Hydrolytic degradation and protein release studies of thermogelling polyurethane copolymers consisting of poly[( R)-3-hydroxybutyrate], poly(ethylene glycol), and poly(propylene glycol

Hydrolytic degradation and protein release studies of thermogelling polyurethane copolymers consisting of poly[( R)-3-hydroxybutyrate], poly(ethylene glycol), and poly(propylene glycol
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  Biomaterials 28 (2007) 4113–4123 Hydrolytic degradation and protein release studies of thermogellingpolyurethane copolymers consisting of poly[( R )-3-hydroxybutyrate],poly(ethylene glycol), and poly(propylene glycol) Xian Jun Loh a,b , Suat Hong Goh b , Jun Li a,c,  a Institute of Materials Research and Engineering (IMRE), National University of Singapore, 3 Research Link, Singapore 117602, Singapore b Department of Chemistry, Faculty of Science, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore c Division of Bioengineering, Faculty of Engineering, National University of Singapore, 7 Engineering Drive 1, Singapore 117574, Singapore Received 21 March 2007; accepted 21 May 2007Available online 26 May 2007 Abstract This paper reports the hydrolytic degradation and protein release studies for a series of newly synthesized thermogelling tri-componentmulti-block poly(ether ester urethane)s consisting of poly[( R )-3-hydroxybutyrate] (PHB), poly(propylene glycol) (PPG), andpoly(ethylene glycol) (PEG). The poly(PEG/PPG/PHB urethane) copolymer hydrogels were hydrolytically degraded in phosphatebuffer at pH 7.4 and 37 1 C for a period of up to 6 months. The mass loss profiles of the copolymer hydrogels were obtained. The hydrogelresidues at different time periods of hydrolysis were visualized by scanning electron microscopy, which exhibited increasing porosity withtime of hydrolysis. The degradation products in the buffer were characterized by GPC,  1 H NMR, MALDI-TOF, and TGA. The resultsshowed that the ester backbone bonds of the PHB segments were broken by random chain scission, resulting in a decrease in themolecular weight. In addition, the constituents of degradation products were found to be 3-hydroxybutyric acid monomer and oligomersof various lengths ( n  ¼  1–5). The protein release profiles of the copolymer hydrogels were obtained using BSA as model protein.The results showed that the release rate was controllable by varying the composition of the poly(ether ester urethane)s or by adjusting theconcentration of the copolymer in the hydrogels. Finally, we studied the correlation between the protein release characteristics of the hydrogels and their hydrolytic degradation. This is the first example that such a correlation has been attempted for a biodegradablethermogelling copolymer system. r 2007 Elsevier Ltd. All rights reserved. Keywords:  Poly(ether ester urethane); Poly[( R )-3-hydroxybutyrate]; Poly(ethylene glycol); Poly(propylene glycol); Hydrolytic degradation; Drug release 1. Introduction In the rapidly developing field of biomaterials, thermo-gelling polymers have exhibited interesting properties thathave made them potential candidates for drug delivery andtissue engineering applications [1–6]. Recently, our labora-tory has synthesized a series of thermogelling multi-blockpoly(ester urethane)s consisting of poly[( R )-3-hydroxybu-tyrate] (PHB), poly(ethylene glycol) (PEG) and poly(pro-pylene glycol) (PPG) using hexamethylene diisocyanate(HDI) as a coupling reagent [7].PHB is a natural biodegradable and biocompatiblepolyester which degrades to  D -3-hydroxybutyrate, anatural non-toxic human blood plasma component [8,9].On the other hand, the PEG–PPG–PEG triblock copoly-mer is a biocompatible polyether that has been approvedby the FDA for biomedical and food applications [10,11].The biostability of polyurethane-based medical implants aswell as the leaching out of potentially toxic degradationproducts are important considerations in the assessment of the suitability of the polymers for biomedical applications[12]. Chain scission of poly(ester urethane)s could eitheroccur at the urethane linkage or the ester linkage, with the ARTICLE IN PRESS$-see front matter r 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.biomaterials.2007.05.016  Corresponding author. Division of Bioengineering, Faculty of Engineering, National University of Singapore, 7 Engineering Drive 1,Singapore 117574, Singapore. Tel.: +6565167273; fax: +6568723069. E-mail address: (J. Li).  ester linkage being the primary site of hydrolysis inpoly(ester urethane)s [13,14].The degradation of such thermogelling copolymers isdifferent from that of chemically crosslinked hydrogels. Ina crosslinked polymer hydrogel, the chemical bond of thecrosslink or polymer backbones must be broken beforeerosion of the polymer fragments can take place [15–17].The rate of degradation of the conventional crosslinkedhydrogel can be controlled by the density and nature of thecrosslink or degradability of the polymer backbone. In aphysical hydrogel of a thermogelling copolymer, theformation of the hydrogel results from the physical packingof the polymeric segments in solution. Surface erosion canoccur with the dissolution of the exposed polymer chains.However, it does not imply that the polymer chains havedegraded into smaller fragments: these chains could havemerely dissolved in the solution. Degradation of thepolymer chains is expected to take place after furtherexposure to the aqueous environment, thus the degradationbehavior is expected to be markedly different from that of chemical hydrogels.Currently, there have been limited studies of thedegradation process of a thermogelling copolymer. Jeonget al. [18] studied the degradation of PEG–PLGA–PEGtriblock hydrogel, while Shim et al. [19] studied thebiodegradability of a sulfonamide-modified poly( e -capro-lactone- co -lactide)–poly(ethylene glycol)–poly( e -caprolac-tone- co -lactide) copolymer. The degradation of thethermogelling copolymers may involve three basic phases:an  incubation  period, a  gel erosion  period, and a  chainscission  period.Besides the biodegradability, thermogelling copolymersare interesting from the viewpoint of sustained drugrelease. PEG–PPG–PEG triblock copolymers have beenthoroughly studied for drug delivery, wound covering andchemosensensitizing for cancer therapy [20–22]. A highconcentration of the copolymer is often needed in thethermogelling formulations (above 15wt%). Such formu-lations exhibit poor resilience and burst effect of release of drugs. These shortcomings have made the system unsui-table for many biomedical applications [23,24]. Moreover,PEG–PPG–PEG triblock copolymers are non-biodegrad-able and have been reported to induce hyperlipidemia andincrease the plasma level of cholesterol in rabbits and rats,suggesting that its use in the human body may not be anattractive option [25,26]. Our thermogelling poly(PEG/PPG/PHB urethane)s require extremely low polymerconcentrations to form hydrogels from aqueous solutionsupon temperature rising. The hydrogels are more robustthan PEG–PPG–PEG triblock copolymer hydrogels, basedon viscosity studies of the hydrogels [7].In this paper, we present a detailed hydrolytic degrada-tion and protein release study of the thermogelling multi-block poly(PEG/PPG/PHB urethane)s, and a discussion onthe correlation between the protein release characteristicsand the hydrolytic degradation. To the best of ourknowledge, this paper represents the first attempt tocorrelate certain features of the protein release withhydrolytic degradation for a biodegradable thermogellingcopolymer system. 2. Experimental methods  2.1. Materials Natural source PHB, PEG, PPG, PEG–PPG–PEG triblock copolymerwith a chain composition of EG 100 PG 65 EG 100  (also known as PluronicF127),  bis (2-methoxyethyl) ether (diglyme, 99%), ethylene glycol (99%),dibutyltin dilaurate (95%) 1,6-hexamethylene diisocyanate (HDI) (98%),methanol, diethyl ether and 1,2-dichloroethane (99.8%) were purchasedfrom Aldrich. The  M  n  and  M  w  of the purified PHB were 8.7  10 4 and2.3  10 5 , respectively. The  M  n  and  M  w  of PEG were found to be 1890 and2060, respectively. The  M  n  and  M  w  of PPG were found to be 2180 and2290, respectively. Diglyme was dried with molecular sieves, and 1,2-dichloroethane was distilled over CaH 2  before use. Bovine serum albumin(BSA) was purchased from Sigma ( M  n  66,000, according to manufac-turer’s specifications). The  M  n  and  M  w  of the starting materials weredetermined by GPC, unless mentioned otherwise.  2.2. Synthesis of multi-block poly(PEG/PPG/PHB urethane)scopolymers Telechelic hydroxylated PHB (PHB-diol) prepolymers ( M  n : 1070) wasprepared by transesterification between the natural source PHB andethylene glycol using dibutyltin dilaurate in diglyme as reported previously[7,27,28]. The yields were about 80%. Poly(PEG/PPG/PHB urethane)swere synthesized from PHB-diol, PEG and PPG with molar ratios of PEG/PPG fixed at 2:1 and PHB content ranging from 5 to 15mol%(calculated from the  M  n  of PHB-diol) using HDI as a coupling reagent.The amount of HDI added was equivalent to the reactive hydroxyl groupsin the solution. Typically, 0.064g of PHB-diol ( M  n  ¼  1070,6.0  10  5 mol), 1.44g of PEG ( M  n  ¼  1890, 7.6  10  4 mol) and 0.82gof PPG ( M  n  ¼  2180, 3.8  10  4 mol) were dried in a 250-mL two-neckflask at 50 1 C under high vacuum overnight. Then, 20mL of anhydrous1,2-dichloroethane was added to the flask and any trace of water in thesystem was removed through azeotropic distillation with only 1mL of 1,2-dichloroethane being left in the flask. When the flask was cooled down to75 1 C, 0.20g of HDI (1.2  10  3 mol) and two drops of dibutyltin dilaurate(  8  10  3 g) were added sequentially. The reaction mixture was stirred at75 1 C under a nitrogen atmosphere for 48h. The resultant copolymer wasprecipitated from diethyl ether, and further purified by redissolving into1,2-dichloroethane followed by precipitation in a mixture of methanol anddiethyl ether to remove remaining dibutyltin dilaurate. A series of poly(PEG/PPG/PHB urethane)s with different compositions of PHB wereprepared, and their number-average molecular weight and polydispersityvalues are given in Table 1 (see Scheme 1). The yield was 80% and above after isolation and purification.  2.3. Hydrolytic degradation of poly(PEG/PPG/PHB urethane)hydrogels Aqueous solutions of 5wt% copolymer were mixed and left toequilibrate overnight at 4 1 C. In a typical preparation, 1mL of polymersolution was injected into a porous cellulose cassette (pore size:   100 m m)and left to equilibrate at 37 1 C to form a polymer gel. The polymer gel haddimensions of 10mm  25mm  4mm. Each hydrogel sample was placedinto 25mL of phosphate buffer solution in a test tube, which wasincubated and shaken at 50rpm in a water bath at 37 1 C. The buffersolution had a pH of 7.4, and contained 8.0g of NaCl, 0.2g of KCl, 1.44gof Na 2 HPO 4 , and 0.24g of K 2 H 2 PO 4  in 1L of solution.The buffer solutions were replaced with fresh ones at predeterminedtime intervals. The process was allowed to proceed for up to 6 months, and ARTICLE IN PRESS X.J. Loh et al. / Biomaterials 28 (2007) 4113–4123 4114  the experiments were done in triplicate. The collected buffer solutions werelyophilized and weighed. In order to determine the weight attributed to thesalt contents of the buffer solution, a blank containing only 25mL of phosphate buffer solution was lyophilized and the residue was weighed togive the weight of the dry salts per 25mL of the buffer solution. The dryweights of the copolymers dissolved and/or degraded into the buffersolutions were obtained by the difference between the samples and blankweights.The dissolved and/or degraded copolymers in the buffer solutions wereextracted using chloroform, followed by evaporation of chloroform anddrying in vacuum at 50 1 C for 1 week. The aqueous phase (buffersolutions) was lyophilized and dried in vacuum at 50 1 C for 1 week to givethe salt residues, which was kept for further analysis.  2.4. Mass loss of poly(PEG/PPG/PHB urethane) hydrogels The mass loss of the copolymer gels after dissolution and degradationwas defined asMass loss  ð % Þ ¼ ½ 1   ð W  t = W  0 Þ   100%, (1)where  W  0  and  W  t  were the initial weight and the weight of the copolymerdissolved or/and degraded in the buffer solution at time  t , respectively.  W  t was obtained after drying the collected buffer solution samples at 50 1 Cunder vacuum for one week.  2.5. Protein release study of poly(PEG/PPG/PHB urethane)hydrogels Aqueous solutions of 5wt% copolymer were mixed and left toequilibrate overnight at 4 1 C. Appropriate amounts of BSA were loadedto make the concentration of BSA in the polymer solution 5mgmL  1 . Forcomparison, 30wt% of EG 100 PG 65 EG 100  triblock copolymer in aqueoussolutions was prepared. The concentration of BSA in these solutions wasalso 5mgmL  1 . In a typical example, 1mL of polymer solution wasinjected into a porous cellulose cassette (pore size:   100 m m) and left toequilibrate at 37 1 C. The polymer hydrogel obtained had dimensions of 10mm  25mm  4mm and was placed in 25mL of phosphate bufferrelease solutions in a test tube, which was incubated and shaken at 50rpmin a water bath at 37 1 C. The buffer solutions were replaced with freshones at predetermined time intervals, and the experiments were done intriplicate. The collected buffer solutions were lyophilized and kept at  80 1 C for further analysis. The BSA content was determined using thePierce BCA Protein Assay kit. Quantitation of BSA was based on acalibration curve, obtained using the BSA standards provided, in therange of 20–2000 m gmL  1 .  2.6. Gel permeation chromatography Molecular weight of the degradation products in the  chloroform-soluble fraction  were determined by gel permeation chromatography (GPC) usinga Shimadzu SCL-10A and LC-8A system equipped with two Phenogel ARTICLE IN PRESS Table 1Molecular weight and composition of the poly(PEG/PPG/PHB urethane) hydrogels before and after degradationMonths EPH(2%) a EPH(5%) a EPH(8%) a M  nb PDI c PHB (wt%)  M  nb PDI c PHB (wt%)  M  nb PDI c PHB (wt%)NMR d TGA e NMR d TGA e NMR d TGA e 0 58200 1.36 2.12 2.49 48100 1.27 5.11 5.71 54700 1.13 8.07 7.241 47600 1.15 2.11 2.01 34700 1.27 5.88 4.59 44300 1.12 8.02 6.112 37500 1.28 1.94 1.92 32400 1.30 5.38 4.11 38000 1.26 6.50 5.593 36700 1.23 0.95 1.19 28400 1.28 3.67 3.63 36300 1.31 3.40 3.816 32400 1.14 0.42 0.38 28500 1.31 3.14 2.58 25700 1.36 2.99 2.53 a Poly(PEG/PPG/PHB urethane)s were denoted by EPH( x %), and the number  x , indicates the nominal weight percentage of the PHB polymer segmentin the copolymer. The samples after degradation are those extracted by chloroform from the phosphate buffer solutions used for the hydrolysis. b As determined from GPC. c PDI: Poly dispersity index. d Calculated from  1 H NMR results. e Calculated from TGA results. NHHNOO ; OOCH 3 CH 3 OmOxOOyOO ; zO ; Poly(PEG/PPG/PHBurethane) O   PPG Thermosensitive PEG Hydrophilic  PHB Hydrophobic  EPH(2%): M  n = 58,200; Yield = 87%EPH(5%): M  n = 48,100; Yield = 83%EPH(8%): M  n = 54,700; Yield = 82% Scheme 1. Structure of the thermogelling poly(PEG/PPG/PHB urethane)s used in this study. Detailed characteristics of the copolymers are given inTable 1. X.J. Loh et al. / Biomaterials 28 (2007) 4113–4123  4115  5 m m 50 and 1000A ˚columns (size: 300  4.6mm) in series and a ShimadzuRID-10A refractive index detector. THF was used as eluent at a flow rateof 0.20mL/min at 45 1 C. Monodispersed PEG standards were used toobtain a calibration curve.  2.7.  1 H NMR spectroscopy The  1 H NMR spectra were recorded on a Bruker AV-400 NMRspectrometer at 400MHz at room temperature. The  1 H NMR measure-ments were carried out with an acquisition time of 3.2s, a pulse repetitiontime of 2.0s, a 30 1  pulse width, 5208Hz spectral width, and 32K datapoints. Chemical shift was referred to the solvent peaks ( d  ¼  7.3 forCHCl 3 ).  2.8. MALDI-TOF mass spectrometry MALDI-TOF was performed on a Bruker (Karlsruhe, Germany)Autoflex MALDI Tandem TOF/TOF mass spectrometer. Dithranol or trans -2-[3-tert-butylphenyl)-2-methyl-2-propenylidene] malononitrile wasused as the matrix and silver trifluoromethanesulfonate as the ion source.  2.9. Fourier transform infrared spectroscopy (FT-IR) FT-IR spectra of the polymer films were recorded on a Bio-Rad 165FT-IR spectrophotometer; 64 scans were signal-averaged with a resolutionof 2cm  1 at room temperature.  2.10. Thermal analysis Thermogravimetric analyses (TGA) were made using a TA InstrumentsSDT 2960. Samples were heated at 20 1 Cmin  1 from room temperature to800 1 C in a dynamic nitrogen atmosphere (flow rate  ¼  70mLmin  1 ).  2.2. Field emission scanning electron microscopy (SEM) SEM images were obtained at acceleration voltage of 5kV on a JSM-6700F microscope (JEOL, Japan). The samples were sputter-coated with athin layer of gold for 15s to make the sample conductive before testing. 3. Results and discussion 3.1. Experimental setup The hydrogel samples were enclosed in a porous cellulosecassette and immersed in a large excess of phosphate buffersolution. The buffer solutions were replaced with freshones at regular time intervals to simulate the dynamic flowof fluids in the body. The hydrolytic degradation experi-ments were carried out at pH 7.4 and 37 1 C to simulatephysiological conditions. The water bath was set in motionat 50rpm to account for bodily movements upon injectionof the gel depot. At various time points, the gel residuesand buffer solutions were collected and lyophilized. Thecontents in the buffer solutions were divided into two parts:the part that could be extracted by chloroform (mainlycopolymer with long chains) and the part that remained inthe aqueous buffer solution phase (mainly salts and low-molecular weight final degradation products). A similarsetup was used for the protein release study with protein-loaded hydrogels. 3.2. Hydrolytic degradation of the poly(PEG/PPG/PHB urethane) hydrogels3.2.1. Mass loss of the hydrogels and the chain scission of the copolymers The hydrolytic degradation process was accompanied bythe mass loss of the hydrogels, as shown in Fig. 1. For allthe gels, an incubation period (period during which therewas little mass loss) was observed. The incubationperiod of the gels decreased with increasing PHBcontent, following the sequence of EPH(2%) 4 E-PH(5%) 4 EPH(8%). The incubation period was followedby a period of steady mass loss. At the end of the erosionprocess, the rate of mass loss was observed to havedecreased and a plateau feature was observed at theterminating end of the erosion profile. The copolymer gelerosion could be controlled by the composition of thecopolymer. With decreasing PHB content, the timerequired for complete erosion increased. EPH(8%) erodescompletely after 30 days, EPH(5%) after 40 days andEPH(2%) after 70 days.Visual examinations of the remained hydrogel sampleswere carried out using SEM in order to observe changes inthe surface structure of the gel. The micrographs are shownin Fig. 2 for EPH(2%), EPH(5%) and EPH(8%) atdegradation time of 0, 14, and 30 days. The surface of the gel residue before erosion was devoid of pores andpacking of the lyophilized gel appeared to be compact.After 14 days of erosion, structural deterioration of the gelwas observed and pores (ca. 5–10 m m) developed on thesurface of the films. After 30 days of erosion, the poresbecame more numerous and an enlargement of the poreswas observed. ARTICLE IN PRESS 020406080100020406080 Period of Hydrolysis (Days)    M  a  s  s   L  o  s  s   (   %   ) Fig. 1. Mass loss (%) of the poly(PEG/PPG/PHB urethane) hydrogels(5wt%) after incubation in PBS at pH 7.4 and 37 1 C ( m : EPH(2%), ’ : EPH(5%),   : EPH(8%)). X.J. Loh et al. / Biomaterials 28 (2007) 4113–4123 4116  FTIR was used to probe the molecular changesoccurring in the polymer segments after various periodsof degradation (Fig. 3). In the original un-degradedsample, the PHB ester peak corresponding to 1721cm  1 can be observed, along with a small peak at 1660cm  1 which corresponds to the –C Q O stretch of the urethanepeak. The gel residue obtained after 1 month of hydrolysisshowed a broadening as well as a shift of the peak to1632cm  1 (attributed to the –C Q O carboxylic stretching)and a concomitant decrease in the height of the 1721cm  1 ester peak was observed. The water-soluble fraction of thehydrolysis products after 1 month show that the ratio of the peak height at 1721cm  1 to the peak height at1632cm  1 greatly decreased. This confirms that the esterbonds were hydrolysed to the carboxylic acid groups. After6 months of hydrolysis, the ratio of the peak height at1721cm  1 to the peak height at 1632cm  1 decreased evenfurther implying further scission of the PHB segments inthe water-soluble fraction after 6 months of hydrolysis.When the PHB segment hydrolyses to form 3-hydroxybu-tyric acid, the number of hydroxyl and carboxylic acidgroups increases. In Figs. 4b–d, two peaks are observed inthe –OH stretching region. The 3500cm  1 peak corre-sponds to the –OH stretch of the hydroxyl moiety while thepeak observed at between 3250 and 3300cm  1 correspondsto the –OH stretch of the carboxylic acid moiety. It can beobserved that the peak corresponding to the –OH stretch of the carboxylic acid moiety is absent in the FTIR spectrumof the srcinal un-degraded polymer sample. 3.2.2. Characterization of the degradation productsextracted by chloroform At various time points during the hydrolytic degradationexperiments, the buffer solutions containing degradationproducts were extracted by chloroform. We hypothesizethat the chloroform extracts mainly contained the copoly-mer degradation products which still have large molecularweight. The very short fragments such as 3-hydroxybutyricacid or its oligomers tend to remain in the buffer solution. ARTICLE IN PRESS EPH (2%)EPH (5%)EPH (8%) Fig. 2. SEM images of hydrogel residues after various periods of degradation in PBS at pH 7.4 and 37 1 C. abdc 1000150020002500300035004000 Wavenumber (cm -1 )1632 cm -1 x 53500 cm -1 3280 cm -1 1721 cm -1 Fig. 3. FTIR spectra of the EPH(5%) samples after different periods of degradation in PBS at pH 7.4 and 37 1 C. (a) Original EPH(5%) sample,(b) gel residue after 1 month of degradation, (c) water-soluble fractionafter 1 month of degradation, (d) water-soluble fraction after 6 months of degradation. X.J. Loh et al. / Biomaterials 28 (2007) 4113–4123  4117
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