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New Biodegradable Thermogelling Copolymers Having Very Low Gelation Concentrations

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New Biodegradable Thermogelling Copolymers Having Very Low Gelation Concentrations
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  New Biodegradable Thermogelling Copolymers Having VeryLow Gelation Concentrations Xian Jun Loh, †,‡ Suat Hong Goh, ‡ and Jun Li* ,†,§ Institute of Materials Research and Engineering, National University of Singapore, 3 Research Link,Singapore 117602, Department of Chemistry, Faculty of Science, National University of Singapore, 3 Science Drive 3, Singapore 117543, and Division of Bioengineering, Faculty of Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117576, Singapore Received August 14, 2006; Revised Manuscript Received October 25, 2006  New biodegradable multiblock amphiphilic and thermosensitive poly(ether ester urethane)s consisting of poly-[(  R )-3-hydroxybutyrate] (PHB), poly(ethylene glycol) (PEG), and poly(propylene glycol) (PPG) blocks weresynthesized, and their aqueous solutions were found to undergo a reversible sol - gel transition upon temperaturechange at very low copolymer concentrations. The multiblock poly(ether ester urethane)s were synthesized fromdiols of PHB, PEG, and PPG using 1,6-hexamethylene diisocyanate as a coupling reagent. The chemical structuresand molecular characteristics of the copolymers were studied by GPC,  1 H NMR,  13 C NMR, and FTIR. The thermalstability of the poly(PEG/PPG/PHB urethane)s was studied by thermogravimetry analysis (TGA), and the PHBcontents were calculated based on the thermal degradation profile. The results were in good agreement with thoseobtained from the  1 H NMR measurements. The poly(PEG/PPG/ PHB urethane)s presented better thermal stabilitythan the PHB precursors. The water soluble poly(ether ester urethane)s had very low critical micellizationconcentration (CMC). Aqueous solutions of the new poly(ether ester urethane)s underwent a sol - gel - sol transitionas the temperature increased from 4 to 80  ° C, and showed a very low critical gelation concentration (CGC)ranging from 2 to 5 wt %. As a result of its multiblock architecture, a novel associated micelle packing modelcan be proposed for the sol - gel transition for the copolymer gels of this system. The new material is thought tobe a promising candidate for injectable drug systems that can be formulated at low temperatures and forms a geldepot  in situ  upon subcutaneous injection. Introduction The synthesis of biodegradable thermogelling polymers haveattracted much attention because of their potential applicationsfor drug delivery and tissue engineering. 1 - 6 Bioactive agentscan be incorporated in the sol state at low temperatures. Thisformulation can be injected into the body where the higher bodytemperature would lead to the formation of a gel depot. Thisdepot can be used for the controlled release of the bioactiveagents. Biodegradable linkages introduced into the polymerbackbone would facilitate the degradation of the copolymer intosmaller fragments and subsequent removal of the polymer fromthe body.As an example of thermogelling polymers, the triblockcopolymers of poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) (PEG-PPG-PEG) have been widely in-vestigated for controlled drug delivery, 6 - 7 wound covering, 8 andchemosensitizing for cancer therapy. 9 However, they generallyhave a high critical gelation concentration (CGC) (15 - 20 wt% or above), exhibiting poor resilience as well as having theburst effect of the release of bioactive agents. These shortcom-ings have made this system unsuitable for many biomedicalapplications. 10 - 11 Moreover, PEG-PPG-PEG triblock copoly-mers are non-biodegradable and have been reported to inducehyperlipidemia and increase the plasma level of cholesterol inrabbits and rats, suggesting that its use in the human body maynot be an attractive option. 12 - 14 Attempts have been made to lower the CGCs of EPE triblockcopolymers. By grafting PEG-PPG-PEG triblocks to poly(acrylicacid), polymers having very low CGCs (0.1 wt %) have beensynthesized. 15 - 18 However, these polymers are non-biodegrad-able, and the excretion from the body could be difficult. Highmolecular weight multiblock PEG-PPG-PEG triblock copoly-mers with a short junction linkages have been synthesized andfound to exhibit lower CGCs than PEG-PPG-PEG triblockcopolymers. 19 - 20 Cohn et al. have synthesized reverse ther-mogelling multiblock copolymers based on PEG, PPG, andPCL. 21 These biodegradable copolymers exhibited CGCs of 10wt %. Interestingly, this work showed that the incorporation of oligo-caprolactone segments lowered the CGCs of the copoly-mers as compared with the PPG-PEG multiblock copolymers.The viscosities of the gels were also lowered compared withthe PPG-PEG multiblock copolymers. On the other hand, PEG-PPG-PEG analogues were developed where the middle PPGblock was replaced by a biodegradable polyester such as poly-(  -caprolactone) or poly( L -lactide), which are of great signifi-cance in biomedical applications because of their biodegrad-ability. However, their CGCs are at a similar range of PEG-PPG-PEG triblock copolymers. 22 Poly[(  R )-3-hydroxybutyrate] (PHB) is a natural biodegradablepolyester, which is highly crystalline and hydrophobic, showinga greater hydrophobicity than either poly(lactic acid) or poly-(  -caprolactone). 23 Herein we hypothesize that incorporatingPHB segments into a PEG-PPG block copolymer would allow * To whom correspondence should be addressed at Division of Bio-engineering, National University of Singapore. Phone:  + 65-6516-7273 or6874-8376. Fax:  + 65-6872-3069. E-mail: bielj@nus.edu.sg or jun-li@imre.a-star.edu.sg. † Institute of Materials Research and Engineering. ‡ Department of Chemistry, Faculty of Science. § Division of Bioengineering, Faculty of Engineering. 585 Biomacromolecules   2007,  8,  585 - 593 10.1021/bm0607933 CCC: $37.00 © 2007 American Chemical SocietyPublished on Web 12/30/2006  the formation of extra physical cross-linking in the hydrogel,increasing its resilience. Additionally, PHB segments wouldprovide the biodegradable segments in the polymer backbone.In this work, we have designed a series of novel thermogellinghigh molecular weight amphiphilic multiblock poly(ether esterurethane)s consisting of PEG, PPG, and PHB blocks (Scheme1). We have shown that this simple synthetic method producesthermogelling copolymers with very low CGCs and tunableproperties, which may be potentially applied as an  in situ forming biodegradable gel depot suitable for sustained drugdelivery. Experimental Section Materials.  Natural source poly[(  R )-3-hydroxybutyrate] (PHB) wassupplied by Aldrich and purified by dissolving in chloroform followedby filtration and subsequent precipitation in hexane before use. The  M  n  and  M  w  of the purified PHB were 8.7  ×  10 4 and 2.3  ×  10 5 ,respectively. Poly(ethylene glycol) (PEG) and poly(propylene glycol)(PPG) with  M  n  of ca. 2000 was purchased from Aldrich. Purificationof the PEG was performed by dissolving in dichloromethane followedby precipitation in diethyl ether and vacuum-dried before use. Purifica-tion of PPG was performed by washing in hexane three times andvacuum-drying before use. The  M  n  and  M  w  of PEG were found to be1890 and 2060, respectively. The  M  n  and  M  w  of PPG were found to be2180 and 2290, respectively. Bis(2-methoxyethyl) ether (diglyme, 99%),ethylene glycol (99%), dibutyltin dilaurate (95%) 1,6-hexamethylenediisocyanate (HDI) (98%), methanol, diethyl ether, 1,2-dichloroethane(99.8%), and 1,6-diphenyl-1,3,5-hexatriene (DPH) were purchased fromAldrich. Diglyme was dried with molecular sieves, and 1,2-dichloro-ethane was distilled over CaH 2  before use. PEG-PPG-PEG triblockcopolymer with a chain composition of EG 100 PG 65 EG 100  (also knownas Pluronic F127) was purchased from Aldrich and used as received. Synthesis of Poly(PEG/PPG/PHB urethane)s.  Telechelic hydroxy-lated PHB (PHB-diol) prepolymers with various molecular weight wereprepared by transesterification between the natural source PHB andethylene glycol using dibutyltin dilaurate in diglyme as reportedpreviously. 24 - 26 The yields were about 80%. Poly(PEG/PPG/PHBurethane)s were synthesized from PHB-diol, PEG, and PPG with molarratios of PEG/PPG fixed at 2:1 and PHB content ranging from 5 to 20mol % (calculated from the  M  n  of PHB-diol) using HDI as a couplingreagent. The amount of HDI added was equivalent to the reactivehydroxyl groups in the solution. Typically, 0.064 g of PHB-diol (  M  n )  1070, 6.0  ×  10 - 5 mol), 1.44 g of PEG (  M  n  )  1890, 7.6  ×  10 - 4 mol), and 0.82 g of PPG (  M  n  )  2180, 3.8  ×  10 - 4 mol) were dried ina 250-mL two-neck flask at 50  ° C under high vacuum overnight. Then,20 mL of anhydrous 1,2-dichloroethane was added to the flask, andany trace of water in the system was removed through azeotropicdistillation with only 1 mL of 1,2-dichloroethane being left in the flask.When the flask was cooled down to 75  ° C, 0.20 g of HDI (1.2 × 10 - 3 mol) and two drops of dibutyltin dilaurate ( ∼ 8 × 10 - 3 g) were addedsequentially. The reaction mixture was stirred at 75  ° C under a nitrogenatmosphere for 48 h. The resultant copolymer was precipitated fromdiethyl ether and further purified by redissolving into 1,2-dichloroethanefollowed by precipitation in a mixture of methanol and diethyl etherto remove remaining dibutyltin dilaurate. A series of poly(PEG/PPG/ PHB urethane)s with different compositions of PHB were prepared,and their number-average molecular weight and polydispersity valuesare given in Table 1. The yield was 80% and above after isolation andpurification.  1 H NMR (CDCl 3 ) of poly(PEG/PPG/PHB urethane)sEPH2:  δ  (ppm) 1.14 (O(C  H  3 )CHCH 2 O), 1.26 (O(C  H  3 )CHCH 2 CO),1.32(OOCNHCH 2 CH 2 C  H  2 C  H  2 CH 2 CH 2 NHCOO),1.48(OOCNHCH 2 C  H  2 -CH 2 CH 2 C  H  2 CH 2 NHCOO), 2.44 - 2.63 (O(CH 3 )CHC  H  2 CO), 3.13(OOCNHC  H  2 CH 2 CH 2 CH 2 CH 2 C  H  2 NHCOO), 3.41 (O(CH 3 )C  H  CH 2 O),3.46 (O(CH 3 )CHC  H  2 O), 3.64 (OC  H  2 C  H  2 O), 4.20 (OOCN  H  CH 2 CH 2 -CH 2 CH 2 CH 2 CH 2 N  H  COO), 5.21 - 5.29 (O(CH 3 )C  H  CH 2 CO).  13 C NMRof EPH2 (CDCl 3 ) of poly(PEG/PPG/PHB urethane)s:  δ  (ppm) 17.77(O( C  H 3 )CHCH 2 O), 20.14 (O( C  H 3 )CHCH 2 CO), 26.69 (OOCNHCH 2 -CH 2 C  H 2 C  H 2 CH 2 CH 2 NHCOO), 30.26 (OOCNHCH 2 C  H 2 CH 2 CH 2 C  H 2 -CH 2 NHCOO), 41.20 (O(CH 3 )CH C  H 2 CO), 64.18 (OOCNH C  H 2 CH 2 -CH 2 CH 2 CH 2 C  H 2 NHCOO),67.99(O(CH 3 ) C  HCH 2 CO),70.94(O C  H 2 C  H 2 O),73.56(O(CH 3 )CH C  H 2 O),75.72(O(CH 3 ) C  HCH 2 O),156.82(OO C  NHCH 2 -CH 2 CH 2 CH 2 CH 2 CH 2 NH C  OO), 169.98 (O(CH 3 )CHCH 2 C  O). Molecular Characterization.  Gel permeation chromatography(GPC) analysis was carried out with a Shimadzu SCL-10A and LC-8A system equipped with two Phenogel 5  µ m 50 and 1000 Å columns(size: 300  ×  4.6 mm) in series and a Shimadzu RID-10A refractiveindex detector. THF was used as eluent at a flow rate of 0.30 mL/min Scheme 1.  Synthesis of PHB-diol and Poly(PEG/PPG/PHB urethane)s 586  Biomacromolecules, Vol. 8, No. 2, 2007   Loh et al.  at 40  ° C. Monodispersed poly(ethylene glycol) standards were used toobtain a calibration curve. The  1 H NMR (400 MHz) and  13 C NMR(100 MHz) spectra were recorded on a Bruker AV-400 NMRspectrometer at room temperature. The  1 H NMR measurements werecarried out with an acquisition time of 3.2 s, a pulse repetition time of 2.0 s, a 30 °  pulse width, 5208 Hz spectral width, and 32K data points.Chemical shift was referred to the solvent peaks ( δ  )  7.3 ppm forCHCl 3 ). Fourier transform infrared (FTIR) spectra of the polymer filmscoated on CaF 2  plate were recorded on a Bio-Rad 165 FT-IRspectrophotometer; 64 scans were signal-averaged with a resolution of 2 cm - 1 at room temperature. Thermal Analysis.  Thermogravimetric analyses (TGA) were carriedout on a TA Instruments SDT 2960. Samples were heated at 20  ° Cmin - 1 from room temperature to 800  ° C in a dynamic nitrogenatmosphere (flow rate  )  70 mL min - 1 ). Critical Micellization Concentration (CMC) Determination.  TheCMC values were determined by using the dye solubilization method. 27,28 The hydrophobic dye 1,6-diphenyl-1,3,5-hexatriene (DPH) was dis-solved in methanol with a concentration of 0.6 mM. 20  µ L of thissolution was mixed with 2.0 mL of copolymer aqueous solution withconcentrations ranging from 0.0001 to 0.5 wt % and equilibratedovernight at 4  ° C. A UV - vis spectrophotometer was used to obtainthe UV - vis spectra in the range of 330 - 430 nm at 25  ° C. The CMCvalue was determined by the plot of the difference in absorbance at378 nm and at 400 nm (  A 378 -  A 400 ) versus logarithmic concentration. Sol - Gel Transition.  The sol - gel transition was determined by atest tube inverting method with temperature increments of 2  ° C perstep. 22a, 29 Each sample of a given concentration was prepared bydissolving the polymer in distilled water in a 2-mL vial. Afterequilibration at 4  ° C for 24 h, the vials containing samples wereimmersed in a water bath at a constant designated temperature for 15min. The gelation temperature was characterized by the formation of a firm gel that remained intact when the tube was inverted by 180 ° . 30 Viscosity Measurements.  Viscosities of the hydrogels were mea-sured at 25  ° C using a Brookfield HADV-III +  digital viscometercoupled to a temperature-controlling unit. The small sample adapterSSA 15/7R was used. The revolution rate of the spindle was set at 20cycles min - 1 and shear rate was set at 9.6 s - 1 . Results and Discussion Synthesis and Characterization of Poly(PEG/PPG/PHBurethane)s.  Previously, we reported the synthesis and biodeg-radation behavior of amphiphilic multiblock poly(ether esterurethane)s consisting of PEG and PHB blocks. 24 - 26 These water-insoluble copolymers could not undergo a sol - gel transitionand were non-thermosensitive. However, in this study, water-soluble and thermosensitive poly(PEG/PPG/PHB urethane)swere synthesized, and for the first time PHB has beenincorporated into a thermogelling copolymer, to enhance thegel properties as well as to make the copolymers biodegradable.Telechelic hydroxylated PHB (PHB-diol) with lower molec-ular weight were obtained through transesterification betweenhigh-molecular-weight natural source PHB and ethylene glycolusing dibutyltin dilaurate as catalyst. 24 The transesterificationreaction was allowed to proceed for a few hours to overnightto produce PHB-diols with  M  n  of 1070 and 2800, respectively,as determined by GPC. The reaction of hydroxyl groups of PHB-diol, PEG, and PPG with isocyanate of 1,6-hexamethlyenediisocyanate (HDI) in the presence of dibutyltin dilaurate ledto formation of poly(PEG/PPG/PHB urethane)s. The proceduresfor the synthesis of PHB-diol and poly(PEG/PPG/PHB ure-thane)s are presented in Scheme 1. Owing to the moisturesensitive nature, any trace of water in the system was removedthrough azeotropic distillation, and the reaction was carried outin dried 1,2-dichloroethane under a nitrogen atmosphere. Thetarget poly(PEG/PPG/PHB urethane)s were isolated and purifiedfrom the reaction mixture by repeated precipitation from amixture of methanol and diethyl ether.A series of random multiblock poly(PEG/PPG/PHB ure-thane)s with different amounts of PHB incorporated weresynthesized, and their molecular weights and molecular weightdistributions were determined by GPC (Table 1). A typical GPCchromatograph for one of the poly(PEG/PPG/PHB urethane)stogether with its corresponding precursors is shown in Figure1. The observation of unimodal peak in GPC chromatographof the purified poly(PEG/PPG/PHB urethane) with non-overlap-ping nature with those of corresponding precursors indicatesthat a complete reaction took place with no unreacted precursor Table 1.  Molecular Characteristics of Poly(PEG/PPG/PHB urethane)sfeed ratio (wt %)composition incopolymer (wt %) c  copolymercharacteristicscopolymer a  M  n  of PHB used b  (g mol - 1 ) PHB PEG PPG PHB PEG PPG  M  n b  ( × 10 3 )  M  w  /  M  n b  cmc d  (g/ mL)EPH1 1070 2.8 61.7 35.5 2.1 64.0 33.9 50.6 1.56 9.79 × 10 - 4 EPH2 1070 5.6 59.9 34.5 5.1 57.0 37.9 45.5 1.38 8.69 × 10 - 4 EPH3 1070 8.7 58.0 33.4 8.1 56.3 35.7 42.5 1.37 5.16 × 10 - 4 EPH4 1070 11.8 55.9 32.2 11.4 61.6 27.0 37.8 1.16 - e  EPH5 2800 6.9 59.1 34.0 7.1 63.3 29.7 39.2 1.18 8.88 × 10 - 4 EPH6 2800 13.6 54.8 31.6 12.7 59.0 28.3 30.0 1.20 - e  a  Poly(PEG/PPG/PHB urethane)s are denoted EPH, E for P E G, P for P P G and H for P H B. The  M  n  of PEG and PPG used for the copolymer synthesiswas 1890 and 2180 g mol - 1 , respectively.  b  Determined by GPC.  c  Calculated from  1 H NMR results.  d  Critical micellization concentration (cmc) in waterdetermined by the dye solubilization technique at 25  ° C.  e  Copolymers not water-soluble. Figure 1.  GPC diagrams of EPH2 and its PHB, PEG, and PPGprecursors: (a) PHB-diol ( M  n  1080); (b) PEG ( M  n  1890); (c) PPG ( M  n 2180); (d) EPH2 ( M  w  62.8  ×  10 3 ,  M  n  45.5  ×  10 3 ,  M  w  /  M  n  1.38). Biodegradable Thermogelling Copolymers  Biomacromolecules, Vol. 8, No. 2, 2007   587  remained. 24 - 26 All the poly(PEG/PPG/PHB urethane)s synthe-sized had narrow molecular weight distribution and highmolecular weight, with polydispersity ranging from 1.16 to 1.56and  M  n  from 3.00 × 10 4 to 5.06 × 10 4 . The results are tabulatedin Table 1.The chemical structure of poly(PEG/PPG/PHB urethane)s wasverified by  1 H NMR and  13 C NMR spectroscopy (Figure 2a,b).Figure 2a shows the  1 H NMR spectrum of EPH2 in CDCl 3 , inwhich all proton signals belonging to both PHB, PEG, and PPGsegments are confirmed. Signals corresponding to methyleneprotons in repeated units of PEG segments are observed at 3.64ppm, the signals at 5.25 ppm are assigned to methine protonsin the repeated unit of PHB segments, 24 - 26 the signals at 1.14ppm are assigned to the methyl protons of PPG. As the contentof HDI among the starting materials is below 1 wt %, thecompositions of the poly(PEG/PPG/PHB urethane)s could bedetermined from the integration ratio of resonances at 1.14, 3.64,and 5.25 ppm within the limits of   1 H NMR precision, and theresults are shown in Table 1.The  13 C NMR was used to ascertain the chemical compositionof the poly(PEG/ PPG/PHB urethane)s. The peak assignmentsof the copolymers were performed by comparison with the  13 CNMR spectra of the precursors. Figure 2b shows the  13 C NMRspectra of EPH2 in CDCl 3 . Briefly, peaks at 17.77 (methyl C),73.56 (methylene C), and 75.72 ppm (methine C) are assignedto the PPG moiety. A peak at 70.94 ppm is assigned to themethylene C of the PEG segment. Peaks at 20.14 (methyl C),41.20 (methylene C), 67.99 (methine C), and 169.98 ppm(carbonyl C) are attributed to the PHB segment. In addition,peaks due to the HDI junction unit could be observed in thespectra (26.69, 30.26, 64.18, and 156.82 ppm).A  13 C NMR spectrum of hexamethylene diisocyanate wasobtained, and the carbonyl carbon peak was observed at 122.85ppm. After the polymerization reaction, the  13 C peak of thecarbonyl carbon of the newly formed urethane linkage wasobserved at 156.82 ppm. This shift was attributed to theattachment of the hydroxyl groups to the isocyanate functionalgroups in the formation of the urethane linkage (NCO  f  NHCOO). This observation, together with the concomitantincrease in the molecular weight of the copolymers indicatesthat the polycondensation reaction was successful.FTIR is useful in the characterization of the functional groupspresent in the polymer. As a typical example, Figure 3 showsFTIR spectra of EPH2 and its PEG, PPG, and PHB precursors.For PPG (Figure 3c) and PEG (Figure 3d), the characteristicC - O - C stretching vibration of the repeated OCH 2 CH 2  unitsis observed at 1102 cm - 1 . An intensive carbonyl stretching bandat 1723 cm - 1 characterizes the FTIR spectrum of pure PHB-diol as shown in Figure 3a. It is clearly seen that in Figure 3b,all the characteristic absorptions for PHB-diol, PEG, and PPGappear in the spectrum of EPH2, which confirms the presenceof the three segments in the poly(PEG/PPG/PHB urethane)s.Furthermore, it can be seen in the profile of EPH2 that the peakascribed to the NCO stretching was not observed in the regionaround 2200 cm - 1 . This provides evidence that the isocyanategroups of the junction units have been reacted and are notpresent in the polymer product. These observations, togetherwith the aforementioned evidence (GPC and NMR results)provide a solid justification for the successful synthesis of themultiblock copolymers. Thermal Properties.  The thermal stability of poly(PEG/PPG/ PHB urethane)s was evaluated using thermogravimetric analysis(TGA). Figure 4 shows the TGA scan results for EPH2compared with its PHB, PEG, and PPG precursors. Thedegradation of pure PHB-diol starts at 218  ° C and completesat 295  ° C (Figure 4a), and PPG starts to degrade at 350  ° C(Figure 4c) while that of pure PEG starts at 400  ° C (Figure4b). EPH2 undergoes a three-step thermal degradation with thefirst step occurring between 227 and 303  ° C and the second Figure 2.  (a) 400 MHz  1 H NMR and (b) 100 MHz  13 C NMR spectraof EPH2 in CDCl 3 . Figure 3.  FTIR spectra of EPH2 and its PHB, PEG, and PPGprecursors: (a) PHB-diol ( M  n  1080); (b) EPH2; (c) PPG ( M  n  2180);(d) PEG ( M  n  1890). 588  Biomacromolecules, Vol. 8, No. 2, 2007   Loh et al.  and third steps between 350 and 433  ° C (Figure 4d). Incomparison with the TGA curves of pure PHB-diol and purePEG, the first weight loss step is attributed to the decompositionof PHB segment and the second and third weight loss step tothe decomposition of both the PEG and PPG segments.However, the second and third weight loss steps are too closefor the accurate determination of the compositions of PPG andPEG separately. Therefore, only the PHB content of EPH2 couldbe determined from the degradation profile. Similar weight losscurves were also observed for other poly(PEG/PPG/PHBurethane)s. The PHB contents estimated from TGA results arein good agreement with those calculated from  1 H NMR. Critical Micellization Concentration (CMC) Determina-tion.  Among the six poly(PEG/PPG/PHB urethane)s, onlyEPH1, EPH2, EPH3, and EPH5 were soluble in water. TheCMC determination was carried out for these four copolymers.This experiment was conducted by varying the aqueous polymerconcentration in the range of 0.0001 to 0.5 wt %, while keepingthe concentration of DPH constant. DPH shows a higherabsorption coefficient in a hydrophobic environment than inwater. Thus, with increasing polymer concentration, the absor-bances at 344, 358, and 378 nm increased (Figure 5a). The pointwhere the absorbance suddenly increases corresponds to theconcentration at which micelles are formed. When the micelleis formed, DPH partitions preferentially into the hydrophobiccore formed in the aqueous solution. 22a,27 - 29 The CMC wasdetermined by extrapolating the absorbance at 378 nm minusthe absorbance at 400 nm (  A 378 -  A 400 ) versus logarithmicconcentration (Figure 5b). The CMC values for the water-solublecopolymers are tabulated in Table 1 and are in the range of 5.16 × 10 - 4 to 9.79 × 10 - 4 g.mL - 1 . Comparing the copolymersof similar molecular weights, the CMC values are much lowerthan that reported by Ahn et al. for a series of multiblock PEG-PPG-PEG copolymers, 19 showing that the incorporation of PHBgreatly increases the hydrophobicity of the copolymers, resultingin a decrease in the CMC values. 13 C NMR was used to investigate the effect of solvent onthe micelle structure. 29,31 - 34 CDCl 3  is a good nonselectivesolvent for PHB, PEG, and PPG while water is a good selectivesolvent for PEG but poor for PPG and PHB. As shown in Figure6, in CDCl 3 , the peaks due to the PHB, PEG, and PPG weresharp and well defined. In D 2 O, PEG is shown as a sharp peakbut the PHB and the PPG peaks are collapsed and broadened.This shows that the molecular motion of PHB and PPG is slowin water, indicating a hydrophobic core structure made up of PHB and PPG with PEG as the outer corona structure,confirming the core-corona structure of the micelle. 31 - 33 How-ever, in the light of the multiblock architecture of the copoly-mers, it is not reasonable to expect that the simple micelles of an ABA-type amphiphilic polymer be formed. Instead, it would Figure 4.  TGA curves of EPH2 and its PHB, PEG and PPGprecursors: (a) PHB-diol ( M  n  1080); (b) PEG ( M  n  1890); (c) PPG ( M  n 2180); (d) EPH2. Figure5.  (a) UV - vis spectra changes of DPH with increasing EPH2copolymer concentration in water at 25  ° C. DPH concentration wasfixed at 6 mM, and the polymer concentration varied between 0.0001and 0.5 wt %. The increase in the absorbance band at 378 nmindicates the formation of a hydrophobic environment in water. (b)CMC determination by extrapolation of the difference in absorbanceat 378 and 400 nm. Figure 6.  13 C NMR spectra of EPH2 (5 wt %) in (a) CDCl 3  and (b)D 2 O at 25  ° C. Biodegradable Thermogelling Copolymers  Biomacromolecules, Vol. 8, No. 2, 2007   589
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