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Synthesis, characterization and hydrolytic degradation of linear and crosslinked poly[(glycino ethyl ester)(allyl amino)phosphazene]
Polymer Degradation and Stability 92 (2007) 795e801 www.elsevier.com/locate/polydegstab
Synthesis, characterization and hydrolytic degradation of linear and crosslinked poly[(glycino ethyl ester)(allyl amino)phosphazene] Lin Yin, Xiaobin Huang*, Xiaozhen Tang School of Chemistry and Chemical Technology, Shanghai Jiao Tong University, Shanghai 200240, China Received 22 November 2006; received in revised form 31 January 2007; accepted 4 February 2007 Available online 14 February 2007
Abstract Polyorganophosphazenes substituted by glycino ethyl ester and allylamine with different ratios were synthesized and their structures were characterized by 1H NMR, 31P NMR and FTIR. Via the crosslink reaction, a novel biodegradable crosslinked polyorganophosphazene material was obtained. DSC and FTIR spectra indicated the occurrence of crosslink. Hydrolysis studies were also performed to compare the crosslinked polymers with linear ones. The co-substituted polyorganophosphazenes with more allylamine at pendant groups exhibited a lower degradation rate than poly[bis(glycino ethyl ester)phosphazene] and crosslinked polyphosphazenes had an even lower degradation rate. SEM photographs characterized the surface of polyphosphazenes films after hydrolytic degradation, confirming that uncrosslinked ones had outstanding hydrolytic evidences at the surface while the crosslinked ones only had sporadic small erosion holes, remaining much smoother. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Polyphosphazenes; Crosslink; Degradation; Glycino ethyl ester; Allylamine
1. Introduction Polyorganophosphazenes are a class of polymers of great potential for biomedical and pharmaceutical applications [1]. These polymers consist of an inorganic backbone of phosphorous and nitrogen with alternating double and single bonds bearing side group substituents at the phosphorus atom [2e5]. Interest in them relies on the fact that their physicale chemical properties are mainly dictated by the properties of the substituents [3,6], so specific polyorganophosphazenes with different substituents exhibit useful properties in many different fields. In order to develop polyorganophosphazenes that could respond to the requisites for biomedical applications, amino acid esters have been suggested as substituents since they have shown their biocompatibility and biodegradability [7], and the hydrolysis products are harmless small molecules
* Corresponding author. Tel.: þ86 21 5474 7142; fax: þ86 21 5474 3264. E-mail address: [email protected] (X. Huang). 0141-3910/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.polymdegradstab.2007.02.001
such as amino acid, phosphoric acid, and ammonia [8,9]. Many different kinds of alkyl or aryl amino acid ester substituted polyphosphazenes have been synthesized, and hydrolysis studies have also been performed to estimate decomposition rates and mechanics of this kind of polymers [10]. They always have broad applications in biomedicine such as absorbable implanted materials and substrates for the controlled release of drugs [11,12]. Polymeric scaffolds implanted at the defective site have great applications in tissue engineering [13], and polyorganophosphazenes have the irresistible advantage that the suitable choice of the side groups allows modulation of mechanical properties, surface characteristics and hydrolytic sensitivity [14]. Thus, these polymeric materials have been successfully used as solid matrices to repair peripheral nerves in rats [15] and to improve bone regeneration [16]. However, poly[(amino acid ester)phosphazenes] always have very high hydrolytic rate [10]. Therefore, hydrolytically insensitive side groups, such as methylphenoxy [11] and phenylphenoxy [17,18], have been introduced to modify the surface hydrophilicity and hydrolysis rate of polyphosphazenes. Another method to
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achieve these goals is by blending polyorganophosphazenes with some traditional biodegradable polymeric materials, such as poly(a-hydroxyester) [19] and polyanhydride [20]. It will also offer the feasibility to fabricate new polymeric materials whose degradation kinetics can be modified by varying the blend composition over a wide range. However, the hydrolytically insensitive groups always contain aromatic rings whose hydrolysis products will be harmful to organic tissue. Meanwhile the compatibility of polyorganophosphazene and other polymers have to be considered if we want to modify them via blending. To overcome these limitations, we designed the novel polyphosphazenes with glycino ethyl ester and allylamine as co-substituent, poly[(glycino ethyl ester)(allyl amino)phosphazene], and tried to prolong the hydrolysis period through crosslink. Allylamine is an important monomer whose polymerized products have widespread applications in microencapsulation and gene transfer vectors as cationic polymers [21e24]. Through the crosslink reaction initiated by BPO, crosslinked polyphosphazenes with modified hydrolytic sensitivity and surface hydrophilicity could be obtained. These modified properties lied on the degree of crosslink, and the degree of crosslink would be dominated by the ratio of glycino ethyl ester and allylamine. Hydrolytic degradation of linear and crosslinked polymers at 37 C were also studied to test the mass loss and hydrolytic surfaces. 2. Materials and methods 2.1. Materials PCl5 was purified by sublimation. NH4Cl was dried at 70 C in a vacuum oven. 1,2,4-Trichlorobenzene was used freshly distilled and THF was distilled twice from Na. Sulfamic acid, calcium sulfate dehydrate, triethylamine and allylamine were used as purchased. Glycine ethyl ester hydrochloride was treated with triethylamine in distilled THF at reflux temperature for 24 h, and then the solution was filtered to remove triethylamine hydrochloride. Resultant glycine ethyl ester solution of THF was stored in a desiccator for use. 2.2. Measurements 1
H NMR (400 MHz), 31P NMR (161.9 MHz) spectra were recorded using a Varian Mercury Plus-400 NMR spectrometer (Varian, USA) in the Fourier transform mode with CDCl3 as solvents. Infrared spectra were measured as films cast on sodium chloride plate with a PerkineElmer 621 instrument (Perkine Elmer, USA). C, H, N elemental analyses were carried out with a Vario EL-III instrument (Elementar, Ger.). Cl elemental analyses were carried out with a Metrohm MIC-IC instrument (Metrohm, Switz.). Gel permeation chromatograms were obtained with the use of a PE Series-200 instrument (PerkineElmer, USA) in DMF as solvent. The glass transition temperatures (Tg) were measured with a PerkineElmer Pyris 2 DSC analyzer (PerkineElmer, USA) at a heating rate of 10 C/min under nitrogen purge. Static water contact angles were measured with an OCA20 instrument (Dataphysics, Ger.). Field emission
scanning electron microscope images were obtained using an S-2150 instrument (Hitachi, Japan) at an activation voltage of 15 kV. 2.3. Syntheses of polymers Poly(dichlorophosphazene) was synthesized directly with PCl5 and NH4Cl in a solution of 1,2,4-trichlorobenzene in the presence of sulfamic acid and calcium sulfate dehydrate [25]. Characteristic 31P NMR (d, ppm): 17.01. IR (cm1): 1207, 1134 (phosphazene backbone), 580, 522 (PeCl). As shown in Scheme 1 (a), excess glycino ethyl ester with triethylamine were added to the solution of poly(dichlorophosphazene) in THF at ambient temperature to substitute all chlorine atoms and get poly[bis(glycino ethyl ester)phosphazene], polymer 1. Characteristic IR (cm1): 3216 (NeH), 2980 (Ce H), 1737 (C]O), 1400 (eCH2e), 1207, 1134 (phosphazene backbone), 1090 (CeO). Molecular weight: Mw ¼ 3.7 104, Mw/Mn ¼ 1.45. As shown in Scheme 1 (b), stoichiometric allylamine and excess glycino ethyl ester, in the presence of excess triethylamine, were sequentially added to the solution of poly(dichlorophosphazene) in THF to get poly[(glycino ethyl ester)(allyl amino)phosphazene], polymer 2 (GE:AA ¼ 2:1 by mole) and polymer 3 (GE:AA ¼ 1:2 by mole). Characteristic IR (cm1): 3216 (NeH), 2980 (CeH), 1737 (C]O), 1649 (C]C), 1400 (eCH2e), 1207, 1134 (phosphazene backbone), 1090 (CeO), 1023, 921 (RCH]CH2). Molecular weight: polymer 2: Mw ¼ 4.6 104, Mw/Mn ¼ 1.84; polymer 3: Mw ¼ 2.5 104, Mw/Mn ¼ 1.75. The insoluble salts were removed by filtration and yellowish, adhesive polymers were obtained by precipitation of the viscous polymer solutions into petroleum ether. Purification of the polymers was accomplished by repeated precipitation from THF into ethanol and petroleum ether. 2.4. Crosslink of co-substituted polymers Polymer 2 (or polymer 3) and initiator BPO (0.5 wt%) were dissolved in THF, cast on sodium chloride plate. Then they were incubated together at 80 C in a vacuum oven for 2e3 h and the infrared spectra of crosslinked polymer were measured. Polymer 2 (or polymer 3) and initiator BPO (0.5 wt%) were dissolved in THF and then made into films using the solution casting method. After volatilization of the solvent, polymers and the initiator were mixed together and became films of about 100 mm in thickness. The films were then incubated at 80 C in a vacuum oven for 2e3 h, obtaining crosslinked polymer films. 2.5. Hydrolytic degradation studies The degradation characteristics of polymers were evaluated by measuring the weight loss. Preweighed polymer films, about 100 mm in thickness, were immersed in phosphate buffer of pH 7.4, at 37 C. Samples were recovered periodically, dried in a vacuum desiccator and then weighted to determine the weight loss. The hydrolytic surfaces of samples were tested by SEM.
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Scheme 1. Reaction equations of (a) poly[(glycino ethyl ester)phosphazene], polymer 1, (b) poly[(glycino ethyl ester)(allyl amino)phosphazene], polymer 2 (GE:AA ¼ 2:1 by mole) and polymer 3 (GE:AA ¼ 1:2 by mole).
3. Results and discussion 3.1. Structural characterization The 1H NMR spectra of polymers 1e3 are shown in Fig. 1. Compared with co-substituted products, polymers 2 and 3, the three peaks a, b and e, which are corresponding to the protons of allylamine, don’t appear at the spectrum of fully substituted product polymer 1. And the integration data indicated that the ratios of two substituents of polymers 2 and 3 were the same as designed.
The 31P NMR spectra of polymers 1e3 are shown in Fig. 2. The spectrum of polymer 1 shows only one broad peak at 2.94 ppm, corresponding to the phosphorous atom with fully substituted glycino ethyl ester as side groups. While the spectra of polymers 2 and 3 show the other broad peak at 3.48 ppm, indicating the occurrence of mixed substitution. The data of elemental analysis are shown in Table 1, which further confirm the polymer structures and the complete substitution of side chlorine of the parent polyphosphazene.
Fig. 1. 1H NMR spectra of polymers 1e3; CDCl3 was used as solvent.
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Fig. 2.
31
P NMR spectra of polymers 1e3; CDCl3 was used as solvent.
3.2. Crosslink of linear polymer chains The linear polymers were yellowish, rubbery, and a little adhesive, had very weak mechanical strength. Crosslink could be a convenient and effective method to modify its physical properties for practical applications. After 2e3 h incubation, crosslinked polymers were not adhesive, and exhibited increased hardness and toughness. But incubation time cannot be too long. Chain scission easily happened after more than 5 h incubation at 80 C, obtaining very adhesive polymer films, which would completely degrade to water-soluble segments in less than one day. 3.2.1. DSC measurements The glass transition temperatures (Tg) of linear polymers 2 and 3 are, respectively, 23.6 C and 19.3 C, while corresponding crosslinked polymers have no outstanding glass transition until 150 C as shown in Fig. 3. This phenomenon confirmed the occurrence of crosslink reaction, and might be caused by the decrease of free volumes along with the increase of crosslinking points, thus the movements of chain segments would be confined and no glass transition could be detected. It was also assumed that the glass transition would not appear at more than 150 C, because even higher temperature might easily result in thermolysis. Table 1 Elemental analyses of polymers 1e3 Polymer 1 2 3
Calcd. Found Calcd. Found Calcd. Found
C (%)
H (%)
N (%)
Cl (%)
38.55 38.88 40.31 40.84 42.63 41.66
6.43 6.08 6.72 6.72 7.10 6.97
16.87 16.66 19.24 19.72 22.38 22.84
0 0.02 0 0 0 0
3.2.2. FTIR spectra Infrared spectroscopy reveals features consistent with the crosslink at double bonds between carbon atoms as shown in Fig. 4. The peak at 1649 cm1 which is corresponding to stretching vibration of C]C becomes smaller obviously compared with the spectra of linear polymers. Another change arises at 1023 cm1 and 921 cm1. The two peaks in this spectral region are corresponding to the out-of-plane bending vibration of RCH]CH2 group and their disappearance proves that the crosslink reaction has occurred. 3.3. Surface wetting properties The surface wetting properties of polymers 1e3 and crosslinked counterparts of polymers 2 and 3 were examined by static water contact angle measurements as shown in Table 2. Co-substitution of allyl amino groups with glycine ethyl ester units increased the surface hydrophobicity and gave the contact angles from 52 to 69 , resulting in the decrease of hydrolytic rate. Compared to the linear ones, crosslinked polymer films had hydrophobic surfaces and gave the contact angles of 91 and 96 . 3.4. Hydrolytic degradation The weight loss curves of polyphosphazenes with different ratios of the two substituents and the crosslinked counterparts of polymers 2 and 3 are shown in Fig. 5. The rate of degradation decreased as the allylamine increases at the pendant groups. In our original design, the different amounts of allylamine substituted at the pendant groups were only used to dominate the degree of crosslink. But the introduction of allylamine had changed the surface hydrophilicity of polymer films, which was examined with contact angle measurements, thus the rate of weight loss decreased.
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Fig. 4. Infrared spectra of (a) linear and (b) crosslinked polymer 2.
was full of cracks comprising of small holes which were caused by hydrolytic degradation, so that the whole film was very easy to fracture into small fragments. The films of linear polymer 2 and polymer 3 also had an outstanding degradation situation. There were clear erosion holes arising at the film surface. But being different from films of polymer 1, the films of cosubstituted polyphosphazenes could hold their shape completely and no obvious cracks appeared though they also became soft after degradation in phosphate buffer of pH 7.4. The SEM photographs of crosslinked polymer films that degraded for 10 days are shown in Fig. 7. The hydrolytic situations were absolutely different from linear ones. There were only sporadic small erosion holes appeared at the film surface and the films could retain their mechanical properties from crosslink, remaining tough and hard. Films with higher crosslinking degree had smoother hydrolytic surfaces because of the surface hydrophobicity resulted from crosslink. 4. Conclusion Fig. 3. (a) DSC thermograms of linear and crosslinked polymer 2 and (b) DSC thermograms of linear and crosslinked polymer 3.
In contrast, the films of crosslinked polymers had a lower degradation rate and the more allylamine substituted at pendant groups, which would result in a higher degree of crosslink, the slower degradation rate was. According to the hydrolyzation mechanism, the route by which poly[(amino acid ester)phosphazenes] hydrolyze always began with the breakdown of the ester unit [10]. Thus, both the glycino ethyl ester at the pendant groups and the polyphosphazene skeleton could be protected against hydrolysis by the crosslinked allyl amino groups, leading to a lower degradation rate. The SEM photographs of linear polymer films degraded for 10 days are given in Fig. 6. The degradation of polyphosphazenes fully substituted by glycino ethyl ester was much more obvious than co-substitution products. The film surface
Polyorganophosphazenes substituted by glycino ethyl ester and allylamine with different ratios were synthesized. DSC and FTIR spectra indicated the occurrence of crosslink reaction. Then via hydrolysis studies, it was confirmed that crosslink was an available method to modify the hydrolytic sensitivity of biodegradable polyorganophosphazenes, and the degradation rate was dominated by the ratio of
Table 2 Static water contact angle on linear polymer films 1e3 and crosslinked polymer films 2 and 3 Polymer
1 2 3
Contact angle ( ) Linear
Crosslinked
52 62 69
e 91 96
800
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Fig. 6. SEM photographs of linear (a) polymer 1, (b) polymer 2, and (c) polymer 3 after hydrolytic degradation in phosphate buffer of pH 7.4 for 10 days.
Fig. 5. Weight loss of (a) polymers 1e3, (b) linear and crosslinked polymer 2, and (c) linear and crosslinked polymer 3 due to hydrolytic degrading in phosphate buffer of pH 7.4.
allylamine at pendant groups. The different hydrolytic erosion surfaces of linear polymeric films and corresponding crosslinked ones were characterized by SEM. Thus, a novel kind of degradable polymers with appropriate degradation rate and adjustable surface hydrolysis property, which can be used for tissue and bone repair scaffolds suited for various interfaces and for other potential biomedical applications, was obtained.
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Fig. 7. SEM photographs of crosslinked (a) polymer 2 and (b) polymer 3 after hydrolytic degradation in phosphate buffer of pH 7.4 for 10 days.
References [1] Crommen J, Vandorpe J, Schacht E. Degradable polyphosphazenes for biomedical applications. J Controlled Release 1993;24(1e3):167e80. [2] Allcock HR, Kugel RL. Synthesis of high polymeric alkoxy- and aryloxyphosphonitriles. J Am Chem Soc 1965;87(18):4216e7. [3] Neilson RH, Neilson PW. Poly(alkyl/arylphosphazenes) and their precursors. Chem Rev 1988;88(3):541e62. [4] Allcock HR. Poly(organophosphazenes) e unusual new high polymers. Angew Chem Int Ed Engl 1977;16(3):147e56. [5] Allcock HR. Rational design and synthesis of new polymeric materials. Science 1992;255:1106e12. [6] Schacht E, Vandorpe J, Dejardin S, Lemmouchi Y, Seymour L. Biomedical applications of degradable polyphosphazenes. Biotechnol Bioeng 1996;52(1):102e8. [7] Allcock HR, Shawn R, Scopelianos AG. Poly[(amino acid ester)phosphazenes] as substrates for the controlled release of small molecules. Biomaterials 1994;15(8):563e9.
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[8] Allcock HR, Fuller TJ, Mack DP, Matsumura K, Smeltz KM. Synthesis of poly[(amino acid alkyl ester)phosphazenes]. Macromolecules 1977;10(4): 824e30. [9] Lemmouchi Y, Schacht E, Dejardin S. Biodegradable poly[(amino acid ester)phosphazenes] for biomedical applications. J Bioact Compat Polym 1998;13(1):4e18. [10] Allcock HR, Pucher SR. Poly[(amino acid ester)phosphazenes]: synthesis, crystallinity, and hydrolytic sensitivity in solution and the solid state. Macromolecules 1994;27(5):1071e5. [11] Laurencin CT, El-Amin SF, Ibim SE, Willoughby DA, Attawia M, Allcock HR, et al. A highly porous 3-dimensional polyphosphazene polymer matrix for skeletal tissue regeneration. J Biomed Mater Res 1996;30(2):133e8. [12] Ibim SM, Ambrosio AA, Larrier D, Allcock HR, Laurencin CT. Controlled macromolecule release from poly(phosphazene) matrices. J Controlled Release 1996;40(1e2):31e9. [13] Grodzinski JJ. Polymers for tissue engineering, medical devices, and regenerative medicine. Concise general review of recent studies. Polym Adv Technol 2006;17(6):395e418. [14] Conconi MT, Lora S, Baiguera S, Boscolo E, Folin M, Scienza R, et al. In vitro culture of rat neuromicrovascular endothelial cells on polymeric scaffolds. J Biomed Mater Res Part A 2004;71(4):669e74. [15] Langone F, Lora S, Veronese FM, Caliceti P, Parnigotto PP, Valenti F, et al. Peripheral nerve repair using a poly(organo)phosphazene tubular prosthesis. Biomaterials 1995;16(5):347e53. [16] Nordstrom P, Pihlajamaki H, Toivonen T, Tormala P, Rokkanen P. Tissue response to polyglycolide and polylevolactide pins in osteotomized cancellous bone. Clin Orthop Relat Res 2001;382:247e57. [17] Sethuraman S, Nair LS, El-Amin S, Farrar R, Nguyen MN, Singh A, et al. In vivo biodegradability and biocompatibility evaluation of novel alanine ester based polyphosphazenes in a rat model. J Biomed Mater Res Part A 2006;77(4):679e87. [18] Singh A, Krogman NR, Sethuraman S, Nair LS, Sturgeon JL, Brown PW, et al. Effect of side group chemistry on the properties of biodegradable Lalanine cosubstituted polyphosphazenes. Biomacromolecules 2006;7(3): 914e8. [19] Ambrosio AMA, Allcock HR, Katti DS, Laurencin CT. Degradable polyphosphazene/poly(a-hydroxyester) blends: degradation studies. Biomaterials 2002;23(7):1667e72. [20] Qiu LY, Zhu KJ. Novel blends of poly[bis(glycine ethyl ester) phosphazene] and polyesters or polyanhydrides: compatibility and degradation characteristics in vitro. Polym Int 2000;49(11):1283e8. [21] Boussif O, Delair T, Brua C, Veron L, Pavirani A, Kolbe HVJ. Synthesis of polyallylamine derivatives and their use as gene transfer vectors in vitro. Bioconjugate Chem 1999;10(5):877e83. [22] Carretti E, Dei L, Baglioni P, Weiss RG. Synthesis and characterization of gels from polyallylamine and carbon dioxide as gellant. J Am Chem Soc 2003;125(17):5121e9. [23] Forzani ES, Solis VM, Calvo EJ. Electrochemical behavior of polyphenol oxidase immobilized in self-assembled structures layer by layer with cationic polyallylamine. Anal Chem 2000;72(21):5300e7. [24] Lu MZ, Lan HL, Wang FF, Chang SJ, Wang YJ. Cell encapsulation with alginate and a-phenoxycinnamylidene-acetylated poly(allylamine). Biotechnol Bioeng 2000;70(5):479e83. [25] Carriedo GA, Garcı´a Alonso FJ, Go´mez-Elipe P, Fidalgo JI, Garcı´a Alvarez JL, Presa-Soto A. A simplified and convenient laboratory-scale preparation of 14N or 15N high molecular weight poly(dichlorophosphazene) directly from PCl5. Chem Eur J 2003;9(16):3833e6.
Synthesis, characterization and hydrolytic degradation of linear and crosslinked poly[(glycino ethyl ester)(allyl amino)phosphazene] Lin Yin, Xiaobin Huang*, Xiaozhen Tang School of Chemistry and Chemical Technology, Shanghai Jiao Tong University, Shanghai 200240, China Received 22 November 2006; received in revised form 31 January 2007; accepted 4 February 2007 Available online 14 February 2007
Abstract Polyorganophosphazenes substituted by glycino ethyl ester and allylamine with different ratios were synthesized and their structures were characterized by 1H NMR, 31P NMR and FTIR. Via the crosslink reaction, a novel biodegradable crosslinked polyorganophosphazene material was obtained. DSC and FTIR spectra indicated the occurrence of crosslink. Hydrolysis studies were also performed to compare the crosslinked polymers with linear ones. The co-substituted polyorganophosphazenes with more allylamine at pendant groups exhibited a lower degradation rate than poly[bis(glycino ethyl ester)phosphazene] and crosslinked polyphosphazenes had an even lower degradation rate. SEM photographs characterized the surface of polyphosphazenes films after hydrolytic degradation, confirming that uncrosslinked ones had outstanding hydrolytic evidences at the surface while the crosslinked ones only had sporadic small erosion holes, remaining much smoother. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Polyphosphazenes; Crosslink; Degradation; Glycino ethyl ester; Allylamine
1. Introduction Polyorganophosphazenes are a class of polymers of great potential for biomedical and pharmaceutical applications [1]. These polymers consist of an inorganic backbone of phosphorous and nitrogen with alternating double and single bonds bearing side group substituents at the phosphorus atom [2e5]. Interest in them relies on the fact that their physicale chemical properties are mainly dictated by the properties of the substituents [3,6], so specific polyorganophosphazenes with different substituents exhibit useful properties in many different fields. In order to develop polyorganophosphazenes that could respond to the requisites for biomedical applications, amino acid esters have been suggested as substituents since they have shown their biocompatibility and biodegradability [7], and the hydrolysis products are harmless small molecules
* Corresponding author. Tel.: þ86 21 5474 7142; fax: þ86 21 5474 3264. E-mail address: [email protected] (X. Huang). 0141-3910/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.polymdegradstab.2007.02.001
such as amino acid, phosphoric acid, and ammonia [8,9]. Many different kinds of alkyl or aryl amino acid ester substituted polyphosphazenes have been synthesized, and hydrolysis studies have also been performed to estimate decomposition rates and mechanics of this kind of polymers [10]. They always have broad applications in biomedicine such as absorbable implanted materials and substrates for the controlled release of drugs [11,12]. Polymeric scaffolds implanted at the defective site have great applications in tissue engineering [13], and polyorganophosphazenes have the irresistible advantage that the suitable choice of the side groups allows modulation of mechanical properties, surface characteristics and hydrolytic sensitivity [14]. Thus, these polymeric materials have been successfully used as solid matrices to repair peripheral nerves in rats [15] and to improve bone regeneration [16]. However, poly[(amino acid ester)phosphazenes] always have very high hydrolytic rate [10]. Therefore, hydrolytically insensitive side groups, such as methylphenoxy [11] and phenylphenoxy [17,18], have been introduced to modify the surface hydrophilicity and hydrolysis rate of polyphosphazenes. Another method to
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achieve these goals is by blending polyorganophosphazenes with some traditional biodegradable polymeric materials, such as poly(a-hydroxyester) [19] and polyanhydride [20]. It will also offer the feasibility to fabricate new polymeric materials whose degradation kinetics can be modified by varying the blend composition over a wide range. However, the hydrolytically insensitive groups always contain aromatic rings whose hydrolysis products will be harmful to organic tissue. Meanwhile the compatibility of polyorganophosphazene and other polymers have to be considered if we want to modify them via blending. To overcome these limitations, we designed the novel polyphosphazenes with glycino ethyl ester and allylamine as co-substituent, poly[(glycino ethyl ester)(allyl amino)phosphazene], and tried to prolong the hydrolysis period through crosslink. Allylamine is an important monomer whose polymerized products have widespread applications in microencapsulation and gene transfer vectors as cationic polymers [21e24]. Through the crosslink reaction initiated by BPO, crosslinked polyphosphazenes with modified hydrolytic sensitivity and surface hydrophilicity could be obtained. These modified properties lied on the degree of crosslink, and the degree of crosslink would be dominated by the ratio of glycino ethyl ester and allylamine. Hydrolytic degradation of linear and crosslinked polymers at 37 C were also studied to test the mass loss and hydrolytic surfaces. 2. Materials and methods 2.1. Materials PCl5 was purified by sublimation. NH4Cl was dried at 70 C in a vacuum oven. 1,2,4-Trichlorobenzene was used freshly distilled and THF was distilled twice from Na. Sulfamic acid, calcium sulfate dehydrate, triethylamine and allylamine were used as purchased. Glycine ethyl ester hydrochloride was treated with triethylamine in distilled THF at reflux temperature for 24 h, and then the solution was filtered to remove triethylamine hydrochloride. Resultant glycine ethyl ester solution of THF was stored in a desiccator for use. 2.2. Measurements 1
H NMR (400 MHz), 31P NMR (161.9 MHz) spectra were recorded using a Varian Mercury Plus-400 NMR spectrometer (Varian, USA) in the Fourier transform mode with CDCl3 as solvents. Infrared spectra were measured as films cast on sodium chloride plate with a PerkineElmer 621 instrument (Perkine Elmer, USA). C, H, N elemental analyses were carried out with a Vario EL-III instrument (Elementar, Ger.). Cl elemental analyses were carried out with a Metrohm MIC-IC instrument (Metrohm, Switz.). Gel permeation chromatograms were obtained with the use of a PE Series-200 instrument (PerkineElmer, USA) in DMF as solvent. The glass transition temperatures (Tg) were measured with a PerkineElmer Pyris 2 DSC analyzer (PerkineElmer, USA) at a heating rate of 10 C/min under nitrogen purge. Static water contact angles were measured with an OCA20 instrument (Dataphysics, Ger.). Field emission
scanning electron microscope images were obtained using an S-2150 instrument (Hitachi, Japan) at an activation voltage of 15 kV. 2.3. Syntheses of polymers Poly(dichlorophosphazene) was synthesized directly with PCl5 and NH4Cl in a solution of 1,2,4-trichlorobenzene in the presence of sulfamic acid and calcium sulfate dehydrate [25]. Characteristic 31P NMR (d, ppm): 17.01. IR (cm1): 1207, 1134 (phosphazene backbone), 580, 522 (PeCl). As shown in Scheme 1 (a), excess glycino ethyl ester with triethylamine were added to the solution of poly(dichlorophosphazene) in THF at ambient temperature to substitute all chlorine atoms and get poly[bis(glycino ethyl ester)phosphazene], polymer 1. Characteristic IR (cm1): 3216 (NeH), 2980 (Ce H), 1737 (C]O), 1400 (eCH2e), 1207, 1134 (phosphazene backbone), 1090 (CeO). Molecular weight: Mw ¼ 3.7 104, Mw/Mn ¼ 1.45. As shown in Scheme 1 (b), stoichiometric allylamine and excess glycino ethyl ester, in the presence of excess triethylamine, were sequentially added to the solution of poly(dichlorophosphazene) in THF to get poly[(glycino ethyl ester)(allyl amino)phosphazene], polymer 2 (GE:AA ¼ 2:1 by mole) and polymer 3 (GE:AA ¼ 1:2 by mole). Characteristic IR (cm1): 3216 (NeH), 2980 (CeH), 1737 (C]O), 1649 (C]C), 1400 (eCH2e), 1207, 1134 (phosphazene backbone), 1090 (CeO), 1023, 921 (RCH]CH2). Molecular weight: polymer 2: Mw ¼ 4.6 104, Mw/Mn ¼ 1.84; polymer 3: Mw ¼ 2.5 104, Mw/Mn ¼ 1.75. The insoluble salts were removed by filtration and yellowish, adhesive polymers were obtained by precipitation of the viscous polymer solutions into petroleum ether. Purification of the polymers was accomplished by repeated precipitation from THF into ethanol and petroleum ether. 2.4. Crosslink of co-substituted polymers Polymer 2 (or polymer 3) and initiator BPO (0.5 wt%) were dissolved in THF, cast on sodium chloride plate. Then they were incubated together at 80 C in a vacuum oven for 2e3 h and the infrared spectra of crosslinked polymer were measured. Polymer 2 (or polymer 3) and initiator BPO (0.5 wt%) were dissolved in THF and then made into films using the solution casting method. After volatilization of the solvent, polymers and the initiator were mixed together and became films of about 100 mm in thickness. The films were then incubated at 80 C in a vacuum oven for 2e3 h, obtaining crosslinked polymer films. 2.5. Hydrolytic degradation studies The degradation characteristics of polymers were evaluated by measuring the weight loss. Preweighed polymer films, about 100 mm in thickness, were immersed in phosphate buffer of pH 7.4, at 37 C. Samples were recovered periodically, dried in a vacuum desiccator and then weighted to determine the weight loss. The hydrolytic surfaces of samples were tested by SEM.
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Scheme 1. Reaction equations of (a) poly[(glycino ethyl ester)phosphazene], polymer 1, (b) poly[(glycino ethyl ester)(allyl amino)phosphazene], polymer 2 (GE:AA ¼ 2:1 by mole) and polymer 3 (GE:AA ¼ 1:2 by mole).
3. Results and discussion 3.1. Structural characterization The 1H NMR spectra of polymers 1e3 are shown in Fig. 1. Compared with co-substituted products, polymers 2 and 3, the three peaks a, b and e, which are corresponding to the protons of allylamine, don’t appear at the spectrum of fully substituted product polymer 1. And the integration data indicated that the ratios of two substituents of polymers 2 and 3 were the same as designed.
The 31P NMR spectra of polymers 1e3 are shown in Fig. 2. The spectrum of polymer 1 shows only one broad peak at 2.94 ppm, corresponding to the phosphorous atom with fully substituted glycino ethyl ester as side groups. While the spectra of polymers 2 and 3 show the other broad peak at 3.48 ppm, indicating the occurrence of mixed substitution. The data of elemental analysis are shown in Table 1, which further confirm the polymer structures and the complete substitution of side chlorine of the parent polyphosphazene.
Fig. 1. 1H NMR spectra of polymers 1e3; CDCl3 was used as solvent.
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Fig. 2.
31
P NMR spectra of polymers 1e3; CDCl3 was used as solvent.
3.2. Crosslink of linear polymer chains The linear polymers were yellowish, rubbery, and a little adhesive, had very weak mechanical strength. Crosslink could be a convenient and effective method to modify its physical properties for practical applications. After 2e3 h incubation, crosslinked polymers were not adhesive, and exhibited increased hardness and toughness. But incubation time cannot be too long. Chain scission easily happened after more than 5 h incubation at 80 C, obtaining very adhesive polymer films, which would completely degrade to water-soluble segments in less than one day. 3.2.1. DSC measurements The glass transition temperatures (Tg) of linear polymers 2 and 3 are, respectively, 23.6 C and 19.3 C, while corresponding crosslinked polymers have no outstanding glass transition until 150 C as shown in Fig. 3. This phenomenon confirmed the occurrence of crosslink reaction, and might be caused by the decrease of free volumes along with the increase of crosslinking points, thus the movements of chain segments would be confined and no glass transition could be detected. It was also assumed that the glass transition would not appear at more than 150 C, because even higher temperature might easily result in thermolysis. Table 1 Elemental analyses of polymers 1e3 Polymer 1 2 3
Calcd. Found Calcd. Found Calcd. Found
C (%)
H (%)
N (%)
Cl (%)
38.55 38.88 40.31 40.84 42.63 41.66
6.43 6.08 6.72 6.72 7.10 6.97
16.87 16.66 19.24 19.72 22.38 22.84
0 0.02 0 0 0 0
3.2.2. FTIR spectra Infrared spectroscopy reveals features consistent with the crosslink at double bonds between carbon atoms as shown in Fig. 4. The peak at 1649 cm1 which is corresponding to stretching vibration of C]C becomes smaller obviously compared with the spectra of linear polymers. Another change arises at 1023 cm1 and 921 cm1. The two peaks in this spectral region are corresponding to the out-of-plane bending vibration of RCH]CH2 group and their disappearance proves that the crosslink reaction has occurred. 3.3. Surface wetting properties The surface wetting properties of polymers 1e3 and crosslinked counterparts of polymers 2 and 3 were examined by static water contact angle measurements as shown in Table 2. Co-substitution of allyl amino groups with glycine ethyl ester units increased the surface hydrophobicity and gave the contact angles from 52 to 69 , resulting in the decrease of hydrolytic rate. Compared to the linear ones, crosslinked polymer films had hydrophobic surfaces and gave the contact angles of 91 and 96 . 3.4. Hydrolytic degradation The weight loss curves of polyphosphazenes with different ratios of the two substituents and the crosslinked counterparts of polymers 2 and 3 are shown in Fig. 5. The rate of degradation decreased as the allylamine increases at the pendant groups. In our original design, the different amounts of allylamine substituted at the pendant groups were only used to dominate the degree of crosslink. But the introduction of allylamine had changed the surface hydrophilicity of polymer films, which was examined with contact angle measurements, thus the rate of weight loss decreased.
L. Yin et al. / Polymer Degradation and Stability 92 (2007) 795e801
799
Fig. 4. Infrared spectra of (a) linear and (b) crosslinked polymer 2.
was full of cracks comprising of small holes which were caused by hydrolytic degradation, so that the whole film was very easy to fracture into small fragments. The films of linear polymer 2 and polymer 3 also had an outstanding degradation situation. There were clear erosion holes arising at the film surface. But being different from films of polymer 1, the films of cosubstituted polyphosphazenes could hold their shape completely and no obvious cracks appeared though they also became soft after degradation in phosphate buffer of pH 7.4. The SEM photographs of crosslinked polymer films that degraded for 10 days are shown in Fig. 7. The hydrolytic situations were absolutely different from linear ones. There were only sporadic small erosion holes appeared at the film surface and the films could retain their mechanical properties from crosslink, remaining tough and hard. Films with higher crosslinking degree had smoother hydrolytic surfaces because of the surface hydrophobicity resulted from crosslink. 4. Conclusion Fig. 3. (a) DSC thermograms of linear and crosslinked polymer 2 and (b) DSC thermograms of linear and crosslinked polymer 3.
In contrast, the films of crosslinked polymers had a lower degradation rate and the more allylamine substituted at pendant groups, which would result in a higher degree of crosslink, the slower degradation rate was. According to the hydrolyzation mechanism, the route by which poly[(amino acid ester)phosphazenes] hydrolyze always began with the breakdown of the ester unit [10]. Thus, both the glycino ethyl ester at the pendant groups and the polyphosphazene skeleton could be protected against hydrolysis by the crosslinked allyl amino groups, leading to a lower degradation rate. The SEM photographs of linear polymer films degraded for 10 days are given in Fig. 6. The degradation of polyphosphazenes fully substituted by glycino ethyl ester was much more obvious than co-substitution products. The film surface
Polyorganophosphazenes substituted by glycino ethyl ester and allylamine with different ratios were synthesized. DSC and FTIR spectra indicated the occurrence of crosslink reaction. Then via hydrolysis studies, it was confirmed that crosslink was an available method to modify the hydrolytic sensitivity of biodegradable polyorganophosphazenes, and the degradation rate was dominated by the ratio of
Table 2 Static water contact angle on linear polymer films 1e3 and crosslinked polymer films 2 and 3 Polymer
1 2 3
Contact angle ( ) Linear
Crosslinked
52 62 69
e 91 96
800
L. Yin et al. / Polymer Degradation and Stability 92 (2007) 795e801
Fig. 6. SEM photographs of linear (a) polymer 1, (b) polymer 2, and (c) polymer 3 after hydrolytic degradation in phosphate buffer of pH 7.4 for 10 days.
Fig. 5. Weight loss of (a) polymers 1e3, (b) linear and crosslinked polymer 2, and (c) linear and crosslinked polymer 3 due to hydrolytic degrading in phosphate buffer of pH 7.4.
allylamine at pendant groups. The different hydrolytic erosion surfaces of linear polymeric films and corresponding crosslinked ones were characterized by SEM. Thus, a novel kind of degradable polymers with appropriate degradation rate and adjustable surface hydrolysis property, which can be used for tissue and bone repair scaffolds suited for various interfaces and for other potential biomedical applications, was obtained.
L. Yin et al. / Polymer Degradation and Stability 92 (2007) 795e801
Fig. 7. SEM photographs of crosslinked (a) polymer 2 and (b) polymer 3 after hydrolytic degradation in phosphate buffer of pH 7.4 for 10 days.
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