2870 �C–Hasym of CH3 groups [27, 49, 50]2930 2930 2930 �C–Hasym of CH2 groups [27, 49, 50]2895sh 2895sh 2895sh �C–Hsym of CH3 groups [27, 49, 50]2872 2868 2869 �C–Hsym of CH2 groups [27, 49, 50]1737 1720 1738sh �C O free of P1 and P2 and carbonyl-bonded for P3 of –NH–COO– [27, 47–50]1726sh 1700sh 1724 �C O carbonyl-bonded of urethane-urethane and urethane-hydroxyl (amide I)1643 1654 1646 �O–H of water [45, 46]1556 1539 1534 �N–C+�N–H amide II; there are two overlapping bands [27, 47–50]1476sh 1476sh 1476sh �C–Hsci of CH2 groups of polyethylene ether segments [27,47-50]1462 1462 1462 �C–Hsci of CH2 groups of polypropylene ether and ricinoleat segments1455 1455 1455 �C–Hasym of CH3 groups1373 1373 1373 �C–Hsym of CH3 groups, is a characteristic band [27, 47–50]1347 1347 1347 �C–H swing vibrations of –O–(CH)3 CH–CH2-groups of polypropylene ether segments [27, 47–50]1300 1300 1300 �C–Ht in plane twisting vibration of CH2 groups [27, 47–50]1282 1282 1282 �C–Hw out-of-plan wagging vibration of –CH2–O-groups [27, 47–50]1251 1251 1251 �N–C+�N–H amide III [27, 47–50]1115 1108 1115 �C–Oasym of CH2–O–CH2 and CH2–O–HC(CH3) [27, 47–50]1012 1017 1010 �C–Osym of ethers [27, 47–50]933 938 932 Hydrogen-bonded O–H out-of-plane bending [49]842 840 844
or solvent composition. The collected data can be used toobtain information on the change in molecules motion, thenature of intermolecular interactions, critical concentrationvalues, or thermodynamic parameters associated with gelformation. In order to observe the possible changes of sig-nals which could be due to the aggregation of molecules,the 1H NMR spectrum of PUs solution was measured as afunction of temperature in deuterated water (Fig. 7). Thetemperatures selected for this study were room tempera-ture (25 �C) and the temperatures close to the human body
Figure 5. �H–O surface absorption as a function of temper-ature in 3685–3080 cm−1 region for P1, P2 and P3 hydrogelsamples.
(37, 38, 39 and 40 �C, respectively), because these are ofinterest for biomaterials.In sol state, PU and water macromolecules move more
freely than in a gel state. During sol–gel transition, thehydrogen bonding between water molecules and polymerschanges, due to the dehydration of hydrophobic domains.Then, the movement of polymer segments is restricted bygelation process. Therefore, the deuterium nuclei will beaffected, leading to the changes in chemical shift of peakswith temperature.58�59 Thus, the proton signals are shiftedtowards the lower chemical shifts with increasing temper-atures. For the P2 and P1 sample, the characteristic peak
Figure 6. Wavenumber of the peaks as a function of the tem-perature for P1, P2 and P3 hydrogel samples.
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Figure 7. 1H-NMR spectra of PUs solutions in D2O at varioustemperatures. Insets show the variations of chemical shifts ofthe main groups with temperature.
of water molecules at � 4.72 ppm at 25 �C shifted to� 4.57 ppm at 37 �C, � 4.56 ppm at 38 �C, � 4.55 ppmat 39 �C, and � 4.54 ppm at 40 �C. For the P3 sam-ple, the water molecules peak is located at � 4.75 ppm at25 �C and is shifted to � 4.60 ppm at 37 �C, � 4.59 ppmat 38 �C, � 4.58 ppm at 39 �C, and � 4.547 ppm at40 �C (see Fig. 7). The change in chemical shift wasmost pronounced during sol–gel transition (between 25and 37 �C recorded spectra), the chemical shift movingwith 0.15 ppm. After the gel is reached, molecules move
more slowly and proton signals (between 37–40 �C) areshifted only with 0.01 ppm. Also, the same trend (decreas-ing chemical shift wit increasing temperature) has thepeaks assigned to methylene (3.3–3.7 ppm) and methyl(1.01–1.34 ppm) protons.In conclusion, the 1H-NMR profile is influenced by
polymer-water interactions, which takes place via thehydrogen bonding interaction between the proton of thepolymer segment and water molecule, leading to the down-field shift with increasing temperature.
Critical Micelle Concentration (CMC) andSurface BehaviourCMC can be determined by surface tensionmeasurements.64 Like surfactants, these PUs exhibit a spe-cific surface tension versus concentration curve.16 Whenthe concentration is below the CMC, the surface tensiondecreases monotonically with increasing concentration ofPUs. This indicates that PU macromolecules remain asfree molecules in solution. When concentration reachesCMC, a sharp decrease in the surface tension is observed.This reveals that PUs will aggregate and form micellesin solution. After reaching CMC, surface tension remainsrelatively constant or changes with a lower slope (Fig. 8).The CMC of PU solutions was determined from theintersection of the two lines fitted to data points. At roomtemperature (25 �C), the synthesized PUs self-assembleinto micelles at a lower CMC (0.3–1.4×10−6 mol/L) com-pared with that of Pluronic P 123 (4�4× 10−6 mol/L).65
According to literature,66–68 CMC decreases and micellarstability increases with the length of the hydrophobicsegment. Thus, these PU samples having a hydrophobicsegment much larger than P123, confer a greater ther-modynamic stability in aqueous solution. In our case,hydrophobicity is given by polyesters (ricinoleat diol)and bifunctional derivative of phosphatidylcholine used insynthesis, as well as aliphatic diisocyanates (HDI, LDI).These PU micelles offer a high potential drug deliverysystem with many advantages as they contain a hydropho-bic core which can be used to encapsulate pharmaceuticalswith poor water solubility.The standard free energies of micellization (�G�) of
PUs are also given in Table III. In the case of a surfactant,the standard free energy change for the transfer of 1 mol ofamphiphilic polymer from solution to the micellar phaseis given by the following equation:
�G� = RT ln��CMC� (8)
where R is the gas constant, T is the absolute tempera-ture and �CMC is the CMC in terms of mole fraction. �G�
of PUs has negative values meaning that their micelliza-tion in aqueous solution is spontaneous.67�69 Free energydecreases (becomes more negative) with the increase intemperature. Thus, the micellization tendency becomesstronger with increasing temperature.70
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Figure 8. Surface tension of PUs hydrogels as a function ofconcentration at (a) 25 �C and (b) 37 �C.
DLS InvestigationsThe main purpose of using DLS is the understanding ofsize distribution, aggregation state and stability of PUsmacromolecules in aqueous solution. The micellar sizeand distribution of PUs in water as a function of temper-ature are illustrated in Figure 9. Since the hydrophobicsegment of PUs is greater than the hydrophilic one, themicelles presented in these systems are anizometric.63�71
This assumption resulted from the micellar distributioncurves (Fig. 9).The observation of bimodal distribution peak at 25 �C
indicates that two types of micelles were found insolution.72 Therefore, at low temperature, small micelleswith a hydrodynamic diameter between 21–27 nm coexist
Figure 9. Size and distribution of PUs micelles in water as afunction of temperature.
with much larger micelles of 74–134 nm in diameter(Table IV). When temperature increases to 37 �C, anizo-metric micelles tend to reorganize to form larger ones.Therefore, the two types of micelles are bridged to formmicellar groups and a monomodal distribution of the peaksis observed. This phenomenon is observed at all PUs
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Table IV. Characteristics of PUs macromolecules in aqueoussolution at different temperatures.
samples and could be assigned to the formation of gelswhich strongly depends on the temperature. After temper-ature rises to 38 �C, the aggregation and packing inter-action between micelles increases, but the mechanism ofaggregation is specific. P2 and P3 systems turned into abimodal particle distribution with one species of 35 and47 nm and another of 68 and 180 nm, respectively. Instead,P1 retains its monomodal curve. With increasing temper-ature, the size distribution curves of the micelles in PUssamples become larger due to the formation of more junc-tions in the network.
The micelles of all PUs samples are not stable withtemperature indicating that their shape changes within themeasured temperature range, because micellar aggregationis the main mechanism for sol–gel transition.73
Polydispersity index (PDI) measurements indicate thewidth of particle size distribution and show value 0 forhighly mono-dispersed sample and 1 or more for highlypoly-dispersed sample.74 Thus, at 25 �C, all PU sampleshave the highest PDI values and represent a broad par-ticle size distribution. This indicates that more than onemicelle types were formed in the solution.75 Then, PDIvalues of PU micelles decreased with increasing tempera-ture (Table IV).
Zeta potential can influence micelles stability in solutionthrough the electrostatic repulsion between them. As thisparameter is a measure of the electric field potential ofthe micelles, it is affected by the size, shape and surfacecharge of the structure. However, low absolute value ofzeta potential is necessary for aggregation of micelles.76�77
Zeta potential values of PU micelles are illustrated inTable IV. Thus, zeta potential of P2 increases from −16.9to −15.5 mV, as the temperature increases from 25 to40 �C. An increase in the zeta potential value can be alsoobserved for P1, from −14.7 to −12.5 mV. For P3 thevalue decreases from −10.1 to −13.2 mV.
From the presented data we can conclude that a restruc-turing of micelles with increase in temperature is observed
for all the samples, but the aggregation mechanism isdifferent.
Sol–Gel Phase Transition of PU SamplesFigure 10 presents the macroscopic observation of aque-ous PU solution (15%wt) in sol, gel and precipitated state(syneresis). At low temperature (15 �C), the PU aqueoussolution exhibits a typical sol behavior. Thus, the PU sam-ple has low viscosity at room temperature, so it can beinjected easily by a 23-gauge needle (inset of the figure).When the solution was heated to 37 �C, the formation ofa stable and clear gel is observed. Then, temperature israised further (50 �C) and the gel changes from translucentto opaque. Now, a macroscopic phase separation occursbetween the polymer and water (syneresis).The sol–gel transition upon temperature change from
low temperature to body temperature was also observedfrom rheological measurements. Thus, the sol–gel phasetransition diagrams of PU samples in aqueous solutionswere determined using this technique.Figure 11 shows the evolution of viscoelastic parame-
ters (G′, G′′, tan�) as a function of temperature for 15%wtaqueous PUs solutions at a heating rate of 0.5 �C/min(1 Pa, 1 rad/s). At the start of experiments (time = 0 s),temperature was around 5 �C and all the samples pre-sented a liquid-like behavior at a temperature below 20 �C,when G′′ > G′ and tan� was high. Around the transitionpoint, viscoelastic parameters change abruptly due to thevery fast answer of macromolecular chains to the ther-mal stimulus. Thus, both moduli elevated rapidly as gela-tion proceeded and the increases rate of G′ was muchhigher than that of G′′, due to the formation of elastichydrogels. For these PU structures, the hydrogel networkformation is due to physical crosslinking such as: hydro-gen bonding, molecular entanglements and hydrophobicinteractions. The difference in increase rates leads to acrossover of G′ and G′′, which could be defined as the gelpoint (t = tgel), indicating transition of the polymer sys-tem from a liquid-phase to a solid-phase, also suggestinga three dimensional (3D) network formation.78 Practically,this crossover point represents the sol–gel transition tem-perature, which was located at the following values: 29,27 and 32 �C for P1, P2 and P3 samples, respectively.The temperature increase leads to gelation, resulting inestablishment of more networks and therefore G′ rapidlyexceeds G′′, where solid-like behavior dominates the vis-coelastic properties of the hydrogel formed. Finally, whenthe interactions between PU macromolecules approaches
Figure 10. Macroscopic observation of the sol–gel–sol phasetransition of the PU sample.
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Figure 11. Evolution of viscoelastic parameters as a func-tion of temperature for polyurethane hydrogels (heating rate:0.5 �C/min, 1 rad/s, 1 Pa).
completion, both G′ and G′′ become constant, suggest-ing the formation of a well-developed hydrogel network.From all the samples, P2 sample presents a wider tempera-ture where the gel state exists. In conclusion, the structure
Figure 12. Evolution of complex viscosity as a function oftime for polyurethane samples (37 �C, 1 rad/s, 1 Pa).
of polyurethane influences both the sharp discontinuity ofparameters and transition temperatures. Thus, the increaseof parameter values occurs in a narrow range of temper-ature and is specific to the PU structure. Also, for P1 therange of temperature increase is around 3 �C and for P2and P3 around 6 �C.Depending on the polymer structure, the samples are
present in gel state in a broad range of temperatures from20 to 50 �C, which corresponds to our interest.Figure 12 illustrates the evolution of complex viscosity
as a function of time for polyurethane samples at 37 �C.At this temperature, i.e., body temperature, the aqueoussolutions of PU samples reach differently the gel state andafter that a stationary state is obtained. Thus, P1 and P3the samples require a longer time to reach the gel state, in370 s and ∼770 s, respectively. For P2, viscosity increasessharply in ∼58 s.
Figure 13. Evolution of complex viscosity as a function oftemperature of the P1 sample, for two heating rates: 0.5 �C/minand 1 �C/min (1 Pa, 1 rad/s).
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In order to elucidate the influence of the heating rateon sol–gel transition, the evolution of complex viscosityas a function of temperature for sample P1 was observed(Fig. 13). For this experiment two different heating rateswere used: 0.5 �C/min and 1 �C/min. The sample shows
Figure 14. SAXS profiles and 2D pattern of 30%wt PU hydro-gels at different temperatures for: (a) P1, (b) P2 and (c) P3.
sol–gel transitions at both heating rates, but the gel domainbeing more extended at the lower heating rate. At a heatingrate of 0.5 �C/min, a sharp discontinuity in viscosity occursat the gelation point, because the macromolecular chainshould have enough time to rearrange the balance betweenhydrophilic and hydrophobic interactions.We can conclude that, as mentioned previously, the con-
formation of isocyanate and phosphatidylcoline structuresinfluences viscoelastic parameters.
SAXS MeasurementsSmall-angle scattering (SAXS) was used to gain a moredetailed insight on the arrangement of structures.79�80
Thus, measurements were made at several temperatures inorder to obtain information on the structural change duringsol-to-gel transitions. The 2D SAXS patterns for PU aque-ous solutions, as well as the integrated scattering curvesrevealed details concerning the evolution of the systemwhen heated from 10 to 35 �C (Fig. 14). Therefore, forboth P1 and P2 (Figs. 14(a) and (b)) no specific structuringof the micelles could be observed at 10 �C. When the tem-perature reached 20 �C both systems showed, within thelow-angle region, a broad peak suggesting a phase tran-sition with formation of an ordered micellar phase witha periodicity distance of d = 8�5 nm.33 Upon heating to25 �C and further to 35 �C, the intensity of this peakincreased considerably and the maximum switched to evenlower s values and d= 9�9 nm. Also, a second peak couldbe observed at s = 0�11 Å−1 and therefore a long rangeorder could be assumed with a strong indication toward aprimitive cubic lattice. In the case of P2 system, broad-ening of the first Bragg peak at temperatures higher than25 �C suggests that the system already starts to switchback to a disordered phase state.By comparison, the P3 system showed a long range
order even at 10 �C having d = 10�2 nm based on thewell represented peak at s = 0�06 Å−1 (Fig. 14(c)). Also,
Figure 15. Bar graph showing viability of rCPCs cultured onthermoreversible hydrogels P1 (black bars) and P2 (blue bars)or in standard conditions (CTRL, red bars). ∗p < 0�05 versus2 Days of culture.
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Figure 16. DiI labelled GFPpos CPCs after four days of cultureon P1 and P2 thermoreversible hydrogels. The same micro-scopic fields are captured by phase contrast and upon fluores-cence excitation, showing green fluorescence protein (green)and DiI (red) corresponding to CPCs. Yellowish fluorescenceis due to the co-localization of GFP and DiI (merge panels).Scale bars= 100 �m.
a barely visible shoulder is present at s = 0�11 Å−1 whichsuggests that the nanostructuring pattern could be assignedto a primitive cubic lattice. When the temperature reached36 �C, Bragg peaks became more pronounced and the scat-tering profile changed consistently showing clearly that themicellar system was packed in a hexagonal phase withd = 12�5 nm.Thus, SAXS experiments revealed formation of ordered
micellar structures in the gel state, results which are inagreement with ATR-FTIR investigations.
In Vitro StudyBoth P1 and P2 thermoreversible hydrogels scaffolds wereeasy to handle and stable for about 10 days in cell cul-ture conditions. Moreover, both scaffolds were suitablefor cell culture since they did not modify the pH of cul-ture medium. As expected,28 compared to standard in vitroconditions, the presence of P1 and P2 hydrogels scaf-folds reduced the number of viable rCPCs (Fig. 15). Oneweek after plating on thermoreversible hydrogel, rCPCscultured on P1 were able to proliferate to a less extent onP2 (Fig. 16). Cell viability was significantly reduced after2 weeks in association with a decrease in gel stability. Weobserved that P1 promoted the formation of clusters ofrCPCs, while cells cultured on P2 maintained a monolayerdistribution.
CONCLUSIONA new series of injectable thermoreversible amphiphilicpolyurethanes hydrogels (PUs) based on different polyolsand aliphatic diisocyanates were synthesized and charac-terized. For these PUs, the chemical structure and molec-ular characteristics were investigated. The variation incontact angles values was ascribed to the mobility of PUmacromolecules from the surfaces and also to the con-tact medium. The new structures decrease the CMC val-ues compared to Pluronic 123. The standard free energies
of micellization (�G�) has negative values and decreases(becomes more negative) with an increase in temperature.The ATR-FTIR study suggests that these PUs struc-
tures formed an ordered gel with water molecules whichdepended on the chemical structure and temperature. Thetemperature-induced micellization is a progressive pro-cess followed by gelation which was due to the orderedpacking of micelles under a cubic or hexagonal phase,as the SAXS experiments revealed. The 1H-NMR profilewas influenced by polymer-water interactions, which tookplace via the hydrogen bonding interaction between theproton of the polymer segment and water molecule, lead-ing to the downfield shift with increasing temperature. TheDLS data reveal that the micelles of aqueous PUs hydro-gels undergo a restructuring with increase in temperature.Aqueous solution of these PUs presents thermore-
versible behavior at a temperature close to the human body(∼32 �C). The sol–gel transitions, gelation points and thetime of gelation are influenced by the chemical structureand heating rate. In vitro studies show that PUs hydro-gels scaffolds are able to proliferate rCPCs cells. All theseproperties allow them to be used as scaffolds in restoringthe heart muscle when the patient suffers a heart attack,which is the main cause of heart damage.
Conflict of InterestThe authors declared no conflict of interest.
Acknowledgments: This work was supported bya Seventh Framework Programme, NMP-2007-2.3-1,BIOSCENT, number 214539/01.01.2009 and a grant ofthe Romanian National Authority for Scientific Research,CNCS-UEFISCDI, project number PN-II-ID-PCE-2011-3-0199 (contract 300/2011).
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